This document describes a multicenter collaborative trial that validated two real-time PCR assays (GeneDisc arrays GD1 and GD2) for detecting Clostridium botulinum types C, D, C-D and D-C. The trial involved eight European laboratories testing the GeneDisc arrays on DNA from 33 C. botulinum isolates and 48 clinical samples. Results showed 99.4-100% concordance between laboratories. The assays demonstrated high reproducibility with low variability (1.1-7.1%). Given the high level of agreement, the GeneDisc PCR arrays were determined to be robust and suitable tools for rapid detection of C. botulinum types C, D and their mosaic variants. This was the first
This document describes a European ring trial study to evaluate a real-time PCR assay for detection and typing of botulinum neurotoxin-producing Clostridia types A, B, E and F in food, feed and environmental samples. A primary evaluation of the real-time PCR method showed 100% accuracy, sensitivity, specificity and selectivity compared to the reference cultural method and mouse bioassay. A ring trial conducted at four European laboratories using 47 strains and 30 clinical and food samples linked to botulism cases showed 95.7% concordance among laboratories. The reproducibility generated a relative standard deviation of 2.18-13.61%. Given the high level of agreement between laboratories, this real-time PCR method is suitable
This document describes the isolation and characterization of a new giant virus called Cedratvirus. Key points:
- Cedratvirus was isolated from an environmental sample in Algeria using Acanthamoeba castellanii.
- It has an ovoid shape with a cork structure at each end, resembling Pithovirus sibericum but with a unique double cork feature.
- The 589kb genome is most closely related to the pithovirus genomes, sharing over 100 genes, but with only 21% of genes involved in best reciprocal hits, indicating genetic distance from known pithoviruses.
This document describes a new method for detecting Staphylococcus bacteria using a specific volatile organic compound (VOC) produced by Staphylococcus called 2-[3-acetoxy-4,4,14-trimethylandrost-8-en-17-yl] propanoic acid (ATMAP). A simple colorimetric assay using methyl red as a pH indicator can specifically detect Staphylococcus by changing color from yellow to orange due to ATMAP production. The assay was tested on liquid cultures and plate cultures to detect various Staphylococcus and other bacterial strains, demonstrating 100% specificity and sensitivity. This new detection method could provide a low-cost and easy-to-use
Real time pcr assay for rapid detection and quantification of campylobacter j...Tiensae Teshome
The document describes the development of a real-time PCR (rtPCR) assay for rapid detection and quantification of Campylobacter jejuni on chicken rinses from poultry processing plants without an enrichment step. Eighty-four chicken rinse samples were collected from three processing plants and tested using both rtPCR and culture methods. Sixty-five samples were positive by rtPCR while 27 were positive by culture, with 27 samples positive by both methods. The rtPCR assay was able to detect C. jejuni with a limit of 1 CFU and provide results within 90 minutes, significantly faster than the 5-7 days required for culture.
This study compared the use of real-time polymerase chain reaction (RT-PCR) and standard isolation techniques for detecting Salmonella in broiler chicks. The incidence of Salmonella was higher in local chicks (21.67%) than imported chicks (11.67%) using standard isolation. Salmonella enteritidis and S. typhimurium were common in local chicks, while S. newport was highest in imported chicks. RT-PCR detected Salmonella in 58.33% of imported and 66.67% of local chicks, higher than standard isolation. RT-PCR is a more rapid, effective method for Salmonella detection but should be used along with standard methods for accurate identification of different
Diversity of O Antigens within the Genus Cronobacter - MartinaPauline Ogrodzki
This study analyzed the diversity of O antigens in the bacterial genus Cronobacter by testing 82 strains representing all Cronobacter species. Restriction fragment length polymorphism analysis of the O-antigen gene cluster identified 11 previously reported and 6 new serotypes. Whole genome sequencing of reference strains confirmed the new serotypes and showed some existing PCR probes did not correctly identify genomic variations. Analysis of lipopolysaccharide phenotypes also differentiated 24 total serotypes among Cronobacter strains. Certain serotypes including C. sakazakii O2, O1, and O4 and C. turicensis O1 were found to predominately cause clinical infections. This work provides an updated systematic classification of Cronobacter serotypes.
This document summarizes rapid detection methods for foodborne pathogen bacteria. It discusses how foodborne illnesses are a major public health problem and rapid detection of pathogens is needed. Several detection methods are outlined, including traditional culturing as well as newer techniques like PCR, real-time PCR, LAMP, and immunoassays. LAMP is highlighted as a new method that can rapidly detect pathogens under isothermal conditions. The document concludes that LAMP is a promising technique for pathogen detection due to its speed, simplicity and accuracy.
This document describes a European ring trial study to evaluate a real-time PCR assay for detection and typing of botulinum neurotoxin-producing Clostridia types A, B, E and F in food, feed and environmental samples. A primary evaluation of the real-time PCR method showed 100% accuracy, sensitivity, specificity and selectivity compared to the reference cultural method and mouse bioassay. A ring trial conducted at four European laboratories using 47 strains and 30 clinical and food samples linked to botulism cases showed 95.7% concordance among laboratories. The reproducibility generated a relative standard deviation of 2.18-13.61%. Given the high level of agreement between laboratories, this real-time PCR method is suitable
This document describes the isolation and characterization of a new giant virus called Cedratvirus. Key points:
- Cedratvirus was isolated from an environmental sample in Algeria using Acanthamoeba castellanii.
- It has an ovoid shape with a cork structure at each end, resembling Pithovirus sibericum but with a unique double cork feature.
- The 589kb genome is most closely related to the pithovirus genomes, sharing over 100 genes, but with only 21% of genes involved in best reciprocal hits, indicating genetic distance from known pithoviruses.
This document describes a new method for detecting Staphylococcus bacteria using a specific volatile organic compound (VOC) produced by Staphylococcus called 2-[3-acetoxy-4,4,14-trimethylandrost-8-en-17-yl] propanoic acid (ATMAP). A simple colorimetric assay using methyl red as a pH indicator can specifically detect Staphylococcus by changing color from yellow to orange due to ATMAP production. The assay was tested on liquid cultures and plate cultures to detect various Staphylococcus and other bacterial strains, demonstrating 100% specificity and sensitivity. This new detection method could provide a low-cost and easy-to-use
Real time pcr assay for rapid detection and quantification of campylobacter j...Tiensae Teshome
The document describes the development of a real-time PCR (rtPCR) assay for rapid detection and quantification of Campylobacter jejuni on chicken rinses from poultry processing plants without an enrichment step. Eighty-four chicken rinse samples were collected from three processing plants and tested using both rtPCR and culture methods. Sixty-five samples were positive by rtPCR while 27 were positive by culture, with 27 samples positive by both methods. The rtPCR assay was able to detect C. jejuni with a limit of 1 CFU and provide results within 90 minutes, significantly faster than the 5-7 days required for culture.
This study compared the use of real-time polymerase chain reaction (RT-PCR) and standard isolation techniques for detecting Salmonella in broiler chicks. The incidence of Salmonella was higher in local chicks (21.67%) than imported chicks (11.67%) using standard isolation. Salmonella enteritidis and S. typhimurium were common in local chicks, while S. newport was highest in imported chicks. RT-PCR detected Salmonella in 58.33% of imported and 66.67% of local chicks, higher than standard isolation. RT-PCR is a more rapid, effective method for Salmonella detection but should be used along with standard methods for accurate identification of different
Diversity of O Antigens within the Genus Cronobacter - MartinaPauline Ogrodzki
This study analyzed the diversity of O antigens in the bacterial genus Cronobacter by testing 82 strains representing all Cronobacter species. Restriction fragment length polymorphism analysis of the O-antigen gene cluster identified 11 previously reported and 6 new serotypes. Whole genome sequencing of reference strains confirmed the new serotypes and showed some existing PCR probes did not correctly identify genomic variations. Analysis of lipopolysaccharide phenotypes also differentiated 24 total serotypes among Cronobacter strains. Certain serotypes including C. sakazakii O2, O1, and O4 and C. turicensis O1 were found to predominately cause clinical infections. This work provides an updated systematic classification of Cronobacter serotypes.
This document summarizes rapid detection methods for foodborne pathogen bacteria. It discusses how foodborne illnesses are a major public health problem and rapid detection of pathogens is needed. Several detection methods are outlined, including traditional culturing as well as newer techniques like PCR, real-time PCR, LAMP, and immunoassays. LAMP is highlighted as a new method that can rapidly detect pathogens under isothermal conditions. The document concludes that LAMP is a promising technique for pathogen detection due to its speed, simplicity and accuracy.
Microbiological tests detect microorganisms or the host immune response to infection. They can identify infectious agents, provide information to guide antimicrobial therapy, and assess drug susceptibility. Test results must be interpreted carefully based on factors like specimen type, test characteristics, clinical findings, and communication between clinician and microbiologist. A variety of methods are used, including microscopy, culture, antigen and antibody detection, and nucleic acid amplification tests.
Customizable pcr microplate array for differential identification of multiple...Tiensae Teshome
1. Customizable PCR-microplate arrays were developed that allow for the simultaneous identification of 10 foodborne pathogens and biothreat agents using pathogen-specific primers.
2. The arrays were tested using genomic DNA from 38 pathogen strains, and specifically identified all pathogens present.
3. In tests with food matrices, the arrays showed detection limits as low as 9 cfu/g for Salmonella Typhimurium in beef hot dogs and 78 cfu/ml in milk. Such microplate arrays could serve as tools for rapid identification of these pathogens during outbreak investigations or for confirmation purposes.
1. The study investigated antibiotic resistance and the presence of the blaCTX-M-15 gene in Enterobacter species isolated from hospitals in Tehran, Iran between 2012-2013.
2. It found high rates of resistance to common antibiotics like Augmentin and high frequencies of the blaCTX-M-15 gene (11.8% of isolates).
3. The blaCTX-M-15 gene was found to be located on conjugative plasmids in one Enterobacter isolate, demonstrating its potential for horizontal transfer between bacteria.
Detection and Subtype Identification of Blastocystis Isolates from Wastewater...gon0603
Presented during the 6th Asian-Pacific Organization for Cell Biology (APOCB) International Congress, EDSA Shangri-La, Manila, Philippines, 25 to 28 February 2011
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
Evaluation of mancozeb activity towards various plant fungal pathogen agents ...UPL
Tests were carried out in petri dishes and concern the evaluation of conidial germination of different pathogen populations of Alternaria spp., V. inaequalis and S. vesicarium (for these last two fungi were analyzed two type of strains: sensitive e resistant to strobilurins according to our previous results (Collina et al, 2007 and Fiaccadori et al, 2011). Mancozeb was used as formulated product (solo) dissolved in sterile distillate water.
This document describes a study that identified a strain of Vibrio owensii as the causative agent of acute hepatopancreatic necrosis disease (AHPND) in cultured shrimp. The key findings are:
1) A Vibrio bacterium, designated strain SH-14, was isolated from diseased shrimp in Shanghai and identified as V. owensii based on genomic sequencing and analysis.
2) Strain SH-14 contains the pirAB genes, which encode toxic proteins PirAB that are responsible for AHPND. The pirAB genes showed 100% similarity to those in known AHPND-causing V. parahaemolyticus strains.
3) Immersion challenge
The document describes a study that used MALDI-TOF MS to identify mycobacterial isolates. It compared two protein extraction protocols (A and B) on reference strains and clinical isolates, finding protocol A identified 92.1% of isolates to the species level compared to 50% for protocol B. Protocol A was then used to identify 27 environmental mycobacterial isolates, with two isolates misidentified by PRA-hsp65 but correctly identified by MALDI-TOF MS. Sequencing of the hsp65 and 16S rRNA genes confirmed the MALDI-TOF MS identifications. The results support the use of MALDI-TOF MS as a rapid and valuable tool for identifying
This document summarizes a study investigating the effectiveness of 16 chemical disinfectants against four human pathogenic viruses (coxsackievirus B3, adenovirus type 5, parainfluenzavirus type 3, and coronavirus 229E) when the viruses were dried on stainless steel disks. Only five disinfectants were found to achieve a 3 log10 or greater reduction in all four viruses tested within 1 minute of exposure, regardless of whether the viruses were suspended in feces or mucin prior to drying. The five effective disinfectants were 2% glutaraldehyde, a mixture containing 0.5% sodium o-benzyl-p-chlorophenate and 0.6% sodium la
This document discusses the development of two diagnostic tools (ELISA and lateral flow device) for the detection of antibodies against ovine and bovine theileriosis. It provides background information on the genus Theileria, which are tick-transmitted protozoan parasites that infect ruminants. It reviews the taxonomy, life cycle, clinical signs, and pathogenic species of Theileria that infect small ruminants. Existing methods for diagnosis of ovine and bovine theileriosis are described. The objectives of this study are to develop a recombinant protein ELISA for detection of T. uilenbergi infection and a lateral flow device for rapid detection of T. annulata infection under field conditions.
detect and identify common human bacterial pathogens in high purity water.Saad Farooqi
A rapid culture independent methodology toquantitatively detect and identify common human bacterial pathogens associated with contaminated high purity water
This study compared the performance of real-time PCR, an enzyme immunoassay (EIA), and culture for detecting Shiga toxin-producing Escherichia coli (STEC) in pediatric patients. The PCR assay detected all 21 STEC-positive samples while the EIA only detected 6. Culture recovered 5 STEC O157 isolates but missed 2 detected by PCR. PCR was more sensitive than EIA or culture, with a detection limit of 102 CFU/ml compared to 106-107 CFU/ml for the other methods. The higher sensitivity of PCR is important for detecting STEC cases since a low bacterial load can still cause disease.
Genotyping and subgenotyping of Trichophyton rubrum isolated from dermatophyt...iosrjce
IOSR Journal of Pharmacy and Biological Sciences(IOSR-JPBS) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of Pharmacy and Biological Science. The journal welcomes publications of high quality papers on theoretical developments and practical applications in Pharmacy and Biological Science. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
The Evaluation of the Speed-Oligo® Mycobacteria Assay for Identification of M...CrimsonpublishersCJMI
The Evaluation of the Speed-Oligo® Mycobacteria Assay for Identification of Mycobacterium spp. from Smear Positive and Negative Sputum Samples by Gülnur Tarhan in ohesive Journal of Microbiology & Infectious Disease
This document summarizes a study that analyzed 267 fecal samples from cattle, goats, and poultry in Botswana for the presence of Cryptococcus neoformans. A total of 72 samples (26.9%) tested positive for C. neoformans, mostly from cattle. The isolates were further analyzed to determine their mating type (MATα, MATa, or hybrids). Mating type analysis revealed the presence of all three types in the isolates from cattle, poultry, and goats, with MATα being most common. The results suggest that veterinary animals can act as reservoirs for C. neoformans and highlight the need to prevent transmission to at-risk human populations.
1. The authors developed a quantitative real-time PCR (Q-PCR) method for detecting Rhodococcus equi, an important horse and emerging human pathogen.
2. The method uses two Q-PCR assays, one targeting the chromosomal choE gene to quantify total R. equi, and the other targeting the virulence plasmid gene vapA to detect the "horse-pathogenic" genotype.
3. Testing on 178 R. equi isolates and 77 non-target bacteria showed the assays were 100% sensitive and specific. The method can accurately quantify R. equi down to 1,000 CFU/ml in bronchoalveolar lavage fluid.
DIAGNOSTIC ADVANCES IN HAEMOPROTOZOAN INFECTIONS OF LIVESTOCKrinkusarawade
The document discusses diagnostic advances for haemoprotozoan infections in livestock. It covers conventional parasitological diagnosis using blood smears as well as molecular diagnosis techniques like PCR, LAMP, and probes. Immunological diagnosis methods such as ELISA, IFAT, CATT, ICT are also summarized. Molecular tools allow specific and sensitive detection of parasites while serological assays are suitable for chronic infections when parasitemia is low. Advanced diagnostics combined with effective treatment can help control haemoprotozoan infections and drug resistance.
This document reviews several studies on the microbiological quality of meat and meat products sold in Tripoli, Libya from 2005-2009. The following key points are made:
- Beef burger samples were highly contaminated with pathogenic bacteria like E. coli (74.5%), E. coli O157:H7 (27.1%), S. aureus (28.8%) and Aeromonas (18.6%).
- Fresh sausage samples were contaminated with E.coli O157:H7 (39.3%) and salmonella (2.1%).
- Chicken burger samples had E. coli (10.9%) and E. coli O157:H7 (4.68%).
-
Characteristics of salmonella spp. isolated from wild birds confiscated in il...racheltrans
1) Salmonella was isolated from 3 of 109 wild birds confiscated from illegal wildlife trade in Rio de Janeiro, Brazil, including one strain of Salmonella Typhimurium and two strains of Salmonella Panama.
2) All Salmonella isolates showed resistance to multiple antimicrobial drugs. PFGE analysis found 100% similarity between the Salmonella Typhimurium strain isolated from a bird and strains from a human outbreak in southern Brazil, indicating potential spread between wildlife and humans.
3) The two Salmonella Panama strains isolated from birds in the same catch showed identical genetic fingerprints, suggesting a common source of infection. However, these strains did not match any isolates in reference databases.
This document describes the development of a multiplex real-time PCR assay that can identify and differentiate B. anthracis from closely related Bacillus species as well as differentiate virulence types of B. anthracis. The assay was designed to target specific markers on the B. anthracis chromosome and plasmids pXO1 and pXO2. It also includes an internal control targeting the B. thuringiensis chromosome. The primers and probes were tested on a panel of B. anthracis strains and shown to correctly identify strains. The assay was also tested in laboratories in three different countries using different real-time PCR instruments. The assay provides a method to simultaneously identify B. anthracis,
The document describes experiments conducted to identify an unknown microorganism. Samples from two patients, labeled Culture A and Culture B, were tested using a Gram stain. Culture A showed pink rod-shaped bacteria, while Culture B showed round purplish-pink bacteria. An antibiotic test found that chloramphenicol and tetracycline were most effective at inhibiting the growth of both bacterial cultures. Based on the morphological and antibiotic test results, the unknown microorganism was identified as Citrobacter Freundii.
Microbiological tests detect microorganisms or the host immune response to infection. They can identify infectious agents, provide information to guide antimicrobial therapy, and assess drug susceptibility. Test results must be interpreted carefully based on factors like specimen type, test characteristics, clinical findings, and communication between clinician and microbiologist. A variety of methods are used, including microscopy, culture, antigen and antibody detection, and nucleic acid amplification tests.
Customizable pcr microplate array for differential identification of multiple...Tiensae Teshome
1. Customizable PCR-microplate arrays were developed that allow for the simultaneous identification of 10 foodborne pathogens and biothreat agents using pathogen-specific primers.
2. The arrays were tested using genomic DNA from 38 pathogen strains, and specifically identified all pathogens present.
3. In tests with food matrices, the arrays showed detection limits as low as 9 cfu/g for Salmonella Typhimurium in beef hot dogs and 78 cfu/ml in milk. Such microplate arrays could serve as tools for rapid identification of these pathogens during outbreak investigations or for confirmation purposes.
1. The study investigated antibiotic resistance and the presence of the blaCTX-M-15 gene in Enterobacter species isolated from hospitals in Tehran, Iran between 2012-2013.
2. It found high rates of resistance to common antibiotics like Augmentin and high frequencies of the blaCTX-M-15 gene (11.8% of isolates).
3. The blaCTX-M-15 gene was found to be located on conjugative plasmids in one Enterobacter isolate, demonstrating its potential for horizontal transfer between bacteria.
Detection and Subtype Identification of Blastocystis Isolates from Wastewater...gon0603
Presented during the 6th Asian-Pacific Organization for Cell Biology (APOCB) International Congress, EDSA Shangri-La, Manila, Philippines, 25 to 28 February 2011
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
Evaluation of mancozeb activity towards various plant fungal pathogen agents ...UPL
Tests were carried out in petri dishes and concern the evaluation of conidial germination of different pathogen populations of Alternaria spp., V. inaequalis and S. vesicarium (for these last two fungi were analyzed two type of strains: sensitive e resistant to strobilurins according to our previous results (Collina et al, 2007 and Fiaccadori et al, 2011). Mancozeb was used as formulated product (solo) dissolved in sterile distillate water.
This document describes a study that identified a strain of Vibrio owensii as the causative agent of acute hepatopancreatic necrosis disease (AHPND) in cultured shrimp. The key findings are:
1) A Vibrio bacterium, designated strain SH-14, was isolated from diseased shrimp in Shanghai and identified as V. owensii based on genomic sequencing and analysis.
2) Strain SH-14 contains the pirAB genes, which encode toxic proteins PirAB that are responsible for AHPND. The pirAB genes showed 100% similarity to those in known AHPND-causing V. parahaemolyticus strains.
3) Immersion challenge
The document describes a study that used MALDI-TOF MS to identify mycobacterial isolates. It compared two protein extraction protocols (A and B) on reference strains and clinical isolates, finding protocol A identified 92.1% of isolates to the species level compared to 50% for protocol B. Protocol A was then used to identify 27 environmental mycobacterial isolates, with two isolates misidentified by PRA-hsp65 but correctly identified by MALDI-TOF MS. Sequencing of the hsp65 and 16S rRNA genes confirmed the MALDI-TOF MS identifications. The results support the use of MALDI-TOF MS as a rapid and valuable tool for identifying
This document summarizes a study investigating the effectiveness of 16 chemical disinfectants against four human pathogenic viruses (coxsackievirus B3, adenovirus type 5, parainfluenzavirus type 3, and coronavirus 229E) when the viruses were dried on stainless steel disks. Only five disinfectants were found to achieve a 3 log10 or greater reduction in all four viruses tested within 1 minute of exposure, regardless of whether the viruses were suspended in feces or mucin prior to drying. The five effective disinfectants were 2% glutaraldehyde, a mixture containing 0.5% sodium o-benzyl-p-chlorophenate and 0.6% sodium la
This document discusses the development of two diagnostic tools (ELISA and lateral flow device) for the detection of antibodies against ovine and bovine theileriosis. It provides background information on the genus Theileria, which are tick-transmitted protozoan parasites that infect ruminants. It reviews the taxonomy, life cycle, clinical signs, and pathogenic species of Theileria that infect small ruminants. Existing methods for diagnosis of ovine and bovine theileriosis are described. The objectives of this study are to develop a recombinant protein ELISA for detection of T. uilenbergi infection and a lateral flow device for rapid detection of T. annulata infection under field conditions.
detect and identify common human bacterial pathogens in high purity water.Saad Farooqi
A rapid culture independent methodology toquantitatively detect and identify common human bacterial pathogens associated with contaminated high purity water
This study compared the performance of real-time PCR, an enzyme immunoassay (EIA), and culture for detecting Shiga toxin-producing Escherichia coli (STEC) in pediatric patients. The PCR assay detected all 21 STEC-positive samples while the EIA only detected 6. Culture recovered 5 STEC O157 isolates but missed 2 detected by PCR. PCR was more sensitive than EIA or culture, with a detection limit of 102 CFU/ml compared to 106-107 CFU/ml for the other methods. The higher sensitivity of PCR is important for detecting STEC cases since a low bacterial load can still cause disease.
Genotyping and subgenotyping of Trichophyton rubrum isolated from dermatophyt...iosrjce
IOSR Journal of Pharmacy and Biological Sciences(IOSR-JPBS) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of Pharmacy and Biological Science. The journal welcomes publications of high quality papers on theoretical developments and practical applications in Pharmacy and Biological Science. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
The Evaluation of the Speed-Oligo® Mycobacteria Assay for Identification of M...CrimsonpublishersCJMI
The Evaluation of the Speed-Oligo® Mycobacteria Assay for Identification of Mycobacterium spp. from Smear Positive and Negative Sputum Samples by Gülnur Tarhan in ohesive Journal of Microbiology & Infectious Disease
This document summarizes a study that analyzed 267 fecal samples from cattle, goats, and poultry in Botswana for the presence of Cryptococcus neoformans. A total of 72 samples (26.9%) tested positive for C. neoformans, mostly from cattle. The isolates were further analyzed to determine their mating type (MATα, MATa, or hybrids). Mating type analysis revealed the presence of all three types in the isolates from cattle, poultry, and goats, with MATα being most common. The results suggest that veterinary animals can act as reservoirs for C. neoformans and highlight the need to prevent transmission to at-risk human populations.
1. The authors developed a quantitative real-time PCR (Q-PCR) method for detecting Rhodococcus equi, an important horse and emerging human pathogen.
2. The method uses two Q-PCR assays, one targeting the chromosomal choE gene to quantify total R. equi, and the other targeting the virulence plasmid gene vapA to detect the "horse-pathogenic" genotype.
3. Testing on 178 R. equi isolates and 77 non-target bacteria showed the assays were 100% sensitive and specific. The method can accurately quantify R. equi down to 1,000 CFU/ml in bronchoalveolar lavage fluid.
DIAGNOSTIC ADVANCES IN HAEMOPROTOZOAN INFECTIONS OF LIVESTOCKrinkusarawade
The document discusses diagnostic advances for haemoprotozoan infections in livestock. It covers conventional parasitological diagnosis using blood smears as well as molecular diagnosis techniques like PCR, LAMP, and probes. Immunological diagnosis methods such as ELISA, IFAT, CATT, ICT are also summarized. Molecular tools allow specific and sensitive detection of parasites while serological assays are suitable for chronic infections when parasitemia is low. Advanced diagnostics combined with effective treatment can help control haemoprotozoan infections and drug resistance.
This document reviews several studies on the microbiological quality of meat and meat products sold in Tripoli, Libya from 2005-2009. The following key points are made:
- Beef burger samples were highly contaminated with pathogenic bacteria like E. coli (74.5%), E. coli O157:H7 (27.1%), S. aureus (28.8%) and Aeromonas (18.6%).
- Fresh sausage samples were contaminated with E.coli O157:H7 (39.3%) and salmonella (2.1%).
- Chicken burger samples had E. coli (10.9%) and E. coli O157:H7 (4.68%).
-
Characteristics of salmonella spp. isolated from wild birds confiscated in il...racheltrans
1) Salmonella was isolated from 3 of 109 wild birds confiscated from illegal wildlife trade in Rio de Janeiro, Brazil, including one strain of Salmonella Typhimurium and two strains of Salmonella Panama.
2) All Salmonella isolates showed resistance to multiple antimicrobial drugs. PFGE analysis found 100% similarity between the Salmonella Typhimurium strain isolated from a bird and strains from a human outbreak in southern Brazil, indicating potential spread between wildlife and humans.
3) The two Salmonella Panama strains isolated from birds in the same catch showed identical genetic fingerprints, suggesting a common source of infection. However, these strains did not match any isolates in reference databases.
This document describes the development of a multiplex real-time PCR assay that can identify and differentiate B. anthracis from closely related Bacillus species as well as differentiate virulence types of B. anthracis. The assay was designed to target specific markers on the B. anthracis chromosome and plasmids pXO1 and pXO2. It also includes an internal control targeting the B. thuringiensis chromosome. The primers and probes were tested on a panel of B. anthracis strains and shown to correctly identify strains. The assay was also tested in laboratories in three different countries using different real-time PCR instruments. The assay provides a method to simultaneously identify B. anthracis,
The document describes experiments conducted to identify an unknown microorganism. Samples from two patients, labeled Culture A and Culture B, were tested using a Gram stain. Culture A showed pink rod-shaped bacteria, while Culture B showed round purplish-pink bacteria. An antibiotic test found that chloramphenicol and tetracycline were most effective at inhibiting the growth of both bacterial cultures. Based on the morphological and antibiotic test results, the unknown microorganism was identified as Citrobacter Freundii.
Dr. Kees van Frankenhuyzen
VU University Amsterdam)
2.3.1. Specificity of Bacillus thuringiensis pesticidal proteins: What we don’t know matters
Dr Kees van Frankenhuyzen (Natural Resources Canada, Canadian Forest Service) presented on the
specificity of Bt proteins. He started by explaining that the specificity of Bt proteins is a key element
in the environmental risk assessment of Bt crops. The specificity determines which organisms could
potentially be affected by the Bt proteins expressed in Bt crops. However, our knowledge on the
specificity of Bt proteins is still incomplete.
Dr van Frankenhuyzen explained that the
This document provides information on using the Coriolis μ air sampler for monitoring biocontamination in veterinary environments. It includes two application notes summarizing studies that used the Coriolis μ to: 1) assess fungal contamination in Portuguese poultry facilities using cultural and molecular methods, finding some toxigenic strains not detected by culture; and 2) demonstrate airborne transmission of swine influenza virus between pigs in an experimental setting, detecting the virus in air samples before infected pigs showed symptoms. It also lists two publications using the Coriolis μ to study Streptococcus suis in swine confinement buildings and Coxiella burnetii in sheep farms. The document encourages joining an online community for sharing Coriolis application
This document summarizes work conducted in Task 5.1 of the EU AniBioThreat project to develop a minimum detection standard for Bacillus anthracis, the bacterium that causes anthrax. Methods for sampling, sample preparation, PCR detection, isolate characterization including whole genome sequencing and a metagenomics approach were evaluated. An inter-laboratory trial of 6 PCR assays showed some published assays had poor selectivity. Collaboration between partners improved diagnostic capabilities and knowledge sharing. The work contributes toward internationally recognized and validated standards for fast and accurate B. anthracis diagnosis in disease investigations and surveillance.
This document provides an overview of global regulatory guidance for ensuring viral safety in biologics production. It discusses three key approaches: preventing contamination through high quality raw materials; detecting contamination through testing cell banks, raw materials, and process intermediates; and evaluating viral clearance in the production process. The summary discusses the types of regulatory documents that provide guidelines on raw materials and cell lines, as well as strategies for preventing contamination, detecting contamination through a variety of assay methods, and limitations of detection assays.
Viral Risk Mitigation - A Global Regulatory PerspectiveMilliporeSigma
Looking for insights into current global regulatory expectations for viral safety? Read the special report from BioProcess International, in collaboration with Martin Wisher, Senior Regulatory Consultant focusing on BioReliance biosafety® services.
This document summarizes a study on the isolation and molecular characterization of human adenovirus. The study found that out of 83 samples collected from eye secretions, 69 (83.13%) tested positive for human adenovirus using rapid tests and PCR. The highest rate of infection was found in individuals aged 16-30 years old (55.04%) and males had a higher rate of infection than females. Human adenovirus was successfully isolated by inoculating samples on chicken embryo fibroblast cell cultures and embryonated eggs, where cytopathic effects were observed. Molecular characterization was also conducted to identify the adenovirus strains present.
Highly Discriminatory Diagnostic Primer Design From Whole Genome DataLeighton Pritchard
Presented at the GMI (Global Microbial Identifier) satellite meeting, sponsored by the UK Department for Environment, Food and Rural Affairs (DEFRA), organised by the Food and Environment Research Agency (FERA), Bedern Hall, York, 10th September 2014.
This document describes the development of a fluorescence-based assay called "ProteAl" to detect the volatile biomarker 2-methylbutanal produced by Proteus bacteria. Gas chromatography-mass spectrometry and Fourier transform infrared spectroscopy were used to identify 2-methylbutanal in the headspace of Proteus cultures. A fluorescent dye, 5-dimethylaminonaphthalene-1-sulfonylhydrazine, was found to react specifically with 2-methylbutanal, producing a distinct green fluorescence. Testing of 95 bacterial strains showed the ProteAl assay can identify Proteus with 100% specificity and sensitivity, providing a simple method for rapid surveillance of this pathogen.
This document summarizes the development and validation of fluorescence polarization immunoassays (FPIAs) for the rapid detection of mycotoxins in wheat. Key points:
- FPIAs were developed for the simultaneous detection of deoxynivalenol (DON) and its modified forms, and T-2/HT-2 toxins and their modified forms in wheat.
- The methods involve simple extraction from wheat samples followed by a 10-15 minute FPIA to quantify mycotoxin levels.
- Validation studies showed the FPIAs met performance criteria for screening methods defined in EU regulations, with cut-off levels accurately distinguishing positive and negative samples.
- The validated
Aquaculture microbiology and biotechnology vol (1)أسعد لحمر
This chapter discusses transgenic fish and the applications of genetic engineering in aquaculture. Specifically, it describes how genetic engineering techniques like chromosome manipulation and hormone treatment are currently used to produce sterile and monosex fish lines. It also outlines how researchers are using transgenic methods to develop fish with desirable traits like increased growth rates, improved feed conversion efficiency, disease resistance, and tolerance to stressful environmental conditions. However, the release of transgenic fish into the environment has raised ecological and human health concerns from some groups. The chapter examines both the potential benefits of transgenic fish for aquaculture as well as some of the criticisms against this technology.
Mrs Hyacinth Bernadette Lobo has over 30 years of experience in laboratory science and science education. She holds an MSc in Medical Molecular Biology and a BSc in Biochemistry/Microbiology. Her work experience includes positions as a science technician at various schools, a microbiology technician for Procter & Gamble, and research roles at University College London investigating dental diseases and rheumatoid arthritis. She has experience in techniques such as cell culture, DNA extraction, gel electrophoresis, and bacterial transformation. Currently she is interested in pursuing opportunities relating to health, nutrition, and molecular biology education.
Microbiological Investigations of Selected Flies of Public Health Importance ...iosrjce
Bacteria associated with flies of public health importance in Nigeria are not well known and their
ecology is also not well understood. We aim to determine the bacteria associated with flies of waste dump site.
Three flies of public health significance were collected from a waste dump site of the Rivers State University of
Science and Technology, Port Harcourt. The three dipterous flies were Luciliasericata, Chrysomyasp and
Musca domestica..The three flies were all of medical importance.The microbial load on three species of flies
was investigated using standard plate count methods. The fly samples were collected from the Post Graduate
Entomology Laboratory was cultured to isolate and identify the microbes associated with them. The samples
were analyzed for total heterotrophic bacteria and fungi counts. The study revealed high heterotrophic bacteria
and fungi counts in all three species of the flies used, with Musca domestica having the highest count of 2.9 X
109Cfu/gram and Chrysomyasp with the least count of 3.4 x 10 5Cfu/g and fungi counts ranged from 3.1 X
103Cfu/g to 2.9 X 105Cfu/g. The bacteria isolated from these samples includes: Escherichia coli,
Pseudomonassp,, Bacillussp, Enterobactersp, Staphylococcussp,Salmonellasp, Proteussp, and Klebsiellasp,
while the fungi species isolated includes: Penicilliumsp,
Aspergillussp,Rhizopussp,Cladosporiumsp,Aspergillusflavus, Aspergillusniger, Fusariumsp and Trichoderma
sp.
This study investigated the prevalence of Toxoplasma gondii infection in 197 pets and stray cats in 4 districts of Khyber Pakhtunkhwa, Pakistan using serological (ELISA) and molecular (PCR) techniques. The results showed that T. gondii infection was significantly higher in stray cats (74.6%) compared to pet cats (25.4%). Infection rates also varied significantly between districts and were highest in Kohat (95.5%). Older cats (>4 years) had significantly higher infection rates (91.66%) than younger cats. Chronic and reactivated chronic infections (58.37%) were more common than acute infections. This research suggests T. gondii is widely
Purpose: To study the demographic characteristics, associated factors, causative agents, of infectious keratitis and develop a diagnostic tool to aid easy diagnosis of keratitis.Methods: Corneal scrapes were collected and subjected to culture, microscopy, considering age, occupation, geographical, Frequency of predisposing ocular conditions, antibiotic susceptibility test, resistance patterns test, drug sensitivity and 16s r-DNAas well as 18s r-DNA based identification was performed and submitted to data bank with accession number. The 16S rDNA sequences of the individual bacteria and fungi were used for a universal primer design and thereby multiplex PCR can be performed.Results: A total of 250 consecutive patients with infective keratitis were evaluated, of which 77 (30.8%) were found to be of bacterial, 67 (26.8%) were fungal, 16 (6.4%) were both fungal and bacterial, and the remaining 90 (36%) were found to be culture negative. Contact lens wear was the main risk factor (80.8%). Ocular surface disease (23.6%), ocular trauma (14.8%), corneal surgery (4.4%) and corneal suture (6.4%) of cases were found in corneal ulcers. Most community acquired bacterial and fungal ulcers resolve with appropriate treatment. 64% of the infections involved positive cultures and 36% involved negative cultures, were found in polymicrobial mode of infection. Fusarium spp. (32.75%) was the most predominant species followed by Aspergillus sp. (20.68%) was found in fungal corneal ulcers and also Staphylococcus sp. was the most common bacteria found in bacterial cultures. The 16S rDNA sequence of the bacteria and fungi cultured from the isolates of the corneal scrapping were performed and the genes were submitted in Genbank.The primers designed for bacteria and fungi gave good results in the multiplex PCR carried out and we suggest this can be used as a diagnostic tool for keratitis.
EU REACH regulation changed the way to do chemical risk assessment. All chemicals marketed or manufactured in the EU must have its own dossier. Non standard methods including alternatives to animal testing are accepted.
Half Italian, half English
This document summarizes key points from a presentation on Clostridium difficile and other healthcare-associated infections. It discusses how C. difficile can cause endemic, epidemic infections in healthcare settings. It also briefly mentions other potential pathogens like norovirus, salmonella, and cryptosporidium. The document then provides more details on C. difficile biology, symptoms, risk factors, diagnostic testing approaches, and strategies for prevention and environmental cleaning.
Syngulon - Breakout session Synthetic Biology June 10, 2022.pdfSyngulon
This document summarizes a presentation given by Dr. Philippe Gabant on the applications of synthetic biology. The presentation discusses Syngulon's work developing bacteriocins and tuning microbiota to address antimicrobial resistance and contamination issues. It provides an overview of Syngulon's technologies, markets in biopharma, cosmetics and more. It also discusses their PARAGEN collection of bacteriocin genes and peptides, and how they are applying synthetic biology to expand this collection. The goal is to balance and control microbial life through developing alternatives to antibiotics.
This document summarizes a quantitative microbial risk assessment (QMRA) that investigated the potential public health risk of transmission of ESBL-producing Escherichia coli and Campylobacter bacteria from poultry farms to humans through flies. The study modeled human exposure based on the fraction of contaminated flies leaving infected poultry farms, the number of bacteria per fly, and the number of positive poultry houses in the Netherlands. It compared the risk estimates to consumption of chicken fillet and found that transmission of both pathogens through flies may be an important transmission route worthy of further modeling and investigation given the potential public health implications.
The document describes the development of a reverse line blot (RLB) hybridization kit that can simultaneously detect four genera of common tick-borne pathogens - Anaplasma, Ehrlichia, Babesia and Theileria. This provides a sensitive and cost-effective diagnostic tool that facilitates the study of tick-borne diseases. The RLB technique involves PCR amplification of the pathogens, followed by hybridization of the products on a membrane containing genus-specific probes. This allows for simultaneous identification of any of the pathogens present in one sample. The kit will support improved diagnosis and epidemiological research on these important diseases affecting both animals and humans.
This study analyzed 846 E. coli isolates from 113 surface water samples and 313 E. coli isolates from 33 wastewater samples in the Netherlands. The researchers found that 26% of surface water E. coli isolates and much higher percentages of wastewater E. coli isolates (31-76%) were resistant to at least one of 8 classes of antimicrobials tested. Multidrug resistance was found in 11% of surface water isolates and significantly higher percentages (19-62%) of wastewater isolates. Median concentrations of multidrug resistant E. coli were highest in wastewater from health care institutions and lowest in surface water. The study indicates that municipal wastewater contributes significantly to the occurrence of antimicrobial resistant E
This document summarizes a study investigating the prevalence of ESBL-producing E. coli bacteria in four Dutch recreational waters and the potential role of nearby wastewater treatment plants (WWTPs) as contamination sources. Samples were taken from recreational waters, WWTP effluents, surface waters upstream and downstream of WWTP discharge points, and additional surface waters not influenced by the WWTPs. ESBL-producing E. coli were detected in all recreational waters and 62% of samples, with concentrations averaging 1.3 CFU/100ml. Isolates with identical genetic characteristics were found in WWTP effluents and surface waters, including occasionally in recreational waters, indicating WWTPs contribute to their presence. However,
This document discusses PCR-based assays for detecting Bacillus anthracis. It begins with an introduction to B. anthracis and the challenges in detecting it due to its similarity to other Bacillus species. It then describes a literature review and in silico analysis of over 300 published PCR primer/probe sequences targeting B. anthracis. Only 4 sequences were found to be 100% specific for B. anthracis based on comparisons to 134 Bacillus genome sequences. An inter-laboratory trial was then conducted among 5 European laboratories to evaluate 6 assays using 90 Bacillus strains. Three assays targeting the lambdaBa03 prophage region performed well without false positives or negatives.
This study found ESBL-producing E. coli bacteria on house flies and blow flies caught at two poultry farms in the Netherlands. At a broiler farm, ESBL-producing E. coli was detected in a pool of blow flies and in fly isolates that matched types found in manure and rinse water at the farm. At a laying hen farm, all fly and manure isolates carried the same ESBL genotype. The results suggest that flies acquire ESBL-producing E. coli at poultry farms and could contribute to the dissemination of these bacteria into the community.
This document describes the development of a multiplex bead-based suspension array assay using Luminex technology to simultaneously genotype 13 phylogenetically informative single nucleotide polymorphisms (SNPs) in Bacillus anthracis. The assay is based on a modified Multiplex Oligonucleotide Ligation-PCR (MOL-PCR) method using dual-priming oligonucleotides for allele-specific probes to reduce cross-reactivity. The 13-plex assay was validated on 73 B. anthracis strains, demonstrating unambiguous SNP calls and lineage identification. An assay limit of detection of 2 ng genomic DNA was determined. The reproducibility and robustness of the method was confirmed in a small-scale proficiency test between four laboratories
A single-reaction quadruplex qPCR assay was developed that can rapidly detect and differentiate Burkholderia mallei and Burkholderia pseudomallei. The assay uses three signature sequences - a multicopy transposase sequence common to both species for sensitive detection, and two unique sequences for species differentiation. It also incorporates an internal control for DNA extraction and amplification using Bacillus thuringiensis. The assay enables detection of less than 1 genome equivalent and differentiation of B. mallei and B. pseudomallei with high sensitivity and reliability for diagnostic and surveillance purposes.
This study found that rat tissues from farms in the Netherlands tested positive for the pla gene, which is a marker for Yersinia pestis. The pla gene sequences from rats were nearly identical to Y. pestis pla but further analysis identified adjacent sequences similar to bacterial replication genes. Attempts to culture or detect other Y. pestis markers from rat tissues were unsuccessful. The findings suggest there are unknown bacteria in rats that contain a pla homolog, which could produce false positive results in Y. pestis detection assays that only target the pla gene. Methods to confirm the presence of Y. pestis should include additional gene targets.
The document describes the development of two suspension microarray assays using different chemistries to detect four biothreat pathogens - Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Coxiella burnetii. Sixteen DNA signatures from the pathogens were amplified using multiplex asymmetric PCR and detected using either direct hybridization (DH) or target-specific primer extension followed by universal hybridization (TSPE-UH). The specificity and sensitivity of both assays were evaluated and compared. The TSPE-UH assay showed lower background signals and was more specific while the DH assay had simpler procedures but lower signal-to-noise ratios. Both assays could detect pathogen DNA from mixed samples and
The document describes the development of multiplex qPCR assays that can rapidly and reliably detect Bacillus anthracis, Francisella tularensis, and Yersinia pestis. The assays target pathogen-specific DNA sequences and include an internal control (B. thuringiensis cry1 gene) to control for DNA extraction and amplification. Validation showed the assays can simultaneously detect 3 pathogen targets with high sensitivity and specificity, and that inclusion of the internal control reduces the risk of false negatives. The assays provide a useful tool for the detection of these high-risk pathogens.
1) Researchers developed a multiplex real-time PCR protocol to specifically detect Bacillus anthracis and differentiate it from closely related Bacillus cereus strains.
2) The protocol simultaneously detects B. anthracis' chromosome and two plasmids (pXO1 and pXO2) as well as an internal control.
3) Evaluation across multiple institutes and PCR platforms showed the protocol generated consistent results, providing a reliable screening method for B. anthracis.
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1) The study analyzed HIV-1 sequences from 37 patients with high numbers (34 or more) of degenerate base codes in their protease/reverse transcriptase sequences, which can indicate either extensive viral evolution or dual infection. 2) Phylogenetic analysis of envelope and gag sequences from these patients found that 16 (43%) had dual HIV-1 infections, representing 1% of all sequences analyzed. 3) Patients with the highest numbers of degenerate base codes were more likely to have dual infections with different HIV-1 subtypes. The study demonstrates that routine HIV genotyping can help detect undiagnosed dual infections.
1. Clinical microbiology
Validation of a real-time PCR based method for detection of Clostridium botulinum
types C, D and their mosaic variants C-D and D-C in a multicenter collaborative
trial
Q2 Cedric Woudstra a
, Hanna Skarin b
, Fabrizio Anniballi c
, Bruna Auricchio c
, Dario De Medici c
, Luca Bano d
,
Ilenia Drigo d
, Trine Hansen e
, Charlotta Löfström e
, Raditijo Hamidjaja f
, Bart J. van Rotterdam f
,
Miriam Koene g
, Marie-Hélène Bäyon-Auboyer h
, Jean-Philippe Buffereau h
, Patrick Fach a,*
a
French Agency for Food, Environmental and Occupational Health and Safety (Anses), Food Safety Laboratory, 23 Av du Général De Gaulle, Fr-94706 Maisons-Alfort, France
b
National Veterinary Institute (SVA), Department of Bacteriology, SE-751 89 Uppsala, Sweden
c
Istituto Superiore di Sanità, Veterinary Public Health and Food Safety Department, National Reference Centre for Botulism, Viale Regina Elena, 299, 00161 Rome, Italy
d
Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), Laboratorio di Treviso, Italy
e
National Food Institute, Technical University of Denmark (DTU), Søborg, Denmark
f
National Institute for Public Health and the Environment (RIVM), Centre for Zoonoses and Environmental Microbiology, Antonie van Leeuwenhoeklaan 9, 3721MA Bilthoven,
The Netherlands
g
Central Veterinary Institute (CVI) of Wageningen University and Research Centre, Edelhertweg 15, 8219PH Lelystad, The Netherlands
h
Analysis and Development Laboratory 22 (LDA22), Ploufragan, France
a r t i c l e i n f o
Article history:
Received 26 December 2012
Received in revised form
19 April 2013
Accepted 1 May 2013
Available online xxx
Keywords:
C. botulinum C and D
Animal botulism
Evaluation trial
GeneDiscÒ
array
a b s t r a c t
Two real-time PCR arrays based on the GeneDiscÒ
cycler platform (Pall-GeneDisc Technologies) were
evaluated in a multicenter collaborative trial for their capacity to specifically detect and discriminate
Clostridium botulinum types C, D and their mosaic variants C-D and D-C that are associated with avian and
mammalian botulism. The GeneDiscÒ
arrays developed as part of the DG Home funded European project
‘AnibioThreat’ were highly sensitive and specific when tested on pure isolates and naturally contaminated
samples (mostlyclinical specimen from avian origin). Results of the multicenter collaborative trial involving
eight laboratories in five European Countries (two laboratories in France, Italy and The Netherlands, one
laboratory in Denmark and Sweden), using DNA extracts issued from 33 pure isolates and 48 naturally
contaminated samples associated with animal botulism cases, demonstrated the robustness of these tests.
Results showed a concordance among the eight laboratories of 99.4%e100% for both arrays. The repro-
ducibility of the tests was high with a relative standard deviation ranging from 1.1% to 7.1%. Considering the
high level of agreement achieved between the laboratories these PCR arrays constitute robust and suitable
tools for rapid detection of C. botulinum types C, D and mosaic types C-D and D-C. These are the first tests for
C. botulinum C and D that have been evaluated in a European multicenter collaborative trial.
Ó 2013 Published by Elsevier Ltd.
1. Introduction
Botulism is a severe flaccid paralytic disease caused by seven
different neuroparalytic toxins subtypes (BoNT A-G) which could
affect animals and humans. Botulinum neurotoxins (BoNTs) are pro-
duced by the anaerobic gram-positive bacteria Clostridium botulinum
group I to IV [1]. Groups I and II BoNT producing Clostridia are mainly
responsible for human botulism whereas toxin produced by group III
(C. botulinum types C and D) are involved in animal botulism world-
wide [2]. BoNT-producing clostridia can affect wild and domesticated
animals such as poultry, birds, cattle, horses, sheep and minks. Out-
breaks with high mortality have become an increasing environmental
and economical problem [3] but the deliberate release of BoNT pro-
ducing clostridia or isolated toxin for bioterrorism purposes is also of
great concern [4,5]. Economical, medical and alimentary conse-
quences could be catastrophic, underlying the necessity of rapid
detection tests. The European project ‘AniBioThreat’ (“Bio-prepared-
ness measures concerning prevention, detection and response to
animal bio-terrorism threats”, www.anibiothreat.com) funded in
2010 for three years by the European commission (DG Home) has
* Corresponding author. Tel.: þ33 (0) 14977 2813; fax: þ33 (0) 14977 9762.
E-mail address: patrick.fach@anses.fr (P. Fach).
Contents lists available at SciVerse ScienceDirect
Anaerobe
journal homepage: www.elsevier.com/locate/anaerobe
1075-9964/$ e see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.anaerobe.2013.05.002
Anaerobe xxx (2013) 1e7
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Please cite this article in press as: Woudstra C, et al., Validation of a real-time PCR based method for detection of Clostridium botulinum types C, D
and their mosaic variants C-D and D-C in a multicenter collaborative trial, Anaerobe (2013), http://dx.doi.org/10.1016/j.anaerobe.2013.05.002
2. been set up for improving prevention, detection and response to
animal bio-terrorism threats. This project focuses on animal bio-
terrorism threats and raised the question of rapid detection of clos-
tridia responsible for animal botulism [6].
The standard method to perform sample analysis of suspected
animal botulism outbreaks is currently the mouse lethality bio-
assay followed by sero-neutralization [7,8]. However this method
presents several drawbacks. It is time consuming, expensive, and
causes ethical considerations. Real-time PCR based assays using
the GeneDiscÒ
technology (Pall, GeneDisc Technologies, Bruz,
France) have recently been developed within the framework of the
European project ‘AniBioThreat’ for neurotoxin gene profiling of
C. botulinum types C and D and their mosaic types C-D and D-C [9].
While not solving the shortcoming of the detection of the neuro-
toxin gene instead of the toxin, these real-time PCR-based assays
have the advantage of being animal free, rapid and easy to use.
Two GeneDiscÒ
arrays were designed as follows: The C. botulinum
types C and D GeneDiscÒ
array (GD1 C&D) and the C. botulinum
types C, D and mosaic GeneDiscÒ
array (GD2 C, D & mosaic). The
first PCR array (GD1) has been developed to detect C. botulinum
types C and type D whereas the second array (GD2) allows for the
differentiation between mosaic C-D and D-C strains from parental
C and D strains. Both arrays include an appropriate external
amplification control (EAC) to monitor PCR inhibitors. The impor-
tance of characterizing the BoNT genes is underlined by the higher
toxic activity of the mosaic types C-D and D-C neurotoxins
compared to normal types C and D [10,11]. There are a limited
number of real-time PCR assays for C. botulinum types C and D that
have been developed and reported in the scientific literature [12e
14]. None of these are able to clearly discriminate C. botulinum
subtypes C, C-D, D and D-C except the GeneDiscÒ
arrays as
described by Woudstra et al., 2012. In addition, none have been
validated for use by a full-scale inter-laboratory collaborative trial.
Proper validation based on consensus criteria is an absolute pre-
requisite for successful adoption of a PCR-based diagnostic meth-
odology [15]. Due to lack of international validation and
standardization protocols for C. botulinum detection, and control of
quality reagents and equipment, the transfer of the assays from
expert to end-user laboratories has met with great difficulties. As a
step toward the recognition of a standard PCR-based method to
detect and subtype C. botulinum C, C-D, D and D-C in clinical
samples, the performance characteristics of the GeneDiscÒ
arrays
should be assessed with a special focus on their reproducibility.
The aim of the present study was to evaluate the two GeneDiscÒ
arrays developed as part of the European Research Project ‘Ani-
BioThreat’ in a multicenter collaborative trial performed at eight
different European laboratories.
2. Materials and methods
2.1. Participating laboratories
The laboratories participating in the evaluation trial were the
French agency for food, environmental and occupational health &
safety (Anses) which is the organizing laboratory; The Analysis and
Development Laboratory 22 (LDA22), Ploufragan, France; The Na-
tional Reference Centre for Botulism (NRCB) at the Istituto Superore
di Sanità, Rome, Italy; The Istituto Zooprofilattico Sperimentale
delle Venezie (IZSVe), Laboratorio di Treviso, Italy; The National
Food Institute, Technical University of Denmark (DTU), Søborg,
Denmark; The Central Veterinary Institute (CVI) of Wageningen
University and Research center, Lelystad, The Netherlands; The
Centre for Zoonoses and Environmental Microbiology, Bilthoven,
RIVM, The Netherlands; and the National Veterinary Institute
(SVA), Uppsala, Sweden. Due to international restrictions on
exchange and sending of BoNT-producing clostridia strains, only
sterile DNAs were shipped to the participants.
2.2. GeneDiscsÒ
arrays
Two GeneDiscsÒ
arrays were used in the ring trial. The Gen-
eDiscÒ
array “GD1 C & D” was used for detection of C. botulinum
types C and D. The GeneDiscÒ
array “GD2 C, D & mosaic” was used
for detection of C. botulinum types C, C-D, D and D-C. Both are
identical to those previously described [9]. Each GeneDisc Ò
array
contains a calibrated EAC provided by Pall GeneDiscÒ
Technologies
which should give a positive signal with a Cq quantification cycle
[16] value around 30. GeneDiscÒ
spotting and manufacturing were
performed by Pall GeneDiscÒ
Technologies (Bruz, France) in one
batch during summer 2011.
2.3. GeneDiscÒ
cycler and software
The V2 GeneDiscÒ
cycler from Pall GeneDiscÒ
technologies was
used by each laboratory for the trial. Technical staffs from each
participating laboratory were trained by Pall GeneDiscÒ
technolo-
gies to use the GeneDiscÒ
cycler and software. Results were
analyzed using the GeneDiscÒ
cycler software (V2.10.12) which is
part of the V2 GeneDiscÒ
cycler. Positive results and Cq values were
recorded according to the GeneDiscÒ
cycler software calculation.
2.4. Bacterial strains
Strains of C. botulinum types C, C-D, D and D-C used in this study
are listed in Tables 1 and 2. The strains consisted of seven
C. botulinum types C, eighteen C. botulinum mosaic types C-D, three
C. botulinum types D and five C. botulinum mosaic types D-C.
C. botulinum strains were grown overnight in Trypticase-Peptone-
Glucose-Yeast (TPGY) extract broth [17], brain heart infusion me-
dium (Difco, Paris, France), or in broth fortified cooked meat me-
dium at and incubated, without homogenization, at 30 C under
anaerobic conditions [18].
2.5. Naturally contaminated samples
A total of 48 naturally contaminated samples (intestinal content,
faeces and organs from birds and cattle), were collected from animal
botulism outbreaks across Europe in the last few years. Each sample
(1 g) was added to 9 ml of pre-reduced tryptone-glucose-yeast
extract (TPGY Q1) and incubated, without homogenization, in anaer-
obic conditions. After 48 h incubation in TPGYat 30 C,1 mL aliquots
of the enrichment broths were collected and centrifuged at 9000 Â g
for 5 min. The supernatant was discarded and the cell pellet sub-
mitted to DNA extraction using either Phenol/Chloroform method
[19], DNeasy blood and tissue kit (Qiagen, Hilden, Germany), Chelex
100 (Bio-Rad Life Science Research, Hercules, CA), and Automatic
system Microlab Starlet (Hamilton, Nevada, USA) employing the
MegMax Total Nucleic Acid Isolation Kit (Ambion, Austin, USA), ac-
cording to manufacturer’s instructions for Gram positive bacteria.
DNA was stored at À20 C until shipment for further analysis.
2.6. Trial organization
Concordance and reproducibility of GD1 and GD2 GeneDiscÒ
arrays were evaluated within a ring trial involving eight European
laboratories. Each laboratory used the real-time PCR platform
“GeneDisc cycler” V2 from Pall GeneDisc technologies for the trial.
Each participating laboratory received 81 identical “blind” coded
DNA samples: 33 originated from C. botulinum strains and 48 from
naturally contaminated samples. All were equally diluted and
C. Woudstra et al. / Anaerobe xxx (2013) 1e72
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YANAE1169_proof ■ 16 May 2013 ■ 2/7
Please cite this article in press as: Woudstra C, et al., Validation of a real-time PCR based method for detection of Clostridium botulinum types C, D
and their mosaic variants C-D and D-C in a multicenter collaborative trial, Anaerobe (2013), http://dx.doi.org/10.1016/j.anaerobe.2013.05.002
3. distributed among the eight participating laboratories in cooled
packages by the organizing laboratory and stored at À20 C until
further analysis. The two GeneDiscÒ
arrays tested were sent to each
laboratory and stored at þ4 C until analysis. Each participant
received a standard operating procedure and a standardized test
report form on which to record the results to return to the trial
leader for analysis.
2.7. Terms and definitions
The following terms and definitions were used in the manuscript:
Relative accuracy (AC) is defined as the degree of correspon-
dence between the response obtained with the PCR tests of Hill
et al. [20] and Kouguchi et al. [21] used as reference for testing
C. botulinum types C and D and the response obtained with the
GeneDiscÒ
arrays on an identical sample.
Relative sensitivity (SE) is the ability of the GeneDiscÒ
arrays to
detect a positive result when the reference real-time PCR methods
as described by Hill et al. (2010) and Kouguchi et al. (2006) also
produced a positive result on an identical sample.
Relative specificity (SP) is the ability of the GeneDiscÒ
arrays to
detect a negative result when the reference real-time PCR methods
as described by Hill et al. (2010) and Kouguchi et al. (2006) pro-
duces a negative result on an identical sample.
Selectivity includes the evaluation of inclusivity and exclusivity.
Inclusivity is the ability of the GeneDiscÒ
arrays to detect the target
bacteria from a wide range of bacterial strains. Exclusivity is the
lack of interference from a relevant range of non-target strains of
the GeneDiscÒ
arrays. The relevant non-target strains used for this
study have been reported in the recent study of Woudstra et al. [9].
Concordance is defined as the percentage chance of finding the
same result, positive or negative, from the identical sample
analyzed by the eight laboratories [22].
Reproducibility refers to the variation in Cq values among the
eight laboratories on identical sample. Reproducibility is expressed
(in %) as the relative standard deviation (RSD%).
3. Results
In a recent study we investigated the selectivity (inclusivity and
exclusivity) of the GeneDiscÒ
arrays using DNA extracts issued from
56 strains of C. botulinum types C, C-D, D, D-C and 63 non-BoNT-
producing Clostridia. Results of selectivity showed 100% of inclu-
sivity on the 56 target strains and 100% of exclusivity on the 63 non-
target strains. Results of 292 real samples analyzed by the reference
real-time PCR assays as described by Hill et al. (2010) and Kouguchi
et al. (2006) and the GeneDiscÒ
arrays gave a relative accuracy (AC)
of 97.9%, relative sensitivity (SE) of 96.8% and relative specificity
(SP) of 99.3% [9].
In the present study the results of a multicenter collaborative trial
performed with eight European laboratories using the GeneDiscÒ
arrays are presented. Tables 1 and 2 show the participants’ results
obtained with 33 C. botulinum isolates using the GeneDiscÒ
arrays
GD1 and GD2 respectively. The results showed a concordance of
Table 1
Strains of C. botulinum tested with the GeneDiscÒ
“GD1, C D”.
Strain Origin GeneDiscÒ
“GD1, C D”
PCR Lab 1 to 8 Cq value EAC Cq value
Mean Cq SD RSD% Mean Cq SD RSD%
1 Intestine Ducka
C þ 8/8 24.41 0.72 2.96 28.60 0.52 1.82
2 Intestine Ducka
C þ8/8 25.67 0.41 1.61 28.78 0.33 1.15
3 Intestine Ducka
C þ8/8 22.74 0.44 1.92 28.56 0.62 2.16
4 Feces Milch-cow D þ8/8 22.67 0.87 3.85 28.72 0.35 1.23
5 Intestine Cow D þ8/8 24.94 0.49 1.95 27.47 1.63 5.93
6 Unknown C þ8/8 25.65 0.41 1.61 27.49 0.43 1.56
7 Unknown C þ8/8 25.97 0.58 2.24 28.47 0.68 2.37
8 Unknown C þ8/8 24.73 0.30 1.22 28.52 0.40 1.39
9 Unknown C þ8/8 23.09 0.57 2.46 28.58 0.39 1.35
10 Unknown C þ8/8 20.49 0.84 4.11 28.46 0.32 1.12
11 Unknown C þ8/8 23.04 0.48 2.10 28.26 0.48 1.69
12 Unknown C þ8/8 24.77 1.76 7.10 27.58 0.44 1.60
13 Unknown C þ8/8 23.83 0.26 1.11 28.48 0.47 1.63
14 Unknown C þ8/8 24.18 0.63 2.62 28.92 1.62 5.61
15 Feed C þ8/8 26.35 0.88 3.35 28.49 0.43 1.52
16 Unknown C þ8/8 24.08 0.90 3.72 28.36 0.37 1.30
17 Unknown
Fox
C þ8/8 21.55 0.86 3.99 28.30 0.43 1.50
18 Unknown C þ8/8 20.13 0.27 1.33 27.43 0.46 1.67
19 Unknown C þ8/8 21.35 0.87 4.06 28.66 0.39 1.36
20 Unknown C þ8/8 26.34 0.76 2.89 28.60 0.65 2.28
21 Unknown Chicken C þ8/8 22.42 1.09 4.87 28.40 0.77 2.70
22 Unknown D þ8/8 23.48 1.11 4.71 28.44 0.63 2.20
23 Unknown Cow D þ8/8 26.30 0.59 2.24 28.25 0.79 2.79
24 Unknown Vaccine strain D þ7/8 23.17 0.67 2.90 27.80 0.37 1.33
25 Unknown D þ8/8 22.95 0.79 3.46 28.42 0.70 2.46
26 Unknown D þ8/8 26.42 0.70 2.64 28.23 0.76 2.68
27 Unknown D þ8/8b
25.30 0.56 2.21 28.56 0.53 1.85
28 Intestine Chicken C þ8/8 26.28 0.93 3.53 28.19 1.11 3.93
29 Unknown C þ8/8 22.74 0.98 4.31 28.31 0.52 1.85
30 Unknown C þ8/8 21.03 0.54 2.56 27.78 0.27 0.96
31 Unknown C þ8/8 27.59 1.40 5.07 28.76 1.17 4.05
32 Unknown C þ7/8 27.42 0.97 3.55 28.52 0.84 2.93
33 Unknown C þ8/8 33.49 1.58 4.72 28.40 1.17 4.11
a
Oxyura leucocephala.
b
One laboratory reported a weak positive result with very low fluorescence signal in regard to the magnitude of fluorescence recorded by other laboratories. Not taken into
account for mean, SD and RSD calculation.
C. Woudstra et al. / Anaerobe xxx (2013) 1e7 3
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YANAE1169_proof ■ 16 May 2013 ■ 3/7
Please cite this article in press as: Woudstra C, et al., Validation of a real-time PCR based method for detection of Clostridium botulinum types C, D
and their mosaic variants C-D and D-C in a multicenter collaborative trial, Anaerobe (2013), http://dx.doi.org/10.1016/j.anaerobe.2013.05.002
4. 99.4% and 100% for the GeneDiscÒ
arrays GD1 and GD2 respectively
among the eight participating laboratories. Data obtained with GD1
showed that two laboratories failed in the identification of one
sample each: laboratory 7 failed to identify strain n24 and labora-
tory 8 failed to identify strain n32. Using GD2 all laboratories
successfully identified the 33 C. botulinum strains. RSDs of the Cq
values obtained for each C. botulinum strain were in the range of
1.1%e7.1%.
Tables 3 and 4 show the participants’ results of the collaborative
trial obtained with DNA from 48 naturally contaminated samples
(clinical specimen mainly gathered from intestinal content, faeces,
organ of birds and cattle) using the GeneDiscÒ
arrays GD1 and GD2
respectively. The results showed 99.5% of concordance between
each participant for GD1 and GD2 GeneDiscÒ
arrays respectively.
Using the GeneDiscÒ
array GD1, two of the laboratories failed to
identify one sample each, laboratory 7 failed to identify sample
n14 and laboratory 3 failed to identify sample n33. Using the
GeneDiscÒ
array GD2, laboratory 7 failed to identify samples n3
and n4. The RSDs of Cq values obtained for each sample were in
the range of 1.3%e4.4%. Regarding the Cq values related to the
external amplification control (EAC), the RSD obtained with GD1
and GD2 on C. botulinum strains and naturally contaminated sam-
ples ranged from 1.0% to 7.9%. Cq values obtained with the EAC
never exceeded the expected value of 30 which indicated the
absence of PCR inhibitors in strains and naturally clinical samples
that were tested in this study.
4. Discussion
Botulism occurs worldwide, both as sporadic cases and massive
outbreaks in wild and domestic animals [23,24]. However, as ani-
mal botulism is not a notifiable disease in all European Countries, it
is probably under-diagnosed and under-reported in Europe. Botu-
lism can cause high mortality, leading rapidly to significant eco-
nomic loss in intensively farmed animals. In case of epizooty, the
economical, medical and alimentary consequences could be cata-
strophic, underlying the necessity of rapid identification of the
source of animal botulism. It is of major importance for veterinar-
ians to quickly characterize the disease and identify the causative
agent in order to take appropriate measures within a limited time
frame. In the first stages of the European project ‘AnibioThreat’, PCR
assays based on the GeneDiscÒ
technology have been developed for
the detection of C. botulinum types C, C-D, D and D-C. The assays
were validated in-house against an extensive list of clostridia and
other bacterial isolates and were shown to be highly specific and
sensitive. The limit of detection of the tests was 38 fg of total DNA,
corresponding to 15 genome copies. Artificially contaminated
samples of cecum showed a limit of detection below 50 spores per
gram [9]. With the aim to further evaluate the robustness and
reproducibility of the assays as a step towards their routine utili-
zation in veterinary laboratories across Europe, a multicenter
collaborative trial was conducted with eight European laboratories
and presented in this study.
Table 2
Strains of C. botulinum tested with the GeneDiscÒ
“GD2, C, D mosaic”.
Strain Origin GeneDiscÒ
“GD2 C, D mosaics C-D, D-C”
PCR Lab 1 to 8 Cq value EAC Cq value
Mean Cq SD RSD% Mean Cq SD RSD%
1 Intestine Ducka
C-D þ 8/8 24.13 0.63 2.61 29.49 1.70 5.76
2 Intestine Ducka
C-D þ8/8 25.28 0.61 2.43 29.33 1.55 5.30
3 Intestine Ducka
C-D þ8/8 22.35 0.81 3.64 29.05 1.39 4.79
4 Feces Milch-cow D-C þ8/8 22.35 1.19 5.35 29.42 1.23 4.19
5 Intestine Cow D-C þ8/8 25.25 0.92 3.64 29.14 1.24 4.25
6 Unknown C-D þ8/8 25.36 0.55 2.18 27.95 1.85 6.64
7 Unknown C-D þ8/8 26.52 1.20 4.54 29.17 1.25 4.29
8 Unknown C-D þ8/8 24.92 1.20 4.81 28.48 2.15 7.54
9 Unknown C-D þ8/8 23.13 0.72 3.11 29.27 1.42 4.86
10 Unknown C-D þ8/8 21.00 0.33 1.58 29.21 1.33 4.56
11 Unknown C-D þ8/8 22.59 0.81 3.57 29.04 1.31 4.52
12 Unknown C-D þ8/8 24.38 0.91 3.73 28.64 1.41 4.92
13 Unknown C-D þ8/8 23.75 0.51 2.15 29.52 1.29 4.36
14 Unknown C-D þ8/8 23.94 0.93 3.88 29.22 1.27 4.34
15 Feed C-D þ8/8 26.67 0.42 1.59 29.30 1.38 4.73
16 Unknown C þ8/8 24.09 0.47 1.94 29.39 1.30 4.42
17 Unknown Fox C-D þ8/8 21.50 0.49 2.26 28.73 1.74 6.05
18 Unknown C-D þ8/8 19.75 0.67 3.38 28.35 1.33 4.70
19 Unknown C þ8/8 21.33 0.49 2.31 29.62 1.25 4.22
20 Unknown C þ8/8 25.50 0.64 2.49 29.32 1.51 5.15
21 Unknown Chicken C-D þ8/8 23.09 0.87 3.75 29.14 1.67 5.74
22 Unknown D þ8/8 23.98 1.49 6.21 28.61 1.96 6.87
23 Unknown Cow D-C þ8/8 26.55 0.91 3.44 28.65 1.61 5.61
24 Unknown Vaccine strain D-C þ8/8 23.17 0.83 3.60 27.60 1.41 5.10
25 Unknown D-C þ8/8 23.39 0.93 3.96 29.19 1.31 4.49
26 Unknown D þ8/8 26.99 0.75 2.80 28.75 1.19 4.14
27 Unknown D þ8/8b
25.64 0.93 3.61 28.81 1.64 5.69
28 Intestine Chickens C-D þ8/8 26.33 0.57 2.17 29.08 1.64 5.63
29 Unknown C þ8/8 22.51 0.55 2.46 28.58 1.70 5.95
30 Unknown C-D þ8/8 20.41 0.70 3.43 28.51 1.29 4.53
31 Unknown C þ8/8 27.17 0.94 3.46 29.56 1.17 3.94
32 Unknown C þ8/8 26.44 0.59 2.24 29.37 1.02 3.46
33 Unknown C þ8/8 33.19 1.66 5.01 29.29 1.55 5.30
a
Oxyura leucocephala.
b
One laboratory reported a weak positive result with very low fluorescence signal in regard to the magnitude of fluorescence recorded by other laboratories. Not taken into
account for mean, SD and RSD calculation.
C. Woudstra et al. / Anaerobe xxx (2013) 1e74
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Please cite this article in press as: Woudstra C, et al., Validation of a real-time PCR based method for detection of Clostridium botulinum types C, D
and their mosaic variants C-D and D-C in a multicenter collaborative trial, Anaerobe (2013), http://dx.doi.org/10.1016/j.anaerobe.2013.05.002
5. Data obtained with DNA extracts from pure isolates and natu-
rally contaminated samples using the GeneDiscÒ
arrays “GD1 C
D” and “GD2 C, D mosaic” were highly concordant, with agree-
ment percentages ranging from 99.4% to 100%. The low (1.1%e7.9%)
RSD of the Cq values recorded by the eight laboratories with both
GD1 and GD2 is another indication of the accuracy and robustness
of the kit prototypes evaluated in this study. RSD values obtained
with the GeneDiscÒ
arrays “GD1 C D” and “GD2 C, D mosaic”
were in the same order of magnitude and often lower than those
reported in previously published evaluation trial using the
GeneDiscÒ
arrays for C. botulinum types A, B, E and F [25]. However,
some drawbacks have been noted during the trial. The software
which is part of the GeneDisc cycler failed to calculate an appro-
priate Cq value for two PCR determinations despite a strong posi-
tive amplification clearly visible when checking the amplification
curves. It seems that the software was unable to calculate the Cq
value due to the very unusual high fluorescence levels obtained for
these samples. A new version of the software is under development
to overcome this inconvenience. Conversely, three PCR de-
terminations gave a positive signal of fluorescence two times lower
Table 3
Naturally contaminated samples tested with the GeneDiscÒ
“GD1, C D”.
Sample Origin GeneDiscÒ
“GD1, C D”
PCR Lab 1 to 8 Cq value EAC Cq value
Mean Cq SD RSD% Mean Cq SD RSD%
1 Liver Pheasant C þ8/8a
25.71 0.50 1.93 28.46 0.42 1.47
2 Spleen Pheasant C þ8/8 27.21 0.53 1.95 28.35 0.63 2.22
3 Intestinal content Pheasant C þ8/8 25.29 0.83 3.29 28.18 1.13 4.00
4 Intestinal content Wild duck C þ8/8 28.35 0.69 2.45 28.41 0.78 2.74
5 Liver Mallard C þ8/8 26.20 0.49 1.89 27.54 1.03 3.75
6 Intestinal content Mallard C þ8/8 27.08 0.35 1.29 27.38 0.85 3.09
7 Soil NA C þ8/8 27.89 0.80 2.88 27.83 1.43 5.15
8 Maggot Egret C þ8/8 28.22 0.98 3.47 28.36 1.03 3.62
9 Liver Egret C þ8/8 29.18 0.79 2.70 28.22 0.76 2.68
10 Intestinal content Egret C þ8/8 28.09 0.65 2.31 28.63 0.37 1.30
11 Intestinal content Wild duck C þ8/8 29.16 0.61 2.09 28.35 0.49 1.74
12 Liver Peewit gull C þ8/8 27.50 0.66 2.39 27.74 0.75 2.69
13 Intestinal content Moorhen C þ8/8 27.25 0.94 3.47 28.68 0.41 1.41
14 Intestinal content Wild duck C þ7/8 26.48 0.64 2.42 28.37 0.64 2.27
15 Intestinal content Wild duck C þ8/8 27.47 0.72 2.61 28.28 0.79 2.80
D þ8/8 30.67 1.19 3.89
16 Intestinal content Wild duck C þ8/8b
28.34 0.61 2.15 28.71 0.67 2.32
17 Intestinal content Peewit gull C þ8/8 27.59 0.37 1.34 28.19 0.60 2.14
18 Liver Herring gull C þ8/8 26.72 0.36 1.34 27.44 0.68 2.46
19 Maggot NA C þ8/8 26.64 0.39 1.45 28.61 0.49 1.70
20 Intestinal content Peewit gull C þ8/8 27.07 1.01 3.73 27.78 1.70 6.13
21 Intestinal content Wild duck C þ 8/8 26.52 0.75 2.83 28.29 0.61 2.17
22 Liver Wild duck C þ8/8c
26.68 0.53 1.98 28.48 0.36 1.27
23 Stomach Mallard C þ8/8 27.63 0.82 2.95 28.34 0.59 2.10
24 Stomach Mallard C þ8/8 25.86 0.57 2.19 27.40 0.89 3.23
25 Liver Rat C þ8/8 25.80 1.01 3.92 28.33 0.67 2.35
26 Intestinal content Rat C þ8/8 26.48 0.49 1.83 28.50 0.44 1.55
27 Liver Coypus C þ8/8 27.63 0.84 3.04 28.18 0.77 2.75
28 Liver Coot C þ8/8 27.77 0.69 2.50 28.42 0.37 1.30
29 Liver Wild duck C þ8/8 29.44 0.84 2.85 28.10 0.40 1.43
30 Intestinal content Wild duck C þ8/8 28.54 0.39 1.37 27.90 0.27 0.97
31 Intestinal content Coypus C þ8/8 26.15 0.77 2.96 28.57 0.45 1.57
32 Feces Dog C þ8/8 29.82 0.80 2.69 28.45 0.54 1.91
D þ8/8 30.24 0.81 2.68
33 Feces Dog C þ8/8 29.56 0.85 2.87 28.08 0.93 3.32
D þ7/8 30.17 1.19 3.95
34 Liver Bovine D þ8/8 28.61 0.76 2.64 28.44 0.69 2.41
35 Feces Bovine D þ8/8 27.57 0.57 2.09 28.40 0.36 1.26
36 Ruminal content Bovine D þ8/8 27.97 0.77 2.77 27.96 0.71 2.53
37 Liver Pochard C þ8/8 29.51 0.66 2.25 28.24 0.67 2.39
38 Intestinal content Coot C þ8/8 28.93 0.71 2.46 28.22 0.68 2.41
39 Feed Mink D þ8/8 28.11 0.99 3.53 27.98 0.79 2.83
40 Intestinal content Cow D þ8/8 28.97 0.77 2.65 28.32 0.88 3.11
41 Control vaccine strain C. botulinum CKIII-4 C þ8/8 24.07 0.66 2.73 28.59 0.50 1.74
42 NA Gallus C þ8/8 31.76 0.74 2.32 28.02 0.33 1.17
43 NA Pheasant C þ8/8 29.65 0.70 2.36 28.54 0.59 2.06
44 Fecal culture Bovin D þ8/8 30.32 0.76 2.52 28.51 0.66 2.31
45 NA Gallus C þ8/8 26.96 0.84 3.11 27.94 1.13 4.04
46 Fecal culture Bovin D þ7/7d
27.18 0.67 2.47 28.43 0.47 1.65
47 Fecal culture Bovin D þ7/7d
28.38 0.64 2.24 27.97 0.80 2.84
48 Fecal culture Bovin 2806 D þ7/7d
28.58 0.85 2.99 27.80 0.47 1.71
NA: not available.
a
One laboratory reported a weak positive result with very low fluorescence signal in regard to the magnitude of fluorescence recorded by other laboratories. Not taken into
account for mean, SD and RSD calculation.
b
One laboratory recorded a positive result with a Cq value significantly different compared with other laboratories.
c
One laboratory showed a positive result but the software failed to calculated a Cq value for the corresponding sample.
d
One laboratory has been unable to test the corresponding samples.
C. Woudstra et al. / Anaerobe xxx (2013) 1e7 5
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Please cite this article in press as: Woudstra C, et al., Validation of a real-time PCR based method for detection of Clostridium botulinum types C, D
and their mosaic variants C-D and D-C in a multicenter collaborative trial, Anaerobe (2013), http://dx.doi.org/10.1016/j.anaerobe.2013.05.002
6. in regards to the other corresponding analyses performed in the
trial. Also, six determinations were found negative despite being
recorded positive by the other seven laboratories. Given that these
false negative results were reported by different participating lab-
oratories, a random degradation of DNA during shipment and
storage can be hypothesized. The degradation pattern of DNA iso-
lated from clinical samples has previously been shown to be
dependent on sample type, target organism and storage tempera-
ture [26]. The false negative results observed were not found
associated with a particular DNA extraction procedure. Primers and
probes degradation could be another potential explanation of the
observed false negative results. Among the 648 PCR determinations
performed on positive samples during this multicenter collabora-
tive trial, only six were recorded as false negative (0.9%). This
clearly indicates there is a very low risk of obtaining false-negative
responses using these tests.
In conclusion the results of this multicenter collaborative trial
showed that the method is effective in detecting C. botulinum types
C, D and their associated mosaic variants in the sample types tested.
The tests are robust, fast, easy to handle and can be readily applied
in microbiological laboratories for the detection of C. botulinum in
putative cases of animal botulism.
Acknowledgments
This research was supported by the framework of the EU-project
AniBioThreat (Grant Agreement: Home/2009/ISEC/AG/191) with
the financial support from the Prevention of and Fight against
Table 4
Naturally contaminated samples tested with the GeneDiscÒ
“GD2, C, D mosaic”.
Sample N
Origin GeneDiscÒ
“GD2 C, D mosaics C-D, D-C”
PCR Lab 1 to 8 Cq value EAC Cq value
Mean Cq SD RSD% Mean Cq SD RSD%
1 Liver Pheasant C-D þ8/8 25.42 0.73 2.87 28.91 1.72 5.96
2 Spleen Pheasant C-D þ8/8 26.43 0.71 2.69 28.97 1.78 6.16
3 Intestinal content Pheasant C-D þ7/8 24.81 0.85 3.44 28.58 2.13 7.46
4 Intestinal content Wild duck C-D þ7/8 28.12 0.64 2.29 29.03 1.72 5.91
5 Liver Mallard C-D þ8/8 25.54 0.94 3.69 28.86 1.16 4.01
6 Intestinal content Mallard C-D þ8/8 26.39 1.02 3.86 28.41 1.43 5.04
7 Soil NA C-D þ8/8 27.64 0.83 3.02 28.95 1.84 6.36
8 Maggot Egret C-D þ8/8 27.80 0.73 2.63 28.88 1.56 5.41
9 Liver Egret C-D þ8/8 28.31 1.09 3.85 29.06 1.68 5.77
10 Intestinal content Egret C-D þ8/8 27.87 0.60 2.17 28.99 1.83 6.30
11 Intestinal content Wild duck C-D þ8/8 28.49 0.76 2.68 28.68 1.94 6.78
12 Liver Peewit gull C-D þ8/8 27.08 0.62 2.29 28.10 1.70 6.03
13 Intestinal content Moorhen C-D þ8/8 27.16 0.66 2.42 29.44 1.31 4.44
14 Intestinal content Wild duck C-D þ8/8 26.07 0.74 2.84 29.02 1.29 4.45
15 Intestinal content Wild duck C-D þ8/8 27.10 0.86 3.17 28.60 1.65 5.78
D-C þ8/8 30.51 1.06 3.48
16 Intestinal content Wild duck C-D þ8/8 27.54 1.58 5.73 28.94 1.87 6.45
17 Intestinal content Peewit gull C-D þ8/8 27.07 0.90 3.34 28.31 1.66 5.88
18 Liver Herring gull C-D þ8/8 26.35 0.50 1.90 28.83 1.04 3.59
19 Maggot NA C-D þ8/8 26.57 0.49 1.86 29.13 1.55 5.33
20 Intestinal content Peewit gull C-D þ8/8 26.38 0.69 2.62 28.46 1.37 4.83
21 Intestinal content Wild duck C-D þ8/8 26.17 0.91 3.47 28.82 2.22 7.71
22 Liver Wild duck C-D þ8/8 26.67 0.56 2.11 29.49 1.46 4.95
23 stomach Mallard C-D þ8/8 27.55 0.77 2.78 29.08 1.75 6.01
24 stomach Mallard C-D þ8/8 25.89 0.51 1.95 28.93 1.40 4.86
25 Liver Rat C-D þ8/8 26.18 0.57 2.20 29.47 1.14 3.87
26 Intestinal content Rat C-D þ8/8 25.98 0.86 3.32 29.10 1.44 4.93
27 Liver Coypus C-D þ8/8 27.63 0.72 2.61 28.87 1.61 5.58
28 Liver Coot C-D þ8/8 27.74 0.60 2.16 29.32 1.28 4.37
29 Liver Wild duck C-D þ8/8 29.10 1.00 3.43 27.89 1.78 6.37
30 Intestinal content Wild duck C-D þ8/8 28.10 0.71 2.51 27.65 1.44 5.22
31 Intestinal content Coypus C-D þ8/8 26.29 0.46 1.74 28.87 1.37 4.76
32 Feces Dog C-D þ8/8 29.41 1.01 3.45 28.23 0.53 1.88
D-C þ8/8 30.15 1.15 3.81
33 Feces Dog C-D þ8/8 29.27 0.94 3.20 28.91 1.80 6.23
D-C þ8/8 29.90 1.17 3.92
34 Liver Bovine D-C þ8/8 28.73 1.25 4.36 29.13 1.25 4.28
35 Feces Bovine D-C þ8/8 27.92 0.91 3.25 28.79 1.23 4.29
36 Ruminal content Bovine D-C þ8/8 28.23 1.03 3.67 27.86 0.88 3.14
37 Liver Pochard C-D þ8/8 28.79 0.66 2.28 29.14 1.32 4.55
38 Intestinal content Coot C-D þ8/8a
28.41 0.96 3.37 28.72 1.59 5.53
39 Feed Mink D-C þ8/8 28.30 1.07 3.79 28.83 1.85 6.43
40 Intestinal content Cow D-C þ8/8 29.28 0.94 3.22 29.46 1.23 4.16
41 Control vaccine strain C. botulinum CKIII-4 C þ8/8 24.03 0.47 1.94 28.87 1.38 4.79
42 NA Gallus C-D þ8/8 31.09 1.23 3.96 28.65 1.31 4.56
43 NA Pheasant C-D þ8/8 29.78 0.68 2.30 28.88 1.95 6.77
44 Fecal culture Bovin D-C þ8/8 30.43 0.91 3.00 28.68 2.26 7.87
45 NA Gallus C-D þ8/8 26.88 0.96 3.58 29.17 1.57 5.39
46 Fecal culture Bovin D-C þ8/8 27.07 0.74 2.72 29.35 1.18 4.01
47 Fecal culture Bovin D-C þ8/8 28.70 0.59 2.06 29.22 1.54 5.28
48 Fecal culture Bovin 2806 D-C þ8/8 28.30 1.11 3.93 28.85 1.07 3.72
NA: Not available.
a
One laboratory observed a positive result but the software failed to calculated a Cq value for the corresponding sample.
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Please cite this article in press as: Woudstra C, et al., Validation of a real-time PCR based method for detection of Clostridium botulinum types C, D
and their mosaic variants C-D and D-C in a multicenter collaborative trial, Anaerobe (2013), http://dx.doi.org/10.1016/j.anaerobe.2013.05.002
7. Crime Programme of the European Union, European Commission e
Directorate General Home Affairs. This publication reflects the
views only of the author, and the European Commission cannot be
held responsible for any use which may be made of the information
contained therein. We also thank the DIM malinf Ile de France for
the financial support of the project. Pia Engelsmann (DTU) is
acknowledged for excellent technical assistance.
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Please cite this article in press as: Woudstra C, et al., Validation of a real-time PCR based method for detection of Clostridium botulinum types C, D
and their mosaic variants C-D and D-C in a multicenter collaborative trial, Anaerobe (2013), http://dx.doi.org/10.1016/j.anaerobe.2013.05.002