This document is regarding the new and improved method of Porcine detection through DNA extraction and also talks about detection in imported meat found in the some of the GCC markets
Enterocin 55 produced by non rabbit-derived strain Enterococcus faecium EF55 ...Agriculture Journal IJOEAR
— Ent55 is produced by poultry strain Enterococcus faecium EF55. It is substance which can be allotted to Class II enterocins; thermo-stable, small peptide. Because producer strain has shown beneficial effect in poultry and broiler rabbits as well, we decided to apply Ent55 in broiler rabbit husbandry. Ent55 showed antimicrobial activity in broiler rabbits by reduction of staphylococci, Clostridiae, pseudomonads and coliforms. Its beneficial effect was demonstrated by stimulation of phagocytic activity as well as by reduction of Eimeria spp. oocysts. GPx values were lower; it means, no oxidative stress was evoked. Moreover, it has not negative influence on growth performance and biochemical parameters. Our results indicated that enterocin produced by not-autochtonous strain can also have protective and beneficial effect in broiler rabbits.
Enterocin 55 produced by non rabbit-derived strain Enterococcus faecium EF55 ...Agriculture Journal IJOEAR
— Ent55 is produced by poultry strain Enterococcus faecium EF55. It is substance which can be allotted to Class II enterocins; thermo-stable, small peptide. Because producer strain has shown beneficial effect in poultry and broiler rabbits as well, we decided to apply Ent55 in broiler rabbit husbandry. Ent55 showed antimicrobial activity in broiler rabbits by reduction of staphylococci, Clostridiae, pseudomonads and coliforms. Its beneficial effect was demonstrated by stimulation of phagocytic activity as well as by reduction of Eimeria spp. oocysts. GPx values were lower; it means, no oxidative stress was evoked. Moreover, it has not negative influence on growth performance and biochemical parameters. Our results indicated that enterocin produced by not-autochtonous strain can also have protective and beneficial effect in broiler rabbits.
Induction of tetraploidy in an ornamental fish koicarp Cyprinus carpio L, usi...researchanimalsciences
Koicarp is potentially an important cultured ornamental fish in freshwater. Moreover there were reports existing on genetic manipulation of koicarp by application of the heat shock. Hence the present study was made to contribute a protocol for induction of tetraploidy by heat shock in the koicarp.Induction of tetraploidy was attempted in Cyprinus carpio L, Koicarp by heat shock. Eggs from five females and milt from five males ok Koicarp were pooled to ensure the required quantity and quality of gametes for fertilization. After insemination the eggs were divided into three batches each experiment based on the post fertilization viz., 25min, 27min and 30min after insemination. Batches of eggs held in plastic containers were exposed to hot water at 38° C, 39° C, 40° C & 41° C for durations of 2min and four min. One batch of the eggs without heat shock treatment was used as control. After treatments, eggs were immediately transferred to incubation troughs. Tetraploidy was ascertained by karyotyping as well as RBC nuclear micro measurements.Heat shock of 41°C for four min, imparted to eggs for 20 min after fertilization induced a maximum of 60± 2% tetraploidy and maximum hatchability of 10± 1.5%. A large proportion of the heat shocked embryos displayed morphological abnormalities such as short and curved tail, destroyed yolksac, deformed vertebral column and malformed cephalic region. A maximum of 60± 2% tetraploids (4n = 156) were obtained when the fertilized eggs (20 min old) were heat shocked at 41° C for four min duration. The tetraploid red blood cells (RBCs) nucleus volume was 2.1 times greater than those of the diploid RBC nucleus.Given that koicarp are such a useful model for other areas of research, perhaps further studies on the induction of tetraploidy in this species will lead to a better understanding of polyploidy induction and the establishment of tetraploid lines of koicarp and other species as well.
Article Citation:
Ananth Kumar and Mohamed Abdul Kadher Haniffa.
Induction of Tetraploidy in an Ornamental Fish Koicarp
Cyprinus carpio L, Using Heat Shock.
Journal of Research in Animal Sciences (2012) 1(1): 013-019.
Full Text:
http://janimalsciences.com/documents/AS0006.pdf
Automated DNA purification from diverse Microbiome samples using dedicated Mi...QIAGEN
This application note demonstrates the automation of QIAGEN’s new line of DNA sample prep kits for the microbiome. The microbiome of samples as diverse as soil, water and stool was purified using dedicated QIAcube compatible kits. Automation on the QIAcube enabled efficient and reliable use of these samples for sensitive downstream applications such as qPCR and NGS. In addition, the CLC Microbial Genomics Module was successfully employed for metagenome sequencing and identification of microbial composition and diversity.
Detection and Surveillance of Antibiotic Resistance Genes From Food and Ferti...QIAGEN
One potential way to acquire antibiotic resistance genes is through the food supply chain. Both livestock and feed may
acquire antibiotic resistant bacteria via different mechanisms. Foodstuffs can be exposed to antibiotic resistant bacteria
through fertilizer originating from waste-water treatment plants. This, in addition to increasing administration of antibiotics
to livestock, can lead to food being a potential source of antibiotic resistance genes. This may lead to horizontal gene
transfer to pathogenic enteropathogens and further to drug resistance in humans. Therefore, the surveillance and prevention
of antibiotic resistance genes in food is important.
To effectively combat the spread of difficult-to-treat bacterial infections, rapid surveillance methods to detect antibiotic
resistance genes are required; in order to monitor both bacterial isolates and metagenomic samples.
Since the gut is known to act as a reservoir for antibiotic resistance genes, a small-scale research study was performed on
5 stool samples isolated from healthy human adults using an antibiotic resistance gene identification PCR array. In addition,
the diversity of antibiotic resistance genes in municipal biosolids was determined using an Antibiotic Resistance Genes
Microbial DNA qPCR Array with DNA extracted from belt-filter, press-cake sewage samples.
22 antibiotic resistance genes were identified from different resistance classifications. Further studies were performed in
beef, chicken, vegetable and pork samples. In conclusion, PCR arrays can be effective tools for detection of antibiotic
resistance genes from food samples and potential fertilizer sources.
DNA Analysis - Basic Research : A Case StudyQIAGEN
Nucleic acid gel electrophoresis is a broadly used technique in all fields of basic life science research. The flexibility and versatility of the QIAxcel allows researchers to streamline and accelerate their molecular biology experiments. The sensitivity and resolution of capillary electrophoresis offers an excellent alternative to long or complex slab gel setup. A wide range of applications in basic research involving microsatellite analysis, mapping mutant genes, linkage analysis, and genotyping transgenes by PCR are all powerful molecular approaches for screening organisms and their genetic profiles. The QIAxcel Advanced provides precise and reliable results to accelerate these analyses and the research projects they are part of.
Hot-start DNA polymerases are commonly used in PCR for genotyping, sequencing, molecular diagnostics, and high-throughput applications. In this presentation, PCR performance of Invitrogen™ Platinum II Taq Hot-Start DNA Polymerase and Invitrogen™ AccuPrime Taq DNA Polymerase is compared in the following areas:
• PCR run time for targets of different lengths
• Amplification of AT-rich and GC-rich sequences
• Tolerance to PCR inhibitors
• Sensitivity in target detection
• Universal protocol for PCR targets of different lengths
• Multiplex PCR of 15 targets
• Product format for direct gel loading
Request a sample of Platinum II Taq enzyme at http://bit.ly/2M4U9cw
Find other PCR enzymes at http://bit.ly/2JIPrzj
Learn more about PCR at http://bit.ly/2y2aSVo
#PCR #PCREducation #Invitrogen #InvitrogenSchoolofMolBio
The enzyme Telomerase maintains telomeres at the ends of
chromosomes. The Telomerase Reverse Transcriptase (TERT)
gene codes for the enzyme’s catalytic domain and is not
expressed in normal somatic cells. As a consequence, normal
cells acquire senescence by shortening of their telomeres
during cell division and eventually undergo apoptosis. In
contrast to normal somatic cells, expression of TERT is
reinstated in cancer cells causing escape from senescence and
apoptosis by maintaining the telomeres. It has recently been
shown that mutations in the TERT promoter region play a key
role in regulating and reinstating TERT expression. Up to 90%
of cancers carry a mutation in the TERT promoter region.
Mutations like C228T and C250T create new binding sites for
the E26 transformation-specific (ETS) transcription factor that
regulates TERT expression (1,2). Experimental evidence
showed that the ETS factor GA-binding protein, alpha subunit
(GABPA) binds to the de novo ETS motif and activates TERT
transcription in cancer cells.
Paratuberculosis (PTB) remains one of the most obstacles limit animal breeding sector all over the world. The current study aimed to detect the etiology of PTB in tissues of clinically suspected small ruminants using histopathological and real-time polymerase chain reaction (RT-PCR) methods. Clinical examination showed 10 (26.4%) PTB suspected cases out of the total (38) examined animals. The suspected cases were euthanized, necropsied, gross lesions were recorded and tissue samples were collected for histopathological and molecular procedures. Grossly intestinal and mesenteric lymph nodes thickening, corrugations and edematous swellings were recorded. Semi-thin sections of the intestine and mesenteric lymph nodes stained with toluidine blue demonstrated MAP organism inside epithelium cells and macrophages. RT-PCR detected MAP IS900 gene in all suspected cases (100%), thus we recommend using RT-PCR as a rapid sensitive method in the diagnosis of PTB.
Key-words: Paratuberculosis, Mycobacterium, Semi thin sections, Toluidine blue, IS900 gene
Gene expression profile of the tumor microenvironment from 40 NSCLC FFPE and ...Thermo Fisher Scientific
The tumor microenvironment (TME) is the intersection between tumor cells and
surrounding non-transformed cells. It contains immune cells, signaling molecules,
stromal and extracellular matrix. Research has shown the TME is often associated
with tumor growth. However, the function and regulatory mechanism of each
constituent is still poorly understood. The presence of PD-L1 is a promising marker
to predict positive response for T cell checkpoint therapy. Current IHC methods to
measure PD-L1 are subjective and highly variable. A higher-throughput and
standardized method that can systematically measure gene expression of cells
present in the TME has emerged to be a more desirable solution.
We applied the OncomineTM Immune Response Research Assay to measure the
expression of 395 genes in non-small cell lung cancer (NSCLC) research samples
from 40 matched FFPE and fresh frozen sample types. This assay covers genes
involved in checkpoint pathway, T cell regulation, cytokine and interferon signaling
pathways, and markers of different tumor infiltrating lymphocyte (TIL) subsets, as
well as tumor markers. With an input requirement of 10 ng of total RNA, libraries
were generated, templated on the Ion ChefTM and sequenced on the Ion S5TM
System. Sequencing data was analyzed and mapped with Torrent Suite Software
and differential expression analysis was conducted with AffymetrixTM Transcriptome
Analysis Console.
Mitochondrial ND-1 gene-specific primer polymerase chain reaction to determin...UniversitasGadjahMada
A specificity method to detect mice meat contamination in beef meatballs using specific primer-polymerase chain reaction (PCR) technique has been developed. The primer ND1-P1 primers were designed using primer-BLAST software using mtDNA of mice as a template. The Primer ND1-P1 forward (5’-CGGCATCCTACAACCATTTGC-3’) and reverse (5’-CGGCTCGTAAAGC-TCCGAA-3’) was able to amplify a 294 bp fragment of ND1 gene in mice mtDNA. The primers have been proven precise with only amplify the target fragment in mice meatball but not in another meatball including beef meatball, chicken meatball, pork meatball, horse meatball, and goat meatball. The present of mice meat in meatballs can be detected at a concentration as low as 5% (w/w). The ND1-P1 primer is potentially used as a specific marker for detection of mice meat in the meat products.
Induction of tetraploidy in an ornamental fish koicarp Cyprinus carpio L, usi...researchanimalsciences
Koicarp is potentially an important cultured ornamental fish in freshwater. Moreover there were reports existing on genetic manipulation of koicarp by application of the heat shock. Hence the present study was made to contribute a protocol for induction of tetraploidy by heat shock in the koicarp.Induction of tetraploidy was attempted in Cyprinus carpio L, Koicarp by heat shock. Eggs from five females and milt from five males ok Koicarp were pooled to ensure the required quantity and quality of gametes for fertilization. After insemination the eggs were divided into three batches each experiment based on the post fertilization viz., 25min, 27min and 30min after insemination. Batches of eggs held in plastic containers were exposed to hot water at 38° C, 39° C, 40° C & 41° C for durations of 2min and four min. One batch of the eggs without heat shock treatment was used as control. After treatments, eggs were immediately transferred to incubation troughs. Tetraploidy was ascertained by karyotyping as well as RBC nuclear micro measurements.Heat shock of 41°C for four min, imparted to eggs for 20 min after fertilization induced a maximum of 60± 2% tetraploidy and maximum hatchability of 10± 1.5%. A large proportion of the heat shocked embryos displayed morphological abnormalities such as short and curved tail, destroyed yolksac, deformed vertebral column and malformed cephalic region. A maximum of 60± 2% tetraploids (4n = 156) were obtained when the fertilized eggs (20 min old) were heat shocked at 41° C for four min duration. The tetraploid red blood cells (RBCs) nucleus volume was 2.1 times greater than those of the diploid RBC nucleus.Given that koicarp are such a useful model for other areas of research, perhaps further studies on the induction of tetraploidy in this species will lead to a better understanding of polyploidy induction and the establishment of tetraploid lines of koicarp and other species as well.
Article Citation:
Ananth Kumar and Mohamed Abdul Kadher Haniffa.
Induction of Tetraploidy in an Ornamental Fish Koicarp
Cyprinus carpio L, Using Heat Shock.
Journal of Research in Animal Sciences (2012) 1(1): 013-019.
Full Text:
http://janimalsciences.com/documents/AS0006.pdf
Automated DNA purification from diverse Microbiome samples using dedicated Mi...QIAGEN
This application note demonstrates the automation of QIAGEN’s new line of DNA sample prep kits for the microbiome. The microbiome of samples as diverse as soil, water and stool was purified using dedicated QIAcube compatible kits. Automation on the QIAcube enabled efficient and reliable use of these samples for sensitive downstream applications such as qPCR and NGS. In addition, the CLC Microbial Genomics Module was successfully employed for metagenome sequencing and identification of microbial composition and diversity.
Detection and Surveillance of Antibiotic Resistance Genes From Food and Ferti...QIAGEN
One potential way to acquire antibiotic resistance genes is through the food supply chain. Both livestock and feed may
acquire antibiotic resistant bacteria via different mechanisms. Foodstuffs can be exposed to antibiotic resistant bacteria
through fertilizer originating from waste-water treatment plants. This, in addition to increasing administration of antibiotics
to livestock, can lead to food being a potential source of antibiotic resistance genes. This may lead to horizontal gene
transfer to pathogenic enteropathogens and further to drug resistance in humans. Therefore, the surveillance and prevention
of antibiotic resistance genes in food is important.
To effectively combat the spread of difficult-to-treat bacterial infections, rapid surveillance methods to detect antibiotic
resistance genes are required; in order to monitor both bacterial isolates and metagenomic samples.
Since the gut is known to act as a reservoir for antibiotic resistance genes, a small-scale research study was performed on
5 stool samples isolated from healthy human adults using an antibiotic resistance gene identification PCR array. In addition,
the diversity of antibiotic resistance genes in municipal biosolids was determined using an Antibiotic Resistance Genes
Microbial DNA qPCR Array with DNA extracted from belt-filter, press-cake sewage samples.
22 antibiotic resistance genes were identified from different resistance classifications. Further studies were performed in
beef, chicken, vegetable and pork samples. In conclusion, PCR arrays can be effective tools for detection of antibiotic
resistance genes from food samples and potential fertilizer sources.
DNA Analysis - Basic Research : A Case StudyQIAGEN
Nucleic acid gel electrophoresis is a broadly used technique in all fields of basic life science research. The flexibility and versatility of the QIAxcel allows researchers to streamline and accelerate their molecular biology experiments. The sensitivity and resolution of capillary electrophoresis offers an excellent alternative to long or complex slab gel setup. A wide range of applications in basic research involving microsatellite analysis, mapping mutant genes, linkage analysis, and genotyping transgenes by PCR are all powerful molecular approaches for screening organisms and their genetic profiles. The QIAxcel Advanced provides precise and reliable results to accelerate these analyses and the research projects they are part of.
Hot-start DNA polymerases are commonly used in PCR for genotyping, sequencing, molecular diagnostics, and high-throughput applications. In this presentation, PCR performance of Invitrogen™ Platinum II Taq Hot-Start DNA Polymerase and Invitrogen™ AccuPrime Taq DNA Polymerase is compared in the following areas:
• PCR run time for targets of different lengths
• Amplification of AT-rich and GC-rich sequences
• Tolerance to PCR inhibitors
• Sensitivity in target detection
• Universal protocol for PCR targets of different lengths
• Multiplex PCR of 15 targets
• Product format for direct gel loading
Request a sample of Platinum II Taq enzyme at http://bit.ly/2M4U9cw
Find other PCR enzymes at http://bit.ly/2JIPrzj
Learn more about PCR at http://bit.ly/2y2aSVo
#PCR #PCREducation #Invitrogen #InvitrogenSchoolofMolBio
The enzyme Telomerase maintains telomeres at the ends of
chromosomes. The Telomerase Reverse Transcriptase (TERT)
gene codes for the enzyme’s catalytic domain and is not
expressed in normal somatic cells. As a consequence, normal
cells acquire senescence by shortening of their telomeres
during cell division and eventually undergo apoptosis. In
contrast to normal somatic cells, expression of TERT is
reinstated in cancer cells causing escape from senescence and
apoptosis by maintaining the telomeres. It has recently been
shown that mutations in the TERT promoter region play a key
role in regulating and reinstating TERT expression. Up to 90%
of cancers carry a mutation in the TERT promoter region.
Mutations like C228T and C250T create new binding sites for
the E26 transformation-specific (ETS) transcription factor that
regulates TERT expression (1,2). Experimental evidence
showed that the ETS factor GA-binding protein, alpha subunit
(GABPA) binds to the de novo ETS motif and activates TERT
transcription in cancer cells.
Paratuberculosis (PTB) remains one of the most obstacles limit animal breeding sector all over the world. The current study aimed to detect the etiology of PTB in tissues of clinically suspected small ruminants using histopathological and real-time polymerase chain reaction (RT-PCR) methods. Clinical examination showed 10 (26.4%) PTB suspected cases out of the total (38) examined animals. The suspected cases were euthanized, necropsied, gross lesions were recorded and tissue samples were collected for histopathological and molecular procedures. Grossly intestinal and mesenteric lymph nodes thickening, corrugations and edematous swellings were recorded. Semi-thin sections of the intestine and mesenteric lymph nodes stained with toluidine blue demonstrated MAP organism inside epithelium cells and macrophages. RT-PCR detected MAP IS900 gene in all suspected cases (100%), thus we recommend using RT-PCR as a rapid sensitive method in the diagnosis of PTB.
Key-words: Paratuberculosis, Mycobacterium, Semi thin sections, Toluidine blue, IS900 gene
Gene expression profile of the tumor microenvironment from 40 NSCLC FFPE and ...Thermo Fisher Scientific
The tumor microenvironment (TME) is the intersection between tumor cells and
surrounding non-transformed cells. It contains immune cells, signaling molecules,
stromal and extracellular matrix. Research has shown the TME is often associated
with tumor growth. However, the function and regulatory mechanism of each
constituent is still poorly understood. The presence of PD-L1 is a promising marker
to predict positive response for T cell checkpoint therapy. Current IHC methods to
measure PD-L1 are subjective and highly variable. A higher-throughput and
standardized method that can systematically measure gene expression of cells
present in the TME has emerged to be a more desirable solution.
We applied the OncomineTM Immune Response Research Assay to measure the
expression of 395 genes in non-small cell lung cancer (NSCLC) research samples
from 40 matched FFPE and fresh frozen sample types. This assay covers genes
involved in checkpoint pathway, T cell regulation, cytokine and interferon signaling
pathways, and markers of different tumor infiltrating lymphocyte (TIL) subsets, as
well as tumor markers. With an input requirement of 10 ng of total RNA, libraries
were generated, templated on the Ion ChefTM and sequenced on the Ion S5TM
System. Sequencing data was analyzed and mapped with Torrent Suite Software
and differential expression analysis was conducted with AffymetrixTM Transcriptome
Analysis Console.
Mitochondrial ND-1 gene-specific primer polymerase chain reaction to determin...UniversitasGadjahMada
A specificity method to detect mice meat contamination in beef meatballs using specific primer-polymerase chain reaction (PCR) technique has been developed. The primer ND1-P1 primers were designed using primer-BLAST software using mtDNA of mice as a template. The Primer ND1-P1 forward (5’-CGGCATCCTACAACCATTTGC-3’) and reverse (5’-CGGCTCGTAAAGC-TCCGAA-3’) was able to amplify a 294 bp fragment of ND1 gene in mice mtDNA. The primers have been proven precise with only amplify the target fragment in mice meatball but not in another meatball including beef meatball, chicken meatball, pork meatball, horse meatball, and goat meatball. The present of mice meat in meatballs can be detected at a concentration as low as 5% (w/w). The ND1-P1 primer is potentially used as a specific marker for detection of mice meat in the meat products.
Johne's disease (JD) or Paratuberculosis (PTB) has gained a great attention by many industrial countries for its severing economic losses and possibly zoonotic concerns. In the current study conventional clinical and direct microscopic examination compared to real time polymerase chain reaction (RT-PCR) were used to diagnose JD in clinically suspected small ruminants. Clinical examination revealed 130 (8.7%) suspected cases that showed history of emaciation and diarrhea out of the total examined (1500) animals. Direct microscopy of Ziehl-Neelsen (ZN) stained smears (130) revealed 62 (47.7%) acid fast bacteria resembled Mycobacterium avium subsp. paratuberculosis (MAP). RT-PCR insertion sequence gene (IS900) detected MAP in 25 (65.8%) out of 38 fecal samples harbored acid fast bacilli. We concluded and recommended that RT-PCR considers the most rapid confirmatory method for screening and diagnosis of the MAP in comparison to low specific conventional phenotypic methods, which still remained valuable techniques in the diagnosis of JD in developing countries.
Key-words: Johne's disease, Paratuberculosis, Acid fast bacteria, Ziehl-Neelsen stain, IS900 gene
This research is carried out in order to improve the production of eggs in indigenous chicken by reducing the
inter-sequence stopped days through use of anti-prolactin agent (Bromocriptine) and serum from laying hen.
Sixty-four indigenous (deshi) chickens of 20-22 weeks of age, were randomly assigned into four groups (i, j, k
and l) and each group consisting of 16 hens. Control was designated as Group I and Bromocriptine orally at a
dose of 641μg/bird/day was used to treat group j, group k was treated with serum of laying kadhaknath hen
serum at a dose of 1 ml intramuscularly/bird/day and group l was treated with both Kadhaknath serum and
Bromocriptine at doses given to group j and k for the period of 15 March, 2019 to 16 June, 2019 and egg
production, stopped days, prolactin level, hematological parameter and egg qualities were observed. A
significant increase (p<0.05) in Egg production was noticed in all treated groups in comparison to the groups
which were in non- treated control and group k showed the highest production. All treatment groups depicted a
significant decrease (p<0.05) in stopped days and prolactin levels and lowest were observed in hens of group l.
In hematological values between the chicken group, no significant differences were noticed. The present study
reveals that combined treatment with Bromocriptine and serum from laying kadhaknath hen increases egg
production without affecting the health of indigenous chickens.
Following the Food Standards Agency’s (FSA) announcement in January that horse and pig DNA had been identified in beef products sold by several supermarket chains, further testing across Europe and beyond has revealed widespread incidences of such contamination.1 However, most testing methods are based on detection of species-specific DNA in meat, using the polymerase chain reaction (PCR) – which does not detect or identify proteins. This is a concern because DNA can be easily disrupted or removed during standard meat processing and food manufacturing. As a result, horse tissue or other contaminants remain undetected in food samples, despite strong presence of the contaminating proteins. An alternative protein-based method, ELISA (enzyme-linked immunosorbent assay), can be used to complement DNA testing, but this method has limitations, including that it detects only one part of the protein and not multiple protein markers.
The LC-MS/MS-based method presented offers a more accurate and reliable approach to meat speciation than PCR or ELISA-based techniques or other indirect methods, and also allows for the detection of veterinary drug residues in the same analysis, which is not possible by ELISA or PCR.
The method was developed using an Eksigent ekspert™ microLC 200 UHPLC system coupled with a SCIEX QTRAP® 5500 LC/MS/MS system. The method uses multiple reaction monitoring (MRM) to detect peptide markers for horse and is capable of providing sequence information by acquiring an enhanced product ion (EPI) scan for each triggering MRM which can be used to further confirm the peptide’s / proteins and therefore the species identity. This gives greater confidence for food testing when distinguishing between species; for example horse and beef proteins may differ by as little as one or two amino acids.
At the same time it is also possible to detect and quantify veterinary drug residues using the same extraction method and LC conditions by simply adding additional MRM transitions to the method. Here the nonsteroidal anti-inflammatory drug (NSAID) BUTE was detected in meat samples.
New rapid method for detection of salmonella in meat and poultry using actero tmSandra Rogoza
Poster Presentation by Dr. Sergiy Olishevskyy, Bsc, PhD of FoodChek Laboratories at IFT 2016.
Despite control and prevention efforts, Salmonella infections arising from contaminated meat and poultry continue to be a significant problem, with millions of cases occurring each year. Conventional methods for the detection of Salmonella can require up to seven days for a positive result and are not appropriate for routine testing of large numbers of samples. Thus, the development and implementation of rapid detection methods of Salmonella are among the most important food safety tasks.
The objective of this study was to develop a sensitive and rapid method for Salmonella detection in meat and poultry, including raw ground beef, raw ground chicken and chicken carcass rinses. This study included validation of a new protocol based on single-step enrichment with Actero™ Salmonella Enrichment Media followed by detection using the DuPont™ BAX® System Real-Time PCR Assay for Salmonella .
At Taste Of Middle East, we believe that food is not just about satisfying hunger, it's about experiencing different cultures and traditions. Our restaurant concept is based on selecting famous dishes from Iran, Turkey, Afghanistan, and other Arabic countries to give our customers an authentic taste of the Middle East
Roti Bank Hyderabad: A Beacon of Hope and NourishmentRoti Bank
One of the top cities of India, Hyderabad is the capital of Telangana and home to some of the biggest companies. But the other aspect of the city is a huge chunk of population that is even deprived of the food and shelter. There are many people in Hyderabad that are not having access to
Key Features of The Italian Restaurants.pdfmenafilo317
Filomena, a renowned Italian restaurant, is renowned for its authentic cuisine, warm environment, and exceptional service. Recognized for its homemade pasta, traditional dishes, and extensive wine selection, we provide a true taste of Italy. Its commitment to quality ingredients and classic recipes has made it a adored dining destination for Italian food enthusiasts.
Ang Chong Yi Navigating Singaporean Flavors: A Journey from Cultural Heritage...Ang Chong Yi
In the heart of Singapore, where tradition meets modernity, He embarks on a culinary adventure that transcends borders. His mission? Ang Chong Yi Exploring the Cultural Heritage and Identity in Singaporean Cuisine. To explore the rich tapestry of flavours that define Singaporean cuisine while embracing innovative plant-based approaches. Join us as we follow his footsteps through bustling markets, hidden hawker stalls, and vibrant street corners.
Piccola Cucina is regarded as the best restaurant in Brooklyn and as the best Italian restaurant in NYC. We offer authentic Italian cuisine with a Sicilian touch that elevates the entire fine dining experience. We’re the first result when someone searches for where to eat in Brooklyn or the best restaurant near me.
Improved DNA Extraction Method for Porcine Contaminants
1. Saudi Journal of Biological Sciences Vol. 15 No (2) December, 2008
225
Improved DNA Extraction Method for Porcine Contaminants, Detection in Imported Meat to The Saudi Market
Ibrahim Abdullah Alaraidh
Biology Department, Teachers College, King Saud University. P. O. 4341, Riyadh 11491, Saudi Arabia.
E-mail: ibrahim1551@hotmail.com
Abstract
A porcine detection methodology based on deoxyribonucleic acid (DNA) extraction and polymerase chain reaction (PCR) amplification of a specific porcine fragment was used in this paper. With the advent of mass globalization and the fast growing and rapidly changing halal industry of the international market it is of vital need that a practical scientific system be applied and established in the Kingdom of Saudi Arabia to detect and monitor the ingredients in food especially porcine contaminants. A simple modified technique was designed to extract DNA from food material. ASL buffer (Qiagen) for lysis and a series of incubation steps were applied prior to PCR and detection limits were established. The optimized method was then used to identify pork in food products obtained from different local hypermarkets. The used method was displayed to be robust and reliable. Out of thirty- three food samples labeled as halal, there were two imported samples; one beef steak and one beef sausage that were positive for porcine contaminants. From the results it can be concluded that sufficient PCR ready DNA can be obtained by this single solution methodology. Although the incubation for the lyses is recommended to be overnight the cost of the experiment and the relative simplicity and reliability of the experiment allows a mass food testing ability and a viable commercialization opportunity in the Kingdom. It is also of absolute importance that a systematic scientific precaution be taken to ensure that imported food products in Saudi Arabia are actually halal, as Allah says in the Holy Qur’an: “Forbidden to you (for food) are: Al-Maitah (the dead animals - cattle - beast not slaughtered), blood, the flesh of swine” (Sûrat Al-Mâ’idah, Verse 3).
Key Words: Porcine, DNA, PCR, Primer, Processed meat, Detection, halal.
Saudi Journal of Biological Sciences 15 (2) 225-229 December 2008
ISSN 1319-562X
The Official Journal of the Saudi Biological Society
htt:www.saudibiosoc.com
Introduction
Preparation of meat products by mixing meats and fats of different origin is illegal. This kind of adulteration is a common procedure in most countries. Adulteration with cheaper ambiguous meats or improper labeling of meat products as “Halal” while it contained pork or pork fat clearly interferes with the religious prohibitions for Muslims around the world. Permissible nature (Halal) of any product including food, accessories and relationships is of particular importance for Muslims, whether in moral or physical life, particularly in the current era of globalization, where a single processed food product could be subject to elements sourced from several nations with its origin doubted or neglected due to ignorance or lack of technology. Therefore, in the current era where free trade and mass market globalization is dominating, certain food producers take advantage of every opportunity to increase profits and neglect proper food safety protocols with respect to religious or cultural beliefs. Hence, several studies are currently being carried out in the field of food detection systems.
Especially with regard to pork in processed or non- processed food products, various methodologies that are equally effective and reliable in their relevant approaches are currently used for detection systems.
For instance the detection of pork and lard as adulterants in processed beef and mutton mixtures is carried out where the mixture of derivative and saturated triglycerides is analyzed by liquid chromatography using a reverse-phase column and a UV detector. Pork fat has larger amounts of triglyceride containing saturated fatty acid at the C- 2 position than does the fat of other meat. The ratio of triglyceride containing saturated fatty acid vs. triglyceride containing unsaturated fatty acid at the same (C-2) position
2. Saudi Journal of Biological Sciences Vol. 15 No (2) December, 2008
226
Ibrahim Abdullah Alaraidh
(SSU/SUS) in a sample is compared with those of pure meats. The presence of pork in the sample causes the ratio to increase compared with ratios for pure beef or mutton. The increase in the SSU/SUS ratio is significant for the addition of 1% pork in beef. In the case of mutton, the addition of 3% pork causes a noticeable change (Saeed, et al ., 1989).
Detection of pork by PCR amplification of nuclear 18S ribosomal RNA and growth hormone gene or Y chromosome has been previously described (Meyer et al., 1995 and Meyer et al., 1994). In other methods, the use of fluorescence sensor capillary electrophoresis allows identification of specific DNA fingerprint species of pork, goat, and beef generated by restriction enzyme digestion using fluorescence–labeling PCR amplification (Al- Rashood et al., 1995). Specific detection of processed pig and cattle materials treated at 134 degrees Celcius in various feed matrices down to a limit of detection of about 0.1% was reported (Fumière et al., 2006). The detection quantification of bovine, porcine, lamb, chicken, turkey, and ostrich DNA in complex samples was achieved by using TaqMan real-time polymerase chain reaction (PCR) systems using minor groove binding (MGB) probes. Species-specific amplification was achieved by combining only two fluorogenic probes and 10 oligonucleotide primers targeting mitochondrial sequences. The limits of detection ranged from 0.03 to 0.80 pg of template DNA. Analysis of experimental mixtures containing two to four different species showed the suitability of the assay for detection of more than 1% of pork, chicken, or turkey and of more than 5% of cattle or lamb. The quantification accuracy in samples containing 10-100% of beef or pork DNA was close to 90% (López-Andreo et al., 2005; Hunt, 1997; Janseen, et al., 1998; Montiel-Sosa, et al., 2000; and Partis, et al, 2000). In a latest research, 3 food samples (2 chocolates and one chicken nugget) were tested positive for containing the 152bp fragments of the porcine leptin (Farouk, et al., 2006).
PCR based methodology, used in this publication where pig-specific primers were used to amplify a 152 bp fragment from the procaine leptin gene that is the homologues of murine obese, is a successful technique of identification. As it is PCR-based, it is the most convenient for critical samples in which DNA is largely degraded such as with processed food (Farouk, et al., 2006; Stratil, et al., 1997; Wintero, et al., 1990, and Wolf, et al., 1999).
The main target of the work was to develop a fast, cheap and reliable molecular technique in foods and to check foods bearing “Halal” tags for porcine contamination by a simple species-specific band identification of PCR products in the Saudi market.
Materials and Methods
Meat Samples
Thirty-three samples of frozen meat (beef steaks, beef sausage, minced beef, beef roast, turkey sausage, chicken sausage, chicken nuggets and chicken balls) were purchased from nearby hypermarkets.
DNA isolation
Frozen meat was thawed and grinded. 30 milligrams of the grounded meat were transferred into 1.5 ml Eppendorf tube. 200 μl ASL buffer (Qiagen) was added into the individual tubes containing the sample material. The mixtures were vortexed for 5 minutes and incubated overnight for sufficient lysis to maximize DNA extraction. After incubation the samples were vortexed for 2 minutes and centrifuged at full speed (13,000 rpm) for 15 minutes using the Biofuge Pico, Germany to remove any solid particles. 40 μl of the supernatant was transferred into a new Eppendorf tubes and stored at -4oC for subsequent PCR application.
DNA Quantification
The concentration and purity of the extracted DNA were measured by absorbance at 260 nm using the Anthelie series UV - visible spectrophotometer (Secoman, France). 10 μl of DNA sample was diluted in 990 μl of nuclease- free water (Promega) for PCR. For both the DNA extraction with the Qiagen kit and the microwave extraction the DNA extracted was measured to be in the range of 1000-1500 ng.
PCR Mix
DNA (200 ng) was amplified using puRe TaqTM Ready- To-GoTM PCR Beads (Amersham, USA). In a final volume of 25μl, each reaction contained 2.5 units of puRe TaqTM DNA polymerase, 10 mM Tris-HCl (pH 9.0 at room temperature), 50 mM KCl, 1.5 mM MgCl2, 200mM dATP, dCTP, dGTP and dTTP, and stabilizer, including BSA. Amplification was performed using 20 pmol of each forward and reverse primer on a thermalcycler (Mastercycler Gradient, Eppendorf, Germany). The forward primer was 5’–TGCAGTCTCTCCTCCAAA-3’ and the reverse primer was 5’–CGATAATTGGATCACATTCTG-‘3 (Meyer et al., 1995). The amplification was run using the following program: 94oC for 3 minutes to denature the template DNA completely, followed by 35 cycles at 94oC for 1 minutes, 55oC for 1 minutes, and 72oC for 1 minute, and closed by the extension step at 72oC for 7 minutes. The amplified DNA was determined by gel electrophoresis (Electrophoresis,
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Improved DNA Extraction Method for Porcine Contaminants
Power Supply EPS 301, amersham Pharmacia biotech) in 1.7% agarose gel of 1x TBE buffer and made visible by staining with ethidium bromide at a constant voltage of 70 for 2 hours. The resulting fragments were visualized by UV transillumination (MacroVue UV-20 Hoefer, Germany).
Results
The gel electrophoresis suggests that by just using ASL buffer for lyses to isolate DNA from meat samples was reliable and subsequently DNA primers could be used to amplify the species-specific 152-bp porcine leptin gene fragment. Showing that DNA primers are able to detect leptin gene in pork samples, the next stage of work aimed at finding out the possibility of using the same protocol to detect porcine contaminants in non-pork food products – as established in Figure 1. The amplification of the 152bp porcine leptin gene fragment as shown in lane 2 “processed pork”, beef sausage sample in lane 4 and beef steak sample in lane 7, illustrates the successful detection of pork contamination or elements in food samples. Whereas lanes 3, 5 and 6 containing other meat products did not show any existence of the porcine leptin gene fragment (Fig.1).
Discussion and Conclusion
In a previous work (Farouk et. al, 2006) the use of the commercial Qiagen Stool Kit (Germany) was an easy, rapid and reliable method to obtain sample extracts suitable to be used for the study of DNA by PCR assays. There are PCR inhibitors in the processed food products having high levels of proteins and fats, which is why different protocols were explored and tested for the preparation of DNA that remove all PCR inhibitors or reduce them in the DNA sample. However the current method of using just the ASL buffer for the lyses step and eventual PCR- ready DNA is cheaper than the use of various different reagents in the commercial kit and faster than the use of the conventional methods.
Comparable to the current methodology another reagent DNAzol® Direct is a universal reagent for processing biological samples for direct PCR. It is manufactured by the Molecular Research Centre Company (Cincinnati, USA). The DNAzol Direct procedure is simple; where a sample is lysed in DNAzol Direct for 15 minutes, and the resulting lysate used in PCR and amplification of a selected DNA fragment(s) is performed (Mackey, et. al., 1997).
According to the manufacturer’s protocol the standard DNAzol Direct procedure supports PCR amplification of DNA fragments up to 8-kb long. The patent-pending DNAzol Direct composition and procedure are based on the use of an alkaline solution containing polyethylene glycol and other additives (Mackey, et. al., 1996). DNAzol Direct quickly and effectively lyses biological samples, releasing DNA into the lysate. The combined effects of the alkaline pH and chaotropic properties of DNAzol Direct sufficiently inactivate PCR inhibitors including proteases and nucleic acid degradation enzymes. After processing a sample in DNAzol Direct, DNA is denatured into a single-stranded form, RNA is hydrolyzed, and proteins are denatured and partially hydrolyzed. Due to its unique composition, the DNAzol Direct lysate does not require neutralisation before its use in PCR. The resulting pH of a PCR mix containing less than 10% of the lysate is within the effective range for PCR which is between 8-8.4. (Chomczynski, et. al.,1997). Thus, from the above references it is of no doubt that single solution reagents can be used for PCR ready DNA.
In conclusion, the detection method, which uses minute DNA quantities and resources, has been established thus, so far out of the food samples tested, 2 samples have been tested positive shown by the amplification 152-bp fragment of the porcine leptin gene. Although this specific methodology using PCR is well known, however it is of utmost importance that this strategy and experiment was successfully done in Saudi Arabia. In addition it is vital to note that this porcine contaminant discovery is just a small quantity from the thousands of different food products in the Kingdom and thus more food has to be tested in future research endeavors and a systematic food screening methodology be established.
This should initiate more systems where various religious, political, educational and scientific bodies of the Kingdom of Saudi Arabia work together to create an awareness program for its citizens with regard to the religious and R&D aspects of the food being consumed. Furthermore specialized initiatives could be implemented to share this technology and knowledge to the higher schools of the Kingdom to develop and spark the talented young minds of the Kingdom to create and foray into the area of food technology to establish the Kingdom as a platform of a
Fig 1. Gel electrophoresis showing the detection of the specific 152bp leptin gene fragments in processed food products. Lane 1: 100bp molecular marker, Lane 2: Pork Sausage, Lane 3: Chicken Nuggets, Lane 4: Beef Sausage (Brand A), Lane 5: Chicken Sausage, Lane 6: Turkey Sausage, Lane 7: Beef Steak (Brand B), Lane 8: negative control.
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Ibrahim Abdullah Alaraidh
Halal-Hub for the Middle East and the Muslin World.
Acknowledgment
The author wish to thank Dr. Abd-ElAziem Farouk Gad, Professor of Molecular Biology and Biotechnology Department of Biology Faculty of Science University of Brunei Darussalam and Mr.Mohamed Faizal bin Noor Batcha (M.Sc (Biotech)) from the Academy of Sciences Malaysia for overall help.
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