RNA Extraction of Peste Des Petits Ruminants Virus (PPRV) from Clinical Samples using Tri-Reagent and Acid Guanidinium Thiocyanate–Phenol–Chloroform Methods
This document describes a study that evaluated two RNA extraction methods, Tri-reagent and Acid guanidinium thiocyanate–phenol–chloroform (AGPC), for detecting Peste Des Petits Ruminants Virus (PPRV) in clinical samples. RNA was extracted from 10 tissue samples that tested positive for PPRV using Immuno-capture ELISA. Both extraction methods produced RNA of sufficient quality and purity for downstream applications. The study found that both Tri-reagent and AGPC are effective methods for extracting RNA from PPRV samples and can enable accurate diagnosis of the disease. Rapid detection of PPRV through nucleic acid-based methods like these helps control outbreaks by facilitating early
Rapid and accurate detection of pathogens under field laboratory conditions is necessary for effective control of veterinary pathogens. Here we describe a prototype, portable, pathogen detection device developed for single tube, real-time, reverse transcription, loop-mediated isothermal amplification (RT-LAMP) using Laem-Singh virus (LSNV) as a model. LSNV is an RNA virus and a component cause of growth retardation in black tiger shrimp. We chose its RNA-dependent RNA polymerase
(RdRp) gene as the target for our tests. The basis for detection was measurement of turbidity arising from formation of a white, insoluble magnesium pyrophosphate precipitate byproduct upon amplification of the RdRp target sequence from 100 ng template RNA extracted from shrimp. The measurement device consisted of a heating block to maintain constant temperature in the RT-LAMP reaction for 8 Eppindorf sample tubes, a light-emitting diode (LED) light source providing red light emission at 650 nm wavelength to pass through sample tubes, a light dependent resistance (LDR) photo-detector and a software program to report turbidity events and could potentially be marketed for under US$3000. The device was connected to a computer to display real-time results in a variety of formats. The optimized protocol for LSNV detection consisted of incubation of the sample tubes at 65uC for 1 h during which turbidity was continuously measured, and quantitative results could be obtained by reaction time measurement. The sensitivity of detection was comparable to that of conventional nested RT-PCR and there was no cross reaction with other common shrimp viruses. The device was used for quantitative measurement of relative copy numbers of LSNV RdRp in 8 shrimp tissues and they were found to be highest in the gills followed in order by the lymphoid organ and hemolymph (p#0.05). This platform can be easily adapted for detection of other pathogens under field laboratory settings.
High-throughput processing to maximize genomic analysis through simultaneous ...Thermo Fisher Scientific
As personalized cancer care evolves, the patient’s nucleic acid becomes ever so important to provide valuable information regarding their genetic makeup and disease state. Common sample types for these analyses include biopsies, which can be very limited in material making the downstream measurement of more than one analyte rather difficult. Obtaining another biopsy, using a different section or splitting the sample can be problematic because of tumor heterogeneity. Even adjacent areas of the same tumor tissue can result in different RNA/DNA profiles so the ability to isolate multiple analytes from the same sample offer a number of benefits, which include preserving samples and data consistency eliminating any sample to sample variation. As more tests are developed to simultaneously monitor genetic alterations, there is a strong need to efficiently isolate both DNA and RNA from the same starting sample in a format compatible with high-throughput processing.
ABSTRACT- Multiple Drug resistance (MDR) tuberculosis timely diagnose is of utmost clinical relevance and needs to be diagnose at initial stages for the proper treatment. The current study was done to detect the several genes for MDR tuberculosis (TB) in clinical isolates by molecular tools. 60 clinical isolates were collected and subjected for AFB smear preparation, Nested PCR (IS6110) for mycobacterium tuberculosis complex detection and MDR TB PCR targeting rpoB, kat G, mab A promoter. 12 came positive for AFB smears, out of which 08 were pulmonary and 04 were extra pulmonary. Nested PCR targeting IS6110 gene was amplified at 123 base pairs with 340 base pairs as IC (internal control) was seen in 25 cases which include 19 pulmonary and 6 extra pulmonary. The Positive TB PCR specimens were subjected for MDRTB PCR Only 06 cases yielded, an amplicon of 315 bp confirming the rpoB gene resistance for resistance for rifampcin drug. In any of the 06 positives none of the other resistance gene other than rpoB was amplified. Targeting multiple genes at once, additional information will be gained from a single test run that otherwise would require several times the reagents and more time to perform. Current study signifies the usage of quick, cost effective, DNA sequences based method for MDR TB detection where disease will be diagnosed earlier and hence treatment would be started at an early stage.
Keywords: Multiple drug resistance, amplicon, Polymerase chain reaction, Nested PCR, Rifampicin.
Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for Rapid...ICRISAT
Rapid detection of pathogens in chickpea has become a pre-requisite to facilitate accurate disease diagnosis and surveillance for better management strategy. Among the diseases affecting chickpea, Fusarium wilt [Fusarium oxysporum f. sp. ciceris (Foc)] can cause 100% yield losses in susceptible cultivars. In this study a method based on the Loop-Mediated Isothermal Amplification (LAMP) assay for the detection of Foc was developed and sensitivity and specificity of the assay was evaluated.
Rapid and accurate detection of pathogens under field laboratory conditions is necessary for effective control of veterinary pathogens. Here we describe a prototype, portable, pathogen detection device developed for single tube, real-time, reverse transcription, loop-mediated isothermal amplification (RT-LAMP) using Laem-Singh virus (LSNV) as a model. LSNV is an RNA virus and a component cause of growth retardation in black tiger shrimp. We chose its RNA-dependent RNA polymerase
(RdRp) gene as the target for our tests. The basis for detection was measurement of turbidity arising from formation of a white, insoluble magnesium pyrophosphate precipitate byproduct upon amplification of the RdRp target sequence from 100 ng template RNA extracted from shrimp. The measurement device consisted of a heating block to maintain constant temperature in the RT-LAMP reaction for 8 Eppindorf sample tubes, a light-emitting diode (LED) light source providing red light emission at 650 nm wavelength to pass through sample tubes, a light dependent resistance (LDR) photo-detector and a software program to report turbidity events and could potentially be marketed for under US$3000. The device was connected to a computer to display real-time results in a variety of formats. The optimized protocol for LSNV detection consisted of incubation of the sample tubes at 65uC for 1 h during which turbidity was continuously measured, and quantitative results could be obtained by reaction time measurement. The sensitivity of detection was comparable to that of conventional nested RT-PCR and there was no cross reaction with other common shrimp viruses. The device was used for quantitative measurement of relative copy numbers of LSNV RdRp in 8 shrimp tissues and they were found to be highest in the gills followed in order by the lymphoid organ and hemolymph (p#0.05). This platform can be easily adapted for detection of other pathogens under field laboratory settings.
High-throughput processing to maximize genomic analysis through simultaneous ...Thermo Fisher Scientific
As personalized cancer care evolves, the patient’s nucleic acid becomes ever so important to provide valuable information regarding their genetic makeup and disease state. Common sample types for these analyses include biopsies, which can be very limited in material making the downstream measurement of more than one analyte rather difficult. Obtaining another biopsy, using a different section or splitting the sample can be problematic because of tumor heterogeneity. Even adjacent areas of the same tumor tissue can result in different RNA/DNA profiles so the ability to isolate multiple analytes from the same sample offer a number of benefits, which include preserving samples and data consistency eliminating any sample to sample variation. As more tests are developed to simultaneously monitor genetic alterations, there is a strong need to efficiently isolate both DNA and RNA from the same starting sample in a format compatible with high-throughput processing.
ABSTRACT- Multiple Drug resistance (MDR) tuberculosis timely diagnose is of utmost clinical relevance and needs to be diagnose at initial stages for the proper treatment. The current study was done to detect the several genes for MDR tuberculosis (TB) in clinical isolates by molecular tools. 60 clinical isolates were collected and subjected for AFB smear preparation, Nested PCR (IS6110) for mycobacterium tuberculosis complex detection and MDR TB PCR targeting rpoB, kat G, mab A promoter. 12 came positive for AFB smears, out of which 08 were pulmonary and 04 were extra pulmonary. Nested PCR targeting IS6110 gene was amplified at 123 base pairs with 340 base pairs as IC (internal control) was seen in 25 cases which include 19 pulmonary and 6 extra pulmonary. The Positive TB PCR specimens were subjected for MDRTB PCR Only 06 cases yielded, an amplicon of 315 bp confirming the rpoB gene resistance for resistance for rifampcin drug. In any of the 06 positives none of the other resistance gene other than rpoB was amplified. Targeting multiple genes at once, additional information will be gained from a single test run that otherwise would require several times the reagents and more time to perform. Current study signifies the usage of quick, cost effective, DNA sequences based method for MDR TB detection where disease will be diagnosed earlier and hence treatment would be started at an early stage.
Keywords: Multiple drug resistance, amplicon, Polymerase chain reaction, Nested PCR, Rifampicin.
Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for Rapid...ICRISAT
Rapid detection of pathogens in chickpea has become a pre-requisite to facilitate accurate disease diagnosis and surveillance for better management strategy. Among the diseases affecting chickpea, Fusarium wilt [Fusarium oxysporum f. sp. ciceris (Foc)] can cause 100% yield losses in susceptible cultivars. In this study a method based on the Loop-Mediated Isothermal Amplification (LAMP) assay for the detection of Foc was developed and sensitivity and specificity of the assay was evaluated.
Introduction to PCR and RT-PCR, RT-PCR
PCR, Contents of PCR, Steps in PCR, PCR VS RT-PCR, Applications
Presented by
N. Ramya
Department of Pharmacology
Cancer is a disease characterized by uncontrolled cell growth and proliferation. Recent advances in molecular medicine and cancer biology have changed the way research clinicians evaluate and consider treatment. Selected tumor biomarkers have been utilized as targets for drug therapy leading to better more effective treatment. Gene expression profiling has been used for identifying new biomarkers for tumor classification and driving decision making for better patient outcome in different tumor types. DNA microarrays have become a key method to acquire a comparative snapshot of the gene expression profile from test samples in a high throughput manner. Quantitative PCR and newer sequencing techniques are popular research alternatives offering highly accurate gene expression measurements, but with limitations due to cost, complex instrumentation and analysis needs. RNA extracted from formalin fixed paraffin embedded tissue (FFPE) creates considerable additional challenges in acquiring accurate gene expression measurements due to the highly fragmented and compromised integrity of FFPE RNA due to the fixation process.
Improved DNA Extraction Method for Porcine ContaminantsIslamic_Finance
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
Corona is here to stay and it is predicted that over 70% of population will get the infection (fortunately not all will fall sick or very sick). (Recovery rate of over 74% & Death rate around 2%).
A lot of confusion exists regarding testing for covid and what test to do, when and how to interpret these tests.
Compiled by Dr. Narendra Malhotra
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
Pcr technology and its importance in covid 19 pandemicAnupam Maity
Since the discovery of the PCR technology, its application in the various fields is increased gradually. Based on to this principle, many variations of the PCR have been established. Year by year, it is upgraded very much. It is established as a most common and accurate technique for the detection of the various diseases in the field of medicine. Now it is a ‘Gold standard’ for the detection of covid-19 also, which is much needed to contain the spread of the virus. Though various detection techniques are there for detection, but real time RT-PCR (variation of PCR) is most reliable. Viral detection is based on a simple principle of nucleic acid (viral) amplification. Various manufacturing companies are manufacturing the PCR instrument. Though the accuracy of the instruments are slightly differ to each other.
A next Generation Sequencing Approach to Detect Large Rearrangements in BRCA1...Thermo Fisher Scientific
We have developed an amplicon-based NGS approach for FFPE
samples that can detect SNVs, small mutations and LRs
simultaneously. We have implemented a comprehensive
bioinformatics algorithm that detects LRs at high sensitivity, even in
the absence of control sample(s). This significantly reduces the cost
and labor for BRCA1/2 genetic analyses.
Noninvasive detection of rare mutations in blood could allow tumor monitoring for
research purposes. Research studies have suggested that cfDNA contains DNA from
tumor cells with somatic mutations that could inform on tumor progression and
therapeutic resistance. Here, we demonstrate a complete workflow from a single tube
of blood through data analysis for research samples down to a 0.1% allelic frequency.
The low abundance tumor mutations found in cfDNA requires sensitive and accurate
mutation detection. We have developed two panels that utilize an amplificationbased
assay that generates tagged DNA copies, which allows detection of low
abundance tumor mutations found in cfDNA. The two panels allow multiplex
interrogation of primary driver and resistance mutations specific to ctDNA from breast
and colon cancer. The Oncomine™ Colon cfDNA panel targets 236 hotspots within
14 genes while the Oncomine Breast cfDNA panel covers 157 hotspot mutations in
10 genes. This workflow was validated from matched single blood tubes, Streck and
K2EDTA. Additionally, the utility for cancer research was demonstrated with
concordance studies using matched FFPE and plasma from oncology samples.
Comparison of Type and Time of Fixation on Tissue DNA Sequencing ResultsThermo Fisher Scientific
The effects of type and duration of tissue fixation were studied using three different
lung (LCa) cancer research samples. Each tissue sample was fixed in five different
fixatives, for three different time points in each fixative. Next generation sequencing
(NGS), tissue morphology analysis (H+E), and antigenicity (IHC) were performed
for each of the resulting samples. The analysis indicates that both time and type of
fixation impact NGS results.
Two main animal pathogenic subspecies of Mycobacterium avium are M. avium avium (Maa) and M. avium paratuberculosis (Map). Their pathogenicity is host-specifi c, Maa causing avian tuberculosis in poultry whereas Map commonly cross-infects to ruminant.Veterinary diagnosis of M. avium infections is microscopic examination of acid-fast bacilli or culture in Löwenstein-Jensen medium,which are time-consuming and low sensitivity. This present study aimed to apply real-time PCR coupled with High-Resolution Melting (HRM) analysis for differential detection of Maa in Thai domestic ducks. Specifi c primer targeting host-expression dependent (hed) region
was designed, PCR product of Maa were amplifi ed from duck’s tissue lesions whereas Map were amplifi ed from cow and deer. HRM real-time PCR was performed and analyzed. Different HRM patterns were showed and melting temperature were analyzed at 83.26 ± 0.12°C for Maa and 84.04 ± 0.09°C for Map. This technique can detect as few as 102 DNA copies and present high specifi city by negative amplifi cation of other pathogenic bacterial species. This technique is sensitive, specifi c, rapid and does not require fl uorescent probes or post-PCR electrophoresis. Our technique is a possible new tool for the detection of Maa and Map infection in tissue specimens.
Implementation of histopathological techniques and transmission electron micr...Agriculture Journal IJOEAR
— Mycoplasma hyopneumoniae is a fastidious bacterium, an important member of swine respiratory disease complex, like Porcine Enzootic Pneumonia, affecting the non-specific defense mechanism of the respiratory tract, high mucociliary system, predisposing the pigs to secondary pathogens. The objective of this study is to implement precise diagnostic techniques for identification of Mycoplasma hyopneumoniae. 19 swine lungs fragments were collected from slaughterhouses and submitted by histopathological techniques. The presence of mucocellular exudate in 78.94% of the samples was observed in the bronchi and bronchioles, absence of eyelashes in 63.15% and lymphoid tissue hyperplasia associated with the bronchus in 42.10%. In pulmonary parenchyma, thickening of alveolar wall and interstitial bronchopneumonia were observed in 68.42%, hemorrhage in 47.36%, which 36.84% had hemosiderin and 15.78% lung consolidation. The presence of mycoplasma by the negative staining technique was identified in all samples, also the labeling of epitopes by colloidal gold immunostaining, using monoclonal antibody. In immunohistochemistry techniques and in situ hybridization, the labeled epitope and genome were observed confirming the presence of Mycoplasma hyopneumoniae in the State of São Paulo. Therefore, the bronchial epithelium is the best tissue to collect the sample for an accurate diagnosis and the best method of diagnosis is the negative staining technique for screening and colloidal gold immunocytochemistry techniques to identify Mycoplasma species.
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.
Ion Torrent™ Next Generation Sequencing-Oncomine™ Lung cfDNA assay detected 0...Thermo Fisher Scientific
Study of genetic Information from cell-free (cf) DNA provide valuable opportunities in cancer research and potentially impact future oncology. As an example, liquid biopsy provide a non-invasive and cost effective solution for future compared to traditional biopsy tests. Here we report the application of research based Ion Torrent™ next-generation sequencing (NGS) Oncomine™ cfDNA assays and associated workflow, which is developed to detect somatic variants at low frequency of 0.1% in cfDNA from plasma.
Orthogonal Verification of Oncomine cfDNA Data with Digital PCR Using TaqMan ...Thermo Fisher Scientific
The discovery of circulating tumor DNA (ctDNA) in blood, urine
and other bodily fluids has led to a new type of non-invasive
method of characterizing cancer-causing mutations, the liquid
biopsy. With NGS technologies becoming increasingly
sensitive, down to a Limit of Detection (LOD) of 0.1%, they are
rapidly gaining traction as a valid assay for cancer genotyping
and have potential to direct cancer treatment plans. The wideangle
view provided by NGS panels, combined with digital
PCR’s zoomed-in precision detection of DNA provide a
comprehensive picture of a cancer’s genetic makeup. By
applying these complementary techniques at the appropriate
time based on the disease type and stage, cancer treatment
becomes quicker, more precise and more cost-effective in the
future. NGS and digital PCR (dPCR) together provide a
complete picture of the cancer genome.
Introduction to PCR and RT-PCR, RT-PCR
PCR, Contents of PCR, Steps in PCR, PCR VS RT-PCR, Applications
Presented by
N. Ramya
Department of Pharmacology
Cancer is a disease characterized by uncontrolled cell growth and proliferation. Recent advances in molecular medicine and cancer biology have changed the way research clinicians evaluate and consider treatment. Selected tumor biomarkers have been utilized as targets for drug therapy leading to better more effective treatment. Gene expression profiling has been used for identifying new biomarkers for tumor classification and driving decision making for better patient outcome in different tumor types. DNA microarrays have become a key method to acquire a comparative snapshot of the gene expression profile from test samples in a high throughput manner. Quantitative PCR and newer sequencing techniques are popular research alternatives offering highly accurate gene expression measurements, but with limitations due to cost, complex instrumentation and analysis needs. RNA extracted from formalin fixed paraffin embedded tissue (FFPE) creates considerable additional challenges in acquiring accurate gene expression measurements due to the highly fragmented and compromised integrity of FFPE RNA due to the fixation process.
Improved DNA Extraction Method for Porcine ContaminantsIslamic_Finance
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
Corona is here to stay and it is predicted that over 70% of population will get the infection (fortunately not all will fall sick or very sick). (Recovery rate of over 74% & Death rate around 2%).
A lot of confusion exists regarding testing for covid and what test to do, when and how to interpret these tests.
Compiled by Dr. Narendra Malhotra
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
Pcr technology and its importance in covid 19 pandemicAnupam Maity
Since the discovery of the PCR technology, its application in the various fields is increased gradually. Based on to this principle, many variations of the PCR have been established. Year by year, it is upgraded very much. It is established as a most common and accurate technique for the detection of the various diseases in the field of medicine. Now it is a ‘Gold standard’ for the detection of covid-19 also, which is much needed to contain the spread of the virus. Though various detection techniques are there for detection, but real time RT-PCR (variation of PCR) is most reliable. Viral detection is based on a simple principle of nucleic acid (viral) amplification. Various manufacturing companies are manufacturing the PCR instrument. Though the accuracy of the instruments are slightly differ to each other.
A next Generation Sequencing Approach to Detect Large Rearrangements in BRCA1...Thermo Fisher Scientific
We have developed an amplicon-based NGS approach for FFPE
samples that can detect SNVs, small mutations and LRs
simultaneously. We have implemented a comprehensive
bioinformatics algorithm that detects LRs at high sensitivity, even in
the absence of control sample(s). This significantly reduces the cost
and labor for BRCA1/2 genetic analyses.
Noninvasive detection of rare mutations in blood could allow tumor monitoring for
research purposes. Research studies have suggested that cfDNA contains DNA from
tumor cells with somatic mutations that could inform on tumor progression and
therapeutic resistance. Here, we demonstrate a complete workflow from a single tube
of blood through data analysis for research samples down to a 0.1% allelic frequency.
The low abundance tumor mutations found in cfDNA requires sensitive and accurate
mutation detection. We have developed two panels that utilize an amplificationbased
assay that generates tagged DNA copies, which allows detection of low
abundance tumor mutations found in cfDNA. The two panels allow multiplex
interrogation of primary driver and resistance mutations specific to ctDNA from breast
and colon cancer. The Oncomine™ Colon cfDNA panel targets 236 hotspots within
14 genes while the Oncomine Breast cfDNA panel covers 157 hotspot mutations in
10 genes. This workflow was validated from matched single blood tubes, Streck and
K2EDTA. Additionally, the utility for cancer research was demonstrated with
concordance studies using matched FFPE and plasma from oncology samples.
Comparison of Type and Time of Fixation on Tissue DNA Sequencing ResultsThermo Fisher Scientific
The effects of type and duration of tissue fixation were studied using three different
lung (LCa) cancer research samples. Each tissue sample was fixed in five different
fixatives, for three different time points in each fixative. Next generation sequencing
(NGS), tissue morphology analysis (H+E), and antigenicity (IHC) were performed
for each of the resulting samples. The analysis indicates that both time and type of
fixation impact NGS results.
Two main animal pathogenic subspecies of Mycobacterium avium are M. avium avium (Maa) and M. avium paratuberculosis (Map). Their pathogenicity is host-specifi c, Maa causing avian tuberculosis in poultry whereas Map commonly cross-infects to ruminant.Veterinary diagnosis of M. avium infections is microscopic examination of acid-fast bacilli or culture in Löwenstein-Jensen medium,which are time-consuming and low sensitivity. This present study aimed to apply real-time PCR coupled with High-Resolution Melting (HRM) analysis for differential detection of Maa in Thai domestic ducks. Specifi c primer targeting host-expression dependent (hed) region
was designed, PCR product of Maa were amplifi ed from duck’s tissue lesions whereas Map were amplifi ed from cow and deer. HRM real-time PCR was performed and analyzed. Different HRM patterns were showed and melting temperature were analyzed at 83.26 ± 0.12°C for Maa and 84.04 ± 0.09°C for Map. This technique can detect as few as 102 DNA copies and present high specifi city by negative amplifi cation of other pathogenic bacterial species. This technique is sensitive, specifi c, rapid and does not require fl uorescent probes or post-PCR electrophoresis. Our technique is a possible new tool for the detection of Maa and Map infection in tissue specimens.
Implementation of histopathological techniques and transmission electron micr...Agriculture Journal IJOEAR
— Mycoplasma hyopneumoniae is a fastidious bacterium, an important member of swine respiratory disease complex, like Porcine Enzootic Pneumonia, affecting the non-specific defense mechanism of the respiratory tract, high mucociliary system, predisposing the pigs to secondary pathogens. The objective of this study is to implement precise diagnostic techniques for identification of Mycoplasma hyopneumoniae. 19 swine lungs fragments were collected from slaughterhouses and submitted by histopathological techniques. The presence of mucocellular exudate in 78.94% of the samples was observed in the bronchi and bronchioles, absence of eyelashes in 63.15% and lymphoid tissue hyperplasia associated with the bronchus in 42.10%. In pulmonary parenchyma, thickening of alveolar wall and interstitial bronchopneumonia were observed in 68.42%, hemorrhage in 47.36%, which 36.84% had hemosiderin and 15.78% lung consolidation. The presence of mycoplasma by the negative staining technique was identified in all samples, also the labeling of epitopes by colloidal gold immunostaining, using monoclonal antibody. In immunohistochemistry techniques and in situ hybridization, the labeled epitope and genome were observed confirming the presence of Mycoplasma hyopneumoniae in the State of São Paulo. Therefore, the bronchial epithelium is the best tissue to collect the sample for an accurate diagnosis and the best method of diagnosis is the negative staining technique for screening and colloidal gold immunocytochemistry techniques to identify Mycoplasma species.
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.
Ion Torrent™ Next Generation Sequencing-Oncomine™ Lung cfDNA assay detected 0...Thermo Fisher Scientific
Study of genetic Information from cell-free (cf) DNA provide valuable opportunities in cancer research and potentially impact future oncology. As an example, liquid biopsy provide a non-invasive and cost effective solution for future compared to traditional biopsy tests. Here we report the application of research based Ion Torrent™ next-generation sequencing (NGS) Oncomine™ cfDNA assays and associated workflow, which is developed to detect somatic variants at low frequency of 0.1% in cfDNA from plasma.
Orthogonal Verification of Oncomine cfDNA Data with Digital PCR Using TaqMan ...Thermo Fisher Scientific
The discovery of circulating tumor DNA (ctDNA) in blood, urine
and other bodily fluids has led to a new type of non-invasive
method of characterizing cancer-causing mutations, the liquid
biopsy. With NGS technologies becoming increasingly
sensitive, down to a Limit of Detection (LOD) of 0.1%, they are
rapidly gaining traction as a valid assay for cancer genotyping
and have potential to direct cancer treatment plans. The wideangle
view provided by NGS panels, combined with digital
PCR’s zoomed-in precision detection of DNA provide a
comprehensive picture of a cancer’s genetic makeup. By
applying these complementary techniques at the appropriate
time based on the disease type and stage, cancer treatment
becomes quicker, more precise and more cost-effective in the
future. NGS and digital PCR (dPCR) together provide a
complete picture of the cancer genome.
Orthogonal Verification of Oncomine cfDNA Data with Digital PCR Using TaqMan ...
Similar to RNA Extraction of Peste Des Petits Ruminants Virus (PPRV) from Clinical Samples using Tri-Reagent and Acid Guanidinium Thiocyanate–Phenol–Chloroform Methods
African Swine Fever (ASF) virus genomics and diagnosticsILRI
Presented by Richard Bishop and Cynthia Onzere at the Closing workshop of the BecA‐ILRI‐CSIRO‐AusAID project on Understanding ASF epidemiology as a basis for control, Nairobi, Kenya, 2‐3 October 2013
Two main animal pathogenic subspecies of Mycobacterium avium are M. avium avium (Maa) and M. avium paratuberculosis (Map). Their pathogenicity is host-specific, Maa causing avian tuberculosis in poultry whereas Map commonly cross-infects to ruminant. Veterinary diagnosis of M. avium infections is microscopic examination of acid-fast bacilli or culture in Löwenstein-Jensen medium,which are time-consuming and low sensitivity. This present study aimed to apply real-time PCR coupled with High-Resolution Melting (HRM) analysis for differential detection of Maa in Thai domestic ducks. Specific primer targeting host-expression dependent (hed) region
was designed, PCR product of Maa were amplified from duck’s tissue lesions whereas Map were amplified from cow and deer. HRM real-time PCR was performed and analyzed. Different HRM patterns were showed and melting temperature were analyzed at 83.26 ± 0.12°C for Maa and 84.04 ± 0.09°C for Map. This technique can detect as few as 102 DNA copies and present high specifi city by negative amplification of other pathogenic bacterial species. This technique is sensitive, specific, rapid and does not require fluorescent probes or
post-PCR electrophoresis. Our technique is a possible new tool for the detection of Maa and Map infection in tissue specimens.
In this slide contains introduction, genomic materials of virus and testing method of covid 19 by using RT-PCR.
Presented by: R.Rekha (Department of pharmacology),
RIPER, anantapur.
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.
Prevalence of Rota Virus Detection by Reverse TranscriptasePolymerase Chain R...IOSRJPBS
The present study was conducted for the period from 1/6/2016 to 20/1/2017 in Baquba city. The study aimed to detection of rotavirus in stool specimens of children fewer than five age and also explore the effects of certain demographic factors on the detection rates by revers transcriptase- polymerase chain reaction. The study included 49 patients with acute diarrhea, 32 were male and 17 were female. The age range was two months to 5 years. Demographic information on the patients regarding age, sex, residence, type of feeding and source of drinking water were collected from their parents. Stool specimens were collected from each patients and. Detection of rotavirus in stool specimens was done by conventional reverse transcriptase polymerase chain reaction (RT-PCR). The results of present study showed that the overall infection rate by rotavirus among patients with acute diarrhea by RT-PCR tests was 93.88%. The highest infection rate was recorded among those >10-≤15 months of age. None of the results showed significantly difference between female and male, PCR (88% vs 96.87%). Likewise, there was insignificantly difference between urban and rural residence, PCR (95.65% vs 92.30%). The results revealed insignificantly higher infection rate among patients (those below 2 years) feed mixing (91.66%) and bottled (100%) compared to that breast feeding (77.77%) by RT-PCR. The rotavirus infection rate was insignificantly higher among patients consuming municipal water for drinking (97.22%) compared to those consuming bottled water (84.61%) by the RT-PCR. The study concluded that rotavirus was detected in high rates among children less than 5 years old with acute diarrhea in Baquba city, particularly those less than 2 year old.
To form the basis of a respiratory disease model in rats by investigating the microbial distribution and composition in the lower respiratory tracts of normal rats. Methods: DNA was extracted from the intestine, trachea, bronchus and lung samples collected from healthy rats under sterile conditions. The 16S rDNA V4-V5 region was sequenced using Illumina high-throughput technology. Results: The sequencing results showed that there was no significant difference in abundance and species diversity of microbiota between the lower respiratory and the intestine. The microbiota structure analysis showed samples from lungs and intestinal shared similarity. However, the dominant species at the levels of phylum, family, and genus diverged. The similarity analysis showed that the lung microbiota were different from the intestines. The linear discriminant analysis showed significantly different species in different tissues; function prediction also showed different microbiota function in different tissues. Conclusions: These results suggest that bacterial colonization depends on the sample’s anatomical location. The human pathogen Acinetobacter lwoffii was also detected in the rat lower respiratory tract samples.
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
RT-PCR and DNA microarray measurement of mRNA cell proliferationIJAEMSJORNAL
For mRNA quantification, RT-PCR and DNA microarrays have been compared in few studies
(RT-PCR). Healing callus of adult and juvenile rats after femur injury was found to be rich in mRNA at
various stages of the healing process. We used both methods to examine ten samples and a total of 26 genes.
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RT-PCR and microarray data for each gene. Meaning became genetically unique because of this diversity.
Relatively lowly expressed genes had the highest r values. The distance between PCR primers and
microarray probes was found to be higher than previously assumed, leading to a drop in agreement between
microarray calls and PCR outcomes. Microarray research showed that RT-PCR expression levels for two
genes had a "floor effect." As a result, PCR primers and microarray probes that overlap in mRNA expression
levels can provide good agreement between these two techniques.
Molecular techniques in food microbiologyNajiyaNaju1
food biotechology
Similar to RNA Extraction of Peste Des Petits Ruminants Virus (PPRV) from Clinical Samples using Tri-Reagent and Acid Guanidinium Thiocyanate–Phenol–Chloroform Methods (20)
RNA Extraction of Peste Des Petits Ruminants Virus (PPRV) from Clinical Samples using Tri-Reagent and Acid Guanidinium Thiocyanate–Phenol–Chloroform Methods
1. 156
Pak. j. life soc. Sci. (2010), 8(2): 156-158
RNA Extraction of Peste Des Petits Ruminants Virus (PPRV) from Clinical
Samples using Tri-Reagent and Acid Guanidinium Thiocyanate–Phenol–
Chloroform Methods
Samina Ashiq, Zulkifal Hussain, Muhammad Abubakar1
, Shamim Saleha, Muhammad Javed
Arshed2
and Qurban Ali2
Department of Microbiology, Kohat University of Science and Technology, Kohat, Pakistan
1
PARC Institute of Advance Studies in Agriculture (PIASA), NARC, Park Road, Islamabad, Pakistan
2
National Veterinary Laboratory, Park Road, Islamabad, Pakistan
Abstract
Peste des petits ruminants (PPR) is an acute and
highly contagious viral disease of small ruminants
such as goats and sheep. It causes economic losses
of Rs. 20.5 billion annually in Pakistan. The
diagnosis of PPR infection in sheep and goats
populations can be synergistically strengthened by
detection of antigen in clinical samples of
susceptible population. In present study, two RNA
extraction methods, i.e., Tri-reagent and Acid
guanidinium thiocyanate–phenol–chloroform
(AGPC) method were investigated for the PPR
virus antigen detection. Both Tri-reagent and Acid
guanidinium thiocyanate–phenol–chloroform
methods used for RNA extraction of PPRV have
equal importance. The RNA of PPRV was
extracted from ten tissue samples that were found
positive by Immuno-capture ELISA. RNA
extracted from these samples was quantified by
spectrophotometric analysis. The rapid detection
by such suitable and appropriate methods of
nucleic acid detection of PPR virus in infected
animals will help in early diagnosis of infection
and subsequent control of the PPR disease in
Pakistan.
Keywords: RNA extraction, Peste des petits
ruminants (PPR), Tri-reagent and AGPC methods.
Introduction
Peste des petits ruminants (PPR) is an acute and
highly contagious viral disease of small ruminants
such as goats and sheep. Abubakar et al., (2008) have
reported dramatic consequences with morbidity of
80-90% and mortality between 50 and 80% due to
infection of PPR virus in small ruminants. PPR is
endemic in Pakistan and has been reported from all
the four provinces. In Pakistan, it causes economic
losses of Rs. 20.5 billion annually. Clinical findings
are fever, anorexia, ocular and nasal discharges, sores
in mouth, diarrhea and pneumonia. The main routes
of PPR virus (PPRV) transmission are oral and
respiratory tracts and oral, nasal and ocular
excretions are the key sources (Couacy-Hymann et
al., 2009).
PPR virus (PPRV) is classified as a member of the
genus Morbillivirus in the family Paramyxoviridae. It
has single-strand negative sense RNA genome of
~16kb (15,948 nucleotides) in length that encodes
eight proteins including six structural proteins
namely: nucleoprotein (N), phosphoprotein (P),
matrix protein (M), fusion protein (F),
haemagglutinin protein (H) and large polymerase
protein (L), and two nonstructural proteins V and C
(Chard et al., 2008).
The small ruminants infected with PPR are routinely
diagnosed on the basis of clinical examination, gross
pathology, histologic findings and laboratory
confirmation. A number of serological and molecular
diagnostic tests are used for the detection of PPR
virus, including isolation on cell culture, agar gel
immunodiffusion (AGID), haemagglutination (HA)
test, haemagglutination inhibition (HI), competitive
enzyme-linked immunosorbent assay (c-ELISA),
immunocapture enzyme-linked immunosorbent assay
(IC-ELISA) and reverse transcriptase polymerase
chain reaction (RT-PCR) (Nussieba et al., 2008;
Forsyth and Barrett, 1995; Libeau et al.,1994).
The main objective of the study was to evaluate two
RNA extraction methods, i.e., Tri-reagent and Acid
guanidinium thiocyanate–phenol–chloroform method
for the PPR virus antigen detection.
Materials and Methods
A total of ten different tissues samples (lung, liver,
spleen, heart and lymph nodes) were collected from
PPR suspected outbreaks in sheep and goats from
different locations of Pakistan (Rawalpindi,
Faisalabad and Lahore).
These samples were tested for antigen detection of
PPRV using Immuno-capture ELISA. IC-ELISA was
Pakistan Journal of
Life and Social Sciences
Corresponding Author: Muhammad Abubakar
PARC Institute of Advance Studies in Agriculture
(PIASA), NARC, Park Road, Islamabad, Pakistan
Email: hayee42@yahoo.com
2. Peste Des Petits Ruminants Virus (PPRV) from Clinical Samples
157
performed using the kits made from BDSL (Pirbright,
UK) as described by Libeau et al., (1994). RNA of
PPRV was extracted from the positive samples by
Tri-reagent and Acid guanidinium thiocyanate–
phenol–chloroform (AGPC) methods.
RNA Extraction
(i) Trireagent Method:
Five samples that were found positive for PPR virus
by IC-ELISA were further processed for isolation of
RNA by Trireagent (Molecular Research Center
Inc.).
(ii) Acid guanidinium thiocyanate–phenol–
chloroform (AGPC) Method:
The same five samples were used for isolation of
RNA by AGPC method as described by
Chomczynski and Sacchi (2006).
RNA Quantification by Spectrophotometry
RNA was quantified by spectrophotometric analysis
using the convention that one absorbance unit at 260
nm wavelength equals 40 μg RNA per ml. The ultra
violet absorbance was checked at 260 and 280 nm for
determination of RNA concentration and purity.
Purity of RNA was judged on the basis of optical
density ratio at 260:280 nm. The following formula
was used to determine RNA concentration of the
original sample:
Concentration of RNA (μg/μl) =
A260 x Dilution factor x 40
1000
Results
The RNA of PPRV was extracted from five samples
that were found positive by IC-ELISA, by Trireagent
and AGPC methods. Same samples were extracted by
Tri-reagent and AGPC methods. RNA extracted from
all samples was quantified by spectrophotometric
analysis. Purity of the extracted RNA (i.e. ratio 1.7-
2.0) was judged on the basis of optical density ratio at
260:280 nm as shown in tables 1 and 2.
Both Tri-reagent and AGPC methods showed equal
compassion in RNA extraction although the AGPC is
standardized in the laboratory while the Tri-reagent is
the commercially standard method. All the samples
were then confirmed for the F-gene of PPRV using
Polymerase Chain Reaction (PCR) (Forsyth and
Barrett, 1995).
Discussion
In this study, the PPRV antigen detection and RNA
extraction was investigated and compared in clinical
samples of small ruminants. The RNA of PPRV was
extracted by Tri-reagent and AGPC methods. Both
Tri-reagent and AGPC methods are recommended as
initial steps in molecular diagnosis of PPRV in
various studies. Farooq et al. (2008) also utilized
AGPC method for RNA extraction in a study on
molecular based diagnosis of Rinderpest and Peste
Des Petits Ruminants Virus in Pakistan.
Balamurugan et al., (2006) utilized Tri-reagent
method in a study while using one-step multiplex
RT-PCR Assay for the detection of Peste des petits
ruminants virus in clinical samples. Results of this
study showed that both RNA extraction methods
Table 1 Optical density ratio at 260:280 nm of RNA samples extracted by Tri-reagent method
Sr # Samples
Dilution factor (500) Dilution factor (200)
RNA µg/µl
260/ 280
RNA µg/µl
260/ 280260nm 280nm 260nm 280nm
1. Sample 1 1.68 1.72 0.97 0.728 0.68 1.07
2. Sample 2 3.38 2.7 1.25 2.656 1.528 1.73
3. Sample 3 1.74 1.64 1.04 0.688 0.648 1.06
4. Sample 4 1.76 1.66 1.06 0.712 0.664 1.07
5. Sample 5 1.78 1.68 1.05 0.64 0.616 1.03
Table 2 Optical density ratio at 260:280 nm of RNA samples extracted by AGPC method
Sr # Samples
Dilution factor (500) Dilution factor (200)
RNA µg/µl
260/280
RNA µg/µl
260/280260 280 260 280
1. Sample 1 0.188 1.74 1.08 0.608 0.576 1.05
2. Sample 2 1.86 1.88 0.98 0.88 0.8 1.1
3. Sample 3 1.76 1.76 1.00 0.832 0.752 1.106
4. Sample 4 1.48 1.46 1.01 0.776 0.72 1.07
5. Sample 5 1.26 1.32 0.95 0.648 0.6 1.08
3. Ashiq et al
158
have equal importance and can be utilized in
extraction of RNA of PPRV. In this study we carried
out the initial steps for molecular diagnosis of PPRV
by Trireagent and AGPC methods and efficiency of
both methods was also compared.
IC-ELISA is used as standard since it has the best
sensitivity and specificity and can be utilized for
samples which are not kept under ideal conditions.
RT-PCR is considered the most precise and sensitive
technique for diagnosis in clinical samples of PPRV
as compared to IC-ELISA, AGID and HA for
increased sensitivity and reduced false positivity
(Farooq et al., 2008).
The diagnosis of PPR infection in sheep and goats
populations can be synergistically strengthened with
detection of antigen in clinical samples of susceptible
population. Thus rapid detection by suitable and
appropriate methods of antigen and nucleic acid
detection of PPRV in infected animals will help in
early diagnosis of infection and subsequently control
of the PPR disease in Pakistan.
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