Protocollo ufficiale per la ricerca e amplificazione di RNA virale mediante tecnica PCR-RealTime per l'individuazione del virus Covid-19
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SARS-CoV-2 (COVID-19) is the virus responsible for respiratory disease caused by a novel (new) coronavirus that was first detected in Wuhan City, Hubei Province, China, later causing a global pandemic.
The virus is contagious and can be spread from humans to humans primarily through the exchange of mucus droplets that are expelled through sneezes or coughs.
The SARS-CoV-2 is a severe acute respiratory syndrome coronavirus. This outbreak is an important reminder that the global community must strengthen national and international programs for detection and response to future disease outbreaks.
This document describes a kit for detecting the human pathogen Mycobacterium tuberculosis using PCR. It discusses how the kit allows for isolation of DNA from samples using a spin column method. It then can detect M. tuberculosis through amplification of a region of its genome using provided master mixes and controls. The kit is designed for testing 24 samples at a time and provides high sensitivity and specificity for detection of this important pathogen.
A field-deployable RT-PCR system performs equivalently to real-time RT-PCR in...Simon Chung - genereach
A field-deployable RT-PCR system was found to perform equivalently to real-time RT-PCR in detecting type 2 porcine reproductive and respiratory syndrome virus (PRRSV). The field-deployable system provided results within 2 hours compared to 2.5 days for laboratory RT-PCR. Testing 50 vaccinated and 50 unvaccinated piglets over 11 weeks showed 96.25% agreement between the two methods. The field-deployable PCR system has potential for timely PRRSV detection and biosecurity management at points of need.
The document discusses the application of polymerase chain reaction (PCR) in detecting infectious diseases. Specifically, it discusses:
1) PCR is a widely used nucleic acid amplification technique that can replicate a specific DNA region millions of times, allowing detection of small amounts of DNA or RNA from infectious pathogens.
2) PCR has revolutionized clinical infectious disease diagnosis by enabling rapid and accurate detection of viruses, bacteria, and other pathogens from patient samples.
3) The document provides examples of how PCR has been used to detect several infectious diseases, including hepatitis B, hepatitis C, human papillomavirus, HIV, influenza, and the novel coronavirus COVID-19.
The importance of controls and novel solutions for successful real-time qPCRQIAGEN
The increasing demand for streamlined, monitored and ultrafast qPCR procedures requires high-performance, real-time quantitative RT and PCR chemistries. Particularly, procedures utilizing generic kits for gene expression analysis should include in-process safety measures to avoid variables and control accuracy of procedures and results. This slidedeck presents innovative solutions for one-step and two-step RT-PCR that significantly enhance performance and reliability in qRT-PCR. The new QuantiNova kit family offers a combination of various integrated safety features to remove variables and prevent artifacts. Internal control RNA, removal of genomic DNA, room temperature set-up capability for RT-PCR and a built-in visual pipetting control verify accurate procedures, ensuring reliable gene expression profiling.
This slidedeck explains the principles of the technologies and shows data demonstrating performance in qRT-PCR. Find out how you can verify accurate performance in qRT-PCR and improve your results!
RT2 Profiler PCR Arrays allow researchers to analyze gene expression profiles for focused panels of genes involved in biological pathways or disease states. The arrays provide laboratory-verified gene assays, integrated controls, and free data analysis software to generate a gene expression profile from a sample in less than 3 hours. Popular arrays analyze pathways such as extracellular matrix and adhesion molecules, WNT signaling, and cancer-related genes. The complete workflow begins with sample preparation and ends with data interpretation and publication of results.
This document provides information on several kits from Norgen Biotek Corp. for detecting HIV and related pathogens. It summarizes 3 kits that detect HIV: 1) an HIV quantitative RT-PCR kit that detects HIV RNA and has a reported linear range of 102-106 copies/μL, 2) an HIV proviral DNA PCR kit that detects HIV DNA with a linear range of 102-106 copies/μL, and 3) a plasma/serum HIV RT-PCR kit that isolates RNA from plasma/serum and detects HIV with a linear range of 10-8x106 VP/μL. It also summarizes kits for detecting Cryptococcus neoformans DNA and Pneumocystis jirove
SARS-CoV-2 (COVID-19) is the virus responsible for respiratory disease caused by a novel (new) coronavirus that was first detected in Wuhan City, Hubei Province, China, later causing a global pandemic.
The virus is contagious and can be spread from humans to humans primarily through the exchange of mucus droplets that are expelled through sneezes or coughs.
The SARS-CoV-2 is a severe acute respiratory syndrome coronavirus. This outbreak is an important reminder that the global community must strengthen national and international programs for detection and response to future disease outbreaks.
This document describes a kit for detecting the human pathogen Mycobacterium tuberculosis using PCR. It discusses how the kit allows for isolation of DNA from samples using a spin column method. It then can detect M. tuberculosis through amplification of a region of its genome using provided master mixes and controls. The kit is designed for testing 24 samples at a time and provides high sensitivity and specificity for detection of this important pathogen.
A field-deployable RT-PCR system performs equivalently to real-time RT-PCR in...Simon Chung - genereach
A field-deployable RT-PCR system was found to perform equivalently to real-time RT-PCR in detecting type 2 porcine reproductive and respiratory syndrome virus (PRRSV). The field-deployable system provided results within 2 hours compared to 2.5 days for laboratory RT-PCR. Testing 50 vaccinated and 50 unvaccinated piglets over 11 weeks showed 96.25% agreement between the two methods. The field-deployable PCR system has potential for timely PRRSV detection and biosecurity management at points of need.
The document discusses the application of polymerase chain reaction (PCR) in detecting infectious diseases. Specifically, it discusses:
1) PCR is a widely used nucleic acid amplification technique that can replicate a specific DNA region millions of times, allowing detection of small amounts of DNA or RNA from infectious pathogens.
2) PCR has revolutionized clinical infectious disease diagnosis by enabling rapid and accurate detection of viruses, bacteria, and other pathogens from patient samples.
3) The document provides examples of how PCR has been used to detect several infectious diseases, including hepatitis B, hepatitis C, human papillomavirus, HIV, influenza, and the novel coronavirus COVID-19.
The importance of controls and novel solutions for successful real-time qPCRQIAGEN
The increasing demand for streamlined, monitored and ultrafast qPCR procedures requires high-performance, real-time quantitative RT and PCR chemistries. Particularly, procedures utilizing generic kits for gene expression analysis should include in-process safety measures to avoid variables and control accuracy of procedures and results. This slidedeck presents innovative solutions for one-step and two-step RT-PCR that significantly enhance performance and reliability in qRT-PCR. The new QuantiNova kit family offers a combination of various integrated safety features to remove variables and prevent artifacts. Internal control RNA, removal of genomic DNA, room temperature set-up capability for RT-PCR and a built-in visual pipetting control verify accurate procedures, ensuring reliable gene expression profiling.
This slidedeck explains the principles of the technologies and shows data demonstrating performance in qRT-PCR. Find out how you can verify accurate performance in qRT-PCR and improve your results!
RT2 Profiler PCR Arrays allow researchers to analyze gene expression profiles for focused panels of genes involved in biological pathways or disease states. The arrays provide laboratory-verified gene assays, integrated controls, and free data analysis software to generate a gene expression profile from a sample in less than 3 hours. Popular arrays analyze pathways such as extracellular matrix and adhesion molecules, WNT signaling, and cancer-related genes. The complete workflow begins with sample preparation and ends with data interpretation and publication of results.
This document provides information on several kits from Norgen Biotek Corp. for detecting HIV and related pathogens. It summarizes 3 kits that detect HIV: 1) an HIV quantitative RT-PCR kit that detects HIV RNA and has a reported linear range of 102-106 copies/μL, 2) an HIV proviral DNA PCR kit that detects HIV DNA with a linear range of 102-106 copies/μL, and 3) a plasma/serum HIV RT-PCR kit that isolates RNA from plasma/serum and detects HIV with a linear range of 10-8x106 VP/μL. It also summarizes kits for detecting Cryptococcus neoformans DNA and Pneumocystis jirove
PCR Array Data Analysis Tutorial: qPCR Technology Webinar Series Part 3QIAGEN
This webinar presentation provides an overview and tutorial on analyzing data from RT2 Profiler PCR Array experiments. It discusses organizing raw Ct value data, performing ΔCt and ΔΔCt calculations to analyze gene expression changes between sample groups, and using the GeneGlobe Data Analysis Center web portal to analyze the data. The webinar highlights new features of the Data Analysis Center including improved data visualization and an upgraded sample manager. It emphasizes following the standard protocol for setting baselines and thresholds when analyzing PCR array data.
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
A Fully Automated POCKIT Central PCR System for Evaluation of the Infectious ...Simon Chung - genereach
This document discusses using PCR to evaluate the uniformity of infectious bronchitis vaccination in chickens. It finds:
1) A POCKIT Central PCR system can detect the IB vaccine virus in tracheal swabs up to 4 weeks after spraying vaccination at hatcheries.
2) Sequence analysis showed nucleic acids detected by the PCR system matched the vaccine used in the study.
3) A PCR positive rate of 40% or higher on day 7 after vaccination could be used as a cutoff for evaluating uniformity, with regular monitoring needed below that threshold.
The study demonstrates PCR is an effective quality control tool for checking IB vaccination uniformity. The portable POCKIT system makes this feasible for poultry grow
RT-PCR is a sensitive technique for detecting and quantifying mRNA. It uses reverse transcriptase to synthesize cDNA from an RNA template, which is then used as a template for PCR amplification using DNA polymerase. RT-PCR can be performed as a one-step or two-step process and is commonly used in clinical microbiology labs to detect RNA viruses from specimens. The COVID-19 RT-PCR test analyzes respiratory specimens for SARS-CoV-2 RNA by amplifying small amounts into DNA to accurately diagnose infections.
Quality Control of RNA Samples - For Gene-Expression Results you Can Rely onQIAGEN
By their very nature RNA molecules, especially mRNA and regulator RNA, are labile and can be highly unstable and sensitive to heat, UV and RNase contamination. The quality, relevance and scientific impact of gene expression results directly depends on the ability to extract RNA without losing any fraction of interest, while preserving the integrity of the biological information it carries. RNA quality control is thus critical to ensure high-quality results and for turning these results into actionable insights with confidence.
In this webinar, we will introduce you to the main parameters influencing RNA-based assays and their respective impact on downstream applications, discuss how to monitor them and cover the advantages of automation for quality control along complex workflows.
A Field-Deployable Insulated Isothermal PCR-Based System for Rapid and Sensit...Simon Chung - genereach
POCKIT Central PCR System for Detecting African Swine Fever Virus in Vietnam
The document describes a study evaluating the POCKIT Central PCR system for detecting African Swine Fever Virus (ASFV) in Vietnam. The system provides fully automated sample-to-answer detection of ASFV in 85 minutes using cartridges that integrate nucleic acid extraction and PCR. The study found the POCKIT Central system performed equivalently to the OIE reference real-time PCR method, with high analytical sensitivity and specificity. The automated system reduces hands-on time and human error compared to traditional PCR methods. It provides a simple workflow for rapid on-site detection of ASFV to help control the ongoing spread of the disease
Nucleic Acid Quantification from FFPE Samples – Are You Doing it Right?QIAGEN
Formalin-fixation and paraffin-embedding is a standard method for long-term preservation of tissue biopsies and these stored samples are a valuable tool for studying diseases such as cancer, especially when they are histologically and pathologically well characterized, and follow-up clinical data is available. The quality of nucleic acids extracted from FFPE samples is influenced by a number of factors, including how the samples were handled before, during and after fixation and embedding. Moreover, there are several difficulties when purifying nucleic acids from FFPE samples as the chemicals and temperature used during the process can degrade the DNA.
In this webinar, we will discuss the variability in quantity and purity of DNA purified from FFPE material. We will show data from different quantification and quality control methods, demonstrate the impact of inaccurate quantification on downstream results and discuss how to overcome these challenges.
RotorGene Q A Rapid, Automatable real-time PCR Instrument for Genotyping and...QIAGEN
The document describes the Rotor-Gene Q real-time PCR instrument. It has a unique centrifugal design that allows for excellent thermal and optical precision compared to block-based instruments. Each reaction tube spins individually, providing very precise temperature uniformity between wells. It also uses LED technology for excitation and filters for detection, allowing for multiple detection channels without need for normalization or reference dyes. Applications mentioned include quantitative PCR, high resolution melt analysis, genotyping, pathogen detection, and multiplex PCR. QIAGEN products are highlighted such as kits, controls, and the QIAgility liquid handler for automated real-time PCR setup.
The document describes RT2 Profiler PCR Arrays, which allow for pathway-focused gene expression profiling using real-time PCR. The system combines the quantitative performance of real-time PCR with the multiple-gene profiling capability of microarrays. Each array profiles the expression of 84 genes relevant to a specific pathway or disease state, along with controls. The arrays have high sensitivity, reproducibility and specificity for accurate comparison of gene expression levels. They provide a reliable tool for scientists to efficiently analyze gene expression of a focused panel of genes related to their research.
Reproducibility, Quality Control and Importance of AutomationQIAGEN
In this webinar, we will introduce you to the key sample quality parameters, discuss their respective impact on downstream applications and how to monitor them, and present the advantages of automating quality control along complex workflows.
RT PCR is too slow for effective control of spread of cov 2 infection, rapid antigen test by giving results in less than 30 minutes can help identify infected persons leading to quick isolation.Lack of sensitivity can be compensated by repeating RAT after a day or so.
A Fully Automated Sample-to-result PCR System for Detecting Infectious DiseasesSimon Chung - genereach
This document describes GeneReach Biotechnology Corporation's POCKIT Central system, a fully automated sample-to-result PCR system for infectious disease detection. The POCKIT Central can detect multiple pathogens from a single sample in less than 2 hours, including dengue virus serotypes. It has been validated against qPCR with equivalent sensitivity and specificity for dengue virus detection and subtyping. The portable and easy-to-use POCKIT Central provides a rapid and accurate molecular diagnostic solution for point-of-care infectious disease testing.
Learn about the power of LNA (Locked Nucleic Acid) technology and QIAGEN's LNA enhanced product portfolio for RNA and DNA research. Download the slide deck!
This is a presentation giving an overview of the GeneXpert DX system for detection of MTB. The assay described in this presentation is the MTB/RIF test.
This slidedeck presents a simple and accurate real-time PCR system for relevant biological pathway- and disease-focused mRNA and long noncoding RNA (lncRNA) expression profiling. Learn about the stringent performance built into the technology to ensure its sensitivity, specificity, reproducibility and reliability. Application examples are also presented.
Critical Factors for Successful Real-Time PCR: Multiplex PCRQIAGEN
Multiplex end-point PCR is a powerful tool for genotyping and many other applications. QIAGEN’s multiplex PCR chemistry is optimized for reliable amplification of many different templates with high variability in copy numbers. Thus it enables very quick establishment of a new lab routine and instant success for your multiplex PCR strategy.
There is a set of critical factors which we recommend to be regarded for planning and performing this kind of PCR. These will be discussed in detail in the webinar. Additionally, our multiplex PCR chemistry has recently been gaining increasing popularity among scientists who are utilizing it for their next-generation sequencing workflows.
10 Tips to maximize your Real Time PCR Success - Download the Technical NoteQIAGEN
This document provides 10 tips for maximizing success with real-time PCR. The tips include using high-quality nucleic acid templates, determining template concentration and purity, checking storage conditions, using the optimal template amount, determining reaction efficiency for each primer pair, properly storing primers and probes, preventing contamination, thoroughly mixing reaction components, performing necessary control reactions, and double checking cycler settings.
New Progress in Pyrosequencing for DNA MethylationQIAGEN
Pyrosequencing is a highly flexible technology that lets you rapidly analyze short- to medium-length sequences fast and quantitatively with high accuracy. The real-time, high-resolution sequence output makes the technology highly suitable for applications including complex mutation analysis, microbial identification and DNA methylation quantification.
The main bottleneck in Pyrosequencing has been limited sequence length, which is critical for some applications. Our new technology, software, and chemistry overcome this bottleneck and give sequence reads that are typically twice as long as those from previous PyroMark systems. The new PyroMark Q24 Advanced system also reduces background noise, improving quantification even at sites distant from the sequencing start. The new system is ideal for applications requiring analysis of longer sequences, such as DNA methylation analysis in epigenetic research, frequency determination in mutation analysis, and various de novo sequencing applications.
In this presentation, we will discuss the following applications and technology improvements:
• DNA methylation analysis at single base resolution at CpG and CpN sites
• Improved quantification of sequence variations at any sequence position
• Easy and improved base calling functionality
Next Generation Sequencing- NGS for COVID19 PPTMesele Tilahun
This document presents a next-generation sequencing (NGS)-based method for diagnosing COVID-19 using a single-step RNA extraction. The method was tested on 336 clinical samples and showed high accuracy compared to RT-PCR, the gold standard method. Key advantages of the NGS approach include higher scalability allowing thousands of samples to be tested simultaneously, and higher sensitivity to detect SARS-CoV-2 RNA compared to RT-PCR. The method provides individual qualitative results for each patient sample along with viral load estimates based on sequencing coverage.
PCR Array Data Analysis Tutorial: qPCR Technology Webinar Series Part 3QIAGEN
This webinar presentation provides an overview and tutorial on analyzing data from RT2 Profiler PCR Array experiments. It discusses organizing raw Ct value data, performing ΔCt and ΔΔCt calculations to analyze gene expression changes between sample groups, and using the GeneGlobe Data Analysis Center web portal to analyze the data. The webinar highlights new features of the Data Analysis Center including improved data visualization and an upgraded sample manager. It emphasizes following the standard protocol for setting baselines and thresholds when analyzing PCR array data.
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
A Fully Automated POCKIT Central PCR System for Evaluation of the Infectious ...Simon Chung - genereach
This document discusses using PCR to evaluate the uniformity of infectious bronchitis vaccination in chickens. It finds:
1) A POCKIT Central PCR system can detect the IB vaccine virus in tracheal swabs up to 4 weeks after spraying vaccination at hatcheries.
2) Sequence analysis showed nucleic acids detected by the PCR system matched the vaccine used in the study.
3) A PCR positive rate of 40% or higher on day 7 after vaccination could be used as a cutoff for evaluating uniformity, with regular monitoring needed below that threshold.
The study demonstrates PCR is an effective quality control tool for checking IB vaccination uniformity. The portable POCKIT system makes this feasible for poultry grow
RT-PCR is a sensitive technique for detecting and quantifying mRNA. It uses reverse transcriptase to synthesize cDNA from an RNA template, which is then used as a template for PCR amplification using DNA polymerase. RT-PCR can be performed as a one-step or two-step process and is commonly used in clinical microbiology labs to detect RNA viruses from specimens. The COVID-19 RT-PCR test analyzes respiratory specimens for SARS-CoV-2 RNA by amplifying small amounts into DNA to accurately diagnose infections.
Quality Control of RNA Samples - For Gene-Expression Results you Can Rely onQIAGEN
By their very nature RNA molecules, especially mRNA and regulator RNA, are labile and can be highly unstable and sensitive to heat, UV and RNase contamination. The quality, relevance and scientific impact of gene expression results directly depends on the ability to extract RNA without losing any fraction of interest, while preserving the integrity of the biological information it carries. RNA quality control is thus critical to ensure high-quality results and for turning these results into actionable insights with confidence.
In this webinar, we will introduce you to the main parameters influencing RNA-based assays and their respective impact on downstream applications, discuss how to monitor them and cover the advantages of automation for quality control along complex workflows.
A Field-Deployable Insulated Isothermal PCR-Based System for Rapid and Sensit...Simon Chung - genereach
POCKIT Central PCR System for Detecting African Swine Fever Virus in Vietnam
The document describes a study evaluating the POCKIT Central PCR system for detecting African Swine Fever Virus (ASFV) in Vietnam. The system provides fully automated sample-to-answer detection of ASFV in 85 minutes using cartridges that integrate nucleic acid extraction and PCR. The study found the POCKIT Central system performed equivalently to the OIE reference real-time PCR method, with high analytical sensitivity and specificity. The automated system reduces hands-on time and human error compared to traditional PCR methods. It provides a simple workflow for rapid on-site detection of ASFV to help control the ongoing spread of the disease
Nucleic Acid Quantification from FFPE Samples – Are You Doing it Right?QIAGEN
Formalin-fixation and paraffin-embedding is a standard method for long-term preservation of tissue biopsies and these stored samples are a valuable tool for studying diseases such as cancer, especially when they are histologically and pathologically well characterized, and follow-up clinical data is available. The quality of nucleic acids extracted from FFPE samples is influenced by a number of factors, including how the samples were handled before, during and after fixation and embedding. Moreover, there are several difficulties when purifying nucleic acids from FFPE samples as the chemicals and temperature used during the process can degrade the DNA.
In this webinar, we will discuss the variability in quantity and purity of DNA purified from FFPE material. We will show data from different quantification and quality control methods, demonstrate the impact of inaccurate quantification on downstream results and discuss how to overcome these challenges.
RotorGene Q A Rapid, Automatable real-time PCR Instrument for Genotyping and...QIAGEN
The document describes the Rotor-Gene Q real-time PCR instrument. It has a unique centrifugal design that allows for excellent thermal and optical precision compared to block-based instruments. Each reaction tube spins individually, providing very precise temperature uniformity between wells. It also uses LED technology for excitation and filters for detection, allowing for multiple detection channels without need for normalization or reference dyes. Applications mentioned include quantitative PCR, high resolution melt analysis, genotyping, pathogen detection, and multiplex PCR. QIAGEN products are highlighted such as kits, controls, and the QIAgility liquid handler for automated real-time PCR setup.
The document describes RT2 Profiler PCR Arrays, which allow for pathway-focused gene expression profiling using real-time PCR. The system combines the quantitative performance of real-time PCR with the multiple-gene profiling capability of microarrays. Each array profiles the expression of 84 genes relevant to a specific pathway or disease state, along with controls. The arrays have high sensitivity, reproducibility and specificity for accurate comparison of gene expression levels. They provide a reliable tool for scientists to efficiently analyze gene expression of a focused panel of genes related to their research.
Reproducibility, Quality Control and Importance of AutomationQIAGEN
In this webinar, we will introduce you to the key sample quality parameters, discuss their respective impact on downstream applications and how to monitor them, and present the advantages of automating quality control along complex workflows.
RT PCR is too slow for effective control of spread of cov 2 infection, rapid antigen test by giving results in less than 30 minutes can help identify infected persons leading to quick isolation.Lack of sensitivity can be compensated by repeating RAT after a day or so.
A Fully Automated Sample-to-result PCR System for Detecting Infectious DiseasesSimon Chung - genereach
This document describes GeneReach Biotechnology Corporation's POCKIT Central system, a fully automated sample-to-result PCR system for infectious disease detection. The POCKIT Central can detect multiple pathogens from a single sample in less than 2 hours, including dengue virus serotypes. It has been validated against qPCR with equivalent sensitivity and specificity for dengue virus detection and subtyping. The portable and easy-to-use POCKIT Central provides a rapid and accurate molecular diagnostic solution for point-of-care infectious disease testing.
Learn about the power of LNA (Locked Nucleic Acid) technology and QIAGEN's LNA enhanced product portfolio for RNA and DNA research. Download the slide deck!
This is a presentation giving an overview of the GeneXpert DX system for detection of MTB. The assay described in this presentation is the MTB/RIF test.
This slidedeck presents a simple and accurate real-time PCR system for relevant biological pathway- and disease-focused mRNA and long noncoding RNA (lncRNA) expression profiling. Learn about the stringent performance built into the technology to ensure its sensitivity, specificity, reproducibility and reliability. Application examples are also presented.
Critical Factors for Successful Real-Time PCR: Multiplex PCRQIAGEN
Multiplex end-point PCR is a powerful tool for genotyping and many other applications. QIAGEN’s multiplex PCR chemistry is optimized for reliable amplification of many different templates with high variability in copy numbers. Thus it enables very quick establishment of a new lab routine and instant success for your multiplex PCR strategy.
There is a set of critical factors which we recommend to be regarded for planning and performing this kind of PCR. These will be discussed in detail in the webinar. Additionally, our multiplex PCR chemistry has recently been gaining increasing popularity among scientists who are utilizing it for their next-generation sequencing workflows.
10 Tips to maximize your Real Time PCR Success - Download the Technical NoteQIAGEN
This document provides 10 tips for maximizing success with real-time PCR. The tips include using high-quality nucleic acid templates, determining template concentration and purity, checking storage conditions, using the optimal template amount, determining reaction efficiency for each primer pair, properly storing primers and probes, preventing contamination, thoroughly mixing reaction components, performing necessary control reactions, and double checking cycler settings.
New Progress in Pyrosequencing for DNA MethylationQIAGEN
Pyrosequencing is a highly flexible technology that lets you rapidly analyze short- to medium-length sequences fast and quantitatively with high accuracy. The real-time, high-resolution sequence output makes the technology highly suitable for applications including complex mutation analysis, microbial identification and DNA methylation quantification.
The main bottleneck in Pyrosequencing has been limited sequence length, which is critical for some applications. Our new technology, software, and chemistry overcome this bottleneck and give sequence reads that are typically twice as long as those from previous PyroMark systems. The new PyroMark Q24 Advanced system also reduces background noise, improving quantification even at sites distant from the sequencing start. The new system is ideal for applications requiring analysis of longer sequences, such as DNA methylation analysis in epigenetic research, frequency determination in mutation analysis, and various de novo sequencing applications.
In this presentation, we will discuss the following applications and technology improvements:
• DNA methylation analysis at single base resolution at CpG and CpN sites
• Improved quantification of sequence variations at any sequence position
• Easy and improved base calling functionality
Next Generation Sequencing- NGS for COVID19 PPTMesele Tilahun
This document presents a next-generation sequencing (NGS)-based method for diagnosing COVID-19 using a single-step RNA extraction. The method was tested on 336 clinical samples and showed high accuracy compared to RT-PCR, the gold standard method. Key advantages of the NGS approach include higher scalability allowing thousands of samples to be tested simultaneously, and higher sensitivity to detect SARS-CoV-2 RNA compared to RT-PCR. The method provides individual qualitative results for each patient sample along with viral load estimates based on sequencing coverage.
The document discusses reverse transcription polymerase chain reaction (RT-PCR) testing for COVID-19. It begins with an introduction to COVID-19 and that RT-PCR is the molecular assay used globally to detect SARS-CoV-2 RNA. It then explains that RT-PCR can detect targeted genetic materials in real time using fluorescent dyes. The document outlines that RT-PCR targets three regions, and detects COVID-19 by monitoring amplification in real time and analyzing cycle threshold data, with results between 37-40 considered positive or negative. It concludes that RT-PCR remains the most sensitive technique for COVID-19 confirmation globally.
This document provides instructions for using the Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-Fluorescence Probing) for the qualitative detection of SARS-CoV-2 in respiratory specimens. The kit uses real-time RT-PCR to detect SARS-CoV-2 RNA and includes positive and negative controls. Test results along with clinical observations are needed for accurate patient diagnosis and management. Strict laboratory procedures and biosafety precautions must be followed when handling specimens and performing the test.
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.
High data quality and accuracy are recognized characteristics of Sanger re-sequencing projects and are primary reasons that next generation sequencing projects compliment their results by capillary electrophoresis data validation. We have developed an on-line tool called Primer Designer™ to streamline the NGS-to-Sanger sequencing workflow by taking the laborious task of PCR primer design out of the hands of the researcher by providing pre-designed assays for the human exome. The primer design tool has been created to enable scientists using next generation sequencing to quickly confirm variants discovered in their work by providing the means to quickly search, order and receive suitable pre-designed PCR primers for Sanger sequencing. Using the Primer Designer™ tool to design M13-tailed and non-tailed PCR primers for Sanger sequencing we will demonstrate validation of 28-variants across 24-amplicons and 19-genes using the BDD, BDTv1.1 and BDTv3.1 sequencing chemistries on the 3500xl Genetic Analyzer capillary electrophoresis platform.
RT2 Profiler PCR Arrays: Pathway-focused Gene Expression Profiling with qRT-P...QIAGEN
This paper evaluates the performance of the newest technique for monitoring the expression of a panel of pathway- or disease-specific genes: the RT2 Profiler PCR Array System. The RT2 Profiler PCR Array System combines the quantitative performance of SYBR® Green real-time PCR with the multiple-gene profiling capabilities of a microarray.
The RT2 Profiler PCR Array is a 96- or 384-well plate containing RT2 qPCR Primer Assays for a set of 84 related genes, plus 5 housekeeping genes and 3 controls. The complete system includes an instrument-specific master mix and an optimized first strand synthesis kit. This paper presents experimental data showing that RT2 Profiler PCR Arrays have the sensitivity, reproducibility, and specificity expected from real-time PCR techniques. As a result, this technology brings focused gene expression profiling to any biological laboratory setting with a real-time PCR instrument.
The COVID-19 pandemic, also known as the corona virus pandemic, is an ongoing pandemic of corona virus disease 2019 (COVID‑19) caused by severe acute respiratory syndrome corona virus 2 (SARS‑CoV‑2).
Though several trials for candidate vaccines and potential therapies are underway, there is currently no cure, and in the absence of either proven effective therapy or a vaccine, diagnostic testing becomes a valuable tool. Testing is our window onto the pandemic and how it is spreading. Without data on who is infected by the virus we have no way of understanding the pandemic. Without this data we cannot know which countries are doing well, and which are just underreporting cases and deaths.
At IntellectPeritus, we have collected around 100 diagnostic kits (used in SARS-COV-2 diagnosis) and around 50 patent documents as sample report and have tried to put value added technology categories to generate specific insights quickly using our deliverable “Innovigence Corona”.
If you are interested in detailed or any customized report for SARS-COV-2 diagnostic kits please feel free to write to us at sales@intellectperitus.com
RT-PCR by Arnab Kumar Samanta(sen-4^J2020)[133].pptxArnabSamanta26
1) RT-PCR is the primary method for detecting SARS-CoV-2, the virus that causes COVID-19. It works by extracting the viral RNA from samples and amplifying specific DNA targets.
2) The RT-PCR process involves reverse transcribing the viral RNA into cDNA, then amplifying the cDNA using PCR. If the virus is present, fluorescent probes will bind to the amplified DNA and be detected in real time.
3) RT-PCR is highly sensitive and specific for detecting SARS-CoV-2, but it also requires specialized equipment and reagents, making it an expensive testing method.
1) Molecular testing for Covid-19 detection includes RT-PCR, TRUE NAT, and CB NAAT tests which detect viral RNA through nucleic acid amplification.
2) Antigen tests detect viral proteins and provide rapid results but have lower sensitivity than molecular tests.
3) Antibody tests detect antibodies produced after infection but cannot be used for diagnosis as they become positive later.
4) Newer modalities under research include multiplex assays, LAMP, CRISPR and other techniques for faster, portable, and higher-throughput Covid-19 testing.
In the detection of Covid-19, the sensitivity is very important for prevention of virus-spreading and aggravation. Here we present a sensitive detection system for Covid-19. I hope, this system contributes to Covid-19 disaster even small degree.
This document summarizes the development and validation of a one-step reverse transcription digital PCR (RT-dPCR) assay for the detection of SARS-CoV-2. Key points:
1. RT-dPCR was found to significantly improve the sensitivity of detection of SARS-CoV-2 in pharyngeal swab samples compared to RT-qPCR, reducing the false negative rate. RT-dPCR detected SARS-CoV-2 in 61 samples that were negative or equivocal by RT-qPCR.
2. When tested on 196 clinical samples, RT-dPCR increased the positive detection rate to 91% compared to 28% for RT-qPCR. RT-dPCR is well-su
This document discusses avian influenza (H5N1) detection and analysis using real-time PCR. It describes the influenza virus, including its structure and types. Avian influenza is highly contagious in birds and can be fatal. Diagnosis involves virus isolation, serology tests, and molecular tests like RT-PCR and real-time RT-PCR. Real-time PCR allows for amplification and detection of targeted DNA sequences in one step and provides amplification curves and results in about 3 hours. The workflow described screens samples for influenza A using a matrix PCR, then checks positive samples for H5 and N1 subtypes using multiplex real-time PCR on the LightCycler system.
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Primer and probe design is important for specificity and efficiency. Primers should be around 18-25 nucleotides, have 50-60% GC content, avoid repeats and polymorphisms, and target intron-exon boundaries when possible.
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2. 2
MIX PREPARATION FOR ALL SEPARATE PRIMER/PROBE COMBINATIONS
All primers and probes described below were validated under the following conditions.
RT-PCR Mix kit:
• Invitrogen Superscript™ III Platinum® One-Step qRT-PCR system (ref: 11732-088)
Real-time PCR equipment:
• LightCycler 480 (96)
Adjustments may be required for the use of other kits or other real-time PCR instruments. All
Assays used the same conditions. Primer and probe sequences, as well as optimized
concentrations are shown in table below. A 25µl reaction was set up containing 5µl of RNA.
Simplex Mix Vol (µl) [final]
H2O PPI 3.60
Reaction mix 2X 12.50 3 mM Mg
MgSO4 (50mM) 0.40 0.8 mM Mg
Forward Primer (10µM) 1.00 0.4 µM
Reverse Primer (10µM) 1.00 0.4 µM
Probe (10µM) 0.50 0.2 µM
SuperscriptIII RT/Platinum Taq Mix 1.00
Final Volume 20.00
Multiplex Mix (nCoV_IP2&IP4) Vol (µl) [final]
H2O PPI 1.3
Reaction mix 2X 12.50 3 mM Mg
MgSO4 (50mM) 0.40 0.8 mM Mg
Forward Primer (10µM) 1.00 0.4 µM
Reverse Primer (10µM) 1.00 0.4 µM
Forward Primer (10µM) 1.00 0.4 µM
Reverse Primer (10µM) 1.00 0.4 µM
Probe (10µM) 0.4 0.16 µM
Probe (10µM) 0.4 0.16 µM
SuperscriptIII RT/Platinum Taq Mix 1.00
Final Volume 20.00
CONTROLS
Each real-time RT-PCR assay includes in addition of unknown samples:
• Two negative samples bracketing unknown samples during RNA extraction (negative
extraction controls)
• Positive controls (in duplicate); when using in vitro synthesized transcripts as controls
include five quantification positive controls (in duplicate) including 105
, 104
and 103
copies genome equivalent (ge) of in vitro synthesized RNA transcripts.
• One negative amplification control.
AMPLIFICATION CYCLES (LIGHTCYCLER SYSTEM)
Reverse transcription 55°C 20 min x1
Denaturation 95°C 3 min x1
Amplification
95°C 15 sec
x50 Acquisition
58°C 30 sec
Cooling 40°C 30 sec x1
3. 3
SENSITIVITY
For the nCoV_IP and E_Sarbeco real-time RT-PCR
Sensitivity, in terms of 95% hit rate is about 100 copies of RNA genome equivalent per reaction
(this amount of target sequences is always detected), the probability to detect lower amounts of
virus decreases, but samples containing 10 copies could be detected with multiplex assay.
Multiplex
(Ct values)
Simplex
(Ct values)
RNA copies
Of
transcript
nCoV_IP2 nCoV_IP4 E_Sarbeco
1,00E+07 21,67 21,97 24,72
1,00E+06 24,97 25,12 28,19
1,00E+05 28,00 27,88 30,96
1,00E+04 31,84 30,51 33,33
Ct values may vary from instrument to instrument by up to 2 cycles, while the interval between two
dilutions steps is constant (∆Ct).
SPECIFICITY
Cross-reactivity with other respiratory viruses was tested with specimens known to be positive for
a panel of respiratory viruses (influenza A(H1N1)pdm09, A(H3N2), B-Victoria, B-Yamagata; influenza
C; RSV A, B; hBoV; hPIV; hMPV; HRV/enterovirus; adenovirus; hCoV (HKU1, OC43, 229E and NL63);
MERS-CoV. None of the tested viruses showed reactivity with PCR2 and PCR4.
POSITIVE CONTROL FOR SARS-CoV-2 REAL-TIME RT-PCR
One specific control has been designated.
Positive control for real-time RT-PCR is an in vitro transcribed RNA derived from strain
BetaCoV_Wuhan_WIV04_2019 (EPI_ISL_402124). The transcript contains the amplification regions of
the RdRp and E gene as positive strand. Each microtube contains 1011
copies of target sequences
diluted in yeast tRNA, and lyophilised.
Reconstitution of transcribed RNA
Add 100 µl of RNase/DNAse-free H2O to obtain a solution at a concentration of 109
copies/µl. Store
at -80°C. Dilute to prepare a master bank at 2x106
copies/µl. Store at -80°C.
From this prepare a working bank of reagent at 2x104
copies/µl in order to avoid freeze/thaw
cycles. Working tubes may be stored at -20°C for less than one week.
Positive controls are available upon request (grippe@pasteur.fr)
Aknowledgements
We gratefully acknowledge the Authors, the Originating and Submitting Laboratories for their sequence
and metadata shared through GISAID (EPI_ISL_402119; EPI_ISL_402121; EPI_ISL_402120;
EPI_ISL_402123; EPI_ISL_402124; EPI_ISL_402125).
Reference
1- Corman VM, Landt O, Kaiser M, et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time
RT-PCR. Euro Surveill 2020;25.