Viral load test
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Viral load assays are changing due to advanced technology to better serve both developed and developing countries. There are two main types of viral load assays: nucleic acid testing (NAT) technologies that detect and quantify viral RNA, and non-nucleic acid testing (NNAT) technologies that detect viral enzymes and proteins as a correlate of viral RNA. Common NAT methods include reverse transcription polymerase chain reaction (RT-PCR), nucleic acid sequence based amplification (NASBA), and branched chain DNA (bDNA). RT-PCR is commonly used to quantify HIV RNA and determine viral load. Examples of commercial viral load tests include Amplicor HIV-1 monitor v1.5, Cobas Taqman, and Real time HIV-1
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Polymerase Chain Reaction: Principles, Applications, and Advancements | The L...
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Polymerase Chain Reaction, often abbreviated as PCR, is a laboratory technique used to amplify specific segments of DNA through a series of temperature-controlled cycles. PCR
This document provides an overview of polymerase chain reaction (PCR) including its history, principles, components, cycle, limitations, advantages, disadvantages, types, and applications. PCR is described as an in vitro method for enzymatically synthesizing specific DNA sequences using two oligonucleotide primers that flank the target region. It allows for exponential amplification of minute amounts of DNA. Real-time PCR is highlighted as an advancement that provides ease of quantification, greater sensitivity, reproducibility, and precision compared to traditional PCR. The document also reviews various applications of PCR in fields such as medicine, forensics, and research.
Polymerase Chain Reaction (PCR) for Mycobacterium.pptx
Polymerase chain reaction (PCR) is a laboratory technique used to make many copies of a specific DNA segment. PCR relies on thermal cycling, heating and cooling the DNA sample to trigger a series of reactions that replicate the target DNA sequence exponentially.
PCR requires DNA template, primers, DNA polymerase enzyme, and nucleotides. Primers flank the target region on DNA to be amplified. In a thermal cycler, the sample undergoes cycles of denaturation (separating DNA strands), annealing (primers bind to flanking regions), and extension (DNA polymerase synthesizes new strand).
After 30-40 cycles, sufficient copies of the target DNA segment have been produced to allow detection and analysis. PCR’s sensitivity allows identifying trace amounts of DNA and has made PCR a foundational tool of molecular biology research and clinical diagnostics.
PCR applications include cloning and sequencing DNA, analyzing forensic samples, detecting viruses or bacteria, DNA fingerprinting, and studying genetic diseases. Quantitative PCR allows quantifying DNA. Reverse transcription PCR (RT-PCR) first transcribes RNA into cDNA for amplification. Real-time PCR monitors amplification as it occurs. Variations alter PCR conditions for specialty applications.
Since its invention in the 1980s, PCR has revolutionized life sciences and biomedical research. Automated thermal cyclers and optimized reagents have made PCR a standard, inexpensive, and readily accessible molecular technique available in all laboratories.
HIV investigaions and diagnosis
The document discusses guidelines for HIV testing and diagnosis. It covers algorithms, timing of laboratory markers, screening tests including ELISA and rapid tests, diagnostic challenges like false negatives and positives, and monitoring of patients including CD4 counts, viral load testing, and STI screening. Key points include using nucleic acid tests to diagnose infants, screening all pregnant women and high-risk groups for STIs, and monitoring HIV patients on ART through regular clinical and laboratory assessments.
Q pcr by meerwais kakar
Quantitative polymerase chain reaction (qPCR) allows real-time amplification and quantification of nucleic acids like RNA or DNA. For gene expression analysis of RNA, the RNA is first converted to complementary DNA (cDNA) via reverse transcription. qPCR is then used to detect changes in expression of genes involved in pathogen response in Arabidopsis thaliana plants infected with a geminivirus compared to uninfected controls. Specific primers are used to quantify levels of mRNA from genes related to pathogen response and control genes. This allows studying the plant's gene expression response to viral infection.
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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
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Viral load test
- 2. Viral load is the concentration of virus in the blood stream. Its used in conjunction with other tests to monitor the progress of patients.
- 3. Its used as a primary indicator of therapeutic efficacy. Used to monitor a change in ARV drugs in drug resistance cases It’s a baseline indicator of disease progression.
- 4. Due to advanced technology viral load assays are dramatically changing to fit the current setting of developed and developing countries. These assays differ in. Sensitivity Dynamic range Target region Extraction, amplification and detection in nucleic acid based assays.
- 5. Nucleic Acid Testing Technologies (NAT)-They detect and quantify Viral RNA Non-Nucleic Testing Technologies( NNAT) They are based on the detection and quantification of HIV viral enzymes and proteins which can be used as a correlate measure of viral RNA.
- 6. Three major methods for detecting and quantifying nucleic acids. Reverse transcription polymerase chain reaction.(RT-PCR) Nucleic Acid Sequence Based Amplification(NASBA) Branched chain DNA (bDNA)
- 7. RT‐PCR is a method of PCR using a Reverse Transcriptase (RT) enzyme to convert viral RNA into complementary DNA (cDNA). The cDNA undergoes replication and detection . RT‐PCR is used to quantify HIV RNA and to dertemine viral load.
- 8. Amplicor HIV-1 monitor v1.5(Roche) Cobas Taqman (Roche) Real time HIV -1 (Abbot) VERSANT HIV-1 RNA ASSAY (kPCR)
- 9. The COBAS Taqman is a real time PCR that targets both the gag and LTR regions of the HIV genome. Coupled with the COBAS Ampliprep, viral load quantification is a fully automated process. It can detect as low as 20cp/ml.
- 10. Can only be accessed by developed countries and a very few places in developing countries. In developed countries it’s the most baseline investigation in HIV positive patients.
- 11. “Science grows like a weed every year”. Kary Mullis THANK YOU!!!!!