Diagnosis Infectious Disease - Nucleic Acid-Based Clinical Assays

7 important questions on Diagnosis Infectious Disease - Nucleic Acid-Based Clinical Assays

How does polymerase chain reaction (PCR) contribute to pathogen identification in clinical laboratories, especially for viruses and intracellular pathogens?

PCR amplifies nucleic acids, forming multiple copies of target sequences. PCR techniques can use primers for a pathogen-specific gene to examine DNA derived from suspected infected tissue, even in the absence of an observable or culturable pathogen. PCR-based tests are widely used for pathogen identification, especially for viruses and intracellular pathogens that are difficult to culture.

Explain the role of nucleic acid hybridization in identifying specific pathogens in patient samples, and how are nucleic acid probes used in this process?

Nucleic acid hybridization is used to identify specific pathogens by employing unique nucleic acid probes to detect the presence of specific DNA sequences. Nucleic acid probes are single-stranded DNA molecules with a sequence complementary to the gene of interest. If a microbe's DNA or RNA sequences match the probe, hybridization occurs, forming a double-stranded molecule. The probe, labeled with a reporter molecule (usually fluorescent), is used to detect the hybridization reaction.

Describe the process of a probe assay, including the sample treatment and detection steps.

In a probe assay, samples are treated with strong alkali (e.g., sodium hydroxide) to lyse cells and partially denature pathogen DNA, forming single-stranded DNA molecules. Incubation at an appropriate temperature facilitates stable duplex formation between target DNA and probe DNA. The extent of hybridization is measured using the reporter molecule attached to the probe. Some assays use two-component probes, functioning as both a reporter and a capture probe, allowing hybridized molecules to be affixed to a matrix for detection.
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How does nucleic acid hybridization play a critical role in PCR-based techniques, and what is the purpose of gene-specific nucleic acid primers in PCR analysis?

Nucleic acid hybridization is crucial in PCR-based techniques used to amplify target DNA or RNA molecules. Gene-specific nucleic acid primers (short oligonucleotides) jump-start DNA polymerase during PCR amplification of pathogen-specific genes. The amplified nucleic acid product (amplicon) is visualized, often using fluorescence, confirming the presence of the pathogen.

What is quantitative real-time PCR (qPCR), and how does it use fluorescent probes to visualize target DNA accumulation?

Quantitative real-time PCR (qPCR) uses fluorescent probes to label PCR amplicons, allowing visualization of target DNA accumulation. Fluorescence increases proportionally as the target DNA is amplified. The probes may be nonspecific or gene-specific, and their fluorescence levels are monitored continuously during qPCR amplification, eliminating the need for gel electrophoresis.

How does reverse transcription PCR (RT-PCR) contribute to the detection of RNA viruses, and what is the role of reverse transcriptase in this process?

Reverse transcription PCR (RT-PCR) uses pathogen-specific RNA to produce complementary DNA (cDNA) directly from patient samples. Reverse transcriptase is used to make a cDNA copy of an RNA sample, which is then amplified using PCR. RT-PCR is especially useful for detecting RNA viruses, including retroviruses like HIV.

What is qualitative PCR, and how does it differ from quantitative real-time PCR (qPCR)?

Qualitative PCR is a diagnostic test that uses a slightly different amplification protocol and an additional step to identify pathogen-associated genes. It incorporates labeled hybridization primers into an amplicon product of a qPCR reaction. The method allows the identification of specific genes associated with pathogens and can be used for rapid diagnosis within hours.

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