Serological Tests in Clinical Diagnosis

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Questions and Answers

Which method is considered a 'gold-standard' approach for the diagnosis of viral infection?

Sequencing

Cell culture (correct)

Real-time PCR

Conventional polymerase chain reaction (cPCR)

What is the main advantage of real-time PCR over cPCR for viral infection diagnosis?

Lower cost

Less risk of contamination

Higher accuracy and sensitivity (correct)

Faster results

How does real-time PCR quantify viral nucleic acids?

By counting the number of cycles

By confirming the amplification using the threshold cycle (correct)

By analyzing the initial sample volume

By measuring the time of amplification

What should be considered when implementing real-time PCR for virus detection in clinical settings?

Appropriate samples, nucleic acid extraction, and selection of diagnostic methods based on sensitivity and specificity targets Signup and view all the answers

What is necessary to evaluate when implementing real-time PCR for virus detection in clinical settings?

Performance of the test protocol by measuring sensitivity, specificity, accuracy, and reproducibility Signup and view all the answers

What is an advantage of the verified real-time PCR method for monitoring diagnosis and treatment outcomes in a clinical setting?

It is an easy, fast, and accurate method Signup and view all the answers

Which method is commonly used for initial IgM antibody detection in serological tests?

ELISA Signup and view all the answers

What is the primary molecular technology used to detect viral DNA or RNA?

PCR Signup and view all the answers

Which test is useful for detecting infections such as hepatitis A, EBV, rubella, and measles?

Serological diagnosis Signup and view all the answers

What method uses two specific antibodies to bind to a viral antigen?

Sandwich ELISA Signup and view all the answers

What type of testing provides high sensitivity and specificity for virus detection and can determine viral load and genotype?

Nucleic acid testing through PCR Signup and view all the answers

Which organization provides guidelines and recommendations for the collection, storage, and management of clinical specimens for viral diseases?

WHO Signup and view all the answers

What is the most crucial step in PCR and real-time PCR?

Isolation of nucleic acids from viruses Signup and view all the answers

How can the quality of nucleic acids be assessed?

Using the 280/260 ratio and 280/230 ratio Signup and view all the answers

What is a widely used molecular diagnostic method for real-time PCR?

Hydrolysis probes Signup and view all the answers

What do the 280/260 ratio and 280/230 ratio indicate about nucleic acid quality?

Close to 2 for optimal PCR efficiency Signup and view all the answers

What is the purpose of hydrolysis probe format in real-time PCR?

To release the fluorescent reporter dye during PCR process Signup and view all the answers

What is molecular diagnostics primarily used for?

Disease diagnosis and treatment, risk detection, and monitoring. Signup and view all the answers

Which method is considered the 'gold-standard' for diagnosing viruses, but is time-consuming and difficult to use for early diagnosis or virus typing?

Virus culture Signup and view all the answers

What is the main advantage of real-time PCR for viral nucleic acid detection?

High accuracy and ability to quantify viral loads Signup and view all the answers

Which viruses can be cultured using viral culture method?

Adenoviruses and cytomegaloviruses Signup and view all the answers

What types of viruses can real-time PCR identify?

A wide range of viruses, including those that are difficult to culture Signup and view all the answers

Why is viral diagnosis important according to the text?

For effective treatment and prevention of viral infections Signup and view all the answers

What is the recommended range for real-time PCR amplification length?

50~150 base pairs Signup and view all the answers

What is the recommended GC ratio range for primers in real-time PCR?

20% - 80% Signup and view all the answers

What is a key consideration for hydrolysis probe design in real-time PCR?

Being close to the forward primer Signup and view all the answers

What is the recommended approach to verify probe and primer specificity in real-time PCR?

Using both in silico tools and experimental evidence Signup and view all the answers

What is a common challenge in multiplex real-time PCR?

$Tm$ variation among primer pairs Signup and view all the answers

What is necessary for accurate results in real-time PCR?

Proper sample storage, nucleic acid preparation, purity, probe selection, and data analysis Signup and view all the answers

Study Notes

The methods used for sampling, storage, and delivery impact diagnostic results in molecular diagnostics, with the isolation of nucleic acids from viruses being the most crucial step in PCR and real-time PCR.
The nucleic acid extraction process can be classified into five methods: boiling, an ion-exchange matrix, DNA precipitation, magnetic glass particles, and a silica membrane.
The amount and type of the specimen and virus determine how the viral nucleic acid extraction should be performed.
Pure nucleic acids are desirable, and their quality can be assessed using the 280/260 ratio and 280/230 ratio, which should be close to 2 for optimal PCR efficiency.
Molecular diagnostics is the application of molecular biology to medical tests, used for disease diagnosis and treatment, risk detection, and monitoring.
Real-time PCR is a fast and sensitive molecular diagnostic technique, which can detect viral DNA or RNA using either nonspecific fluorescent dyes or sequence-specific probes.
Probe-based real-time PCR is a widely used molecular diagnostic method, with hydrolysis probes being a common format.
The hydrolysis probe format uses a probe labeled with a fluorescent reporter and quencher dyes. During the PCR process, the polymerase cleaves the probe, releasing the fluorescent reporter dye.
Molecular beacon probes and hybridization probes are other formats for real-time PCR detection.
Clinical trials use a real-time PCR platform in combination with three other nucleic acid detection methods.
Real-time PCR has been researched for multiplexing using probes like peptide nucleic acids and locked nucleic acids.
The hydrolysis probe format is commonly used clinically, easy to design, and has high sensitivity and specificity.
The design of a hydrolysis probe follows certain rules.
Real-time PCR amplification length should be between 50~150 base pairs.
GC ratio of primers should be between 20% and 80%.
Primers' melting points (Tm) should be similar and have high specificity.
Hydrolysis probe should not contain a G at the 5' end or have more Gs than Cs, and should be close to the forward primer.
The difference between target and reference gene database access numbers, exon location, and sequence of other viruses or types should be considered in primer design.
In silico tools like BLAST and experimental evidence from gel electrophoresis, melting profiles, DNA sequencing, amplicon size, and restriction enzyme digestion are used to verify probe and primer specificity.
The Tm of oligonucleotides can be predicted using algorithms for initial design, but optimum annealing temperature should be determined experimentally.
Real-time PCR reagents used in clinical trials undergo performance evaluations.
Competition for limited detector channels is common in multiplex real-time PCR, and all primer pairs should be optimized to operate at the same temperature.
Real-time PCR instrument should have a limited number of channels to minimize inter-channel interference.
Proper sample storage, nucleic acid preparation, purity, probe selection, and data analysis are necessary for accurate results.
The MIQE guidelines are the most promising validation methods for real-time PCR.
Minimum detection limit of 95% and repeated measurements for analytical sensitivity evaluation.
Two duplicate measurements for sensitivity evaluation.
Validation methods include probit analysis, absolute quantification, and detection limit evaluation.

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Description

Learn about the use of serological tests for identifying antibodies and antigens in clinical diagnosis. Understand how antibodies that react specifically with the virus are utilized to establish a serological diagnosis.