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Molecular-Genetic Diagnostic Methods

Q: What temperature stages comprise a single cycle of classical polymerase chain reaction?

Denaturation — heating the mixture to 92–95 °C to separate DNA strands.Annealing — cooling the mixture to 37–60 °C for complementary primer binding.Elongation — heating the mixture to 63–75 °C for synthesis of the new strand using thermostable DNA polymerase.

For medical students2 min readUpdated 2026-10-10

Molecular-genetic testing is a modern laboratory diagnostic method aimed at the direct detection of specific pathogen DNA or RNA sequences in pathological specimens. It is based on the quantification of nucleic acids and is characterized by extremely high sensitivity and specificity.

Method PrincipleDetection of unique nucleotide sequences

ResolutionDifferentiation down to strain and serotype level

MultiplexingSimultaneous detection of multiple microorganisms in a single sample

LimitationLow clinical significance in opportunistic infections

Principles and Main Types

Molecular-genetic diagnostics relies on identifying unique genetic markers of an infectious agent. Unlike classical culture methods, microbial viability is not required, as the target is directly the genetic material (DNA or RNA).

Key types of molecular-genetic assays include:

Polymerase chain reaction (PCR) — the most demanded and widespread variant.
Reverse transcription PCR (RT-PCR) — used for RNA-containing agents.
Restriction fragment length polymorphism (RFLP) analysis.
Sequencing — decoding the nucleotide sequence.
Southern blotting.
Plasmid profiling of the pathogen.

Characteristics of Polymerase Chain Reaction (PCR)

PCR is the foundation of modern molecular diagnostics. The essence of this assay is the targeted search for a small, strictly specific DNA fragment inherent only to a particular species or strain of a pathogen.

The method possesses outstanding differentiating capability. Microbiologists can use it to classify identified microorganisms at several levels:

At the genus level.
At the species level.
At the serotype level.
Distinguishing pathogenic strains from non-pathogenic ones within the same species.

Advantages and Capabilities

The integration of genetic technologies has fundamentally changed approaches to laboratory pathogen detection due to three key advantages:

Multiplexing. The technology allows simultaneous screening for the genetic material of several different pathogens in a single biological sample, significantly saving time.
Ultra-high sensitivity. The method can detect even unculturable pathogen forms — microorganisms that cannot be grown on standard microbiological culture media.
Real-time monitoring. Modern modifications (real-time PCR) not only confirm microbial presence but also quantify viral or bacterial load and perform dynamic monitoring of the infectious process.

Applications and Limitations

Molecular-genetic tests are actively used for primary screening, monitoring therapeutic efficacy, and combined with other laboratory approaches in diagnosing viral, bacterial, and parasitic infections.

However, the method has significant limitations that define its role in modern medicine:

For primary pathogens: Despite all obvious advantages, the reliability of current test systems is still considered insufficient to completely replace classical (culture and microscopic) microbiological methods.
For opportunistic infections: At the current stage of laboratory development, the clinical significance of molecular-genetic methods in diagnosing diseases caused by opportunistic flora is evaluated as very low.

Mnemonic

To quickly remember the key advantages of PCR, use the rule "S-M-R": Sensitivity (finds unculturable forms), Multiplexing (multiple pathogens in one tube), Real-time (quantitative monitoring).

Frequently asked questions

What temperature stages comprise a single cycle of classical polymerase chain reaction?

Denaturation — heating the mixture to 92–95 °C to separate DNA strands.
Annealing — cooling the mixture to 37–60 °C for complementary primer binding.
Elongation — heating the mixture to 63–75 °C for synthesis of the new strand using thermostable DNA polymerase.

What components are essential in a PCR reaction mixture?

Polymerase chain reaction requires the following mandatory components:

DNA template — the target pathogen DNA region.
Primers — short oligonucleotides complementary to the 3' ends of the target gene.
Thermostable DNA polymerase — the enzyme for synthesizing the new strand.
Free nucleotides — building blocks for chain assembly.

How does classical PCR methodologically differ from real-time PCR?

The key methodological difference is that in real-time PCR, amplification and detection occur simultaneously rather than sequentially. Additionally, a special molecular probe is added to the real-time reaction mix, generating a fluorescent signal upon binding to the amplified strand.

Which enzymes are used for reverse transcription PCR (RT-PCR)?

Based on the source materials, the following enzymes are used for RT-PCR:

Reverse transcriptase (reversase) — synthesizes DNA on an RNA template.
DNA polymerase — used to amplify the DNA segment in PCR.

Why is the clinical significance of PCR in opportunistic infections evaluated as low?

At the current stage, the clinical significance of molecular-genetic methods in diagnosing opportunistic infections is assessed as low and is not recognized as clinically diagnostic on its own.

What exactly does PCR detect in a sample?

The reaction targets a small, specific DNA fragment that serves as a unique target for a particular pathogen.

Can cultures be completely replaced by molecular diagnostics?

No. Despite high accuracy, the reliability of genetic test systems is currently insufficient to completely displace classical methods when searching for primary pathogens.

How effective is PCR for infections caused by opportunistic microflora?

Currently, the clinical utility of molecular-genetic methods in diagnosing opportunistic infections is not considered significant and is evaluated as low.

Go deeper

Characteristics of Reverse Transcription PCR (RT-PCR)
Principles and Stages of Restriction Fragment Length Polymorphism (RFLP) Analysis
Sequencing: Decoding Nucleotide Sequences
Southern Blotting Hybridization Technique
Diagnostic Value of Plasmid Profiling