Molecular diagnostic techniques

Abstract

Clinical microbiology laboratories increasingly rely on molecular diagnostic techniques. The various formats of nucleic acid amplification are the most frequently used molecular tests in the diagnosis of infectious diseases. In many clinical settings, polymerase chain reaction (PCR) is clearly the method of choice due to its exquisite sensitivity and specificity. Today, many conventional PCR methods are being replaced by real-time PCR, which allows more rapid detection and quantification of the PCR product, as well as detection of different strains of the pathogen by melting curve analysis. The ability to measure the quantity of microbe by quantitative PCR has become increasingly important, providing information on the progression and prognosis of disease, and effectiveness of treatment. Other widely used molecular diagnostic techniques are isothermal amplification methods and nucleic acid hybridization techniques. Microarray is a technique which holds promise and has an exceptional sensitivity and the capacity to detect several pathogens simultaneously. However, microarrays are currently too expensive to be adapted for routine diagnostics, and their diagnostic use requires broad-based nucleic acid amplification prior to analysis which is not well established. Several molecular methods can be used for genotyping, which allows the identification of different subtypes of the pathogen; genotyping plays a role in the risk assessment and management of infections. Clinicians need to recognize the enhanced accuracy and speed of the molecular diagnostic techniques for the diagnosis of infections, but also to understand their limitations. Laboratory results should always be interpreted in the context of the clinical presentation of the patient, and appropriate site, quality, and timing of specimen collection are required for reliable test results.

Keywords: genotype; mass spectrometry; microarray analysis; nucleic acid amplification techniques; nucleic acid hybridization.

Figure 1

Principle of polymerase chain reaction (PCR)

Figure 2

Principle of nucleic acid sequence-based amplification

Figure 3

This example is a modification of a microarray for the detection of human papilloma virus. In this example, biotin-labeled PCR products are hybridized with immobilized oligonucleotides. The chip is incubated in an anti-biotin antibody-conjugated gold nanoparticle solution. The silver enhancement by the gold nanoparticles bound to the biotin precipitates silver metal particles at the chip surfaces. This blocks light irradiated from above, which can be measured, allowing detection and quantitative analysis of the target DNA.