Molecular Detection Of Microorganisms

INTRODUCTION

Molecular microbiology has emerged as the leading area in the clinical microbiology laboratory and created new opportunities for laboratory diagnosis to affect patient care. Molecular methods have been found to be advantageous in situations in which conventional methods are slow, insensitive, expensive, or not available.

MOLECULAR METHODS

Molecular methods are mainly of two major types, amplified methods, and non-amplified methods.
  1. Amplified Methods
  2. Non-amplified Method

1. Amplified Methods

  • Polymerase chain reaction (PCR)
  • Transcription mediated amplification(TMA)
  • Nucleic acid sequence-based amplification (NASBA)
  • Ligase chain reaction (LCR)

2. Non-amplified Method

  • Nucleic acid probes

1. Amplified Methods

Amplification of nucleic acid is the basis of all the amplified methods. The amplified nucleic acid may be either DNA or RNA.

1. Polymerase Chain Reaction (PCR)

It is the target amplification system. PCR permits the synthesis of essentially limitless quantities of a target nucleic acid sequence. Other target amplification techniques include TMA and NASBA. Besides originally described PCR, other types of PCR include reverse transcriptase PCR (RT-PCR), nested PCR, multiplex PCR, and real-time PCR.

A. RT-PCR

As originally described, PCR was a technique for DNA amplification. In this process, complementary DNA (cDNA) is first produced from RNA with the help of enzyme reverse transcriptase and then cDNA is amplified by PCR.

B. Nested PCR

Nested PCR was developed to increase the sensitivity and specificity of the PCR technique. It uses two pairs of amplification primers. One primer pair is used in the first round of PCR to amplify the desired sequence. The amplified product of the first round is then subjected to the second round of PCR with the second set of primers which anneal to the sequences found only in the first round products.

Multiplex PCR

Two or more primer sets designed for amplification of different targets are included in this technique. This will help in the amplification of more than one target sequence in a clinical specimen. Multiplex PCRs are usually less sensitive than PCRs with a single set of primers. PCR has been applied in the clinical laboratory for the diagnosis of various infectious agents

Real-Time PCR

Real-time PCR is a major breakthrough for the detection of PCR products. In real-time PCR, amplified products (amplicons) are detected as they accumulate after each cycle, in contrast to standard PCR where amplicons are detected at the end of the procedure.

Thus, a positive result can be obtained quickly, often while the assay is still running. The imaging system for the detection of the amplicon is a part of the real-time instrument. Real-time PCR can also be used to quantitate nucleic acids, which is useful for monitoring certain diseases, such as HIV and hepatitis.

Transcription Mediated Amplification (TMA)

Transcription mediated amplification (TMA) is an isothermal RNA amplification method. RNA target is reverse transcribed into cDNA and then RNA copies are synthesized with the help of RNA polymerase, tuberculosis, C.  trachomatis N. gonorrhea, HCV, and HIV-1.

Nucleic Acid Sequence-Based Amplification (NASBA)

Like TMA, it is also an isothermal RNA amplification method. The method is similar to TMA, RNA target is reverse transcribed into cDNA and then RNA copies are synthesized with the help of RNA polymerase.

Ligase Chain Reaction (LCR)

Ligase chain reaction (1CR) is a probe amplification method in contrast to the above-mentioned methods which are target amplification methods. The probe amplification method differs from the target, the amplification method in that amplified products contains only a sequence present in the initial probes. LCR based amplification has been used for M. tuberculosis, N. gonorrhea, and C. trachomatis.

Non-Amplified Method

Nucleic Acid Probes


Probes for identification of group A streptococci, group B streptococci, enterococci, Haemophilus influenza, mycobacteria, N.gonorrhoeae, Staphylococcus aureus, Streptococcus pneumonia, Campylobacter sp., Histoplasma capsulatum, Blastomyces dermatitis, and Coccidioides mitis isolated in culture are also available. DNA probes for the detection of LT and ST toxins of Esch. coli are also available.

SIGNIFICANCE IN CLINICAL LABORATORY

Molecular methods have a significant role in the following situations in the clinical microbiology laboratory.
  • Detection of uncultivable and slow-growing microorganisms.
  • Role in clinical virology
  • Disease prognosis
  • Response to treatment

Detection of Uncultivable and Slow Growing Microorganisms

A number of microorganisms have been first identified directly from clinical specimens by molecular methods. HCV, Bartonella henselae, Sin Nobre virus, and Human herpesvirus 8 (HAV-8) are some examples of human pathogens first identified from clinical specimens using molecular methods. These molecular methods are also useful for fastidious microorganisms that may die in transit or may be overgrown by Contaminants when cultured.

N. gonorrhoeae is one such example whose nucleic acid can be detected under circumstances in which it cannot be cultured.

Role in Clinical Virology

Molecular approaches are often faster, more sensitive, and more cost-effective than the conventional approaches in clinical virology. Enteroviral meningitis, HSV encephalitis and CMV infections in immunocompromised patients are examples for which nucleic acid-based tests are relevant and cost-effective for diagnosis.

Disease Prognosis

Molecular methods provide important information that may predict disease progression. HIV-I viral load as a predictor of progression to AIDS is probably the best example.

Response to Treatment

Molecular methods have been developed to detect the genes responsible for drug resistance. These methods have been used to supplement conventional antimicrobial susceptibility testing for the detection of methicillin resistance in staphylococci, vancomycin resistance in enterococci, and rifampicin resistance in Mycobacterium tuberculosis.

Molecular techniques have a significant role in predicting and monitoring patient responses to antiviral therapy. HIV-1 viral load assays have been developed to monitor the response of antiretroviral therapy.
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