Molecular Methods in Microbiology

 

INTRODUCTION

 

The DNA, RNA or proteins of an infectious agent in a clinical sample can be used to help identify the agent in many cases. New techniques and applications of the techniques are being developed for the analysis of infectious agents. It also helps in disease prognosis and monitoring the response to treatment. The advantages of molecular techniques are their sensitivity, specificity and safety. 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 classified into three categories :

      A.    Hybridization

      B.     Amplification

      C.     Sequencing and enzymatic digestion of nucleic acids.

A. Hybridization

DNA probes can be used like antibodies as sensitive and specific tools to detect, locate and quantitate specific nucleic acid sequences in clinical specimens. Nucleic acid probes are segments of DNA or RNA labelled with radioisotopes or enzymes or fluorescent dyes that can hybridize to complementary nucleic acid with high degree of specificity.

A number of DNA probes have been developed for direct detection of micro-organisms in clinical specimens and for identification of organisms after isolation of culture. Applications of DNA probe technology in microbiology are:

•             Nucleic acid probes for direct detection of groupA streptococci, Chlamydia trachomatis and Neisseria gonorrhoeae are available.
•         Probes for identification of group A streptococci, group B streptococci, enterococci, Haemophilus influenzae, Mycobacteria, N. gonorrhoeae, Staphylococcus aureus, Streptococcus pneumoniae, Campylobacter sp., Histoplasma capsulatum, Blastomyces dermatidis and Coccidioides immitis isolated in culture are also available.
•                 DNA probes for detection of LT and ST toxins of E. coli are available.

B. Amplified Methods

1.     Polymerase chain reaction (PCR)

2.     Transcription mediated amplification (TMA)

3.     Nucleic acid sequence amplification (NASBA)

4.     Ligase chain reaction (LCR)

 

1. Polymerase Chain Reaction (PCR)

It is the target amplification system. The polymerase chain reaction (PCR) can detect single copies of viral DNA by amplifying the DNA many million-fold and is one of the newest techniques of genetic analysis.

In this technique, a sample is incubated with two short DNA oligomers, termed primers, that are complementary to the ends of a known genetic sequence of the viral DNA, a heat-stable DNA polymerase (Taq or other polymerases obtained from thermophilic bacteria), nucleotides, and buffers. The oligomers hybridize to the appropriate sequence of DNA and act as primers for the polymerase, which copies that segment of the DNA. The sample is then heated to denature the DNA (separating the strands of the double helix) and cooled to allow hybridization of the primers to the new DNA. Each copy of DNA becomes a new template. The process is repeated many (30-50) times to amplify the original DNA sequence in an exponential manner. A target sequence can be amplified a million-fold in a few hours using this method. PCR has been applied in clinical laboratory for diagnosis of various infectious agents.

Besides originally described PCR, other types of PCR include reverse-transcriptase PCR (RT-PCR), nested PCR and multiplex PCR.

i. RT-PCR: Reverse transcription PCR (RT-PCR) amplifies an RNA target. In this technique, target is RNA instead of DNA. The unique step to this procedure is the use of the enzyme reverse transcriptase that directs synthesis of DNA from the viral RNA template. Once the DNA has been produced, relatively routine PCR technology is applied to obtain amplification.

ii. Nested PCR: Nested PCR involves the sequential use of two primer sets. The first set is used to amplify a target sequence. The amplicon obtained is then used as the target sequence for a second amplification using primers internal to those of the first amplicon. Essentially, this is an amplification of a sequence internal to an amplicon. The advantage of this approach is extreme sensitivity and confirmed specificity without the need for using probes.

iii. Multiplex PCR: Multiplex PCR is a method by which more than one primer pair is included in the PCR mixture. This will help in amplification of more than one target sequence in a clinical specimen. The control amplicon should always be detectable after PCR. Multiplex PCRs are usually less sensitive than PCRs with single set of primers.

iv. Arbitrary primed PCR: Arbitrary primed PCR uses short primers that are not specifically complementary to a particular sequence of a target DNA. By comparing fragment migration patterns following agarose gel electrophoresis, strains or isolates can be judged to be the same, similar or unrelated.

v. Quantitative PCR: Quantitative PCR is an approach that combines the power of PCR for the detection and identification of infectious agents with the ability to quantitate the actual number of targets originally in the clinical specimen. It can be used for studying and understanding the disease state (e.g., acquired immunodeficiency syndrome [AIDS]), the prognosis of certain infections, and the effectiveness of antimicrobial therapy.

vi. Real time PCR: Real time PCR combines rapid thermo cycling with the ability to detect target by fluorescently labeled probes as the hybrids are formed, i.e. in real time. This technology allows for high throughput of samples, multiplexing reactions, quantitation of target and on-line monitoring.

 

2. Transcription Mediated Amplification (TMA)

Transcription mediated amplification (TMA) is an isothermal RNA amplification method and use three enzymes: Reverse Transcriptase (RT), RNAase H and T7 DNA dependent RNA polymerase. RNA target is reverse transcribed into cDNA and then RNA copies are synthesized with the help of RNA polymerase. A 10⁹ fold amplification of the target RNA can be achieved in about 2 hours.

Advantages of TMA include no requirement for a thermal cycler and contamination risk is minimized TMA based assays are available for detection of M. tuberculosis, C trachomatis, N gonorrhoeae, Hepatitis virus (HCV) and Human Immunodeficiency Virus 1 (HIV-1).

 

3. Nucleic Acid Sequence-Based Amplification (NASBA)

Both TMA and NASBA are examples of transcription-mediated amplification. These isothermal assays use three enzymes: Reverse Transcriptase (RT), RNAase H, and T7 DNA dependent RNA polymerase. 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. It also does not require thermal cycler. NASBA based kits for detection and quantitation of HIV-1 RNA and CMV RNA are available.

 

4. Ligase Chain Reaction (LCR)

Ligase chain reaction (LCR) is an amplification of probe nucleic acid rather than target nucleic acid. By this approach, an amplified probe is the final reaction product to be detected, while the target sequence is neither amplified nor incorporated into this product.

LCR uses two pairs of probes that span the target sequence of interest. Once annealed to the target sequence, a space remains between the probes that is enzymatically closed using a ligase (i.e., a ligation reaction). On heating, the joined probes are released as a single strand that is complementary to the target nucleic acid. These newly synthesized strands are then used as the template for subsequent cycles of probe annealing and ligations. Through the process, probe DNA is amplified to a level readily detectable using assays similar to those described for the biotin-avidin system. Like PCR, LCR also requires thermal cycler.

LCR based amplification has been used to detect Chlamydia trachomatis and Neisseria gonorrhoeae.

v Applications of Molecular Methods in Clinical Laboratory

Molecular methods have a significant role in the following situations in clinical microbiology laboratory.

1.     Detection of uncultivable and slow growing  micro-organisms.

2.     Role in clinical virology.

3.     Disease prognosis.

4.     Response to treatment and Detection of drug resistance

5.     Genotyping of microorganism

1. Detection of Uncultivable and Slow Growing Micro-organisms

The greatest advantage of molecular methods has been in the discovery of previously unrecognized or uncultivable organisms. Molecular methods have been used to detect previously unknown agents directly in clinical specimens by using broad-range primers for a number of micro-organisms. HCV, and Human Herpes Virus 8 (HHV-8), Bartonella henselae are some examples of human pathogens first identified from clinical specimens using molecular methods.

Molecular methods have the ability to detect nonviable organisms that are not retrievable by cultivation-based methods. These methods are also useful for fastidious micro-organisms which 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. The use of improper collection, inappropriate transport conditions or delay in transport can reduce the viability of the organism but do not affect the nucleic acid detection.

These can detect and identify organisms that cannot be grown in culture or are extremely difficult to grow (e.g., hepatitis B virus and the agent of Whipple's disease)and also more rapid detection and identification of organisms that grow slowly (e.g., mycobacteria, certain fungi).

2. Role in Clinical Virology

Molecular methods to replace culture for detection of bacteria in routine practice are limited because of need to isolate the organisms for antibiotic sensitivity testing. These methods can actually replace the culture only in those micro-organisms which have predictable antibiotic susceptibility and consequently, routine susceptibility testing is not performed.

Culture-based methods in virology are costly and antiviral susceptibility testing is not routinely done in clinical virology. Molecular approaches are often faster, more sensitive and more cost-effective than the conventional approaches. Seasonal Influenza A(H1N1)pdm09(Swine flu), 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.

3. Disease Prognosis

Molecular methods are able to quantitate infectious agent burden directly in patient specimens , an application that has particular importance for managing Human Immunodeficiency Virus (HIV) infections. Thus, it provides important information which may predict disease progression.

Molecular methods can be used for subtyping of certain viruses which may provide information about the severity of infection. HPV causes dysplasia, neoplasia and carcinoma of cervix in women. HPV types 16 and 18 are associated with a high risk of progression to neoplasia, whereas HPV types 6 and 11 have a low risk.

4. Response to Treatment and Drug Resistance

Molecular methods have been developed to detect the genes responsible for drug resistance that may not always readily be detected by phenotypic methods. Examples include detection of the van genes, which mediate vancomycin resistance among enterococci, and the mec gene, which encodes resistance among staphylococci 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. Viral load assays have also been used in monitoring the response to therapy in patients who are chronically infected with HBV and HCV.

Molecular methods can be used to detect drug resistance mutations in RT and protease genes ofHIV-1. These mutations lead to lower levels of sensitivity to antiretroviral drugs and are important causes of treatment failure. This helps to determine an appropriate treatment in patients who do not respond to therapy.

 

5.     Genotyping of microorganism

It is epidemiologically important to know circulating genotypes of microorganisms particularly for viruses in a particular region and also severity of disease is different in different genotypes, e.g. Dengue infection is very severe with type 2 Dengue virus. We can also trace common  source of infection with help of genotyping.