POLYMERASE CHAIN REACTION

Types

Randomly amplified polymorphic DNA (RAPD) typing

This is used for the epidemiological identification of bacterial isolates. At least one short primer of variable length is arbitrarily picked, and allowed to strengthen to the template DNA at low stringency. PCR amplification is established, and the products resolved electrophoretically to yield DNA fingerprints which differs according to the degree of relatedness of the strains under investigation

Reverse Transcriptase (RT) PCR

This approach uses RNA as a first pattern. RNA is converted to cDNA by a retroviral reverse transcriptase. The cDNA is amplified, and the amplified band discovered. A recombinant modified form of Thermus thermophilus DNA polymerase gene expressed in E.coli, the enzyme rTth, has a productive reverse transcriptase activity in the presence of manganese, and DNA polymerase activity in the presence of magnesium. The use of this enzyme has enabled the determination of cellular mRNA expression by a single-step procedure. The importance of RT PCR lies in its ability to detect RNA Viruses (e.g. HIV), study intracellular signals (e.g. intracellular interleukin expression), and therapeutic to quantify viral loads (e.g. semi-quantitative RT PCR for HIV loads to monitor response to antiretroviral therapy).

Multiplex PCR

A third variation is known as multiplex PCR, which is perfect for when you want to amplify several different DNA sequences within the same sample, which would be very time consuming and challenging to complete with multiple regular PCRs in turn. Simply put, this is achieved by using various primers at the same time. However, care must be taken to ensure that there is minimal overlap between primer specificity, as cross-template binding can spell disaster for the process. It is beneficial to use short primers, typically only ~18-22 bases in size, such that the chance of any cross-hybridization is minimized. Furthermore, the different primers all must respond and anneal at similar temperatures during the thermal cycling process. If these conditions are met, then it is possible to duplicate vast amounts of different DNA in a single PCR process, vastly reducing both the time and money required to complete the process.

Ligase chain reaction(LCR)

This uses four primers so designed that on annealing the primers bind so that they are immediately adjacent and completely cover the target sequence. The DNA ligase present joins both the fragments, which can then be detected

DIAGNOSIS

Some Common uses of PCR in Infectious disease diagnosis

This procedure can be used in a variety of applications. DNA copies produced through PCR amplification can be used in a large number of medical and forensic applications. It can likewise be used in the identification and detection of infectious diseases and for a wide variety of research purposes in the field of molecular genetics.

Tuberculosis

With an increasing incidence of both HIV infection and multidrug-resistant strains of M. tuberculosis, early detection is vital for diagnosis and therapy. Classical techniques for the discovery of the disease have a drawback in that the organism is fastidious and grows slowly. Many molecular strategies for the detection of mycobacteria have been developed. Important ones are PCR, transcription-mediated amplification, nucleic acid sequence-based amplification, and ligase chain reaction. Mycobacterial speciation, which is time-consuming and exacting by conventional techniques, can be differentiated relatively easily by Multiplex PCR. Molecular susceptibility testing for first-line drugs INH and rifampicin is based on the fact that there is a mutation in the cat gene and the rpoB gene. This can be detected by a PCR amplification of the gene fragment followed by simple electrophoresis in denaturing gels for single-stranded conformational polymorphism (SSCP) analysis. This technique is sensitive and can detect even point mutations. The above procedures lead to early diagnosis of infections by M tuberculosis and initiation of appropriate therapy.

Streptococcal pharyngitis

In developed countries, PCR diagnosis of Gp A hemolytic streptococci by PCR (Gp A Streptococcus direct test or GASD) is gaining importance since its definitive exclusion could curtail empirical treatment of pharyngitis, decrease the use of beta-lactam antibiotics, and reduce overall costs of therapy.

Ulcerative urogenital infections

A multiplex PCR for differentiating etiological causes of ulcerative urogenital infections due to Haemophilus ducreyi, T. pallidum, HSV has been reported.

Atypical pneumonia

A multiplex PCR based strategy wherein rapid diagnosis of infections caused by chlamydia pneumonia, mycoplasma pneumonia, and legionellae could help reshape the management of atypical cases of pneumonia.

Persistent illness

Persistent infections by HSV, CMV, EBV, VZV, HHV, JC virus, measles, hepatitis, rarely respect organ boundaries, are challenging to diagnose and treat. In the immune-competent host, these diseases usually present as an acute illness, and a durable protective immunity follows recovery. However, in the immunocompromised host, these infections may be reactivated. They cause not only a diagnostic dilemma but also the detection of their nucleic acid by amplification procedures that may not be of clinical relevance. Quantitative assays for their presence are essential and have been developed.

Acute febrile illnesses like falciparum malaria, salmonellosis, babesiosis, have been identified using PCR. Especially with falciparum infections, use of a single PCR reaction, and hybridization assays with various probes are used in species identification.