The Polymerase Chain reaction

Traditional methods of confirming the diagnosis of tuberculosis by smear and culture are insensitive and slow. Only approximately one half of pulmonary tuberculosis is sputum smear positive and bacteria can take weeks to grow on culture medium. Even the use of liquid media can take many days to confirm the diagnosis. The polymerase chain reaction (PCR),is a method of DNA augmentation, which can rapidly multiply the bacterial DNA potentially giving a "culture" result within hours of taking a specimen from the patient. The essentials of the PCR reaction are shown in Fig 4.

Figure 4
Diagram showing a simplified account of the chemical steps involved in the polymerase chain reaction. Two DNA strands are created from one after a single cycle.

A particular sequence of DNA specific to M.tuberculosis is first extracted using restriction enzymes. The target DNA is then denatured into two separate strands by heating. The binding of specific primers can then take place. Series of base pairs can be added from the tips of the primers to eventually build up the whole template. Thus two strands of DNA are made from one. If this process is repeated 30 times approximately 1,000,000 strands of DNA will result. Use of gel electrophoresis and a gene probe can then identify the particular DNA sequence chosen on the genome specific for M.tuberculosis.
Commercial kits (such as the Accuprobe system from Gen-Probe) can identify a culture as being from M. tuberculosis or M. avium within 2 hours.
In practice there have been a number of teething problems with the technique. Contamination can result in false positivity. Sensitivity is not as good as traditional solid media based culture and the method is comparatively expensive.
The Federal Drug administration of the USA has licensed the technique only for identifying the organism in the AFB sputum smear positive patient. Its use for non-respiratory tuberculosis particularly for the examination of CSF in tuberculosis meningitis has not been thoroughly worked out.
It is hoped that new generations of kits will improve sensitivity.

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New TB lab test method cuts diagnosis time