ends, forming a duplex with a 5' overhang at each end. The 5' overhang of the primer contains a recognition sequence (5'-GTTGAC-3') for the restriction enzyme Hinc II. An exonuclease-deficient form of the large fragment of Esherichia coli DNA polymerase I (exo' Klenow polymerase) extends the 3' ends of the duplex using dGTP, dCTP, TTP, and dATP (α S), which produces a hemiphosphorothioate recognition site. Hinc II nicks the unprotected primer strand of the hemiphosphorothioate recognition site, leaving intact the modified complementary strand. The exo' Klenow polymerase extends the 3' end at the nick and displaces the downstream complement of the target strand. The polymerization/displacement step regenerates a nickable Hinc II recognition site. Nicking and polymerization/displacement steps cycle continuously producing single-stranded complements of the target strand. The SDA assay can produce a 107- to 108- fold amplification in about 2 hours. The specificity of the SDA reaction is based on the choice of primers to direct the DNA synthesis.
3-4. Ligase chain reaction (LCR)
The LCR target nucleic acid sequence is found within the single copy chromosomal gene of M. tuberculosis which encodes for protein antigen b (PAB). This gene sequence appears to be specific to the M. tuberculosis complex and has been detected in all M. tuberculosis complex strains examined. The four oligonucleotide probes are designed in pairs that hybridize to complementary single-stranded M. tuberculosis target sequences exposed in sample preparation. When a pair of probes has hybridized to a target sequence on a single strand of DNA, there is a gap of a few nucleotides between the probes. Polymerase acts to fill in this gap with the nucleotides in the LCR reaction mixture. Once the gap is filled, ligase can covalently join the pair of probes to form an amplification product that is complementary to the original target sequence and can itself serve as a target in subsequent rounds of amplification. Amplification occurs when the LCR reaction mixture and the sample are incubated in a DNA thermal cycler.
3-5. Detection of M. tuberculosis in clinical samples
The polymerase chain reaction using oligonucleotides based on the repetitive sequence (IS6110) of M. tuberculosis as a primer and the Gen-Probe MTD, which combines an M. tuberculosis rRNA amplification method with the hybridization protection assay format, were evaluated for detection of M. tuberculosis in clinical samples16). The detection limits of these two assay systems which are based on nucleic acid amplification for cultured M. tuberculosis, were less than 10 cells per reaction. A total of 135 sputum specimens were examined by the two assay systems. The PCR and the MTD systems for detection of M. tuberculosis gave overall positivity rates of 84.2% and 91.9%, respectively, as compared with 71.9% by smear and 96.9% by culture in the liquid medium Septi-Chek AFB. Although the sensitivities of the PCR and MTD appeared to be similar to that of culture with the Septi-Chek system, the two methods should be quite useful for rapid detection of M.tuberculosis infections. On the other hand, two cooperative blind studies conducted between 6 to 9 laboratories to estimate the reliability and reproducibility of these two commercially available kits revealed the necessity of good laboratory practice and the development of reference reagents to monitor the performance of the whole assay, including pretreatment of clinical specimens.
4. Drug resistanse in M. tuberuclosis strains
Short course chemotherapy under direct observation has been a highly effective method for tuberculosis control. Unfortunately, this strategy is not effective in patient with rifampin mono-resistant tuberculosis or in patients with multi-drug resistant (MDR) disease. Failure to rapidly detect and appropriately treat patients with multi-drug resistant tuberculosis leads to increased mortality rates, and has resulted in serious nosocomial outbreaks. Therefore, there is an urgent need to develop a rapid, simple, and accurate assay to assess drug resistance in M. tuberculosis.
