Application of PCR-based restriction fragment length polymorphism for the identification of mycobacterial isolates.
OBJECTIVE
Conventional identification of mycobacteria is achieved by standard biochemical tests that are time consuming, laborious and is not always conclusive. This study was thus undertaken to standardize a simple, rapid and cost-effective polymerase chain reaction based restriction fragment length polymorphism (PCR-RFLP) using primers coding for the 16S - 23S rRNA spacer region to identify the mycobacterial isolates to the species level.


METHODS
The PCR with primers targeting the 16S-23S rRNA spacer region was standardized using the standard mycobacterial strains and applied on 51 clinical isolates. The PCR amplified products were subjected to RFLP using the restriction enzymes, Hae III, MspI and BstXI. The results obtained were compared with those of conventional biochemical tests.


RESULTS
PCR was sensitive to detect 2.5 pg of H37Rv DNA (370 bp for slow grower mycobacteria) and 1.5 pg of M. fortuitum DNA (450 bp for rapid grower mycobacteria). Based on the PCRRFLP products obtained the 51 mycobacterial isolates were classified into 41 slow growers and 10 rapid growers. Among the 41 slow growers, 40 were identified as M. tuberculosis, one as M. xenopi and 10 rapid growers as M. fortuitum.


CONCLUSIONS
PCR using primers targeting the 16S-23S rRNA spacer region was a reliable tool for rapid identification of mycobacterial isolates into slow and rapid growers within 4 h of isolation and further speciation by PCR-RFLP within 6-8 h.
Deepa
P
P
L and T Microbiology Research Centre, Vision Research Foundation, Sankara Nethralaya, Chennai, India.
Therese
K L
KL
Madhavan
H N
HN
D017931:DNA Primers; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D019003:Deoxyribonuclease HpaII; D015252:Deoxyribonucleases, Type II Site-Specific; D004587:Electrophoresis, Agar Gel; D009159:Mycobacteriaceae / Q000145:classification / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length
D017931:DNA Primers; D021903:DNA, Ribosomal Spacer; D019003:Deoxyribonuclease HpaII; C047564:CCANNNNNNTGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases