Development of a new sensitive and efficient multiplex polymerase chain reaction (PCR) for identification and differentiation of different mycobacterial species.
For early detection and species differentiation of mycobacteria, polymerase chain reaction (PCR) techniques are currently in wide use. However, individual techniques using amplification of different targets with appropriate primers still have some limitations, which have to be overcome. The ideal technique would use DNA sequences which should be present in all mycobacteria and absent in others and would be able to discriminate one species from the other, as non-tuberculous mycobacteria (NTM) are on rise in terms of frequency of detection. We developed a multiplex PCR based on amplification of 165, 365 and 541 bp target fragments of unrelated genes, hsp 65 coding for 65 kDa antigen, dnaJ gene of mycobacteria and insertion element IS 6110 of Mycobacterium tuberculosis, respectively. This multiplex PCR was tested over 5 years from 1996 to 2001 with 411 clinical specimens from suspected cases of tuberculosis and mycobacterioses and compared with standard laboratory techniques. The multiplex PCR was positive for 379 cases compared with 280 cases by standard techniques (P < 0.0001). It could distinguish between strains of the M. tuberculosis complex and NTM; the results are comparable with standard techniques. Thus the multiplex PCR can be useful in early detection, species differentiation and epidemiology.
Bhattacharya
Basudev
B
University College of Medicine, Kolkata, India. bbasudev@rediffmail.com
Karak
Kalpana
K
Ghosal
Alok Gopal
AG
Roy
Atanu
A
Das
Shyamal
S
Dandapat
Premanshu
P
Khetawat
Dimple
D
Mondal
Dibya Kanti
DK
Bhattacharya
Sujit
S
Chakrabarti
Sekhar
S
D015373:Bacterial Typing Techniques / Q000379:methods* / Q000592:standards; D001826:Body Fluids / Q000382:microbiology; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D005784:Gene Amplification; D005798:Genes, Bacterial; D006801:Humans; D009161:Mycobacterium / Q000145:classification* / Q000302:isolation & purification; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D013492:Suppuration / Q000382:microbiology; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology
D017931:DNA Primers; D004269:DNA, Bacterial