Membrane Reverse Dot Blot Hybridization For Rapid Identification Of Mycobacterium Species And Drug-resistant Mycobacterium Tuberculosis Genotype

Objective To establish a simple, rapid, sensitive and specific method for identification of mycobacterial species, and to evaluate the clinical value of polymerase chain reaction-membrane reverse dot blot hybridization technique in identifying rapidly mycobacterial species.Methods 253 clinical isolates of mycobacteria were identified by 16S rRNA polymerase chain reaction-single stranded conformation polymorphism(PCR-SSCP) analysis. The oligonucleotide probes of 16S rRNA used for identification of mycobacterial species were prepared and droped on nitrocellulose membrane. The target DNA fragments of 28 mycobacterial standard strains, 9 non-mycobacterial strains and 253 mycobacterial isolates were labeled with biotin by PCR amplification, and then hybridized with oligonucleotide probes on membrane.Results The standard strains of 28 mycobacteria and 9 nonmycobacteria were analyzed with membrane-reverse dot blot hybridization, the results showed that the oligonucleotide probe was specific. Of 253 clinical isolates of mycobacteria, 198 strains were identified as Mycobacterium tuberculosis complex with PCR-SSCP,, and were also positive hybridization with probe al with reverse dot blot hybridization; 36 strains were identified as non-tuberculous mycobacteria with PCR-SSCP, and their species were identified with specific probes as follow: 11 were M. kansasii, M. gastri, M. scrofulaceum, M. simiae with probe d1, 6 were M. inreacellulare with probe cl, 4 were M. smegmatis with probe ql, 3 were M. vaccae with probe yl, 3 were M. chelonae with probe p1, 2 were M. gordonae with probe k1, 2 were M. fortuitum with probe r1', 2 were M. tuberculosis and inreacellulare complex strains with probe al and c1, 1 was M. avium-inreacellulare with b1 and c1, 1 was M. terrae with probe i1', 1 was M. avium with probe bl. 19 strains were negative hybridization with all probes. The 16S rRNA PCR-membrane reverse dot blot hybridization technique showed that the sensitivity was 92.49%, and the specificity was 100%.The more ideal results could be obtained at the following experimental conditions: 0.22 μ. m nitrocellulose membrane with specific probes was used; the concentration of probe was 1.5pmol/μl; the temperature and time of hybridization and washing...