Rapid Detection And Identification Of Mycobacterium Species By Amplification Of RpoB Squences And Reverse Dot Blot Hybridization Assay

rpoB is the encoding gene of the β subunit of RNA polymerase. The DNA-dependent RNA polymerase of Mycobacterium tuberculosis is a complex oligomer containing four different subunits(α,β,β',δ)and assembled in two major forms: a core enzyme(α₂ββ') and a holoenzyme(α,β,β',δ). The core enzyme can perform RNA polymerization but requires a 8 subunit to initiate site-specific transcription at promoter sites. The gene encoding subunits α₂ββ'and 8 have been designated rpoA,rpoB,rpoC,and rpoD.The β subunit of M. tuberculosis H₃7RV have 1178 amino acids, and the ORF(open reading frame) encoding it contains 3534bp. We choose a 360bp fragment to design oligonucleotide probes. At the beginning and end of this fragment the sequence is conserved,and it contains two polymorphic regions.Primer I and primer II were used to amplify a 360bp of the rpoB gene of 24 mycobacterium species type strains and 37 mycobacterium strains isolated from PLA tuberculosis research center .To test the specificity of the primers,we use them to amplify eight nonmycobacterium strains,among them two strains(alpha hemolytic streptococcus and corynebacterium pseudodiphtheriticum)have been amplified.According the sequence of the 360bp rpoB gene fragment of different mycobacterium strains,we designed 21 probes to identify mycobacterium strains. Except the probe-M.fortuitum(pFor) hybridize with M.marinum, all the other probes only identify the corresponding strains. And the probe-M.marinura(pMar) do not hybridize with M.fortuitum, so it can also identify these two strains. And the two nonmycobacterium strains(alpha hemolytic streptococcus and corynebacterium pseudodiphtheriticum)do not hybridize with the 21 probes.Among the 37 clinical isolates, 12 are identified to be M.tuberculosis,7 M.intracellulare,7 M.abscessus, 6 M. scro- fulaceum, 2 M.kansasii, 1 M.gordonae, 1 M.fortuitum,and 1Mycobacterium genus.There are 7 results do not coincide with the traditional clinical test results. Through sequencing the 7strains' 360bp fragments,the final results coincide with our hybridization test.According the polymorphic region of the 360-bp fragment of rpoB,the primers and probes are designed . Through our test,these primers and probes have good specificity and sensitivity.lt can identify M.kansasii from M.gastri,which can not be identified by 16SrDNA sequence. M.szulgai and M.malmoense, M.chelonae and M.abscessus , they have high degree of sequence similarity in 16SrDNA,which can also be identified by the rpoB-PCR- reverse dot blot hybridization assay.lt has a broad application perspective in clinical mycobacterium species identification.