Detection of gene mutation of rifampin-resistant Mycobacterium tuberculosis by reverse dot-blot hybridization membrane chip

  Objective   A new method, reverse dot-blothybridization membrane chip, was used to rapidly detect rpoB mutations in Mycobacterium trberculosis.

  Methods   Probes were designed according to the sequence ofMycobacterium tuberculosis rpoB gene and probes were spotted and fixed on nylon membrane.The DNA fragment which contained rpoB gene was amplified with biotin-labelled primers by PCR, and then hybridized wtih probes on membrane chip.The results of membrane chip were compared with the results of traditional drug susceptibility testing and DNA sequencing.

  Results   63 Mycobacterium tuberculosis isolates were detected by membrane chips.No mutation was detected among 10 RFP-susceptive isolates.48 out of 53 RFP-resistant isolates, were detected rpoB gene mutation.The sensitivity and specificity of membrane chip detecting isolates was 90.6% (48/53)and 100% (10/10)respectively.Positive predictive value and negative predictive vlue was 100% and 66.7% respectively.27 sputa specimens of TB patients and 17 sputa specimens of other patients were detected by membrane chips.The rersults showed that 11 specimens were detected rpoB gene mutation among 12 RFP-resistant speciments, the concordance with the traditional drug susceptivility test of mycobacterium tuberculosis was 92.3%.6 specimens were detected mutation by membrane chip among 15 RFP-susceptive specimens and 1 specimen was detcted mutation among 17 sputa specimens from other patients.The sensitivity and specificity of membrane chip detecting sputa specimens was 91.7% (11/12)and 78.1% (25/32)respectively, positive predictive value and negative predictive value was 61.11% and 96.1% respectively.

  Conclusion   rpoB gene mutations in most of the rifampicin-resitant v Mycobacterium tuberculosis can be rapidly, simply, and relagtive accurately detected by using reverse dot-blothybridization membrane chip.