Detection Of Isoniazid And Rifampin Resistance By Reverse Dot Blot In China

Part Ⅰ Establishment ofReverse Dot BlotObjective: To establish a simple, rapid and sensitive hybridization method (ReverseDot Blot, RDB) for detecting drug resistance relevant gene mutation in mycobacteriumtuberculosis1. Methods: The probes detecting drug resistance were as follows:5wild-type and7mutant probes for rifamping,2wild-type and3mutant probes forisoniazid and immobilized on nylonmembrane strips. PCR using biotin labeled primersand thereafter the PCR product was hybridized with probes on membrane,which includeforty-eight DNA samples with the TB wild type and mutant types from the NationalInstitute for the Control of Pharmaceutical and Biological Products (NICPBP)(Beijing,China). All samples were further tested by DNA clonal sequencing analysis. Results:Forty-eight DNA samples from the NICPBP were successfully detected by RDB.Conclusions: RDB is a rapid and sensitive method to detect the rpoB,katG and inhAgenemutations. PartⅡ Detection ofClinical Samples by Reverse Dot Blot in ChinaObjective: To establish a simple, rapid and sensitive hybridization method fordetecting drug resistance relevant gene mutation of clinical samples in china. Methods:The probes detecting drug resistance were as follows:5wild-type and7mutant probesfor rifamping.2wild-type and3mutant probes for isoniazid and immobilized onnylonmembrane strips. PCR using biotin labeled primers and thereafter the PCRproduct was hybridized with probes on membrane,which include (1)Forty-six clinicalsamples came from the﹡﹡Hospital of Zibo (Zibo,China). The diagnosis of TBinfection was based on radiography, smear of sputum, culture based method of L-Jstandard culture-based drug sensitivity testing system.All patients were treated withanti-tuberculosis drugs, all clinically suspected resistant samples were further tested byDNA clonal sequencing analysis.(2)Eighty of the culture based method bacterias fromGuangzhou﹡﹡Hospital (Guangzhou,China) were used to test this method.Forty ofthe samples were drug resistant, others were drug-susceptible. The results of reverse dot blot hybridization were compared with the results of drug sensitivity test andsequencing. Results: Fourty drug resistant mycobacterium tuberculosis isolates andfourty drup sensitive isolates were analyzed. The gene mutations were consistent withthe DNA sequencing and the in vitro susceptibility test in37/40(92.5%)and40/40(100%)of the isolates respectively.46patients of the﹡﹡Hospital of Zibo Citywere detected, including5subjects (10.9%)were not successfully detected and41of thesamples were successfully detected by RDB. Conclusions: Reverse dot blothybridization is a rapid and sensitive method to detect the rpoB,katG and inhAgenemutations, which may be used in early detection of the resistance of TB to rifampinand isoniazid.