噬菌体生物扩增法快速检测结核分枝杆菌方法学研究

Study on the method for rapid detection of Mycobacterium tuberculosis by phage amplified biologically assay

目的 建立噬菌体生物扩增法快速检测结核分枝杆菌试验方法 ,探讨最佳检测条件。方法 应用分枝杆菌噬菌体感染结核分枝杆菌 ,比较各种检测条件对测定结果的影响 ,并将所建方法用于痰标本检测 ,结果与BIOTECLab公司产品比较。结果 1× 10 9噬菌斑形成单位 (PFU) /ml的噬菌体 ,37℃感染 6 0min可检出 2 0 0～ 5 0 0条 /ml结核分枝杆菌。杀毒剂 10 0mmol/ml,室温作用 5min可完全灭活受试噬菌体。指示细胞浓度在 1× 10 8条 /ml时 ,形成的噬菌斑清晰 ,便于计数。加热灭活的细菌和指示细胞不被噬菌体感染。H3 7Rv、H3 7Ra和牛分枝杆菌参考菌株检测结果均为阳性 ,7种非分枝杆菌和 16种常见非结核分枝杆菌检测结果为阴性 ,4种非结核分枝杆菌 (偶然、胞内、金色、草分枝杆菌 )在高浓度时 (>1× 10 5条 /ml)检测结果为阳性。重复试验结果表明 ,本法批间和批内变异系数均<15 %。痰标本氢氧化钠 半胱氨酸液化方式的阳性检出率为 94 % (49/ 5 2 ) ,显著高于氢氧化钠液化结果的 6 2 % (32 / 5 2 ,χ2 =15 0 6 ,P <0 0 1) ;预培养 1d的阳性检出率为 6 5 % (5 1/ 78) ,显著高于未预培养结果的 4 0 % (31/ 78,χ2 =18 0 5 ,P <0 0 1)。临床标本检测结果表明 ,我们的试剂与进口试剂检测结果基本相同。结论 噬菌体?

Objective To set up a method for rapid detection of Mycobacterium tuberculosis (MTB) by phage amplified biologically (PhaB) assay and to investigate the optimal test condition Methods Various test conditions were compared in order to observe the influence on detective results after MTB was infected by Mycobacteriophage The test condition established was used for detection of sputum samples, and the results were compared with BIOTEC Lab test Results The bacterial concentration of MTB in 200 500/ml was detected by PhaB assay at 1×10 9 plaque forming unite (PFU) /ml of Mycobacteriophage , 37℃ for 60 min The optimal concent ration of virucidal for inactivation of Mycobacteriophage was 100 mmol /ml for 5 min at room temperature The bacteriolytic plaque was clear at the concentration of 1×10 8/ml indicator cells Bacterium inactivated by heat can not be infected by Mycobacteriophage Positive result was observed for control strains of H 37 Rv, H 37 Ra and M bovis while negative result was obtained for 7 strains of non Mycobacterium and 16 control strains of non Tuberculosis Mycobacteria (NTM) The 4 strains of NTM( M fortuitum, M intrcellulare, M aurum, M phlei )showed positive reaction at higher concentrations (>1×10 5/ml) The repetition test showed that the differentiation coefficient in batch and inner was all under 15% There was a significantly difference( P <0 01)in positive rate between two digestion decontamination procedure with N acetyl cysteine NaOH liquefacient(94%) and NaOH liquefacient(62%) The positive rate of the samples cultured one day(65%) was significantly higher than that of the samples without preculture(40%) The results for detection of clinical samples by two reagents, ours and BIOTEC Lab, were nearly the same Conclusion Because its rapidity, simplicity, and sensitivity, PhaB assay can be used for rapid detection of MTB, but the condition of test is very important