结核杆菌耐药基因膜芯片检测

目的建立一种快速检测结核杆菌对异烟肼、利福平、链霉素、乙胺丁醇、吡嗪酰胺、喹诺酮类耐药的基因的方法。方法应用Oligo 6.0设计12对引物、54条探针,构建多重聚合酶链式反应(multiplex polymerase chain reac-tion,多重PCR)结合反向斑点杂交膜芯片检测结核耐药基因的方法,并对52株结核杆菌临床分离株进行检测。结果12对引物分4个反应管建立了同一条件下PCR反应体系,54条探针组成的膜芯片中包含36条野生型检测探针、16条突变型检测探针、阳性和阴性对照探针各1条;利用膜芯片检测结核杆菌耐药性的灵敏度为95.4%(41/43),特异度为100%,与药敏试验耐药种类的完全一致率为53.5%(23/43)。结论多重PCR联合膜芯片技术能有效地检测结核杆菌耐药基因,并有助于结核杆菌耐药性判断,具有灵敏度高、特异性好、简便、快速等优点,适合于基层应用。

Membrane chip for detection of drug resistance gene of Mycobacterium tuberculosis

Objective To develop a rapid method for the detection of Mycobacterium tuberculosis(MTB) durg-resistant genes of isonizai,rifampicin,streptomycin,ethambutol,pyrazinamide,and quinolones.Methods We designed 12 pairs primers and 54 probes by Oligo6.0,constructed a gene membrane chip for MTB drug-resistant gene detection by the combinatiion of multiplex PCR and reverse dot blot hybridization,and detected 52 clinical isolates of MTB.Results The 12 primes were divided into 4 reactons to establish a multiplex PCR reaction system under the same conditions.Then with reverse dot blot hybridization,a gene membrane chip of 54 oligonucleotide probes was developed and the chip included 36 wild-type probes,16 mutant probes,and a positive and a negative probe.The sensitivity of the MTB drug-resistant gene detection with the chip was 95.4%(41/43) and the specificity was 100%.Conclusion The gene membrane chip developed with the combination of multiplex PCR and reverse dot blot hybridization could be used to detect MTB drug-resistant gene effectively,and the method is rapid and convenient,and with good sensitivity and specificity for grassroot application.