适用于结核分枝杆菌微测序耐药基因芯片的多重PCR体系的优化与评价

目的优化结核分枝杆菌耐药相关基因多重PCR反应体系并评估其优缺点。方法利用改进的方法提取结核分枝杆菌基因组DNA,优化适合扩增rpoB,katG,mabA/inhA,pncA,embB,gyrA,rpsL,rrs和eis耐药基因片段的多重PCR体系,经测序确定所扩增出的基因片段,计算PCR操作过程所用的时间和材料成本等,分析多重PCR反应体系存在的优缺点。结果经过优化煺火温度、引物浓度以及添加NH 4^+和二甲基亚砜可以较好的扩增出5重和4重PCR产物,达到两管同时扩增9个结核分枝杆菌耐药相关基因的目的。利用该条件可以扩增出全部培养出的135株菌株DNA样本和80.56%的痰菌DNA样本,并且可以缩短PCR操作时间、降低试剂耗材成本,减少操作失误。结论优化的两管多重PCR法在同时扩增9个结核分枝杆菌耐药相关基因,具有明显的优势,具有良好的实用价值。

Improvement and evaluation of the multiplex PCR system suit for micro-sequencing gene chip in detecting drug resistance of Mycobacterium tuberculosis

Objective To upgrade the multiplex PCR reaction system of related gene for drug resistance of Mycobacterium tuberculosis(M.tb),and to evaluate its advantages and disadvantages.Methods Genome DNA was extracted from M.tb clinical isolates and sputum samples by modified method.The multiplex PCR systems were improved by optimizing the PCR reaction system for amplification of 9 drug resistant fragments,rpoB,katG,mabA/inhA,pncA,embB,gyrA,rpsL,rrs and eis.After amplified fragments were confirmed by sequencing,the success rates of amplification by multiplex PCR were analyzed.The advantages and shortages of the established multiplex PCR were analyzed on the operating hours and cost.Results Those 9 drug resistant gene fragments were successfully amplified in two multiplex PCR systems by optimizing the annealing temperature,primer concentrations and supplementing NH4+ and dimethyl sulfoxide.The success rates of amplification were 100% for DNA from 135 cultured strains and 80.56% for the 36 genome DNA form the sputum.This improved multiplex PCR method could shorten the operation time,reduce the cost,and minus the operation errors.Conclusion The optimized two-tube multiplex PCR method has obvious advantages in amplifying 9 drug resistance-related genes of M.tb,which shows high practical value.