PCR-SSCP检测耐多药结核菌基因与细菌培养结果对比分析

目的应用PCR-SSCP检测常规及BACTEC培养耐INH、RFP、SM的耐多药结核分枝杆菌临床分离株KatG、rpoB、rpsL基因突变,分析其敏感性和特异性,评价其在检测结核分枝杆菌耐药性方面的价值和临床实用性。方法22株常规培养和8株BACTEC培养检出的高浓度及低浓度耐INH、RFP、SM耐多药结核分枝杆菌分离株,分别用PCR-SSCP检测KatG、rpoB、rpsL基因,观察其电泳条带,并与结核菌标准株H37RV对比。结果22株常规培养耐INH、RFP、SM的耐多药结核分枝杆菌分离株中,KatG、rpoB、rpsLPCR扩增产物,用SSCP分别检出KatG基因16株(72.7%)、rpoB基因19株(86.4%)和rpsL基因14株(63.6%)。8株BACTEC培养耐INH、RFP、SM的耐多药结核分枝杆菌分离株中分别检出KatG基因5株(62.5%),rpoB基因6株(75%)、rpsL基因5株(62.5%)。综合两者KatG、rpoB和rpsL基因检出率分别为70.0%(21/30)、83.3%(25/30)和63.3%(19/30)。高浓度耐药与低浓度耐药分离株中的检出率有显著差异(P<0.05),常规培养与BACTEC培养分离株中的检出率无显著差异(P>0.05),与结核菌标准株H37RV电泳条带对比,特异性为100%,敏感性97%。结论PCR-SSCP敏感、特异,可快速检测结核分枝杆菌的KatG、rpoB和rpsL基因,可用于耐多药结核分枝杆菌的临床检测。

A Comparative Study on the Detection of Multi-drug Resistant Mycobacteria by PCR-SSCP and BACTEC System

Objective To determine the mutation of multi-drug resistant loci KatG,rpoB and rpsL by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), and the drug resistance (INH, RFP and SM) of clinical isolates of Mycobacterium tuberculosis (MTB) in BACTEC culture. The aim is to evaluate these two methods for rapid detection of drug resistant mycobacteria. Methods 22 isolates obtained from conventional culture medium and 8 drug-resistant isolates obtained from BACTEC culture were used in this study. PCR-SSCP was used to determine the mutation in KatG, rpoB and rpsL loci. The mycobacterium H37RV strain was used for comparison. Results Among the 22 INH-, RFP- and SM-resistant isolates, KatG, rpoB and rpsL genes were detected in 16 (72.7%), 19 (86.4%), and 14 (63.6%) isolates, respectively. For the 8 isolates obtained from BACTEC culture, KatG, rpoB and rpsL genes were detected in 5 (62.5%), 6 (75%) and 5 (62.5%) isolates, respectively. Altogether, the detection rate for KatG, rpoB and rpsL genes were 70.0%?83.3% and 63.3%, respectively. Difference in the detection rate was seen between the groups of highly drug-resistant mycobacteria and the group of less drug-resistant mycobacteria (P<0.05). No difference in the detection rate between the group of mycobacteria obtained from conventional culture medium and from the BACTEC culture. When compared with H37RV, SSCP is highly specific (100%) and sensitive (97%). Conclusion PCR-SSCP is a rapid, sensitive and specific method for the detection of KatG,rpoB and rpsL genes. It can be used to detect multi-drug resistant Mycobacterium tuberculosis.