半巢式PCR和PCR-RFLP法诊断耐青霉素肺炎链球菌的比较

Comparison of seminested-PCR and PCR-RFLP for detection of penicillin-resistant Streptococcus pneumoniae isolates

目的评价半巢式PCR和PCR-RFLP法用于诊断肺炎链球菌青霉素耐药性的价值。方法收集130株肺炎链球菌临床分离株和50株健康人定植株,琼脂稀释法测定青霉素最低抑菌浓度(MIC),半巢式PCR、PCR-RFLP法测定对青霉素耐药性。结果健康人定植株均对青霉素敏感,临床分离株青霉素敏感率、中介率、耐药率分别为25.4%、23.1%和51.5%;当MIC为0.25～0.5μg/ml时,半巢式PCR法检测青霉素耐药性的敏感性、特异性、阳性预测值和阴性预测值分别为86.7%、98.6%、86.7%和98.6%;当MIC≥1μg/ml时,对应值分别为96.2%、97.6%、97.4%和96.4%。而PCR-RFLP法诊断中介株的敏感性仅34.2%;诊断耐药株的阳性预测值仅67.5%。结论半巢式PCR法优于PCR-RFLP法,可用于对耐青霉素肺炎链球菌的快速检测;PCR-RFLP敏感性不高,可用于分子流行病学分析。

Objective To find out a rapid and reliable method to detect penicillin-resistant Streptococcus pneumoniae（PRSP） and its drug-resistance,and evaluate the application of seminested-PCR and PCR-RFLP for this assay.Methods One hundred and thirty clinical S.pneumoniae isolates and 50 isolates from healthy carriers were collected in Nanjing between March 2005 and June 2007.The minimum inhibitory concentration（MIC） to penicillin was measured by agar dilution test.The genome of S.pneumoniae was extracted and amplified by seminested-PCR and PCR-RFLP to detect the resistance to penicillin.Results All isolates from healthy carriers were susceptible to penicillin.Among the clinical isolates,the susceptible,intermediate and resistant rates to penicillin were 25.4%,23.1% and 51.5% respectively.When MIC were between 0.25-0.5 μg/ml,the sensitivity,specificity,positive and negative predictive values in seminested-PCR assay were 86.7%,98.6%,86.7% and 98.6% respectively,while they were 96.2%,97.6%,97.4% and 96.4% when MIC were greater than or equal to 1 μg/ml.By PCR-RFLP the sensitivity for intermediate isolates was 34.2% and the positive predictive values was 67.5%,thus only susceptible isolates were detectable.Conclusion For rapid diagnosis of PRSP,seminested-PCR should be better than PCR-RFLP which may be applied in epidemiological analysis for its poor sensitivity.