多重实时PCR检测腹膜透析相关性腹膜炎致病菌的应用分析

摘要

目的：探讨多重实时PCR( RT-PCR)检测腹膜透析相关性腹膜炎( PDAP)致病菌的应用价值。方法收集70例患者的腹透液标本,分别行传统细菌培养与荧光染料法细菌通用引物RT-PCR检测。根据70例样本培养结果及相关参考文献,选取最常见的6种PDAP致病菌作为检测菌,设计各检测菌特异引物,分别采用单一及多重荧光探针法 RT-PCR,针对通用RT-PCR法检出的阳性样本进行检测。结果70例标本中传统培养46例阳性,阳性率为65．71%；通用RT-PCR法57例阳性,阳性率为81．43%,两种检测方法阳性率差异有统计学意义( P<0．05)。在6种常见致病菌检测中,对通用RT-PCR法检出的57例阳性样本行单一及多重RT-PCR检测,两者均检出38例样本在6种检测菌范围内,两种方法检测结果一致,1例RT-PCR结果与培养结果不符,剩余19例菌种在6种检测菌范围以外。通用 RT-PCR可在4~6h内明确是否存在细菌感染；针对6种致病菌的检测,单一RT-PCR可在6~9 h内完成,多重RT-PCR仅需3 h即可明确菌种。结论与传统培养法比较, RT-PCR方法有较高灵敏性和特异性,多重RT-PCR因可同时检测多种致病菌,较单一RT-PCR更加迅速、简便、经济。%Objective To estimate the clinical value of bacterial detection in peritoneal dialysis-associated peritoni-tis( PDAP) by multiplex real-time polymerase chain reaction ( RT-PCR ) . Methods Seventy peritoneal dialysate specimens were collected, conventional bacterial culture and SYBR Green RT-PCR detection of the bacterial uni-versal primers were used respectively. According to references and the bacterial culture results of these 70 speci-mens, six common bacteria of PDAP were selected in this assay,multiplex and monoplex RT-PCR were used re-spectively to exam the positive specimens by SYBR Green RT-PCR detection. Results The positive rate of tradi-tional culture among the 70 cases was 65. 71%. The SYBR Green RT-PCR detection results showed that 70 speci-mens total positive rate was 81. 43%, and there was statistical difference between the two methods(P<0. 05). In the detection of these 6 common pathogenic bacteria, both multiplex and monoplex RT-PCR assays found 38 cases of positive samples among the 57 specimens detected by SYBR Green RT-PCR. The results of the two methods were completely identical. One of the positive samples examined by RT-PCR was different form classical bacterial cul-ture, and the remaining 19 cases failed to clear strains of pathogenic bacteria. SYBR Green PCR for detecting path-ogenic bacteria could show results in 4~6 hours, and in the experiments of the 6 common bacteria, monoplex RT-PCR could be finished in 6~9 hours,but multiplex RT-PCR just needed at most 3 hours. Conclusion Compared with the traditional culture method, all of the three RT-PCR assays are sensitive, specific, and more rapidly. The multiplex RT-PCR can detect several kinds of bacteria simultaneously, be more practical,convenient and economi-cal than that of the monoplex RT-PCR.