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  

Application analysis of multiplex real-time PCR assays for detection of pathogenic bacterium in peritoneal dialysis-associated peritonitis

作　　者：燕雯雯 沈继录[2] 李晓峰 吴永贵[1] 

机构地区：[1]安徽医科大学第一附属医院肾内科,合肥230022 [2]Dept of Clinical Laboratory,The First Affiliated Hospital of Anhui Medical University,hefei230022

出　　处：《安徽医科大学学报》2016年第5期712-717,共6页Acta Universitatis Medicinalis Anhui

基　　金：安徽省自然科学基金资助(编号:1408085MH183)

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6~9h内完成,多重RT-PCR仅需3h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.

关 键 词：腹膜透析相关性腹膜炎 致病菌 实时荧光PCR 传统培养 

分 类 号：R459.51[医药卫生—治疗学] R372[医药卫生—临床医学]