3种条件性致病菌三重PCR检测方法的建立及初步应用

本研究旨在建立一种能同时检测金黄色葡萄球菌(Staphylococcus aureus,S.aureus)、绿脓杆菌(Pseudomonas aeruginosa,P.aeruginosa)、肺炎克雷伯杆菌(Klebsiella pneumoniae,K.pneumoniae)的三重PCR检测方法.根据金黄色葡萄球菌nuc基因、绿脓杆菌toxR基因、肺炎克雷伯杆菌PhoE基因设计并合成引物,在单一PCR条件基础上优化建立三重PCR反应条件,并进行特异性、敏感性和重复性分析及与细菌分离培养的比对试验.结果显示,3对引物均能特异性扩增出目的条带,大小分别为484、278和368 bp.最佳退火温度在56~59 ℃之间,引物浓度均为0.2 μmol/L,dNTP浓度为200 μmol/L,Mg²⁺浓度为2.5 mmol/L.3种细菌间无交叉反应.对支气管鲍特杆菌等其他8种实验动物常见致病菌均无交叉反应.最低能同时检测到10^-5 ng/μL的细菌基因组DNA.3次重复结果一致,表明建立的三重PCR方法重复性好.同时采用细菌分离培养法和三重PCR方法对30份实验小鼠样本进行检测,对比结果显示三重PCR方法检出率略高于细菌分离培养法,细菌分离培养法呈阳性的样品,三重PCR方法均能检出.结果表明,本试验建立的三重PCR检测方法具有特异、敏感、高效等优点,为实验动物细菌快速检测和流行病学调查提供了技术支持.

Establishment and Preliminary Application of Triple PCR to Identify Three Kinds of Conditional Pathogenic Bacteria

The aim of this study was to establish a simultaneous triple PCR detection method for Staphylococcus aureus (S.aureus),Pseudomonas aeruginosa (P.aeruginosa) and Klebsiella pneumoniae (K.pneumoniae).Three pairs of specific primers had been designed according to nuc gene of S.aureus,toxR gene of P.aeruginosa and PhoE gene of K.pneumoniae.The triple PCR reaction conditions were optimized on the basis of single PCR methods.At the same time,specificity,sensitivity and repeatability tests of the triple PCR method were studied,and the results of bacteria isolation and culture and the triple PCR method were compared.The results showed that the amplification product sizes were 484,278 and 368 bp,respectively.The optimal annealing temperature was 56 to 59 ℃,the concentrations of primers were all 0.2 μmol/L,dNTP concentration was 200 μmol/L,Mg²⁺ concentration was 2.5 mmol/L.The specificity test showed that there was no cross reaction between these three bacteria templates and other eight kinds of common bacteria templates,such as Bordetella bronchiseptica.The minimum of simultaneous detection of three bacteria genomic DNA was 10^-5 ng/μL.The results of three repeatability tests of triple PCR were the same which indicated the repeatability was good.30 samples from mice were detected by bacteria isolation and culture and the triple PCR method,the detection rate of triple PCR was slightly higher than the bacteria isolation and culture.The positive samples detected by bacteria isolation and culture were also positive detected by triple PCR.The results showed that a specific,sensitive and efficient triple PCR system had been established and could provide the technical support for bacteria detection and epidemiological investigation of laboratory animals.