摘要
目的 建立金黄色葡萄球菌、肺炎克雷伯杆菌、绿脓假单胞菌三种条件性致病菌的多重荧光定量 PCR(qPCR)检测方法.方法 选取金黄色葡萄球菌 nuc基因、肺炎克雷伯杆菌 phoE 基因、绿脓假单胞菌 gyrB 基因设计特异性引物及探针,建立多重 qPCR方法,并测试其特异性、灵敏度、标准曲线及重复性等性能指标.用该方法检测 30 个临床粪便样品、6 个阳性参考品,检测结果与分离培养法结果进行对比,计算符合率.结果 多重 qPCR 检测方法对金黄色葡萄球菌、肺炎克雷伯杆菌、绿脓假单胞菌的标准菌株基因组 DNA 均能稳定检出;对鼠伤寒沙门菌、嗜肺巴斯德杆菌、鼠棒状杆菌、支气管鲍特杆菌等病原微生物项目无非特异性扩增;针对金黄色葡萄球菌、肺炎克雷伯杆菌、绿脓假单胞菌标准菌株基因组 DNA的灵敏度分别为 1.0×10-5、1.0×10-4、1.0×10-5ng/μL;批间差异、批内差异均小于 2%,重复性良好;临床样品和阳性参考品的检测结果显示多重 qPCR 法与分离培养法的检测结果一致.结论 本研究成功建立了一种可同时、快速检测金黄色葡萄球菌、肺炎克雷伯杆菌、绿脓假单胞菌的多重qPCR检测方法.
Abstract
Objective A multiplex qPCR(Fluorescence quantitative PCR,qPCR)test method for three conditional pathogens,Staphylococcus aureus,Klebsiella pneumoniae,and Pseudomonas aeruginosa,was established.Method The nuc gene of Staphylococcus aureus,the phoE gene of Klebsiella pneumoniae,and the gyrB gene of Pseudomonas aeruginosa were selected to design specific primers and probes,and a multiplex qPCR method was established.Its performance indicators such as specificity,sensitivity,standard curve,and repeatability were tested.This method was used to test 30 clinical stool samples and 6 positive references.The test result were compared with the result of the culture method,and the compliance rate was calculated.Result The multiplex qPCR method can stably test the genomic DNA of standard strains of Staphylococcus aureus,Klebsiella pneumoniae,and Pseudomonas aeruginosa;there is no nonspecific amplification of pathogenic microorganisms such as Salmonella typhimurium,Pasteurella pneumotropica,Corynebacterium kutscheri,and Bordetella bronchiseptica;the sensitivity of the genomic DNA of standard strains of Staphylococcus aureus,Klebsiella pneumoniae,and Pseudomonas aeruginosa is 1.0×10-5,1.0×10-4,and 1.0×10-5 ng/μL,respectively;the inter-batch and intra-batch differences are less than 2%,and the repeatability is good;the test result of clinical samples and positive references show that the test result of the multiplex qPCR method are consistent with those of the isolation and culture method.Conclusion This study successfully established a multiplex qPCR method for simultaneous and rapid detection of Staphylococcus aureus,Klebsiella pneumoniae,and Pseudomonas aeruginosa.
NETL
NSTL
万方数据