金黄色葡萄球菌荧光定量PCR检测方法的建立及其在大鼠、小鼠粪便检测中的应用  

Establishment of Fluorescence qPCR Method for Detection of Staphylococcus Aureus and Its Application in Feces Detection of Rats and Mice

作　　者：于灵芝 谢建芸 冯丽萍 魏晓锋 YU Lingzhi;XIE Jianyun;FENG Liping;WEI Xiaofeng(Shanghai Laboratory Animal Research Center,Shanghai 201203,China)

机构地区：[1]上海实验动物研究中心,上海201203

出　　处：《实验动物与比较医学》2023年第5期566-573,共8页Laboratory Animal and Comparative Medicine

基　　金：上海市科技创新行动计划“实验猪病毒多联检测技术的质量控制体系研究”(20140900302);上海市科技计划项目“上海市水生实验动物专业技术服务平台”(22DZ2291200)。

摘　　要：目的建立一种快速灵敏的金黄色葡萄球菌(Staphylococcus aureus)检测方法。方法选择金黄色葡萄球菌特异性基因nuc作为靶基因,设计并合成一对特异性引物和一条TaqMan探针,利用荧光定量PCR技术建立nuc基因的核酸检测方法,并在大鼠、小鼠粪便样本检测中进行应用。结果对金黄色葡萄球菌和其他非金黄色葡萄球菌菌株中提取的DNA进行荧光定量PCR检测,结果显示金黄色葡萄球菌出现特异性扩增曲线,而其他非金黄色葡萄球菌未出现,表明设计的引物和探针对金黄色葡萄球菌检测具有特异性。将提取的金黄色葡萄球菌DNA进行10倍梯度稀释后测定其灵敏度,结果显示最低检出限是10 fg的DNA量,比普通PCR方法高2个数量级。本研究共检测91份样品,有4份来自同一设施的大鼠样品,扩增曲线为典型的S曲线;将该PCR产物测序并进行BLAST比对,该样本的基因序列与金黄色葡萄球菌的基因序列相似度为100%,表明该样本为金黄色葡萄球菌nuc基因核酸阳性,阳性率为4.40%,与细菌培养法的检测结果一致。核酸提取采用全自动核酸纯化仪,从核酸提取到检测结果判定快速,所需时间小于1.5 h。结论建立的以nuc为靶基因鉴定金黄色葡萄球菌的qPCR方法,具有快速、灵敏度高和特异性强的优点,可用于实验动物大鼠、小鼠粪便中金黄色葡萄球菌的检测。Objective To establish a method for rapid and sensitive detection of Staphylococcus aureus.Methods The specific gene nucof Staphylococcusaureuswas selected as the target gene.A pair of specific primers and a TaqMan probe were designed and synthesized according to the published sequence of the nucgene.Establish a nucleic acid detection method for nucgene using fluorescence quantitative PCR technology,and apply it clinically in the detection of fecal samples from rats and mice.Results The DNA extracted from Staphylococcusaureusand other non-Staphylococcusaureusstrains was detected by qPCR.The results showed that Staphylococcusaureus had a specific amplification curve,while other non-Staphylococcusaureus did not,indicating that the designed primers and probes were specific for Staphylococcusaureus.The sensitivity of this method was determined by diluting the DNA of Staphylococcusaureusby 10 times.The results showed that the detection limit of this method was 10 fg DNA,which was 2 orders of magnitude higher than that of ordinary PCR method.A total of 91 clinical samples were detected in this study,of which 4 rat samples from the same facility had a typical S-curve.The PCR products were sequenced and BLAST compared.The gene sequence of this sample was 100%similar to that of Staphylococcusaureus,indicating that the sample was positive for the nucleic acid of Staphylococcus aureus nuc gene,with a positive rate of 4.40%.The result was consistent with that obtained by bacterial culture method.The nucleic acid extraction adopted a full-automatic nucleic acid purification instrument,and the time required from nucleic acid extraction to detection result determination was less than 1.5 h.Conclusion The qPCR method established in this study to identify Staphylococcusaureuswith nucgene as the target gene has the advantages of fast,high sensitivity and specificity,and can be used for the detection of Staphylococcusaureusin feces of rats and mice.

关 键 词：金黄色葡萄球菌 检测方法 荧光定量PCR 粪便 大鼠 小鼠 

分 类 号：Q95-33[生物学—动物学]