qPCR快速检测金黄色葡萄球菌及MRSA方法的建立及评价

目的建立q PCR法快速检测金黄色葡萄球菌及其mec A基因,以期快速精准诊断金黄色葡萄球菌感染及初步判断耐药情况。方法从NCBI数据库下载金黄色葡萄球菌nuc、atl、ica B、fnb A、hla、srap基因序列用于鉴定标志筛选,mec A基因序列用于MRSA标志筛选;经DNA MAN比对后,选择各基因保守区分别设计1~2套引物、探针,建立单重及双重q PCR,用临床分离株及标准菌株筛选出检测性能最好的基因片段作为检测标志物,建立金黄色葡萄球菌鉴定和耐药双重q PCR检测方法,并进行性能评价。结果经筛选,金黄色葡萄球菌atl基因(CP009361.1:1010217~1010341)、mec A基因(KF058908.1:1715~1843)两片段检测性能最优,将其作为标志物建立双重q PCR法。该方法的检测下限均低至4 copies/反应;扩增线性范围均达2.0×102~8copies/m L。335份金黄色葡萄球菌(含94份MRSA)培养阳性的患者样本中,q PCR法分别检出SA 335份,MRSA 94份;95份金黄色葡萄球菌培养阴性的患者样本中,q PCR法分别检出SA 17份,MRSA 4份,经PCR产物测序,与标准菌株同源性均≥90%。双重q PCR法从样品处理到报告结果≤2.0 h。结论 q PCR法方法简便、快速、灵敏度高、特异性好,可望提高金黄色葡萄球菌感染的诊断能力并实现快速检测,为尽早精准治疗赢得时间。

Establishment and evaluation of a quantitative real time PCR assay for rapid detection of Staphylococcus aureus and methicillin resistant Staphy lococcus aureus

ObjectiveTo establish a quantitative real time polymerase chain reaction（qPCR） assay for rapid detection of Staphylococcus aureus (SA) and its mecA gene, rapidly and accurately diagnose SA infection as well as preliminarily determine its drug resistance.MethodsSequences of nuc, atl, icaB, fnbA, hla, and srap genes of SA were downloaded from NCBI database for screening of markers, and sequence of mecA gene was used for screening of methicillin resistant SA (MRSA); sequence alignment was conducted by DNA MAN, conserved region of each gene sequence was employed for designing primers and fluorogenic probes. Simplex and duplex qPCR were established, and gene sequences with the best detection performance were screened by clinical and standard strains as markers, a duplex qPCR system for identifying SA and drug resistance was developed and evaluated.ResultsThe atl gene（CP009361.1：1010217-1010341）and mecA gene （KF058908.1：1715-1843）possessed the best detection performance, and were used as detection markers in the duplex qPCR system. qPCR system could amplify both targets in a range of 2.0×102 8 copies/mL with a strong linear relationship and lower detection limits for both targets reached 4 copies/PCR. Among 335 positive culture specimens for SA (including 94 MRSA) from patients, 335 and 94 specimens were detected SA and MRSA by duplex qPCR respectively; among 95 negative culture specimens for SA from patients, 17 and 4 specimens were detected SA and MRSA by duplex qPCR respectively, PCR products were sequenced, the homology with standard strain were all≥90%. Time from specimen processing to result reporting was≤2.0 hours by duplex qPCR method.ConclusionqPCR method is simple, rapid, sensitive, and specific, it’s a promising way to improve the diagnostic efficacy of SA infection and achieve the rapid detection, which contributes to the early precision treatmen