基于中部同序引物的多重PCR联合核酸质谱分析技术检测常见血流感染病原菌

Diagnosis of common blood stream infection pathogens based on central homo-sequence primer by multiplex PCR combined with MALDI-TOF MS

目的基于多重聚合酶链式反应(PCR)与基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)联用的高通量检测技术,构建不同菌种的特征性单核苷酸多态性图谱以建立血流感染病原菌快速、准确、高灵敏的诊断方法。方法选取血流感染常见的大肠埃希菌等7种病原菌作为检测对象,优化多重PCR条件,采用MALDI-TOF MS检测各目标菌特征峰,建立多重PCR联合质谱检测体系;分别设计普通引物对及中部同序引物对,分析引物二聚体的形成情况;采用模拟的细菌感染血液样本,测定上述建立体系的特异度及灵敏度;收集2020年6—9月解放军总医院检验科疑似菌血症患者血液样本150份,并将上述体系的鉴定结果与临床应用的传统鉴定方法结果使用χ²检验进行比较。结果本研究设计的中部同序引物对的引物二聚体循环阈值(Ct)值在38以上,比普通引物对延迟出现6~10个循环;建立的联合质谱检测体系可同时检测分为两组的7种细菌,目标菌均可检测到特异的产物峰,除目标菌外的临床菌株只有引物峰,所有图谱无非特异的杂峰;大肠埃希菌的灵敏度可以达到50 CFU/ml,其余各菌的检测限为100 CFU/ml;对150例患者的血液样本进行检测,传统方法鉴定出阳性46例,阳性检出率为30.67%(46/150),包括2例混合感染,联合质谱方法鉴定出阳性48例,阳性检出率为32.0%(48/150),包括3例混合感染;阴性符合率为100%(101/101),核酸质谱敏感度为97.82%(45/46),特异度为97.11%(101/104),一致性检验Kappa=0.938(P=0.625),2种方法检测一致性良好。结论所建立的检测体系不仅能快速、准确地鉴定引起血流感染的7种常见病原菌,有效缩短传统培养鉴定所需时间,还可检测多重细菌混合感染,弥补混合感染漏检可能。该方法也可用于其他病原菌的鉴定。

Objective Based on the high-throughput detection technique of multiplex PCR combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,constructing the characteristic SNP profiles of different strains,and establishing a rapid,accurate and highly sensitive method for the diagnosis of bloodstream infection pathogens.Methods Seven kinds of pathogens such as common Escherichia coli were selected as target.The multiple PCR reaction conditions was optimized,and the characteristic peaks of each target bacteria were detected by MALDI-TOF MS to establish the joint detect system.Common primer pairs and central homo-sequence primer pairs were designed to analyse the formation of primer dimer.Using simulated bacterial infection blood samples with detection system to determine specificity and sensitivity.One hundred and fifty blood samples from suspected bacteremia patients were collected from June to September 2020 in a hospital in Beijing,and the identification results were compared to traditional identification method of clinical application that are usingχ2 test.Results The cycle threshold(Ct)value of the central homo-sequence primers that were designed were more than 38,with a delay of 6-10 cycles.The joint mass spectrometry detection system could detect seven kinds of bacteria divided into two groups at the same time.The target bacteria can be detected specific product of the peak,and the clinical strains other than the target strains only had primer peaks.All maps had non-specific miscellaneous peaks.The sensitivity of Escherichia coli could reach 50 CFU/ml,and the detection limit of other bacteria was 100 CFU/ml.The detection results of 150 patients showed that 46 cases were positive by traditional method.The positive rate was 30.67%(46/150),including two cases of mixed infection.Forty-eight cases were positive by mass spectrometry,and the positive rate was 32.0%(48/150),including three cases of mixed infections.The negative coincidence rate was 100%(101/101).The comparison of the two methods showed that the P=0.625>0.01,the Kappa=0.938,the sensitivity and specificity was 97.82%(45/46)and 97.11%(101/104),respectively.There was no significant difference between the two methods,and the results of nucleic acid mass spectrometry could also be used in clinic.Conclusions The established detection system can not only quickly and accurately detect seven common pathogens causing bloodstream infection,and effectively shorten the time needed for traditional culture and identification,but also can detect multiple bacterial mixed infections at the same time to make up for the possibility of missed detection.Besides,the method can also be used to identify other bacteria.