CAJ | 학술논문

目的建立多重Taqman探针实时RT-PCR法同步检测肠道病毒71型(EV71)、柯萨奇病毒A16型(CA16)和包括EV71、CA16的肠道病毒通用型(EV).方法回顾性研究.根据EV71、CA16和EV的保守序列,设计并合成相应的引物和探针,对该体系进行灵敏度、特异性和重复性验证.收集2012年4至7月南京医科大学附属南京儿童医院收治的176例疑似手足口病患儿咽拭子标本作为实验组,另收集10名在此期间到我院体检的健康儿童、10例门诊流感样患儿和90例我院呼吸科收治的呼吸系统疾病患儿咽拭子标本作为对照组.应用该方法对以上286份标本进行EV71/CA16/EV的同步检测.应用SPSS 13.0统计软件进行统计分析.结果该方法EV71、CA16和EV3个通道的检测限均为1.0×103拷贝/ml；对9株肠道病毒和3株非肠道病毒毒株核酸的检测结果均正确,特异度100％；对1.0×103拷贝/ml、1.0× 104拷贝/ml、1.0×105拷贝/ml的重组质粒进行重复性实验,其变异系数分别为0.44％～ 1.04％,0.38％～0.73％,0.46％～0.90％；运用多重Taqman探针实时RT-PCR法与实时RT-PCR法对176例疑似手足口病患儿临床标本进行检测和结果比对,两种方法的一致性为97.2％ (171/176),Kappa=0.94.结论建立了同步检测EV71/CA16/EV的多重Taqman探针实时RT-PCR法,该方法灵敏度高,特异性好,可为临床快速诊断EV71、CA16和EV感染提供依据,有较强的临床及科研应用前景.
목적 건립다중Taqman탐침실시RT-PCR법동보검측장도병독71형(EV71)、가살기병독A16형(CA16)화포괄EV71、CA16적장도병독통용형(EV).방법 회고성연구.근거EV71、CA16화EV적보수서렬,설계병합성상응적인물화탐침,대해체계진행령민도、특이성화중복성험증.수집2012년4지7월남경의과대학부속남경인동의원수치적176례의사수족구병환인인식자표본작위실험조,령수집10명재차기간도아원체검적건강인동、10례문진류감양환인화90례아원호흡과수치적호흡계통질병환인인식자표본작위대조조.응용해방법대이상286빈표본진행EV71/CA16/EV적동보검측.응용SPSS 13.0통계연건진행통계분석.결과 해방법EV71、CA16화EV3개통도적검측한균위1.0×103고패/ml；대9주장도병독화3주비장도병독독주핵산적검측결과균정학,특이도100％；대1.0×103고패/ml、1.0× 104고패/ml、1.0×105고패/ml적중조질립진행중복성실험,기변이계수분별위0.44％～ 1.04％,0.38％～0.73％,0.46％～0.90％；운용다중Taqman탐침실시RT-PCR법여실시RT-PCR법대176례의사수족구병환인림상표본진행검측화결과비대,량충방법적일치성위97.2％ (171/176),Kappa=0.94.결론 건립료동보검측EV71/CA16/EV적다중Taqman탐침실시RT-PCR법,해방법령민도고,특이성호,가위림상쾌속진단EV71、CA16화EV감염제공의거,유교강적림상급과연응용전경.
Objective To establish the triplex Taqman probes real-time RT-PCR method for simultaneously detecting of EV71,CA16 and EV.Methods Retrospective study.Specific primers and probes were designed based on conserved regions of EV71,CA16 and EV.The sensitivity,specificity and reproducibility were assessed by the optimized reaction system.A total of 176 throat swabs as the experimental group were collected from children with suspected hand foot mouth disease (HFMD),who admitted from April 2012 to July 2012 in Nanjing Children's Hospital affiliated to Nanjing Medical University.During this time,10 cases of healthy children,10 cases of outpatients with flu-like symptoms and 90 cases of inpatients in pneumology department of our hospital were recruited as control group,whose throat swabs were also collected.All of 286 samples were tested by the triplex Taqman probes real-time RT-PCR for simultaneously detecting EV71,CA16 and EV.SPSS13.0 was used to analyze the results.Results The sensitivities of the triplex Taqman probes real-time RT-PCR was 1.0 × 103 copies per milliliter for EV71,CA16 and EV.It showed 100％ specificity for 9 enterovirus and 3 non-enterovirus.Analysis with 1.0 × 103-1.0 × 105 copies per milliliter constructed plasmids demonstrated high reproducibility with coefficient of variation of 0.44％-1.04％ for EV71,0.38％-0.73％ for CA16,and 0.46％-0.90％ for EV.More over 176 samples collected from children with suspected HFMD were detected by triplex Taqman probes real-time RTPCR and real-time RT-PCR.The results showed 97.2％ (171/176)agreement and 0.94 Kappa value with high concordance.Conclusions The triplex Taqman probes real-time RT-PCR detecting EV71,CA16 and EV simultaneously has been established successfully.The assay,with high sensitivity and specificity,provide good basis for the rapid clinical diagnosis of EV71,CA16 and EV and open up broad prospects for clinical and relevant researches.