摘要
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.
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 eases 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 x 103 copies per milliliter for EVT1, CA16 and EV. It showed 100% specificity for 9 enterovirus and 3 non-enterovirus. Analysis with 1.0 x 103- 1.0 x 105 copies per milliliter constructed plasmids demonstrated high reproducibility with coefficient of variation of 0. 44% -1.04% for EV71,0. 38% -0. 73~/o for CA16 ,and 0. 46% -0.90% for E~. More over 176 samples collected from children with suspected HFMD were detected by triplex Taqman probes real-time RT- PCR and real-time RT-PCR. The results showed 97.2% （171/176） agreement and 0. 94 Kappa value withhigh 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.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2013年第9期845-849,共5页
Chinese Journal of Laboratory Medicine
基金
南京市医学科技发展资金资助项目（南京市卫生青年人才培养工程QRX11323）
关键词
手足口病
肠道病毒A型
人
聚合酶链反应
Hand, foot and mouth disease
Enterovirus A, human
Polymerase chain reaction
