[1]陈勉乔,于浩洋,卢秋梅,等.EV71-CA16肠道病毒荧光定量RT-PCR诊断试剂盒的研制[J].现代检验医学杂志,2015,30(06):27-31.[doi:10.3969/j.issn.1671-7414.2015.06.008]
　CHEN Mian-qiao,YU Hao-yang,LU Qiu-mei,et al.Development of EV71-CA16 Enterovirus Fluorescence Qualitative RT-PCR Diagnostic Kit[J].Journal of Modern Laboratory Medicine,2015,30(06):27-31.[doi:10.3969/j.issn.1671-7414.2015.06.008]
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EV71-CA16肠道病毒荧光定量RT-PCR诊断试剂盒的研制()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第30卷
期数:
2015年06期
页码:
27-31
栏目:
论著
出版日期:
2015-12-30

文章信息/Info

Title:
Development of EV71-CA16 Enterovirus Fluorescence Qualitative RT-PCR Diagnostic Kit
作者:
陈勉乔1; 于浩洋1; 卢秋梅2; 陈立嘉3; 刘晓青4
1.深圳市晋百慧生物有限公司，广东深圳518055； 2.广州市药品检验所，广州510160；3.白云山何济公制药厂，广州510410； 4.汕尾逸挥基金医院，广东汕尾516000
Author(s):
CHEN Mian-qiao1; YU Hao-yang1; LU Qiu-mei2; CHEN Li-jia3; LIU Xiao-qing4
1.Shenzhen GeneBioHealth Co.LTD，Guangdong Shenzhen 518055，China； 2.Guangzhou Institute for Drug Control，Guangzhou 510160，China； 3.Baiyun Mountain He Jigong Pharmaoeutical Factory，Guangzhou 510410，China； 4.Shanwei Yihui Foundation Hospital，Guangdong Shanwei 516000，China
关键词:
荧光定量RT-PCR; EV71; CA16肠道病毒; 快速检测
分类号:
Q503
DOI:
10.3969/j.issn.1671-7414.2015.06.008
文献标志码:
A
摘要:
目的研制一种能同时检测EV71，CA16肠道病毒的二联实时荧光定量PCR检测试剂盒，主要用于EV71，CA16肠道病毒的快速检测和流行病学监测。方法设计特异度的EV71型、CA16型基因的引物和探针，优化实时荧光定量PCR检测体系，并研究产品的灵敏度、精密度、稳定性和检测线性范围；同时对2014年5月份收集的26份样品进行检测。结果试验得到了阳性重组质粒，线性范围在5*102 copies/μl～105 copies/μl，该范围内检测结果良好；优化后肠道病毒CA16上下游引物和探针浓度分别为0.48，0.24μmol/L，EV71上下游引物和探针浓度分别为0．40，0.20 μmol/L。敏感度达到500 copies/μl，重复性变异系数CV≤5％；该荧光定量PCR检测试剂盒稳定性强，在-20℃下可以保存一年，对EV71，CA16肠道病毒具有良好的特异度。用该方法检测20份临床阳性样品和6份阴性样品，阳性检出率为100％(20/20)，阴性检出率为100％(6/6)。结论试验建立的EV71，CA16肠道病毒实时荧光定量RT-PCR检测方法可用于EV71，CA16肠道病毒的临床诊断。
Abstract:
ObjectiveA novel real-time RT-PCR kit was developed to detect EV71 and CoxA16 enteroviruses simultaneously，which used for hand，foot and mouth disease in the clinical diagnosis and epidemiological surveillance．MethodsDesigned specific primers and probes of EV71 and CA16，optimized the detection system of real-time quantitative PCR and research the sensitivity，precision，stability of the products and the linear range of detection．Then tested 26 samples which were collected on May 2014．ResultsThe results showed that this experiment obtained positive recombinant plasmid，the range of the linear relation was from 5*102 to 105 copies/μl，and the detection result within this range was fine．The optimal concentrations of CA16 upstream and downstream primers were 0.48 μmol/L and the probes were 0.24 μmol/L，and EV71 upstream and downstream primers were 0.40 μmol/L and the probes were 0.20 μmol/L．The detection sensitivity reached to 500 copies/μl，the CV of the repeatability test was no more than 5％，the fluorescence quantitive PCR Kit was stable and could be saved for one year at -20℃ environment，and this Kit had high specificity for EV71 and CA16 enteroviruses．20 linical positive samples and 6 negative samples were detected in this experiment，detection rate of positive samples was 100% (20/20) and negative samples was 100% (6/6) by fluorescent quantitative PCR detection method．ConclusionThe experiment demonstrated that the detection method of the fluorescent quantitative PCR for EV71 and CA16 enteroviruses could be used for the diagnosis in clinical application．

参考文献/References:

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备注/Memo

备注/Memo:
基金项目：深圳市技术创新计划(CYZZ20140605154742155)。 作者简介：陈勉乔(1983-)，男，硕士研究生，主要从事疾病快速诊断试剂盒的研发，Tel：15914069189，E-mail：mianqiaochen@yeah.net。 通讯作者：于浩洋(1971-)，男，德籍博士，高级工程师，主要从事医学分子学研究，E-mail：guixuanyu@hotmail.com。
更新日期/Last Update: 1900-01-01