[1]许先凯,邱鸿杰,卢秋梅.基于MGB探针法荧光PCR检测手足口病病原的研究[J].现代检验医学杂志,2018,33(01):124-127.[doi:10.3969/j.issn.1671-7414.2018.01.001]
　XU Xian-kai,QIU Hong-jie,LU Qiu-mei.Development of Fluorescence PCR for Detection of Hand Foot Mouth Viruses with MGB Probe[J].Journal of Modern Laboratory Medicine,2018,33(01):124-127.[doi:10.3969/j.issn.1671-7414.2018.01.001]
点击复制

基于MGB探针法荧光PCR检测手足口病病原的研究()

《现代检验医学杂志》[ISSN:/CN:]

卷:
第33卷
期数:
2018年01期
页码:
124-127
栏目:
论著
出版日期:
2018-02-05

文章信息/Info

Title:
Development of Fluorescence PCR for Detection of Hand Foot Mouth Viruses with MGB Probe
文章编号:
1671-7414(2018)01-124-04
作者:
许先凯1,邱鸿杰1,卢秋梅2
1.汕头市龙湖区动物防疫监督所,广东汕头 515041; 2.广州市药品检验所,广州 510160
Author(s):
XU Xian-kai1,QIU Hong-jie1,LU Qiu-mei2
1.Animal Prevention Inspection of Longhu District,Guangdong Shantou 515041,China; 2.Guangzhou Institute for Drug Control,Guangzhou 510160,China
关键词:
MGB探针;  荧光PCR;  手足口病病原
分类号:
Q503; R373.2
DOI:
10.3969/j.issn.1671-7414.2018.01.001
文献标志码:
A
摘要:
目的 建立一种能同时检测EV71型、CA16型及其它肠道病毒的荧光PCR检测方法,主要用于手足口病病原的快速检测。方法 针对肠道病毒的保守基因设计特异性引物和探针序列,优化荧光RT-PCR反应体系,构建标准的阳性质控模板,建立标准曲线,研究产品的检测限、重复性、特异性; 同时对2015年6月份收集的26份阳性样品和10份阴性样品进行检测。结果 试验得到了阳性重组质粒,线性范围在8×102 copies/μl～8×108 copies/μl范围内检测结果良好; 优化后肠道病毒EVUN上下游引物和MGB探针浓度分别为0.50,0.50,0.30 μmol/L,RT-PCR反应条件为:42℃30 min,95℃3 min; 95℃5 s,60℃35 s,45个循环。检测限达到800 copies/μl,变异系数CV≤5%,重复性好,特异性良好,与其他传染病毒无交叉反应; 用该检测方法检测26份临床阳性样本和10份阴性样本,阳性检出率为100%(26/26),阴性检出率为100%(10/10)。结论 试验所建立的荧光PCR检测方法,可用于临床对手足口病病原的快速检测。
Abstract:
Abstract:Objective A fluorescence PCR methods was developed to detect EV71 and CoxAl6 and other enteroviruses simultaneously,which usedfor hand,foot and mouth disease(HFMD)viruses in the clinical rapid diagnosis.Methods Designed specific primers and probes of the enteroviruses gene which represents one of the highly conserved regions of the virus gene,and optimized the detection system of real-time quantitative RT-PCR.The positive control template and the standard curve were constructed.Researchedthe limit of detection,repeatability and specificity of the products,and tested 26 positive samples and 10 negative samples which collected on June 2015.Results The results showed that this experiment obtained positive recombinant plasmid,the range of the linear relation was from 8×102 to8×108 copies/μl,and the detection result within this range was fine.The optimal concentrations of EVUN upstream and downstream primers were 0.50 μmol/Land the MGB probes were 0.30 μmol/L,RT-PCR reaction conditions as follows:42℃ 30 min,95℃ 3 min.95℃ 5 s,60℃ 35 s,45 cycle.The limit of detection reached to 800 copies/μl.The CV of the repeatability assay was no more than5%.Specificity was good,and no cross reaction with other infectious viruses.26 clinical positive samples and 10 negative samples were detected in this experiment,detection rate of positive samples was 100%(26/26)and negative samples was 100%(10/10)by fluorescent quantitative RT-PCR detection method.Conclusion The experiment demonstrated that the detection methodof the fluorescent quantitative PCR for hand-foot-mouth viruses could be usedfor the rapid diagnosis in clinical application.

参考文献/References:

[1] Hwang S,Kang B,Hong J,et al.Development of duplex real-timeRT-PCR based on Taqman technology for detecting simultaneously the genome of pan-enterovirus and enterovirus 71[J].Journal of Medical Virology,2013,85(7):1274-1279.
[2] 解 娟,袁 军,张 艳,等.血清淀粉样蛋白A(SAA)在儿童手足口病中的临床应用[J].现代检验医学杂志,2016,31(6):102-104. Xie J,Yuan J,Zhang Y,et al.Clinical application of serum amyloida(SAA)in children with hand foot and mouth disease[J].Journal of Modern Laboratory Medicine,2016,31(6):102-104.
[3] Sun LL,Wang JK,Cui XQ,et al.Association of viral replication capacitywith the pathogenicity of enterovirus 71[J].Virus Res,2014(189):1-7.
[4] Yan XF,Gao S,Xia JF,et al.Epidemic characteristics of hand,foot,andmouth disease in Shanghai from 2009 to 2010:Enterovirus 71 subgenotype C4 as the primary causative agent and a high incidence of mixed infections with coxsackievirus A16[J].Scandinavian Journal of Infectious Diseases,2012,44(4):297-305.
[5] Yi EJ,Shin YJ,Kim JH,et al.Enterovirus 71 infection and vaccines[J].Clinical and Experimental Vaccine Research,2017,6(1):4-14.
[6] Zhang LJ,Huang GL,Cai QX,et al.Optimize the interactions at S4 with efficient inhibitors targeting 3C proteinase from enterovirus 71[J].Journal ofMolecular Recognition,2016,29(11):520-527.
[7] Zhang YJ,Mao HY,Yan JY,et al.Development of no-vel all Glo-probe-based one-step multiplex qRT-PCR assay for rapid identification of avian influenza virus H7N9[J].Arch Virol,2014,159(7):1707-1713.
[8] 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(6):73-76. Chen MQ,Yu HY,Lu QM,et al.Development of EV71-CA16 enterovirusfluorescence qualitative RT-PCR diagnostic kit[J].Journal of Modern Laboratory Medicine,2015,30(6):73-76.
[9] Yang TC,Xu GZ,Dong HJ,et al.A case-control study of risk factors forsevere hand foot mouth disease among children in Ningbo,China,2010-2011[J].European Journal of Pediatrics,2012,171(9):1359-1364.
[10] Xing W,Liao Q,Viboud C,et al.Hand,foot and mouth disease inChina,2008-12:an epidemiological study[J].Lancet Infect Dis,2014,14(4):308-318.
[11] 于浩洋,陈勉乔,邵 曼.用于检测肠道病毒的核酸及其应用.中国,201410251945.0[P].食品伙伴网知识产权服务中心,2015-05-06. Chen HY,Chen MQ,Shao M.Application for detection of enteroviruses nucleic acid:China,201410251945.0[P].Foodmate IPr service Center,2015-05-06.
[12] Wang Y,Zou G,Xia AM,et al.Enterovirus 71 infection in children withhand,foot,and mouth disease in Shanghai,China:epidemiology,clinical featureand diagnosis[J].Virology Journal,2015,12(1):83.
[13] Ben-Chetrit E,Wiener-Well Y,Shulman LM,et al.Coxsackievirus A6-related hand foot and mouth disease: skin manifestations in a cluster of adult patients[J].J Clin Virol,2014,59(3):201-203. 收稿日期:2017-08-22 修回日期:2017-10-10

相似文献/References:

[1]陈 蕾,刘志忠,贾淑芬,等.双管单色荧光PCR法与基因芯片法检测CYP2C19基因多态性的比较研究[J].现代检验医学杂志,2016,31(04):51.[doi:10.3969/j.issn.16717-414.2016.04.013]
　CHEN Lei,LIU Zhi-zhong,JIA Shu-fen,et al.Comparing Study of Double Tube and One-Color Fluorescent PCR Technology and Gene Chip Method in Detecting the CYP2C19 Gene Polymorphisms[J].Journal of Modern Laboratory Medicine,2016,31(01):51.[doi:10.3969/j.issn.16717-414.2016.04.013]

备注/Memo

备注/Memo:
作者简介:许先凯(1984-),男,大学本科,主要从事动物疫病预防控制工作,E-mail:kesen066@163.com。 通讯作者:卢秋梅(1988-),女,大学本科,主要从事药物医学的检验工作,E-mail:670747691@qq.com。
更新日期/Last Update: 2018-02-07