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预防医学  2016, Vol. 28 Issue (3): 217-220    
  论著 本期目录 | 过刊浏览 | 高级检索 |
EV71、CA16和肠道病毒通用型三重荧光RT-PCR检测技术的建立
徐昌平1, 2, 郑春红1, 严菊英2, 葛琼2, 龚黎明2, 郭江峰1
1.浙江理工大学生命科学学院,浙江 杭州 310018;
2.浙江省疾病预防控制中心
A study on the development of a triplex real-time RT-PCR for simultaneous detection of enteroviruses and differentiation of EV 71 and CA 16
XU Chang-ping, ZHENG Chun-hong, YAN Ju-ying, GE Qiong, GONG Li-ming, GUO Jiang-feng
College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, Zhejiang, 310018, China
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摘要 目的 建立一种可以同时检测EV71、CA16和肠道病毒通用型的三重荧光RT-PCR检测方法,为手足口病监测与应急诊断提供高通量快速检测技术。方法 分别在VP1和5’UTR基因保守区设计EV71、CA16和肠道病毒通用型的特异性检测引物以及Taqman探针,建立并评估三重荧光RT-PCR技术的特异性和敏感性,并对91份疑似手足口病患儿临床标本进行核酸检测与效果验证。结果 建立的三重荧光RT-PCR技术可同时对EV71、CA16和肠道病毒通用型进行特异性检测区分,敏感性均可达到0.1 TCID50/mL,从核酸提取至完成检测仅需2~3 h。91份疑似手足口病患儿临床标本采用三重荧光RT-PCR方法检测,EV71、CA16和其他型别肠道病毒的检出率分别为30.77%、9.89%和5.49%;荧光RT-PCR方法的检出率高于培养分离法。结论 建立的三重荧光RT-PCR技术能同时准确分型EV71、CA16和其他型别肠道病毒。
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徐昌平
郑春红
严菊英
葛琼
龚黎明
郭江峰
关键词 手足口病EV71CA16荧光RT-PCR    
AbstractObjective To develop a multiplex real-time RT-PCR assay for simultaneous detection of enteroviruses and differentiation of EV71 and CA16. Methods Specific primers and probes were designed for enteroviruses,EV71 and CA16.The probes labeled with various fluorescent reporter dyes, and a triplex real-time RT-PCR technique was developed to simultaneously detect these viruses. A total of 91 clinical specimens with suspected HFMD were analyzed by this method. Results This assay could simultaneously detect enterovirus and differentiation of EV71 and CA16,and the sensitivity of the assay was up to 0.1 TCID50/mL, and only need 2 to 3 hours for completing the detection. A total of 91 clinical specimens were detected by this assay in 28 of the 91(30.77%) specimens contained EV71, 9 of the 91(9.89%) contained CA16,and 5 of the 91 (5.49%) contained other enteroviruses. Conclusion This assay would be a useful molecular diagnostic tool for large-scale screening of clinical samples, especially at the peroid of HFMD outbreaks.
Key wordsHand    foot and mouth disease    EV 71    CA 16    Real-time RT-PCR
收稿日期: 2015-10-19      出版日期: 2017-12-07
ZTFLH:  R725.1  
基金资助:浙江省医学重点学科群建设项目(XKQ-009-003)
通信作者: 郭江峰,E-mail:jfguo@zstu.edu.cn   
作者简介: 徐昌平,硕士,主管技师,主要从事微生物检验工作
引用本文:   
徐昌平, 郑春红, 严菊英, 葛琼, 龚黎明, 郭江峰. EV71、CA16和肠道病毒通用型三重荧光RT-PCR检测技术的建立[J]. 预防医学, 2016, 28(3): 217-220.
XU Chang-ping, ZHENG Chun-hong, YAN Ju-ying, GE Qiong, GONG Li-ming, GUO Jiang-feng. A study on the development of a triplex real-time RT-PCR for simultaneous detection of enteroviruses and differentiation of EV 71 and CA 16. Preventive Medicine, 2016, 28(3): 217-220.
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http://www.zjyfyxzz.com/CN/      或      http://www.zjyfyxzz.com/CN/Y2016/V28/I3/217
[1] LIU Y, WANG X, SUN D, et al. Detecting spatial-temporal clusters of HFMD from 2007 to 2011 in Shandong Province, China[J]. PLoS One, 2013, 8(5):e63447.
[2] DWIBEDI B, KAR B R, KAR S K.Hand, foot and mouth disease (HFMD): a newly emerging infection in Orissa, India[J]. Natl Med J India, 2010, 23(5):313.
[3] ABUBAKAR S, CHEE H Y, AL-KOBAISI M F, et al. Identification of enterovirus 71 isolates from an outbreak of hand, foot and mouth disease (HFMD) with fatal cases of encephalomyelitis in Malaysia[J]. Virus Res, 1999, 61(1):1-9.
[4] 顾红岩,刘志达,张玲,等.2013年北京地区手足口病住院患儿的病原分布及临床特点[J].中华儿科杂志, 2015, 53(6):459-463.
[5] 周俊,吴亦栋,陈晓玲,等.2013年杭州市手足口病患儿及其接触人群流行病学调查[J].浙江大学学报(医学版),2014,3(2): 212-217.
[6] 杨智宏,朱启镕,李秀珠,等. 2002年上海儿童手足口病病例中肠道病毒71型和柯萨奇病毒A组16型的调查[J].中华儿科杂志, 2005, 43(9):648-652.
[7] CUI A, XU C, TAN X, et al. The development and application of the two real-time RT-PCR assays to detect the pathogen of HFMD[J]. PLoS One, 2013, 8(4):e61451.
[8] LI W, YI L, SU J, et al. Seroprevalence of human enterovirus 71 and coxsackievirus A16 in Guangdong, China, in pre- and post-2010 HFMD epidemic period[J]. PLoS One, 2013, 8(12):e80515.
[9] 刘兰芬,张国平,张俊棉,等.河北省2008年手足口病病毒的分离和鉴定[J].中国卫生检验杂志, 2009,19(4): 837-838.
[10] 骆玲飞,王小光,刘继倩,等.疑似重症手足口病病原学检测及流行病学分析[J].中国卫生检验杂志,2011,21(4):971-972.
[11] 许建明,段向英,张念之,等.三重实时荧光RT-PCR检测三种鱼类弹状病毒的研究[J].检验检疫科学,2009,19(1):5-10.
[12] 梁晓艳,万东山,郭兰英.多重荧光RT-PCR技术检测猪流感病毒、猪乙型脑炎病毒、猪瘟病毒、猪繁殖与呼吸综合征病毒方法的建立[J].中国动物检疫,2015,32(4):68-71.
[13] 谢席平,黄莲珍,刘让万,等.检测柯萨奇病毒A组6、10、16型的多重荧光RT-PCR建立及应用[J].临床检验杂志,2014,32(10):721-725.
[14] WANG T, LIU J H, ZHANG J, et al. A multiplex allele-specific real-time PCR assay for screening of ESR1 mutations in metastatic breast cancer[J]. Exp Mol Pathol, 2015, 98(2):152-157.
[15] SINGH P,MUSTAPHA A.Multiplex real-time PCR assays for detection of eight shiga toxin-producing escherichia coli in food samples by melting curve analysis[J]. Int J Food Microbiol, 2015, 215(12):101-108.
[16] DUITAMA J, KUMAR D M, HEMPHILL E, et al. Primer hunter: a primer design tool for PCR-based virus subtype identification[J]. Nucleic Acids Res, 2009, 37(8):2483-2492.
[17] XIAO X L, HE Y Q, YU Y G, et al. Simultaneous detection of human enterovirus 71 and coxsackievirus A16 in clinical specimens by multiplex real-time PCR with an internal amplification control[J]. Arch Virol, 2009, 154(1):121-125.
[18] 倪楠,侯佩强,张荣强,等.肠道病毒、EV71及CoxA16多重PCR检测方法的研究[J].中国病原生物学杂志,2013, 8(5):439-441.
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