犬瘟热病毒原液TaqMan荧光定量RT-PCR检测方法的建立

doi:10.3969/j.issn.1004-1524.2017.11.07

浙江农业学报 ›› 2017, Vol. 29 ›› Issue (11): 1819-1826.DOI: 10.3969/j.issn.1004-1524.2017.11.07

犬瘟热病毒原液TaqMan荧光定量RT-PCR检测方法的建立

魏斌¹, 田一男¹, 李平², 石梅³, 曹雪峰¹, 肖启程¹, 涂蕊¹, 但佳明¹, 杨亭玉¹, 彭广能¹, 钟志军^{1, *}

1.四川农业大学动物医学院动物疫病与人类健康四川省重点实验室,四川成都 611130;
2.邛崃市农业和林业局,四川邛崃 611530;
3.绵阳市经开区动物疫病预防控制中心,四川绵阳 621000

收稿日期:2017-04-26 出版日期:2017-11-20 发布日期:2017-12-05
通讯作者: 钟志军,E-mail:zhongzhijun488@126.com
作者简介:魏斌(1992—),女,山西平遥人,硕士研究生,从事兽医临床病理学与分子诊断学。E-mail:865360412@qq.com
基金资助:
国家重点研发计划(2016YFD0501009); 国家自然科学基金项目(31000548); 成都大熊猫繁育研究基金会项目(CPF2015-4)

Establishment of TaqMan fluorescent quantitative RT-PCR assay for detection of Canine distemper virus without nucleic acid extraction

WEI Bin¹, TIAN Yi’nan¹, LI Ping², SHI Mei³, CAO Xuefeng¹, XIAO Qicheng¹, TU Rui¹, DAN Jiaming¹, YANG Tingyu¹, PENG Guangneng¹, ZHONG Zhijun^{1, *}

1. Key Laboratory of Animal Disease and Human Health of Sichuan Province, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China;
2. Agricultural and Forestry Bureau of Qionglai, Qionglai 611530, China;
3. Center for Disease Control and Prevention of Mianyang, Mianyang 621000, China

Received:2017-04-26 Online:2017-11-20 Published:2017-12-05

摘要/Abstract

摘要： 为建立一种不提取病毒RNA,直接采用TaqMan荧光定量RT-PCR检测样本上清液中犬瘟热病毒的方法。通过扩增CDV的NP基因部分片段构建重组质粒,建立TaqMan荧光定量PCR方法,对所建立的方法进行特异性、敏感性、重复性检测;比较了提取核酸后进行荧光定量RT-PCR与不提取核酸对原液进行荧光定量RT-PCR检测的结果,最后对疑似CDV病料进行检测。结果显示,建立的CDV质粒TaqMan荧光定量PCR检测灵敏度比普通PCR灵敏度高1 000倍。将核酸提取液和病毒原液稀释后用该研究建立的方法进行检测,最小检测稀释倍数分别为10⁷和10⁵,表明提取核酸后样本中的有效cDNA浓度比直接使用病毒原液检测高100倍。特异性和重复性检测结果表明,该方法特异性良好且重复性较高。对97份临床病料进行检测,建立的方法共检出60份阳性,阳性检出率高于普通RT-PCR和胶体金快速检测试纸板,提取核酸法与原液法的一致率为100%。标准曲线分析表明,当病毒含量高于10²拷贝·μL^-1时,与普通RT-PCR以及胶体金快速检测试纸板相比,CDV原液TaqMan荧光定量RT-PCR检测技术阳性检出率更高、准确性更好,值得临床推广应用。

关键词: 犬瘟热病毒, 病毒原液, TaqMan荧光定量RT-PCR, 特异性, 重复性

Abstract: To establish a direct TaqMan fluorescent quantitative RT-PCR (direct-qRT-PCR) method without extracting nucleic acid for detecting canine distemper virus(CDV). Specificity, sensitivity and reproducibility of the method were tested by using constructed recombinant plasmid. Fluorescence quantitative RT-PCR was used to compare the detection limitation in both the nucleic acid extraction and the viral stock. Clinical samples were also collected and applied for verification the established method. Our results showed that the sensitivity of this method was 1 000 times higher than ordinary PCR. Nucleic acid extraction and viral stock was detected by the established method, the minimum detectable dilution times were 10⁷ and 10⁵, respectively, indicated that the effective cDNA concentration in the sample after nucleic acid extraction was 100 times higher than that using viral stock to test. Specificity and reproducibility results showed that the method had good specificity and reproducibility. 97 clinical samples were detected by this method, 60 samples showed positive. The detectable rate of this method was higher than those of ordinary RT-PCR and colloidal gold rapid detection plate. The positive detectable rate using nucleic acid extraction was perfectly matched with that using viral stock. Compared with ordinary RT-PCR and colloidal gold rapid detection plate, standard curve showed direct-qRT-PCR had high accuracy and positive detectable rate while the virus concentration was higher than 10² copies·μL^-1, and it was worthy of clinical application.

Key words: canine distemper virus, viral stock, TaqMan quantitative RT-PCR, specificity, reproducibility

中图分类号:

S855.3

魏斌, 田一男, 李平, 石梅, 曹雪峰, 肖启程, 涂蕊, 但佳明, 杨亭玉, 彭广能, 钟志军. 犬瘟热病毒原液TaqMan荧光定量RT-PCR检测方法的建立[J]. 浙江农业学报, 2017, 29(11): 1819-1826.

WEI Bin, TIAN Yi’nan, LI Ping, SHI Mei, CAO Xuefeng, XIAO Qicheng, TU Rui, DAN Jiaming, YANG Tingyu, PENG Guangneng, ZHONG Zhijun. Establishment of TaqMan fluorescent quantitative RT-PCR assay for detection of Canine distemper virus without nucleic acid extraction[J]. , 2017, 29(11): 1819-1826.

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犬瘟热病毒原液TaqMan荧光定量RT-PCR检测方法的建立

Establishment of TaqMan fluorescent quantitative RT-PCR assay for detection of Canine distemper virus without nucleic acid extraction

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