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2025年4月5日 星期六
  2016, Vol. 24 Issue (12): 1964-1972    
  研究资源与技术改进 本期目录 | 过刊浏览 | 高级检索 |
鸭呼肠孤病毒基因Ⅰ型与Ⅱ型鉴别诊断RT-PCR方法的建立与应用
云涛,张存,华炯钢,余斌,叶伟成,陈柳,倪征
浙江省农业科学院 畜牧兽医研究所
Establishment and Application of RT-PCR for Identification and Diagnosis of Duck reovirus Genotype Ⅰ and Ⅱ
2, 2, 2, 2, 2, 2
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摘要 临床发病鸭(Anas platyrhynchos domestica)群中存在两种基因型鸭呼肠孤病毒(Duck reovirus, DRV)的感染。为建立一种能鉴别诊断DRVⅠ型(classical DRV, C-DRV)与Ⅱ型(novel DRV, N-DRV)的一步法RT-PCR检测方法,本研究通过对GenBank已公布的现有C-DRV和N-DRV S1和S4基因片段序列进行比对分析,设计合成两对特异性引物,并对引物浓度、退火温度和循环次数等扩增条件进行优化,建立一种能鉴别诊断C-DRV和N-DRV的一步法RT-PCR检测方法。结果表明,一步法RT-PCR最佳的反应体系为:PrimeScript 1 Step Enzyme Mix 1 μL,2×1 Step Buffer 12.5 μL,RNA模板1.5 μL,上下游引物(20 μmol/L)各1 μL, RNase Free dH2O补足25 μL;最佳反应条件:50 ℃ 反转录30 min;94 ℃预变性2 min;94 ℃变性30 s,55 ℃退火30 s,72 ℃延伸30 s,共30个循环。该方法可从C-DRV和N-DRV基因组中扩增出大小分别为249和505 bp单一特异性片段,从两种病毒基因组混合物中可同时扩增出两条特异性片段,而对鸭源常见病毒及正常细胞基因组在同等条件下检测均为阴性;该方法具有较高的敏感性,其最低病毒检出量分别为0.47和0.62个半数组织培养感染剂量(50% tissue culture infective dose, TCID50);对不同代次、不同时间提取的病毒RNA样品重复检验3次,结果均一致。对浙江省2011~2015年采集的239份疑似临床病料进行检测,C-DRV与N-DRV的阳性率分别为2.5%(6/239)和31.4%(75/239);通过对阳性病料P10和P18扩增序列的测序也证实检测结果的准确性。本研究建立的双重一步法RT-PCR可以有效区分两种基因型DRV,且临床样品检测表明N-DRV是浙江省流行的优势基因型。本研究为鉴别诊断C-DRV与N-DRV提供了一个快速、灵敏性高及低成本的实验室诊断方法,该方法的建立可为DRV 的分子流行病毒学调查提供有效的技术支持。
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作者相关文章
云涛
张存
华炯钢
余斌
叶伟成
陈柳
倪征
关键词 鸭呼肠孤病毒(DRV)多重一步法RT-PCR基因型    
Abstract:There are two genotypes (ⅠandⅡ) of Duck reovirus (DRV) in clinical infection ducks (Anas platyrhynchos domestica). To establish a multiplex one-step RT-PCR method for the simultaneous detection and differentiation of DRV genotypeⅠ(classical DRV, C-DRV) and genotype Ⅱ (novel DRV, N-DRV) from clinical samples, two sets of primers were designed for a RT-PCR assay system according to S1 and S4 non-structural protein encoding sequences of two genotypes viruses. The amplification conditions, including primer concentration, annealing temperature and cycle times, were optimized. The results showed that the most effective and precise reaction system was as follow: PrimeScript 1 Step Enzyme Mix 1 μL, 2×1 Step Buffer 12.5 μL, each primer (20 μmol/L) 1 μL, RNA 1.5 μL, adding RNase Free dH2O to a final volume of 25 μL; The optimal reaction procedure was as follow: Reverse transcription for 30 min at 50 ℃; Pre-denaturation for 2 min at 94 ℃; Denaturation temperature for 2 min at 94 ℃, annealing temperature for 30 s at 55 ℃, extension for 30 s at 72 ℃, total of 30 cycles. These genotype-specific primer pairs could amplify 249 and 505 bp PCR products corresponding to C-DRV and N-DRV, respectively, and 2 fragments of 249 and 505 bp were simultaneously amplified from the mixed RNA sample of C-DRV and N-DRV. The assay exhibited high specificity as all negative controls and other duck pathogens, such as the Duck tembusu virus (DTMUV), Duck plague virus (DPV), Newcastle disease virus (NDV), Muscovy duck parvovirus (MDPV), Duck hepatitis A virus (DHAV-1) and H9N2 Avian influenza virus (AIV). The sensitivity of this method was determined to be 0.47 50% tissue culture infective dose (TCID50) for C-DRV and 0.62 TCID50 for N-DRV. Virus RNAs extracted from different generations and different times were detected by the developed multiplex one-step RT-PCR. The result showed that the method was found to be highly reproducible. In addition, RT-PCR assays of DRV diseased fowl samples (total 239) collected from different regions of Zhejiang province during 2011~2015 were taken. The detected results showed that the positive rates of C-DRV and N-DRV were 2.5% (6/239) and 31.4% (75/239), respectively. Six C-DRV positive samples were all Muscovy duck. In 75 N-DRV positive samples, there were 54 Muscovy duck (Cairna moschata) samples (positive rate 72%), 5 goose (Anser anser) samples (positive rate 6.7%) and 16 other breed duck samples (positive rate 21.3%). The results indicated that N-DRV was the currently prevalence genotype in Zhejiang province. P10 and P18 sequence of positive tissues were analyzed to confirm the accuracy of the test results. In addition, the multiplex one-step RT-PCR method provides a rapid, highly sensitive, and cost-effective laboratory diagnosis for C-DRV and N-DRV infections in duck, and the assay can provide effective technical support for molecular epidemiological survey of DRV.
Key wordsDuck reovirus(DRV)    Multiplex one-step RT-PCR    Genotype
收稿日期: 2016-06-03      出版日期: 2016-11-07
基金资助:鸭坦布苏病毒 E 蛋白毒力相关位点的研究
通讯作者: 云涛     E-mail: yt-t@163.com
引用本文:   
云涛 张存 华炯钢 余斌 叶伟成 陈柳 倪征. 鸭呼肠孤病毒基因Ⅰ型与Ⅱ型鉴别诊断RT-PCR方法的建立与应用[J]. , 2016, 24(12): 1964-1972.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2016/V24/I12/1964
 
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