水貂阿留申病毒、肠炎病毒与犬瘟热病毒多重PCR检测方法的建立与应用
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摘要
目的水貂阿留申病、病毒性肠炎与犬瘟热并称影响水貂健康的三大疫病,拟建立一种可同时检测三种病毒的多重PCR检测方法。方法针对三种病毒基因保守区分别设计了3对特异性引物,对貂阿留申病毒(ADV)和水貂肠炎病毒(MEV)的DNA模板和犬瘟热病毒(CDV)的RNA模板进行了多重PCR扩增和扩增条件优化。结果 PCR可同时扩增出601 bp(ADV)、205 bp(MEV)和451 bp(CDV)的特异性目的条带,敏感性试验表明最低核酸检出量为ADV每微升2.67×10~4拷贝、MEV每微升3.02×10~4拷贝和CDV每微升1.72×10~5拷贝。临床样品检测结果表明多重PCR和单一PCR检测结果一致。结论建立的多重PCR检测方法可快速地检测ADV、MEV和CDV单一或混合感染的临床样品。
Objective Aleutian disease,mink enteritis and canine distemper are the three major diseases affecting health of mink. This study intends to establish a multiplex PCR assay for simultaneously detecting of these three viruses. Methods According to the conservative sequences reported in Gen Bank,three pairs of specific primers were designed to amplify the DNA templates of Aleutian mink disease parvovirus( ADV),mink enteritis parvovirus( MEV),and RNA templates of canine distemper virus( CDV),and optimized the amplifying conditions. Results The specific objective strips of 601 bp( ADV),205 bp( MEV) and 451 bp( CDV) were amplified simultaneously. The sensitivity test showed that the lowest nucleic acid detection limits were 2. 67 × 10~4 copies per μL for ADV,3. 02 × 10~4 copies per μL for MEV,and 1. 72 × 10~5 copies per μL for CDV. The results of test of the clinical samples showed that the multiple PCR and single PCR assay were consistent. Conclusions The established multiplex PCR assay in this study can be used to rapidly detect the clinical samples of ADV,MEV and CDV single or mixed infections.
Objective Aleutian disease,mink enteritis and canine distemper are the three major diseases affecting health of mink. This study intends to establish a multiplex PCR assay for simultaneously detecting of these three viruses. Methods According to the conservative sequences reported in Gen Bank,three pairs of specific primers were designed to amplify the DNA templates of Aleutian mink disease parvovirus( ADV),mink enteritis parvovirus( MEV),and RNA templates of canine distemper virus( CDV),and optimized the amplifying conditions. Results The specific objective strips of 601 bp( ADV),205 bp( MEV) and 451 bp( CDV) were amplified simultaneously. The sensitivity test showed that the lowest nucleic acid detection limits were 2. 67 × 10~4 copies per μL for ADV,3. 02 × 10~4 copies per μL for MEV,and 1. 72 × 10~5 copies per μL for CDV. The results of test of the clinical samples showed that the multiple PCR and single PCR assay were consistent. Conclusions The established multiplex PCR assay in this study can be used to rapidly detect the clinical samples of ADV,MEV and CDV single or mixed infections.
引文
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[13]尹全,李忆,邓强,等.一种可同步检测3种外源基因的多重PCR方法[J].山西农业科学,2017,17(1):37-40,88.
[14]潘德芹,朱瑞良,李奕芙,等.水貂主要病原的分子生物学鉴定及多重感染的病原分析[J].中国兽医学报,2015,16(2):218-224.
[15]杜雄伟,李叶,胡传伟.检测动物产品中水貂阿留申病毒和犬细小病毒复合PCR方法的建立和应用[J].中国动物检疫,2012,12(8):43-45.
[16]刘飞,张宝存,张晓华,等.对虾6种病毒多重PCR检测方法的建立[J].渔业科学进展,2014,19(1):60-67.
[2]张卓.水貂阿留申病毒分离鉴定及其致病性研究和诊断方法的建立[D].东北农业大学,2015.
[3]钟世勋,迟珊珊,王振,等.山东规模化养殖场毛皮动物多重感染病原学分析[J].中国兽医学报,2014,11:1770-1777.
[4]潘德芹.水貂多重感染的病原学分析及绿脓杆菌松花粉多糖佐剂灭活苗的研制[D].山东农业大学,2014.
[5]Farid AH,Zillig ML,Finley GG,et al.Prevalence of the Aleutian mink disease virus infection in Nova Scotia,Canada[J].Prev Vet Med,2012,106:32-40.
[6]Porter DD,Larsen AE,Porter HG.Aleutian disease of mink[J].Adv Immunol,1980,29:61-86.
[7]王洋,胡博,马凡舒,等.水貂病毒性肠炎研究进展[J].特产研究,2016,8(3):67-71.
[8]Mao Y,Su J,Wang J.Roles of three amino acids of capsid proteins in mink enteritis parvovirus replication[J].Virus Res,2016,222:24-28.
[9]王宇菲,和彦良,孟相秋,等.水貂犬瘟热的流行、症状及预防[J].山东畜牧兽医,2016,11(3):32.
[10]庄金秋,梅建国,王金良,等.水貂犬瘟热病毒LD-1株的分离与鉴定[J].中国畜牧兽医,2014,9(4):226-232.
[11]何亚鹏,张琪,史怀平,等.绵羊痘病毒、山羊痘病毒及羊口疮病毒多重PCR检测方法的建立和应用[J].动物医学进展,2017,19(3):11-15.
[12]王苗苗,张凡庆,王曼,等.猪五种繁殖障碍性疫病病原体多重PCR的建立及初步应用[J].动物医学进展,2017,22(3):27-31.
[13]尹全,李忆,邓强,等.一种可同步检测3种外源基因的多重PCR方法[J].山西农业科学,2017,17(1):37-40,88.
[14]潘德芹,朱瑞良,李奕芙,等.水貂主要病原的分子生物学鉴定及多重感染的病原分析[J].中国兽医学报,2015,16(2):218-224.
[15]杜雄伟,李叶,胡传伟.检测动物产品中水貂阿留申病毒和犬细小病毒复合PCR方法的建立和应用[J].中国动物检疫,2012,12(8):43-45.
[16]刘飞,张宝存,张晓华,等.对虾6种病毒多重PCR检测方法的建立[J].渔业科学进展,2014,19(1):60-67.