[1]姜一曈,王昭华,鑫 婷,等. 基于重组酶聚合酶扩增技术的犬细小病毒快速检测方法的建立[J].中国预防兽医学报,2018,(10):926-929.[doi:10.3969/j.issn.1008-0589.201809043]
 JIANG Yi-tong,WANG Zhao-hua,XIN Ting,et al. Development of the recombinase polymerase amplification assay for rapid detection of canine parvovirus[J].Chinese journal of preventive veterinary medicine,2018,(10):926-929.[doi:10.3969/j.issn.1008-0589.201809043]
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基于重组酶聚合酶扩增技术的犬细小病毒快速检测方法的建立

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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2018年10
页码:
926-929
栏目:
诊断和检测技术
出版日期:
2018-11-15

文章信息/Info

Title:
 

Development of the recombinase polymerase amplification assay for rapid detection of canine parvovirus

文章编号:
1008-0589(2018)10-0926-04
作者:
 

姜一曈王昭华鑫 婷贾 红郭晓宇朱鸿飞*侯绍华*

 

(中国农业科学院 北京畜牧兽医研究所,北京 100081)

Author(s):
 

JIANG Yi-tong WANG Zhao-hua XIN Ting JIA Hong GUO Xiao-yu ZHU Hong-fei* HOU Shao-hua*

 

(Institute of Animal Sciences of Chinese Academy of Agricultural Sciences, Beijing 100081, China)



关键词:
犬细小病毒重组酶聚合酶扩增技术快速检测
Keywords:
 

canine parvovirus recombinase polymerase amplification rapid detection

分类号:
S852.65
DOI:
10.3969/j.issn.1008-0589.201809043
文献标志码:
A
摘要:
 

为建立一种快速、简便检测犬细小病毒(CPV)的方法,本研究基于CPV VP2基因的保守序列,设计合成引物,通过对反应条件的优化,建立了CPV的重组酶聚合酶(RPA)检测方法。该方法在38 ℃恒温反应15 min即可快速、特异性的检测出CPV。特异性试验结果显示,除CPV (BJ-2b株、BJ-2c株)外,犬瘟热病毒、犬冠状病毒、犬腺病毒、犬副流感病毒等检测均为阴性;敏感性试验结果显示,以重组质粒pEASY-CPV-VP2为模板,RPA的检测下限为1×101拷贝/μL,其敏感度比普通PCR方法高10倍。利用建立的RPA方法对疑似CPV感染的临床样品的阳性检出率为88 %,高于普通PCR的检出率(82 %)和胶体金的检出率(76 %)。本研究建立的CPV RPA检测方法操作简单,反应灵敏,结果确实可靠,适用于CPV的快速检测。

  

Abstract:
 

To establish a rapid and simple detection method for canine parvovirus (CPV), the recombinase polymerase amplification (RPA) assay was developed with the primers designed according to the conserved sequence of CPV VP2 gene. The detection procedure of RPA assay was completed within 15 min at 38℃. The assay was specific for the target sequence (135bp) amplification from CPV, and had no any amplification from other viral pathogens. Based on pEASY-CPV-VP2 as template, the analytical sensitivity of the RPA assay was 101copies/ reaction of a standard DNA template, which was 10 times more sensitive than the common PCR method. The positive detection rate of clinical samples with suspected CPV infection using the established RPA method was 88%, which was significantly higher than the detection rate of conventional PCR (80%) and the detection rate of colloidal gold (76%). The RPA assay established in this study was a simple, rapid, reliable and affordable method that could potentially be applied for the detection of CPV in the research laboratory and on site diagnosis.

参考文献/References:

 

[1]Calderon M G, Romanutti C, D Antuono A, et al. Evolution of canine parvovirus in Argentina between years 2003 and 2010: CPV2c has become the predominant variant affecting the domestic dog population [J]. Virus Res, 2011, 157(1): 106-110.

[2]Perez R, Calleros L, Marandino A, et al. Phylogenetic and genome-wide deep-sequencing analyses of canine parvovirus reveal co-infection with field variants and emergence of a recent recombinant strain [J]. PLoS One, 2014, 9(11): e111779.

[3]Timurkan M, Oguzoglu T. Molecular characterization of canine parvovirus(CPV) infection in dogs in Turkey [J]. Vet Ital, 2015, 51(1): 39-44.

[4]Castro T X, Costa E M, Leite J P, et al. Partial VP2 sequencing of canine parvovirus (CPV) strains circulating in the state of Riode Janeiro, Brazil: detection of the new variant CPV-2c [J]. Braz J Microbiol, 2010, 41(4): 1093-1098.

[5]Decaro N, Desario C, Campolo M, et al. Clinical and virological findings in pups naturally infected by canine parvovirus type 2 Glu-426 mutant [J]. J Vet Diagn Invest, 2005, 17(2): 133- 138.

[6]Decaro N, Buonavoglia C. Canine parvovirus-a review of epidemiological and diagnostic aspects, with emphasis on type 2c [J]. Vet Microbiol, 2012, 155(1): 1-12.

[7]Desario C, Decaro N, Campolo M. Canine parvovirus infection: which diagnostic test for virus? [J]. J Virol Methods, 2005, 126(1-2): 179-185.

(本文编辑:彭永刚)

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更新日期/Last Update: 2018-11-28