Ⅰ群禽腺病毒套式PCR检测方法的建立及应用

中国畜牧兽医 ›› 2013, Vol. 40 ›› Issue (8): 30-33.

Ⅰ群禽腺病毒套式PCR检测方法的建立及应用

索南卓玛, 增巴

青海省海南州同德县畜牧兽医工作站, 青海同德 813200

收稿日期:2012-12-24 出版日期:2013-08-20 发布日期:2013-08-16
作者简介:索南卓玛(1972- ),女,青海人,学士,兽医师,研究方向:畜牧兽医。

Establishment and Application of a Nested PCR Assay for Detection of Fowl Adenovirus Group Ⅰ

SUO NAN Zhuo-ma, ZENG Ba

The Station of Animal Husbandry and Veterinary in Tongde County Hainan Prefecture of Qinghai Province, Tongde 813200, China

Received:2012-12-24 Online:2013-08-20 Published:2013-08-16

摘要/Abstract

摘要： 根据Ⅰ群禽腺病毒(fowl adenovirus Ⅰ,FAVⅠ)六邻体(hexon)基因序列,设计合成2对引物,建立了适合FAVⅠ快速检测的套式PCR方法(nested PCR)。应用该方法对FAVⅠ阳性毒株及临床病料进行检测,2步PCR扩增片段大小分别为475和237 bp,而其他7种禽源病毒的扩增结果均为阴性;该套式PCR方法扩增的敏感性为100 pg和1 fg,敏感性提高了10⁵倍;所建立的FAVⅠ套式PCR方法大大提高了常规PCR的特异性和敏感性,重复性好,可以检测出极低含量的FAVⅠ,可用于FAVⅠ的临床诊断和分子流行病学调查等。

关键词: Ⅰ群禽腺病毒; 六邻体基因; 套式PCR; 检测

Abstract: According to the sequence of hexon gene of fowl adenovirus groupⅠ(FAVⅠ) strain published in GenBank,two pairs of primers were designed and synthesized.The outer primers amplified a fragment of 475 bp in length, and the inner primers amplification fragment was 237 bp in length. A nested PCR assay for rapid detection of FAVⅠ was established.A specific 237 bp fragment was amplified from DNA templates of FAVⅠstrain,but no bands were amplified with templates extracted respectively from avian influenza virus (AIV) subtype H9,Newcastle disease virus (NDV), infectious bursal disease virus (IBDV),duck plague virus (DPV), reticuloendotheliosis (REV), avian reovirus (ARV), Marek's disease virus (MDV). Sensitivity of the 1st and 2nd amplifications by the nested PCR assay were 100 pg and 1 fg,respectively.The sensitivite of the 2nd amplifications increased by 10⁵ times.The results showed that the nested PCR was specific,sensitive,rapid,accurate,and could be used as a routine assay for the detection of FAVⅠ.This method had good reproducibility, specificity and sensitivity, and might detect low content FAVⅠ accurately and rapidly. This method could be used as a method for the diagnosis and detection of clinical cases,and molecular epidemiological investigation of FAVⅠ.

Key words: fowl adenovirus groupⅠ; hexon gene; nested PCR; detection

中图分类号:

索南卓玛, 增巴. Ⅰ群禽腺病毒套式PCR检测方法的建立及应用[J]. 中国畜牧兽医, 2013, 40(8): 30-33.

SUO NAN Zhuo-ma, ZENG Ba. Establishment and Application of a Nested PCR Assay for Detection of Fowl Adenovirus Group Ⅰ[J]. , 2013, 40(8): 30-33.

参考文献

1 王素华.腺病毒载体及其在基因治疗中的应用[J]. 中国畜牧兽医,2009,36(9):119~121.

2 何秀苗,张科,秦爱建,等.Ⅰ群禽腺病毒江苏分离株(FAVⅠ-JS)的鉴定[J].中国预防兽医学报,2005(1):42~45.

3 罗思思,谢芝勋,邓显文,等.Ⅰ群禽腺病毒五邻体基因的克隆及原核表达[J].广东农业科学,2011,38(7):154~157.

4 殷震,刘景华.动物病毒学[M].第2版.北京:科学出版社,1997.

5 谢芝勋,Khan M I.应用PCR检测腺病毒[J].中国兽医学报,2000,20(4):332~334.

6 Corredor J C,Garceac A,Krell P J,et al.Sequence comparison of the right end of fowl adenovirus genomes[J].Virus Genes,2008,36(2):331~344.

7 Domanska-Blicharz K,Tomczyk G,Smietanka K,et al.Molecular characterization of fowl adenoviruses isolated from chickens with gizzard erosions[J].Poult Sci,2011,90(5):983~989.

8 Domermuth C H,Weston C R,Cowen B S,et al.Incidence and distribution of avian adenovirus group splenomegaly of chickens[J].Avian Dis,1980,24(3):591~599.

9 Hess M.Detection and differentiation of avian adenovirus:A review[J].Avian Pathol,2000,29(3):195~206.

10 Kim J N,Byun S H,Kim M J,et al.Outbreaks of hydropericardium syndrome and molecular characterization of Korean fowl adenoviral isolates[J].Avian Dis,2008,52(3):526~530.

11 Pichla-Gollon S L,Drinker M,Zhou X Y,et al.Structure-based identification of a major neutralizing site in an adenovirus hexon[J].J Viro1,2007,81(4):1680~1689.

12 Raj G D,Ramapraba S,Matheswaran K,et al.Comparison of hemagglutination inhibition test and ELISA in quantification of antibodies to egg drop syndrome virus[J].Acta Virol,2004,48(3):183~187.

13 Saifuddin M,Wilks C R.Development of an enzyme linked imnmnosorbent assay to detect and quantify adenovirus in chicken tissues[J].Avian Dis,1990,34(2):239~245.

14 Sentles-Cue C G,Wills R W,Stayer P A.Epidemiology and effect on production parameters of an outbreak of inclusion body hepatitis in broilers[J].Avian Dis,2010,54(1):74~78.

15 Xie Z X,Fadi A A,Girshick T,et al.Detection of avian adenovirus by polymerase chain reaction[J].Avian Dis,1999,43(1):98~105.

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