犬Ⅰ型腺病毒荧光定量PCR检测方法的建立
|
摘要
通过分析对比GenBank公布的几株犬Ⅰ型腺病毒序列,针对犬Ⅰ型腺病毒特有的E3区,设计了1对特异性引物。对引物浓度、退火温度、扩增体系等条件优化后,建立了犬Ⅰ型腺病毒荧光定量PCR检测方法,并对40份CAV-Ⅰ人工感染银黑狐的粪便样品进行了检测。结果表明,与普通PCR相比,建立的犬Ⅰ型腺病毒荧光定量PCR检测方法具有特异性好、灵敏度高、重复性强的特点。该检测方法可用于Ⅰ型犬腺病毒的快速诊断与监测,并能够定量分析CAV-Ⅰ的感染程度。
In order to establish a real-time fluorescent quantitative PCR assay for rapid detection of canine adenovirus typeⅠ(CAV-Ⅰ),the special primers were designed for E3 conserved region based on the sequences of several strains of canine adenovirus type Ⅰin GenBank,and the concentration of primer,Tm value and volume of amplification were optimized.We used this established assay to detect the CAV-Ⅰin forty fecal samples from artificially infected silver foxes.Compared to the general PCR assay,results showed that the real-time fluorescent quantitative PCR assay for CAV-Ⅰis more specific,highly sensitive and repeatable.Therefore,it could be applied for rapid detection,monitor and quantitative analysis of canine adenovirus typeⅠ.
In order to establish a real-time fluorescent quantitative PCR assay for rapid detection of canine adenovirus typeⅠ(CAV-Ⅰ),the special primers were designed for E3 conserved region based on the sequences of several strains of canine adenovirus type Ⅰin GenBank,and the concentration of primer,Tm value and volume of amplification were optimized.We used this established assay to detect the CAV-Ⅰin forty fecal samples from artificially infected silver foxes.Compared to the general PCR assay,results showed that the real-time fluorescent quantitative PCR assay for CAV-Ⅰis more specific,highly sensitive and repeatable.Therefore,it could be applied for rapid detection,monitor and quantitative analysis of canine adenovirus typeⅠ.
引文
[1]殷震,刘景华.动物病毒学[M].2版.北京:科学出版社,1997.
[2]王东.犬Ⅰ型腺病毒强毒株的分离与鉴定及其对犬的实验感染初步研究[P].2009.
[3]郑志福.狐狸脑炎诊断与防治[J].中国畜禽种业,2017,13(3):31-33.
[4]钟志宏,夏咸柱,赵奕,等.狐狸脑炎病毒的分离鉴定和流行病学调查[J].兽医大学学报,1990,10(2):111-119.
[5]罗国良,闫喜军,钟伟.狐狸传染性脑炎病原学研究进展[J].动物医学进展,2008,29(8):63-66.
[6]罗国良,柴秀丽,闫喜军,等.犬腺病毒间接免疫荧光检测方法的建立[J].特产研究,2008,30(1):10-15.
[7]王洋.水貂肠炎细小病毒在体内分布规律及相关细胞因子的变化[P].2015.
[8]李安,谢金文,魏加贵,等.荧光定量PCR技术在分子检测上的研究进展[J].中国畜牧兽医,2009,36(4):73-75.
[9]于永乐,张传美,杨海燕,等.水貂肠炎病毒SYBR GreenⅠ荧光定量PCR检测方法的建立[J].兽类学报,2015,35(1):102-109.
[10]郭洪梅.狐源犬Ⅰ型腺病毒分离鉴定及其母源抗体消长规律测定[P].2007.
[11]Dowgier G,Lahoreau J,Lanave G,et al.Sequential circulation of canine adenoviruses 1and 2in captive wild carnivores,France[J].Vet Microbiol,2018,221:67-73.
[12]Balboni A,Dondi F,Agnoli C,et al.Novel sequence variants of viral hexon and fibre genes in two dogs with canine adenovirus type 1-associated disease[J].Vet J,2017,223:73-75.
[2]王东.犬Ⅰ型腺病毒强毒株的分离与鉴定及其对犬的实验感染初步研究[P].2009.
[3]郑志福.狐狸脑炎诊断与防治[J].中国畜禽种业,2017,13(3):31-33.
[4]钟志宏,夏咸柱,赵奕,等.狐狸脑炎病毒的分离鉴定和流行病学调查[J].兽医大学学报,1990,10(2):111-119.
[5]罗国良,闫喜军,钟伟.狐狸传染性脑炎病原学研究进展[J].动物医学进展,2008,29(8):63-66.
[6]罗国良,柴秀丽,闫喜军,等.犬腺病毒间接免疫荧光检测方法的建立[J].特产研究,2008,30(1):10-15.
[7]王洋.水貂肠炎细小病毒在体内分布规律及相关细胞因子的变化[P].2015.
[8]李安,谢金文,魏加贵,等.荧光定量PCR技术在分子检测上的研究进展[J].中国畜牧兽医,2009,36(4):73-75.
[9]于永乐,张传美,杨海燕,等.水貂肠炎病毒SYBR GreenⅠ荧光定量PCR检测方法的建立[J].兽类学报,2015,35(1):102-109.
[10]郭洪梅.狐源犬Ⅰ型腺病毒分离鉴定及其母源抗体消长规律测定[P].2007.
[11]Dowgier G,Lahoreau J,Lanave G,et al.Sequential circulation of canine adenoviruses 1and 2in captive wild carnivores,France[J].Vet Microbiol,2018,221:67-73.
[12]Balboni A,Dondi F,Agnoli C,et al.Novel sequence variants of viral hexon and fibre genes in two dogs with canine adenovirus type 1-associated disease[J].Vet J,2017,223:73-75.