H3N2亚型犬流感病毒RT-LAMP快速检测方法的建立及应用
|
摘要
为了快速诊断H3N2亚型犬流感病毒(CIV)感染,根据H3N2亚型CIV的M和HA基因序列,设计了3组LAMP引物,优化反应条件,建立了H3N2亚型CIV的RT-LAMP检测方法。结果表明,RT-LAMP方法能在63℃恒温条件下,在45min内完成扩增,通过反应管中预加的荧光染料颜色的变化实现目视化判定。该方法对犬的常见病原无交叉反应,具有较好特异性;灵敏度是常规RT-PCR方法的100倍。该方法操作简单、快速灵敏、特异性强,适合现场应用,对H3N2亚型CIV的检测具有实际的应用价值。
To diagnose H3N2 Canine Influenza Virus(CIV)rapidly,according to the high conservitive sequences of the M and HA genes of H3N2 CIV,three sets of primers were designed.A rapid visable RTLAMP were developped by optimizing the reaction system and conditions.The results showed that the optimzing reaction temperature and time of CIV RT-LAMP were 63℃and 45 minutes,respectively.RT-LAMP amplification products could be visually inspected according to the colour changing of the fluorescent dyes previously added in.Moreover,that no cross-reactivity was found to other common pathogenic microorganisms proved it's good specificity.The sensitivity of this method was 100 times higher than that of a standard RT-PCR.The above-mentioned results showed that the developed RT-LAMP in this study was more convenient,rapid,highly sensitive and specific than traditional methods.It is more suitable for field testing and has good pactical application value for diagnosing H3N2 CIV.
To diagnose H3N2 Canine Influenza Virus(CIV)rapidly,according to the high conservitive sequences of the M and HA genes of H3N2 CIV,three sets of primers were designed.A rapid visable RTLAMP were developped by optimizing the reaction system and conditions.The results showed that the optimzing reaction temperature and time of CIV RT-LAMP were 63℃and 45 minutes,respectively.RT-LAMP amplification products could be visually inspected according to the colour changing of the fluorescent dyes previously added in.Moreover,that no cross-reactivity was found to other common pathogenic microorganisms proved it's good specificity.The sensitivity of this method was 100 times higher than that of a standard RT-PCR.The above-mentioned results showed that the developed RT-LAMP in this study was more convenient,rapid,highly sensitive and specific than traditional methods.It is more suitable for field testing and has good pactical application value for diagnosing H3N2 CIV.
引文
[1]Li S J,Shi Z H,Jiao P R,et al.Avian-origin H3N2canine influenza A viruses in Southern China[J].Infect Genet Evol,2010,10(8):1286-1288.
[2]陈樱,莫彦宁,周华波,等.类人H3N2犬流感病毒广西分离株的鉴定及其遗传演化分析[J].中国预防兽医学报,2015,37(5):401-404.
[3]于志君,朱晓文,刘红,等.A型流感病毒跨种传播和致病性相关蛋白研究进展[J].动物医学进展,2011,32(11):90-94.
[4]Song D,Kang B,Lee C,et al.Transmission of avian influenza virus(H3N2)to dogs[J].Emerg Infect Dis,2008,14(5):741-746.
[5]Su S,Chen Y,Zhao F R,et al.Avian-origin H3N2canine influenza virus circulating in farmed dogs in Guangdong,China[J].Infect Genet Evol,2013,19:251-256.
[6]秦海斌,张汇东.犬流感研究进展[J].动物医学进展,2010,31(6):96-99.
[7]Lyoo K S,Kim J K,Kang B,et al.Comparative analysis of virulence of a novel,avian-origin H3N2canine influenza virus in various host species[J].Virus Res,2015,195(2):135-140.
[8]段旭彤,荣广玉,田巧珍,等.H3N2亚型犬流感病毒反向遗传操作系统的建立[J].中国动物传染病学报,2017,25(3):7-11.
[9]唐连飞,禹思宇,周宇.数字RT-PCT技术检测H3N2亚型猪流感病毒方法的建立[J].中国畜牧兽医,2017,44(6):1847-1853.
[10]Sun Y,Shen Y,Zhang X,et al.A serological survey of canine H3N2,pandemic H1N1/09and human seasonal H3N2influenza viruses in dogs in China[J].Vet Microbiol,2014,168(1):193-196.
[11]Moon H,Hong M,Kim J K,et al.H3N2canine influenza virus with the matrix gene from the pandemic A/H1N1virus:infection dynamics in dogs and ferrets[J].Epidemiol Infect,2015,143(4):772-780.
[12]Na W,Lyoo K S,Song E J,et al.Viral dominance of reassortants between canine influenza H3N2and pandemic(2009)H1N1viruses from a naturally co-infected dog[J].Virology,2015,12:134.
[13]Hong M,Na W,Yeom M,et al.Complete genome sequences of H3N2canine influenza virus with the matrix gene from the pandemic A/H1N1virus[J].GenomeAnnounc,2014,2(5):1010-1014.
[14]Song D,Moon H J,An D J,et al.A novel reassortant canine H3N1influenza virus between pandemic H1N1and canine H3N2influenza viruses in Korea[J].J Gen Virol,2012,93(3):551-554.
[15]Ozawa M,Kawaok Y.Cross talk between animal and human influenza viruses[J].Anim Biosci,2013,1(1):21-42.
[16]宋晶伟,王晨曦,张谞霄,等.北美H3N8亚型犬流感病毒双RT-PCR检测方法的建立[J].中国兽医杂志,2017,53(8):26-31.
[17]高晓宇,曹荣峰,张桂红,等.犬流感病毒RT-LAMP快速检测方法的建立[J].中国预防兽医学报,2010,32(1):36-39.
[18]姜睿姣,邬旭龙,张鹏飞,等.非洲马瘟病毒RT-LAMP检测方法的建立[J].动物医学进展,2017,38(12):1-5.
[19]梅晨,李淑芳,徐玉智,等.副鸡禽杆菌LAMP检测方法的建立[J].动物医学进展,2018,39(1):47-51.
[20]周杨,万强,蔡芷荷,等.环介导等温扩增技术在食品中痢疾志贺氏菌快速检测[J].微生物学通报,2017,44(9):2247-2254.
[21]罗思思,谢芝勋,谢丽基,等.H7亚型和N9亚型禽流感病毒RT-LAMP可视化检测方法的建立[J].畜牧兽医学报,2015,46(7):1176-1183.
[2]陈樱,莫彦宁,周华波,等.类人H3N2犬流感病毒广西分离株的鉴定及其遗传演化分析[J].中国预防兽医学报,2015,37(5):401-404.
[3]于志君,朱晓文,刘红,等.A型流感病毒跨种传播和致病性相关蛋白研究进展[J].动物医学进展,2011,32(11):90-94.
[4]Song D,Kang B,Lee C,et al.Transmission of avian influenza virus(H3N2)to dogs[J].Emerg Infect Dis,2008,14(5):741-746.
[5]Su S,Chen Y,Zhao F R,et al.Avian-origin H3N2canine influenza virus circulating in farmed dogs in Guangdong,China[J].Infect Genet Evol,2013,19:251-256.
[6]秦海斌,张汇东.犬流感研究进展[J].动物医学进展,2010,31(6):96-99.
[7]Lyoo K S,Kim J K,Kang B,et al.Comparative analysis of virulence of a novel,avian-origin H3N2canine influenza virus in various host species[J].Virus Res,2015,195(2):135-140.
[8]段旭彤,荣广玉,田巧珍,等.H3N2亚型犬流感病毒反向遗传操作系统的建立[J].中国动物传染病学报,2017,25(3):7-11.
[9]唐连飞,禹思宇,周宇.数字RT-PCT技术检测H3N2亚型猪流感病毒方法的建立[J].中国畜牧兽医,2017,44(6):1847-1853.
[10]Sun Y,Shen Y,Zhang X,et al.A serological survey of canine H3N2,pandemic H1N1/09and human seasonal H3N2influenza viruses in dogs in China[J].Vet Microbiol,2014,168(1):193-196.
[11]Moon H,Hong M,Kim J K,et al.H3N2canine influenza virus with the matrix gene from the pandemic A/H1N1virus:infection dynamics in dogs and ferrets[J].Epidemiol Infect,2015,143(4):772-780.
[12]Na W,Lyoo K S,Song E J,et al.Viral dominance of reassortants between canine influenza H3N2and pandemic(2009)H1N1viruses from a naturally co-infected dog[J].Virology,2015,12:134.
[13]Hong M,Na W,Yeom M,et al.Complete genome sequences of H3N2canine influenza virus with the matrix gene from the pandemic A/H1N1virus[J].GenomeAnnounc,2014,2(5):1010-1014.
[14]Song D,Moon H J,An D J,et al.A novel reassortant canine H3N1influenza virus between pandemic H1N1and canine H3N2influenza viruses in Korea[J].J Gen Virol,2012,93(3):551-554.
[15]Ozawa M,Kawaok Y.Cross talk between animal and human influenza viruses[J].Anim Biosci,2013,1(1):21-42.
[16]宋晶伟,王晨曦,张谞霄,等.北美H3N8亚型犬流感病毒双RT-PCR检测方法的建立[J].中国兽医杂志,2017,53(8):26-31.
[17]高晓宇,曹荣峰,张桂红,等.犬流感病毒RT-LAMP快速检测方法的建立[J].中国预防兽医学报,2010,32(1):36-39.
[18]姜睿姣,邬旭龙,张鹏飞,等.非洲马瘟病毒RT-LAMP检测方法的建立[J].动物医学进展,2017,38(12):1-5.
[19]梅晨,李淑芳,徐玉智,等.副鸡禽杆菌LAMP检测方法的建立[J].动物医学进展,2018,39(1):47-51.
[20]周杨,万强,蔡芷荷,等.环介导等温扩增技术在食品中痢疾志贺氏菌快速检测[J].微生物学通报,2017,44(9):2247-2254.
[21]罗思思,谢芝勋,谢丽基,等.H7亚型和N9亚型禽流感病毒RT-LAMP可视化检测方法的建立[J].畜牧兽医学报,2015,46(7):1176-1183.