荧光定量PCR检测鹅细小病毒体系的建立和应用
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摘要
为解决在养殖过程中多种疫病混合感染造成鹅细小病毒(Goose parvovirus,GPV)的临床及实验室诊断困难的问题,利用分离得到的GPV GDGZh1设计合成1对特异性引物GPV-rt PCRF/GPV-rt PCRR,建立了GPV SYBR Green I实时荧光定量PCR检测方法,并进行了临床样品及GPV在种鹅体内定植规律的检测.结果表明,该方法对质粒的检测灵敏度为3.5×10~2copies/μL,GPV具有组织广泛嗜性,种鹅的直肠、脾脏、心脏、肾脏和卵巢中病毒含量在攻毒后第5天最高,肝脏中病毒含量在攻毒后第9天最高,心脏和肾脏在攻毒后第17天检测不到病毒,直肠、肝脏、脾脏和卵巢在攻毒后第25天还能检测到病毒.这说明种鹅感染GPV后,病毒可以在其体内长期存在,并不断向外排毒,还能通过垂直传播的方式将病毒传递给雏鹅.
In order to solve the difficult clinical and laboratory diagnosis of Goose parvovirus( GPV)caused by co-infection with various pathogens in the breeding process,one pair of specific primers GPVrt PCRF/GPV-rt PCRR were designed by using GPV( GDGZh1) and a SYBR Green I real-time PCR was established to detect clinical samples and colonization of the virus. The results showed that the sensitivity of method established in this study was high to detect 3. 5 × 10~2 copies/μL gene fragments. The geese were inoculated with goose parvovirus and samples were collected at different times to detect the virus content in different organs by using the real-time PCR method. The result showed that the goose organ in the challenge group had virus colonization. Virus content in the rectum,spleen,heart,kidneys,and ovaries was highest on the 5 th day after challenge,and virus content in the liver was highest on the 9 th day after the challenge. On the 17 th day after challenge,no virus was detected in heart and kidney,however,the virus could be detected in rectum,liver,spleen,and ovary on the 25 th day after the challenge.The result suggested that the GPV could proliferate in goose and continually transmit to other goslings through vertical transmission.
In order to solve the difficult clinical and laboratory diagnosis of Goose parvovirus( GPV)caused by co-infection with various pathogens in the breeding process,one pair of specific primers GPVrt PCRF/GPV-rt PCRR were designed by using GPV( GDGZh1) and a SYBR Green I real-time PCR was established to detect clinical samples and colonization of the virus. The results showed that the sensitivity of method established in this study was high to detect 3. 5 × 10~2 copies/μL gene fragments. The geese were inoculated with goose parvovirus and samples were collected at different times to detect the virus content in different organs by using the real-time PCR method. The result showed that the goose organ in the challenge group had virus colonization. Virus content in the rectum,spleen,heart,kidneys,and ovaries was highest on the 5 th day after challenge,and virus content in the liver was highest on the 9 th day after the challenge. On the 17 th day after challenge,no virus was detected in heart and kidney,however,the virus could be detected in rectum,liver,spleen,and ovary on the 25 th day after the challenge.The result suggested that the GPV could proliferate in goose and continually transmit to other goslings through vertical transmission.
引文
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[14]沈美艳,于海,袁东芳,等.商品肉鸡场细菌超广谱β-内酰胺酶基因的荧光定量PCR检测[J].中国家禽,2017(6):20-23.
[15]刘婷婷,谢芝勋,宋德贵,等.H3和H4亚型禽流感病毒双重实时荧光定量PCR检测方法的建立[J].中国家禽,2015(7):20-24.
[16]刘玲凤,黎振标,司兴奎.鸭圆环病毒实时荧光定量PCR检测方法的建立及初步应用[J].中国家禽,2017(7):15-19.
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[18]WANG J,WANG J,CUI Y,et al.Development of a taqmanbased real-time PCR assay for the rapid and specific detection of novel duck-origin goose parvovirus[J].Mol Cell Prob,2017,34:56-58.
[19]徐楠楠.应用FQ-PCR检测GPV在产蛋种鹅体内定植规律的研究[D].长春:吉林农业大学,2011.
[20]马磊.GPV原位PCR和免疫荧光检测方法的建立及其在雏鹅体内定植研究[D].长春:吉林农业大学,2011.
[2]CALNEK B W.禽病学[M].10版.北京:中国农业大学出版社,1999:988-995.
[3]殷震,刘景华.动物病毒学[M].2版.北京:科学出版社,1997:165-168.
[4]秦爱建.免疫鹅琼脂扩散实验在小鹅瘟诊断中的应用[J].中国预防兽医学报,1993,15(1):30-31.
[5]YANG J,CHEN H,WANG Z,et al.Development of a quantitative loop-mediated isothermal amplification assay for the rapid detection of novel goose parvovirus[J].Front Micr,2017,8:2472.
[6]FAN J,ZOU Y,YANG Z,et al.The development of an indirect ELISA for the detection of antibodies to goose parvovirus in blood serum[J].Lett Appl Micr,2013,57(1):26-32
[7]WANG Q,JU H,LI Y,et al.Development and evaluation of a competitive ELISA using a monoclonal antibody for antibody detection after goose parvovirus virus-like particles(VLPs)and vaccine immunization in goose sera[J].J Virol Meth,2014,209:69-75
[8]LI P,ZHANG R,CHEN J,et al.Development of a duplex seminested PCR assay for detection of classical goose parvovirus and novel goose parvovirus-related virus in sick or dead ducks with short beak and dwarfism syndrome[J].J Virol Meth,2017,249:165-169.
[9]HIGUCHI R,DOLLINGER G,WALSH P S,et al.Simultaneous amplification and detection of specific DNA sequences[J].Biotechnology(N Y),1992,10(4):413-417.
[10]HIGUCHI R,FOCKLER C,DOLLINGER G,et al.Kinetic PCR analysis:real-time monitoring of DNA amplification reactions[J].Biotechnology(N Y),1993,11(9):1026-1030.
[11]DALSECCO L S,PALHARES R M,OLIVEIRA P C,et al.A Fast and Reliable Real-Time PCR Method for Detection of Ten Animal Species in Meat Products[J].J Food Sci,2018,83(2):258-265.
[12]BUSTIN S A.Absolute quantification of m RNA using real-time reverse transcription polymerase chain reaction assays[J].J Mol End,2000,25(2):169-193.
[13]胡海,钱琨,张丹阳,等.禽白血病病毒衣壳蛋白p27基因绝对定量PCR方法的建立及应用[J].中国家禽,2015(20):16-20.
[14]沈美艳,于海,袁东芳,等.商品肉鸡场细菌超广谱β-内酰胺酶基因的荧光定量PCR检测[J].中国家禽,2017(6):20-23.
[15]刘婷婷,谢芝勋,宋德贵,等.H3和H4亚型禽流感病毒双重实时荧光定量PCR检测方法的建立[J].中国家禽,2015(7):20-24.
[16]刘玲凤,黎振标,司兴奎.鸭圆环病毒实时荧光定量PCR检测方法的建立及初步应用[J].中国家禽,2017(7):15-19.
[17]王晓峰,钱勇.现代家禽产业技术体系——行业发展的科技支撑[J].中国禽业导刊,2009(5):2-12.
[18]WANG J,WANG J,CUI Y,et al.Development of a taqmanbased real-time PCR assay for the rapid and specific detection of novel duck-origin goose parvovirus[J].Mol Cell Prob,2017,34:56-58.
[19]徐楠楠.应用FQ-PCR检测GPV在产蛋种鹅体内定植规律的研究[D].长春:吉林农业大学,2011.
[20]马磊.GPV原位PCR和免疫荧光检测方法的建立及其在雏鹅体内定植研究[D].长春:吉林农业大学,2011.