鲤浮肿病毒和锦鲤疱疹病毒三重PCR检测方法的建立及初步应用
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摘要
鲤浮肿病毒(Carp Edema Virus,CEV)和锦鲤疱疹病毒(Kio Herpes Virus,KHV)皆可感染锦鲤,对锦鲤养殖造成严重危害。本研究旨在建立一种可同时检测CEV和KHV的三重PCR检测方法。参考日本学者Oyamatsu等建立的nest-PCR方法和锦鲤疱疹病毒行业标准SC/T 7212.1—2011《鲤疱疹病毒检测方法第1部分:锦鲤疱疹病毒》,分别选取扩增CEV P4a基因的548 bp片段,KHV TK基因的409 bp片段和KHV Sph基因的292 bp片段的3对特异性引物进行组合,考虑多重PCR扩增过程中的影响因素,优化退火温度和引物浓度,进行敏感性和特异性试验。结果显示,该方法能特异地扩增出3条目的条带,大小分别是548 bp、409 bp和292 bp,最佳退火温度为53℃,引物浓度均为0.3μmol/L。该方法对CEV和KHV的检测限均为300 copies/μL,同时与其他水生动物病毒均无交叉反应。因此,本研究建立的方法可用于锦鲤样品中CEV和KHV的快速检测。
Both carp edema virus and koi herpesvirus can infect koi, which may result in high losses in koibreeding. The purpose of this study is to establish a triplex PCR detection method of CEV and KHV.With reference to the nest-PCR method established by Oyamatsu and the SC/T 7212.1—2011 "Methods for Detection of Cyprinus Herpesvirus-Part 1: Koi Herpesvirus", three pairs of specific primers which amplified the 548 bp fragment of the CEV P4 a gene, the 409 bp fragment of the KHV TK gene and the 292 bp fragment of the KHV Sph gene, were selected to combine. Considering the influencing factors in the process of multiplex PCR amplification, we optimized the annealing temperature and primer concentration,and tested the sensitivity and specificity. The results showed that the method can be used for the specific amplification of three target bands, which are 548 bp, 409 bp and 292 bp, respectively, and the optimal annealing temperature was 53℃, the concentrations of primers were all 0.3 μmol/L. The limit of detection of this method was 300 copies/μL, and there was no cross-reaction with other aquatic viruses. Therefore,the method established could be used for rapid detection of CEV and KHV in koi samples.
Both carp edema virus and koi herpesvirus can infect koi, which may result in high losses in koibreeding. The purpose of this study is to establish a triplex PCR detection method of CEV and KHV.With reference to the nest-PCR method established by Oyamatsu and the SC/T 7212.1—2011 "Methods for Detection of Cyprinus Herpesvirus-Part 1: Koi Herpesvirus", three pairs of specific primers which amplified the 548 bp fragment of the CEV P4 a gene, the 409 bp fragment of the KHV TK gene and the 292 bp fragment of the KHV Sph gene, were selected to combine. Considering the influencing factors in the process of multiplex PCR amplification, we optimized the annealing temperature and primer concentration,and tested the sensitivity and specificity. The results showed that the method can be used for the specific amplification of three target bands, which are 548 bp, 409 bp and 292 bp, respectively, and the optimal annealing temperature was 53℃, the concentrations of primers were all 0.3 μmol/L. The limit of detection of this method was 300 copies/μL, and there was no cross-reaction with other aquatic viruses. Therefore,the method established could be used for rapid detection of CEV and KHV in koi samples.
引文
[1]苏建通.锦鲤的养殖与鉴赏[M].北京:中国农业出版社,2011.
[2]农业部渔业渔政管理局.中国渔业统计年鉴2016[M].北京:中国农业出版社,2016.
[3]温智清,刘莹,唐绍林,等.云南锦鲤养殖场鲤浮肿病毒的鉴定及基因型分析[J].病毒学报,2017,33(6):905-913.
[4]徐立蒲,张文,王姝,等.河南沿黄滩区鲤浮肿病和锦鲤疱疹病毒初步调查研究[J].科学养鱼,2017(9):59-61.
[5]徐立蒲,王小亮,张文,等.养殖锦鲤鲤鱼浮肿病的检测与鉴定[J].中国畜牧兽医,2017,44(2):613-618.
[6]Miwa S,Ito T,Sano M.Morphogenesis of Koi Herpesvirus Observed by Electron Microscopy[J].Journal of Fish Diseases,2007,30(12):715-722.
[7]Miyazaki T,Kuzuya Y,Yasumoto S,et al.Histopathologlical an Ultrastructural Features of Koi Herpesvirus(KHV)-infected Carp Cyprinus Carpio,and the Morphology and Morphogenesis of KHV[J].Diseases of Aquatic Organisms,2008,80(1):1-11.
[8]王小亮,徐立蒲,王姝,等.养殖鲤和锦鲤中的鲤浮肿病毒检测与分析[J].中国畜牧兽医,2017,44(10):3084-3089.
[9]王姝,徐立蒲,王小亮,等.鲤浮肿病毒混合感染的监测与分析[J].检验检疫学刊,2018,28(4):6-9.
[10]Oyamatsu T,Hata N,Yamada K,et al.An Etiological Study on Mass Mortality of Cultured Colorcarp Juveniles Showing Edema[J].Fish Pathology,1997,32(2):81-88.
[11]吕晓楠,徐立蒲,王姝,等.鲤浮肿病研究进展[J].中国动物检疫,2018,35(5):75-80.
[12]郑树城,王庆,李莹莹,等.鲤疱疹病毒3型研究进展[J].病毒学报,2016,32(1):108-120.
[13]谭爱萍,叶星,姜兰,等.鲤疱疹病毒检测方法第1部分:锦鲤疱疹病毒[M].北京:中国农业出版社,2011.
[14]乌日琴,陈芳,张艺宜,等.多重PCR方法检测锦鲤疱疹病毒基因[J].中国动物检疫,2011,28(11):39-43.
[15]Swaminathan T R,Kumar R,Dharmaratnam A,et al.Emergence of Carp Edema Virus(CEV)in Cultured Ornamental Koi Carp,Cyprinuscarpio Koi in India[J].Journal of General Virology,2016,97(12):3392-3399.
[16]徐立蒲,王静波,张文,等.鲤鱼浮肿病流行情况的初步研究[J].检验检疫学刊,2016,26(5):14-16.
[17]Zhang X D,Ni Y Y,Ye J,et al.Carp Edema Virus,an Emerging Threat to the Carp(Cyprinus Carpio)Industry in China[J].Aquaculture,2017,474:34-39.
[18]徐立蒲,王小亮,李清,等.我国北方地区鲤浮肿病毒的流行情况与监测分析[J].水生生物学报,2018,42(5):935-941.
[19]Matras M,Borzym E,Stone D,et al.Carp Edema Virus in Polish Aquaculture-evidence of Significant Sequence Divergence and a New Lineage in Common Carp Cyprinuscarp(L.)[J].Journal of Fish Diseases,2017,40(3):319-325.
[20]Adamek M,Matras M,Junq-Schroers V,et al.Comparison of PCRMethod for the Detection of Genetic Variants of Carp Edema Virus[J].Diseases of Aquatic Organisms,2017,126(1):75-81.
[21]刘继超,姜铁民,陈历俊,等.多重PCR检测原料乳中沙门氏菌、无乳链球菌和金黄色葡萄球菌的研究[J].中国食品科学,2010,10(6):173-179.
[22]杨明柳,吴斌,汤细彪,等.致猪水肿病大肠杆菌毒力因子二重PCR检测方法的建立及应用[J].中国兽医学报,2008,28(9):996-999.
[23]李伟杰,赵耘,魏财文,等.致猪水肿病大肠埃希菌多重PCR检测方法的建立及应用[J].中国畜牧兽医,2015,42(3):537-543.
[24]王楠,王剑,尹丹韩,等.三重PCR检测草莓灰霉病菌、炭疽病菌和黄萎病菌-三重草莓灰霉[J].中国农业科学,2010,43(21):4392-4400.
[2]农业部渔业渔政管理局.中国渔业统计年鉴2016[M].北京:中国农业出版社,2016.
[3]温智清,刘莹,唐绍林,等.云南锦鲤养殖场鲤浮肿病毒的鉴定及基因型分析[J].病毒学报,2017,33(6):905-913.
[4]徐立蒲,张文,王姝,等.河南沿黄滩区鲤浮肿病和锦鲤疱疹病毒初步调查研究[J].科学养鱼,2017(9):59-61.
[5]徐立蒲,王小亮,张文,等.养殖锦鲤鲤鱼浮肿病的检测与鉴定[J].中国畜牧兽医,2017,44(2):613-618.
[6]Miwa S,Ito T,Sano M.Morphogenesis of Koi Herpesvirus Observed by Electron Microscopy[J].Journal of Fish Diseases,2007,30(12):715-722.
[7]Miyazaki T,Kuzuya Y,Yasumoto S,et al.Histopathologlical an Ultrastructural Features of Koi Herpesvirus(KHV)-infected Carp Cyprinus Carpio,and the Morphology and Morphogenesis of KHV[J].Diseases of Aquatic Organisms,2008,80(1):1-11.
[8]王小亮,徐立蒲,王姝,等.养殖鲤和锦鲤中的鲤浮肿病毒检测与分析[J].中国畜牧兽医,2017,44(10):3084-3089.
[9]王姝,徐立蒲,王小亮,等.鲤浮肿病毒混合感染的监测与分析[J].检验检疫学刊,2018,28(4):6-9.
[10]Oyamatsu T,Hata N,Yamada K,et al.An Etiological Study on Mass Mortality of Cultured Colorcarp Juveniles Showing Edema[J].Fish Pathology,1997,32(2):81-88.
[11]吕晓楠,徐立蒲,王姝,等.鲤浮肿病研究进展[J].中国动物检疫,2018,35(5):75-80.
[12]郑树城,王庆,李莹莹,等.鲤疱疹病毒3型研究进展[J].病毒学报,2016,32(1):108-120.
[13]谭爱萍,叶星,姜兰,等.鲤疱疹病毒检测方法第1部分:锦鲤疱疹病毒[M].北京:中国农业出版社,2011.
[14]乌日琴,陈芳,张艺宜,等.多重PCR方法检测锦鲤疱疹病毒基因[J].中国动物检疫,2011,28(11):39-43.
[15]Swaminathan T R,Kumar R,Dharmaratnam A,et al.Emergence of Carp Edema Virus(CEV)in Cultured Ornamental Koi Carp,Cyprinuscarpio Koi in India[J].Journal of General Virology,2016,97(12):3392-3399.
[16]徐立蒲,王静波,张文,等.鲤鱼浮肿病流行情况的初步研究[J].检验检疫学刊,2016,26(5):14-16.
[17]Zhang X D,Ni Y Y,Ye J,et al.Carp Edema Virus,an Emerging Threat to the Carp(Cyprinus Carpio)Industry in China[J].Aquaculture,2017,474:34-39.
[18]徐立蒲,王小亮,李清,等.我国北方地区鲤浮肿病毒的流行情况与监测分析[J].水生生物学报,2018,42(5):935-941.
[19]Matras M,Borzym E,Stone D,et al.Carp Edema Virus in Polish Aquaculture-evidence of Significant Sequence Divergence and a New Lineage in Common Carp Cyprinuscarp(L.)[J].Journal of Fish Diseases,2017,40(3):319-325.
[20]Adamek M,Matras M,Junq-Schroers V,et al.Comparison of PCRMethod for the Detection of Genetic Variants of Carp Edema Virus[J].Diseases of Aquatic Organisms,2017,126(1):75-81.
[21]刘继超,姜铁民,陈历俊,等.多重PCR检测原料乳中沙门氏菌、无乳链球菌和金黄色葡萄球菌的研究[J].中国食品科学,2010,10(6):173-179.
[22]杨明柳,吴斌,汤细彪,等.致猪水肿病大肠杆菌毒力因子二重PCR检测方法的建立及应用[J].中国兽医学报,2008,28(9):996-999.
[23]李伟杰,赵耘,魏财文,等.致猪水肿病大肠埃希菌多重PCR检测方法的建立及应用[J].中国畜牧兽医,2015,42(3):537-543.
[24]王楠,王剑,尹丹韩,等.三重PCR检测草莓灰霉病菌、炭疽病菌和黄萎病菌-三重草莓灰霉[J].中国农业科学,2010,43(21):4392-4400.