IHHNV PCR检测方法改进及广西流行株基因型分析
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摘要
【目的】建立并优化传染性皮下及造血组织坏死病毒(IHHNV)检测及其基因分型的套式PCR,并确定IHHNV在广西流行的基因型,为有效防控广西对虾传染性皮下及造血组织坏死病(IHHN)提供参考依据。【方法】在以PCR检测IHHNV现有标准的基础上,于389F/R和309F/R两对引物扩增片段之外的绝对保守区域设计外引物IHHNVWF/WR,优化退火温度和引物浓度后建立IHHNV检测及基因分型的套式PCR,并通过与一步法PCR对比以验证其优越性;采用建立的套式PCR对546株2010—2018年收集的IHHNV广西流行株进行基因型分析,确定IHHNV在广西流行的基因型。【结果】优化后的第一轮PCR反应体系20.0μL:2×F8 FastLong PCR MasterMix 10.0μL,上、下游引物(20μmol/L)各0.8μL,DNA模板2.0μL,无核菌酶灭菌水补足至20.0μL。扩增程序:94.0℃预变性3 min;94.0℃10 s,59.0℃15 s,72.0℃15 s,进行35个循环;72.0℃延伸5 min。套式PCR检测IHHNV的灵敏度较一步法PCR提高100倍;对100份IHHNV阳性临床样品的检测结果与一步法PCR的检测结果一致,符合率达100%。从546株IHHNV广西流行株中均能扩增获得309 bp的目的条带,全部为感染型(基因1型和基因2型)。【结论】在PCR检测IHHNV现有标准基础上建立的IHHNV检测及基因分型套式PCR,其灵敏度较一步法PCR提高100倍,尤其适用于检测IHHNV含量低的样品,可为疫病监测及进出口检疫提供更敏感的技术手段。当前广西流行的IHHNV均为感染型(基因1型和基因2型)。
【Objective】The nested PCR detection methods were developed in order to provide technical support for detection and genotypes analysis of infectious hypodermal and hematopoietic necrosis virus(IHHNV),and the genotypes of IHHNV epidemic in Guangxi were investigated to offer a reference for effective prevention and control of infectious subcutaneous and hematopoietic necrosis virus(IHHN)for prawn in Guangxi.【Method】A pair of outer primers named IHHNV-WF/WR were designed on the conserved domain which located at both ends of primers 389 F/R and 309 F/R used in the present standards for PCR detection of IHHNV. Then the nested PCR detection methods were developed following optimization of annealing temperature and primers concentration. The superiority of the method was evaluated by comparing with former one-step PCR. A total of 546 IHHNV strains from Guangxi during 2010-2018 were conducted genotypes analysis using the nested PCR. The epidemic strains of IHHNV in Guangxi were identified.【Result】The optimized first-round PCR ampli?cation reactions 20.0 μL included 10.0 μL of 2×F8 FastLong PCR Master Mix,0.8 μL of upstream and downstream primers(20 μmol/L),2.0 μL of the DNA template and sterile water to a ?nal volume of 20.0 μL. PCR ampli?cation was carried out as follows:3 min initial denaturation step at 94 ℃;followed by 35 cycles of 94 ℃ for 10 s,59 ℃ for 15 s and 72 ℃ for 15 s;with a ?nal extension step of 5 min at 72 ℃. The nested PCR methods developed were 100 times more sensitive than former one-step PCR methods. The nested PCR was used to detect 100 IHHNV-positive clinical samples,the results were consistent with former one-step PCR,coincidence rate was 100%. All 546 IHHNV epidemic strains from Guangxi could amplify 309 bp target bands,and they were infectious genotypes(genotype 1 and genotype 2).【Conclusion】The nested PCR methods developed in this study on the current basis for detection and genotypes analysis of IHHNV are 100 times more sensitive than former one-step PCR methods,and can provide more sensitive means for detecting IHHNV in surveillance and quarantine samples which contain small amount of IHHNV. IHHNV epidemic strains in Guangxi are all infectious genotypes(genotype 1 and genotype 2)at present.
【Objective】The nested PCR detection methods were developed in order to provide technical support for detection and genotypes analysis of infectious hypodermal and hematopoietic necrosis virus(IHHNV),and the genotypes of IHHNV epidemic in Guangxi were investigated to offer a reference for effective prevention and control of infectious subcutaneous and hematopoietic necrosis virus(IHHN)for prawn in Guangxi.【Method】A pair of outer primers named IHHNV-WF/WR were designed on the conserved domain which located at both ends of primers 389 F/R and 309 F/R used in the present standards for PCR detection of IHHNV. Then the nested PCR detection methods were developed following optimization of annealing temperature and primers concentration. The superiority of the method was evaluated by comparing with former one-step PCR. A total of 546 IHHNV strains from Guangxi during 2010-2018 were conducted genotypes analysis using the nested PCR. The epidemic strains of IHHNV in Guangxi were identified.【Result】The optimized first-round PCR ampli?cation reactions 20.0 μL included 10.0 μL of 2×F8 FastLong PCR Master Mix,0.8 μL of upstream and downstream primers(20 μmol/L),2.0 μL of the DNA template and sterile water to a ?nal volume of 20.0 μL. PCR ampli?cation was carried out as follows:3 min initial denaturation step at 94 ℃;followed by 35 cycles of 94 ℃ for 10 s,59 ℃ for 15 s and 72 ℃ for 15 s;with a ?nal extension step of 5 min at 72 ℃. The nested PCR methods developed were 100 times more sensitive than former one-step PCR methods. The nested PCR was used to detect 100 IHHNV-positive clinical samples,the results were consistent with former one-step PCR,coincidence rate was 100%. All 546 IHHNV epidemic strains from Guangxi could amplify 309 bp target bands,and they were infectious genotypes(genotype 1 and genotype 2).【Conclusion】The nested PCR methods developed in this study on the current basis for detection and genotypes analysis of IHHNV are 100 times more sensitive than former one-step PCR methods,and can provide more sensitive means for detecting IHHNV in surveillance and quarantine samples which contain small amount of IHHNV. IHHNV epidemic strains in Guangxi are all infectious genotypes(genotype 1 and genotype 2)at present.
引文
白丽蓉,赵志英.2012.对虾传染性皮下与造血组织坏死病毒(IHHNV)的研究进展[J].中国农学通报,28(14):114-119.[Bai L R,Zhao Z Y.2012.Research progress of infectious hypodermal and hematopoietic necrosis virus(IHH-NV)in penaeid shrimp[J].Chinese Agricultural Science Bulletin,28(14):114-119.]
邓威,许杰,刘群,韩进刚,李军,刘健,杨凯,张丽.2017.2015-2016年天津市对虾传染性皮下及造血组织坏死病毒感染调查[J].中国动物检疫,34(9):23-25.[Deng W,Xu J,Liu Q,Han J G,Li J,Liu J,Yang K,Zhang L.2017.Investigation on Penaeus IHHNV infection in Tianjin City from 2015 to 2016[J].China Animal Health Inspection,34(9):23-25.]
梁静真,马沙,肖双燕,韩书煜,黄艳华,黄德生,张振豪,吕忠坚,覃志彪,黄钧.2018.2013-2016年广西沿海养殖凡纳滨对虾2种病毒的流行情况调查[J].西南农业学报,31(5):1088-1096.[Liang J Z,Ma S,Xiao S Y,Han SY,Huang Y H,Huang D S,Zhang Z H,LüZ J,Qin Z B,Huang J.2018.Epidemiology of two viruses in cultural Penaeus vannamei from Guangxi coastal areas from 2013to 2016[J].Southwest China Journal of Agricultural Sciences,31(5):1088-1096.]
林婧楠,赵景壮,卢彤岩,徐黎明.2018.鲤春病毒血症病毒的研究进展[J].水产学杂志,31(5):44-49.[Lin J N,Zhao J Z,Lu T Y,Xu L M.2018.Research progress on carp spring virernia virus[J].Chinese Journal of Fisheries,31(5):44-49.]
童桂香,韦信贤,吴伟军,吴祥庆,黄国秋,黄玉柳,叶欣宇,黎小正.2013.广西凡纳滨对虾IHHNV感染情况的调查与分析[J].南方农业学报,44(12):2089-2093.[Tong GX,Wei X X,Wu W J,Wu X Q,Huang G Q,Huang Y L,Ye X Y,Li X Z.2013.Investigation and analysis of infectious hypodermal and hematopoietic necrosis virus infection in Penaeus vannamei in Guangxi[J].Journal of Southern Agriculture,44(12):2089-2093.]
王博雅,王力,刘美如,叶仕根,黎睿君,李华,李强.2017.凡纳滨对虾3种主要病毒和虾肝肠胞虫在辽宁地区的流行情况分析[J].大连海洋大学学报,32(2):150-154.[Wang B Y,Wang L,Liu M R,Ye S G,Li R J,Li H,Li Q.2017.Epidemiological investigation of three major viruses and Enterocytozoon hepatopenaei(EHP)in Pacific white leg shrimp Litopenaeus vannamei in Liaoning Province[J].Journal of Dalian Ocean University,32(2):150-154.]
王筱珊,胡智博,费荣梅.2017.江苏地区对虾3种病毒病的流行病学调查及5株IHHNV的编码区基因序列分析[J].水产学报,41(10):1623-1630.[Wang X S,Hu Z B,Fei RM.2017.Prevalence of three viruses in Pacific white leg shrimp in Jiangsu Province and the analysis of coding sequence of five strains of infectious hypodermal and hematopoietic necrosis virus[J].Journal of Fisheries of China,41(10):1623-1630.]
韦信贤,童桂香,谢宗升,吴祥庆,黄国秋,黄玉柳,廖永志,黎小正.2011.Taq Man-LNA探针荧光定量PCR快速检测对虾传染性皮下及造血组织坏死病毒[J].南方农业学报,42(12):1545-1549.[Wei X X,Tong G X,Xie Z S,Wu X Q,Huang G Q,Huang Y L,Liao Y Z,Li X Z.2011.Rapid detection of infectious hypodermal and hematopoietic necrosis virus in shrimps using Taq ManLNA probe real-time PCR[J].Journal of Southern Agriculture,42(12):1545-1549.]
谢数涛,何建国,杨晓明,吕玲,江静波.2001.套式PCR检测斑节对虾白斑症病毒(WSSV)[J].青岛海洋大学学报,31(2):220-224.[Xie S T,He J G,Yang X M,LüL,Jiang J B.2001.Detection of white spot syndrome virus(WSSV)in Penaeus monodon using nested PCR[J].Journal of Ocean University of Qingdao,31(2):220-224.]
袁颜颜,杨冰,万晓媛,刘笋,刘天齐,黄倢.2015.采用OIE标准检测养殖对虾中传染性皮下及造血组织坏死病毒(IHHNV)的PCR检出类型[J].渔业科学进展,36(1):67-73.[Yuan Y Y,Yang B,Wan X Y,Liu S,Liu T Q,Huang J.2015.PCR detecting types by the OIE standards for infectious hypodermal and hematopoietic necrosis virus(IHHNV)in farmed shrimp[J].Progress in Fishery Sciences,36(1):67-73.]
曾地刚,陈秀荔,谢达祥,赵永贞,黎铭,陈晓汉.2013.利用高通量测序技术分析IHHNV感染凡纳滨对虾的基因差异表达[J].南方农业学报,44(11):1899-1903.[Zeng D G,Chen X L,Xie D X,Zhao Y Z,Li M,Chen X H.2013.Analysis on differential gene expression in shrimp Litopenaeus vannamei induced by IHHNV infection using highthroughput sequencing[J].Journal of Southern Agriculture,44(11):1899-1903.]
Motte E,Yugcha E,Luzardo J,Castro F,Leclercq G,Rodriguez J,Miranda P,Borja O,Serrano J,Terreros M.2003.Prevention of IHHNV vertical transmission in the white shrimp Litopenaeus vannamei[J].Aquaculture,219(1-4):57-70.
Saksmerprome V,Puiprom O,Noonin C,Flegel T W.2010.Detection of infectious hypodermal and haematopoietic necrosis virus(IHHNV)in farmed Australian Penaeus monodon by PCR analysis and DNA sequencing[J].Aquaculture,298(3-4):190-193.
Tang K F J,Lightner D V.2006.Infectious hypodermal and hematopoietic necrosis virus(IHHNV)-related sequences in the genome of the black tiger prawn Penaeus monodon from Africa and Australia[J].Virus Research,118(1-2):185-191.
Tang K F J,Navarro S A,Lightner D V.2007.PCR assay for discriminating between infectious hypodermal and hematopoietic necrosis virus(IHHNV)and virus-related sequences in the genome of Penaeus monodon[J].Diseases of Aquatic Organisms,74(2):165-170.
Tang K F J,Poulos B T,Wang J,Redman M,Shih H H,Lightner D V.2003.Geographic variations among infectious hypodermal and hematopoietic necrosis virus(IHH-NV)isolates and characteristics of their infection[J].Diseases of Aquatic Organisms,53(2):91-99.
Tang K F,Durand S V,White B L,Redman R M,Pantoja CR,Lightner D V.2000.Postlarvae and juveniles of a selected line of Penaeus stylirostris are resistant to infectious hypodermal and hematopoietic necrosis virus infection[J].Aquaculture,190(3-4):203-210.
邓威,许杰,刘群,韩进刚,李军,刘健,杨凯,张丽.2017.2015-2016年天津市对虾传染性皮下及造血组织坏死病毒感染调查[J].中国动物检疫,34(9):23-25.[Deng W,Xu J,Liu Q,Han J G,Li J,Liu J,Yang K,Zhang L.2017.Investigation on Penaeus IHHNV infection in Tianjin City from 2015 to 2016[J].China Animal Health Inspection,34(9):23-25.]
梁静真,马沙,肖双燕,韩书煜,黄艳华,黄德生,张振豪,吕忠坚,覃志彪,黄钧.2018.2013-2016年广西沿海养殖凡纳滨对虾2种病毒的流行情况调查[J].西南农业学报,31(5):1088-1096.[Liang J Z,Ma S,Xiao S Y,Han SY,Huang Y H,Huang D S,Zhang Z H,LüZ J,Qin Z B,Huang J.2018.Epidemiology of two viruses in cultural Penaeus vannamei from Guangxi coastal areas from 2013to 2016[J].Southwest China Journal of Agricultural Sciences,31(5):1088-1096.]
林婧楠,赵景壮,卢彤岩,徐黎明.2018.鲤春病毒血症病毒的研究进展[J].水产学杂志,31(5):44-49.[Lin J N,Zhao J Z,Lu T Y,Xu L M.2018.Research progress on carp spring virernia virus[J].Chinese Journal of Fisheries,31(5):44-49.]
童桂香,韦信贤,吴伟军,吴祥庆,黄国秋,黄玉柳,叶欣宇,黎小正.2013.广西凡纳滨对虾IHHNV感染情况的调查与分析[J].南方农业学报,44(12):2089-2093.[Tong GX,Wei X X,Wu W J,Wu X Q,Huang G Q,Huang Y L,Ye X Y,Li X Z.2013.Investigation and analysis of infectious hypodermal and hematopoietic necrosis virus infection in Penaeus vannamei in Guangxi[J].Journal of Southern Agriculture,44(12):2089-2093.]
王博雅,王力,刘美如,叶仕根,黎睿君,李华,李强.2017.凡纳滨对虾3种主要病毒和虾肝肠胞虫在辽宁地区的流行情况分析[J].大连海洋大学学报,32(2):150-154.[Wang B Y,Wang L,Liu M R,Ye S G,Li R J,Li H,Li Q.2017.Epidemiological investigation of three major viruses and Enterocytozoon hepatopenaei(EHP)in Pacific white leg shrimp Litopenaeus vannamei in Liaoning Province[J].Journal of Dalian Ocean University,32(2):150-154.]
王筱珊,胡智博,费荣梅.2017.江苏地区对虾3种病毒病的流行病学调查及5株IHHNV的编码区基因序列分析[J].水产学报,41(10):1623-1630.[Wang X S,Hu Z B,Fei RM.2017.Prevalence of three viruses in Pacific white leg shrimp in Jiangsu Province and the analysis of coding sequence of five strains of infectious hypodermal and hematopoietic necrosis virus[J].Journal of Fisheries of China,41(10):1623-1630.]
韦信贤,童桂香,谢宗升,吴祥庆,黄国秋,黄玉柳,廖永志,黎小正.2011.Taq Man-LNA探针荧光定量PCR快速检测对虾传染性皮下及造血组织坏死病毒[J].南方农业学报,42(12):1545-1549.[Wei X X,Tong G X,Xie Z S,Wu X Q,Huang G Q,Huang Y L,Liao Y Z,Li X Z.2011.Rapid detection of infectious hypodermal and hematopoietic necrosis virus in shrimps using Taq ManLNA probe real-time PCR[J].Journal of Southern Agriculture,42(12):1545-1549.]
谢数涛,何建国,杨晓明,吕玲,江静波.2001.套式PCR检测斑节对虾白斑症病毒(WSSV)[J].青岛海洋大学学报,31(2):220-224.[Xie S T,He J G,Yang X M,LüL,Jiang J B.2001.Detection of white spot syndrome virus(WSSV)in Penaeus monodon using nested PCR[J].Journal of Ocean University of Qingdao,31(2):220-224.]
袁颜颜,杨冰,万晓媛,刘笋,刘天齐,黄倢.2015.采用OIE标准检测养殖对虾中传染性皮下及造血组织坏死病毒(IHHNV)的PCR检出类型[J].渔业科学进展,36(1):67-73.[Yuan Y Y,Yang B,Wan X Y,Liu S,Liu T Q,Huang J.2015.PCR detecting types by the OIE standards for infectious hypodermal and hematopoietic necrosis virus(IHHNV)in farmed shrimp[J].Progress in Fishery Sciences,36(1):67-73.]
曾地刚,陈秀荔,谢达祥,赵永贞,黎铭,陈晓汉.2013.利用高通量测序技术分析IHHNV感染凡纳滨对虾的基因差异表达[J].南方农业学报,44(11):1899-1903.[Zeng D G,Chen X L,Xie D X,Zhao Y Z,Li M,Chen X H.2013.Analysis on differential gene expression in shrimp Litopenaeus vannamei induced by IHHNV infection using highthroughput sequencing[J].Journal of Southern Agriculture,44(11):1899-1903.]
Motte E,Yugcha E,Luzardo J,Castro F,Leclercq G,Rodriguez J,Miranda P,Borja O,Serrano J,Terreros M.2003.Prevention of IHHNV vertical transmission in the white shrimp Litopenaeus vannamei[J].Aquaculture,219(1-4):57-70.
Saksmerprome V,Puiprom O,Noonin C,Flegel T W.2010.Detection of infectious hypodermal and haematopoietic necrosis virus(IHHNV)in farmed Australian Penaeus monodon by PCR analysis and DNA sequencing[J].Aquaculture,298(3-4):190-193.
Tang K F J,Lightner D V.2006.Infectious hypodermal and hematopoietic necrosis virus(IHHNV)-related sequences in the genome of the black tiger prawn Penaeus monodon from Africa and Australia[J].Virus Research,118(1-2):185-191.
Tang K F J,Navarro S A,Lightner D V.2007.PCR assay for discriminating between infectious hypodermal and hematopoietic necrosis virus(IHHNV)and virus-related sequences in the genome of Penaeus monodon[J].Diseases of Aquatic Organisms,74(2):165-170.
Tang K F J,Poulos B T,Wang J,Redman M,Shih H H,Lightner D V.2003.Geographic variations among infectious hypodermal and hematopoietic necrosis virus(IHH-NV)isolates and characteristics of their infection[J].Diseases of Aquatic Organisms,53(2):91-99.
Tang K F,Durand S V,White B L,Redman R M,Pantoja CR,Lightner D V.2000.Postlarvae and juveniles of a selected line of Penaeus stylirostris are resistant to infectious hypodermal and hematopoietic necrosis virus infection[J].Aquaculture,190(3-4):203-210.