Acute hepatopancreatic necrosis disease(AHPND),also called early mortality syndrome(EMS),has caused massive economic loss in aquaculture since 2009.It was considered that the disease was caused by a novel type of Vibr...Acute hepatopancreatic necrosis disease(AHPND),also called early mortality syndrome(EMS),has caused massive economic loss in aquaculture since 2009.It was considered that the disease was caused by a novel type of Vibrio parahaemolyticus which harbors a large plasmid.Two insecticidal Photorhabdus insect-related(pir)genes were found in the plasmid.In this study,81 isolates have been collected from a shrimp farm in Maoming,Guangdong Province,PRC.Suspected isolates were identified using 3 pairs of primer recently made public.Both the 16S rRNA gene and pir gene were sequenced.ERIC-PCR was employed for analysis of strain diversity.12 isolates were amplified by PCR and identified as Vibrio parahaemolyticus by 16S rRNA sequence alignment.Sequence alignments of the pir gene showed few mutations in the 12 strains of V.parahaemolyticus.The 12 strains separated into 3 clusters at a cut of 90%similarity by ERIC-PCR.The virulence gene expression and toxicity profile showed differences between strains.This study generated comprehensive identification of pathogenic bacteria and microbial molecular resources,which will contribute to future research of AHPND.展开更多
为了建立一种敏感、特异的致对虾急性肝胰腺坏死病(AHPND)的一类弧菌(VpAHPND)的荧光定量PCR(qPCR)检测方法,本研究根据Gen Bank中该类弧菌(本研究所用的VpAHPND为副溶血弧菌变异株,下同)PirB毒素基因(KU145397),设计q PCR扩增引物,并...为了建立一种敏感、特异的致对虾急性肝胰腺坏死病(AHPND)的一类弧菌(VpAHPND)的荧光定量PCR(qPCR)检测方法,本研究根据Gen Bank中该类弧菌(本研究所用的VpAHPND为副溶血弧菌变异株,下同)PirB毒素基因(KU145397),设计q PCR扩增引物,并采用该引物经PCR扩增PirB基因片段,构建重组质粒p MD18-T-PirB并经PCR和测序鉴定正确后作为质粒标准品。以10倍倍比稀释的重组质粒标准品进行q PCR扩增,建立标准曲线,并经反应条件优化初步建立了检测该类VpAHPND的SYBR Green I q PCR方法。以白斑综合征病毒、传染性皮下及造血组织坏死病毒、虾虹彩病毒、虾肝肠胞虫及该VpAHPND的基因组DNA为模板,采用本研究建立的SYBR Green I q PCR方法检测,评估该方法的特异性;以8.3×10^(1)拷贝/μL~8.3×10^(8)拷贝/μL的质粒标准品作为模板,利用本研究建立的SYBR Green I q PCR以及常规PCR检测,比较两种方法的敏感性;以3个不同浓度的质粒标准品作为模板,利用该方法分别进行批内和批间重复性试验,评估该方法的重复性。建立的该q PCR方法的标准曲线显示,各浓度的质粒标准品的拷贝数与其Ct值均呈良好的线性关系,相关系数R^(2)为0.998。特异性试验结果显示,该方法仅能检出VpAHPND,而其他病原均为阴性结果,特异性强;敏感性试验结果显示,该方法对质粒标准品的检测限为83拷贝/μL,敏感性是水产行业标准常规PCR的1000倍。重复性试验结果显示,批内和批间重复性试验变异系数分别在0.31%~0.81%和2.05%~4.34%,重复性好。利用该方法对61株虾源弧菌样品检测,结果显示阳性检出率为29.5%(18/61),阴性样品检测率为70.5%(43/61),与行业标准的常规PCR检测方法结果完全一致,二者的阳性符合率为100%,总符合率为100%。本研究建立了一类致对虾VpAHPND的SYBR Green I q PCR检测方法,该方法特异性强、敏感性高、重复性好,为AHPND的诊断、监测与流行病学调查提供技术支撑。展开更多
Vibrio parahaemolyticus carrying a plasmid encoding the PirAvp and PirBvp toxins is the causative agent of acute hepatopancreatic necrosis disease(AHPND)in penaeid shrimps.In the Pacific white shrimp farming industry,...Vibrio parahaemolyticus carrying a plasmid encoding the PirAvp and PirBvp toxins is the causative agent of acute hepatopancreatic necrosis disease(AHPND)in penaeid shrimps.In the Pacific white shrimp farming industry,one possible strategy to reduce economic loss due to AHPND is the development of a shrimp line resistant to the disease.In this study,we identified single nucleotide polymorphisms(SNPs)in the Litopenaeus vannamei anti-lipopolysaccharide factor 1(nLvALF1)and penaeidin 2-1(PEN2-1)genes,and we analyzed the associations between these SNPs and resistance/susceptibility to V.parahaemolyticus infection in the Pacific white shrimp.Postlarvae(PL20)shrimp from a local hatchery in Prachuap Khiri Khan Province were challenged with an isolate of VPAHPND and mortality was observed for 14 days.DNA was extracted from susceptible(died within 6 days)and resistant(survived the challenge)shrimp(45 individuals/group)and used for PCR amplifications of nLvALF1(397 bp)and PEN2-1(637 bp)gene fragments.PCR products were sequenced by direct sequencing and SNPs were identified from sequencing chromatograms.Nine and seven SNPs were identified in nLvALF1 and PEN2-1 gene fragments,respectively.Analyses of allele frequencies in susceptible and resistant samples using Chi-square tests revealed that four and six SNPs in nLvALF1 and PEN2-1,respectively,were associated with resistance/susceptibility to V.parahaemolyticus infection(P<0.05).The SNPs in the candidate genes identified here are potential DNA markers for breeding V.parahaemolyticus-resistant Pacific white shrimp in the study population;however,further validation will be required if these SNPs are to be used across populations.展开更多
基金中国东盟海上合作基金项目(2016-2018)青岛海洋科学与技术试点国家实验室主任基金(QNLM201706)+10 种基金中国水产科学研究院中央级公益性科研院所基本科研业务费专项资金(2017HY-ZD10)中国水产科学研究院黄海水产研究所中央级公益性科研院所基本科研业务费专项资金(20603022016012)现代农业产业技术体系(CARS-47)共同资助supported by China ASEAN Maritime Cooperation Fund Project (2016-2018)Pilot National Laboratory for Marine Science and Technology (Qingdao) (QNLM201706)Central Public-Interest Scientific Institution Basal Research Fund CAFS (2017HY-ZD10)Central Public-interest Scientific Institution Basal Research Fund YSFRI CAFS (20603022016012)China Agriculture Research System (CARS-47)
基金This work was supported by the National Natural Science Foundation of China[3157101641]the project of Science and Technology Commission of Shanghai Municipality[14DZ1205100,14320502100]+1 种基金Key Project of Shanghai Agriculture Prosperity through Science and Technology[2015(4e8)],Shanghai Engineering Research Center of Aquatic-Product Processing&Preservation[11DZ2280300]the“Dawn”Program of Shanghai Education Commission[15SG48].
文摘Acute hepatopancreatic necrosis disease(AHPND),also called early mortality syndrome(EMS),has caused massive economic loss in aquaculture since 2009.It was considered that the disease was caused by a novel type of Vibrio parahaemolyticus which harbors a large plasmid.Two insecticidal Photorhabdus insect-related(pir)genes were found in the plasmid.In this study,81 isolates have been collected from a shrimp farm in Maoming,Guangdong Province,PRC.Suspected isolates were identified using 3 pairs of primer recently made public.Both the 16S rRNA gene and pir gene were sequenced.ERIC-PCR was employed for analysis of strain diversity.12 isolates were amplified by PCR and identified as Vibrio parahaemolyticus by 16S rRNA sequence alignment.Sequence alignments of the pir gene showed few mutations in the 12 strains of V.parahaemolyticus.The 12 strains separated into 3 clusters at a cut of 90%similarity by ERIC-PCR.The virulence gene expression and toxicity profile showed differences between strains.This study generated comprehensive identification of pathogenic bacteria and microbial molecular resources,which will contribute to future research of AHPND.
文摘为了建立一种敏感、特异的致对虾急性肝胰腺坏死病(AHPND)的一类弧菌(VpAHPND)的荧光定量PCR(qPCR)检测方法,本研究根据Gen Bank中该类弧菌(本研究所用的VpAHPND为副溶血弧菌变异株,下同)PirB毒素基因(KU145397),设计q PCR扩增引物,并采用该引物经PCR扩增PirB基因片段,构建重组质粒p MD18-T-PirB并经PCR和测序鉴定正确后作为质粒标准品。以10倍倍比稀释的重组质粒标准品进行q PCR扩增,建立标准曲线,并经反应条件优化初步建立了检测该类VpAHPND的SYBR Green I q PCR方法。以白斑综合征病毒、传染性皮下及造血组织坏死病毒、虾虹彩病毒、虾肝肠胞虫及该VpAHPND的基因组DNA为模板,采用本研究建立的SYBR Green I q PCR方法检测,评估该方法的特异性;以8.3×10^(1)拷贝/μL~8.3×10^(8)拷贝/μL的质粒标准品作为模板,利用本研究建立的SYBR Green I q PCR以及常规PCR检测,比较两种方法的敏感性;以3个不同浓度的质粒标准品作为模板,利用该方法分别进行批内和批间重复性试验,评估该方法的重复性。建立的该q PCR方法的标准曲线显示,各浓度的质粒标准品的拷贝数与其Ct值均呈良好的线性关系,相关系数R^(2)为0.998。特异性试验结果显示,该方法仅能检出VpAHPND,而其他病原均为阴性结果,特异性强;敏感性试验结果显示,该方法对质粒标准品的检测限为83拷贝/μL,敏感性是水产行业标准常规PCR的1000倍。重复性试验结果显示,批内和批间重复性试验变异系数分别在0.31%~0.81%和2.05%~4.34%,重复性好。利用该方法对61株虾源弧菌样品检测,结果显示阳性检出率为29.5%(18/61),阴性样品检测率为70.5%(43/61),与行业标准的常规PCR检测方法结果完全一致,二者的阳性符合率为100%,总符合率为100%。本研究建立了一类致对虾VpAHPND的SYBR Green I q PCR检测方法,该方法特异性强、敏感性高、重复性好,为AHPND的诊断、监测与流行病学调查提供技术支撑。
基金Funding for this research was provided by the National Science and Technology Development Agency,Ministry of Higher Education,Science,Research and Innovation.
文摘Vibrio parahaemolyticus carrying a plasmid encoding the PirAvp and PirBvp toxins is the causative agent of acute hepatopancreatic necrosis disease(AHPND)in penaeid shrimps.In the Pacific white shrimp farming industry,one possible strategy to reduce economic loss due to AHPND is the development of a shrimp line resistant to the disease.In this study,we identified single nucleotide polymorphisms(SNPs)in the Litopenaeus vannamei anti-lipopolysaccharide factor 1(nLvALF1)and penaeidin 2-1(PEN2-1)genes,and we analyzed the associations between these SNPs and resistance/susceptibility to V.parahaemolyticus infection in the Pacific white shrimp.Postlarvae(PL20)shrimp from a local hatchery in Prachuap Khiri Khan Province were challenged with an isolate of VPAHPND and mortality was observed for 14 days.DNA was extracted from susceptible(died within 6 days)and resistant(survived the challenge)shrimp(45 individuals/group)and used for PCR amplifications of nLvALF1(397 bp)and PEN2-1(637 bp)gene fragments.PCR products were sequenced by direct sequencing and SNPs were identified from sequencing chromatograms.Nine and seven SNPs were identified in nLvALF1 and PEN2-1 gene fragments,respectively.Analyses of allele frequencies in susceptible and resistant samples using Chi-square tests revealed that four and six SNPs in nLvALF1 and PEN2-1,respectively,were associated with resistance/susceptibility to V.parahaemolyticus infection(P<0.05).The SNPs in the candidate genes identified here are potential DNA markers for breeding V.parahaemolyticus-resistant Pacific white shrimp in the study population;however,further validation will be required if these SNPs are to be used across populations.
