实时荧光定量PCR扩增特异性vapA基因检测杀鲑气单胞菌
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摘要
为实现杀鲑气单胞菌早期快速准确定量检测,研究旨在建立杀鲑气单胞菌的SYBR Green Ⅰ实时荧光定量PCR(Real-time PCR)检测方法。根据GenBank中杀鲑气单胞菌毒力阵列蛋白基因(vapA)保守序列设计并合成一对特异性引物,对其特异性、灵敏度、可重复性和应用性进行评价。结果显示,研究设计的引物具有良好的种间特异性,仅对杀鲑气单胞菌及其亚种有阳性扩增,与其他细菌不发生交叉反应。构建的Real-time PCR标准曲线质粒拷贝数与循环阈值呈良好的线性关系,扩增所得标准曲线分别为y=–4.8345x+42.535,相关系数R~2为0.998,最低检测限为34拷贝/μL,较常规PCR的灵敏度高出约1000倍。应用建立的方法检测人工感染的虹鳟病样,15个被检样品呈阳性反应,与细菌常规鉴定方法结果一致。研究表明,所建立的基于实时荧光定量PCR技术的杀鲑气单胞菌检测方法快速、特异、灵敏,可用于临床诊断和疫病监测。
In order to implement the early and quick quantitative determination of Aeromonas salmonicida, a SYBR Green Ⅰ Real-time PCR method of A. salmonicida was established based on the pathogen sequence information. Based on the vapA of virulence array protein gene sequence of A. salmonicida, a pair of primers was designed and used in a real time quantitative polymerase chain reaction(qPCR) assay. The specificity, sensitivity,repeatability and application of the system were also evaluated. The results showed that A. salmonicida and its subspecies can be clearly discriminated from the other 10 bacteria species by SYBR Green I Real-time qPCR,which indicated that the primer pair has good inter-species specificity. The standard curve established by recombinant plasmid showed a fine linear relationship between initial templates and threshold cycle, which can be described as y=–4.8345 x+42.535(R~2=0.998). The sensitivity analysis showed that the detection limit was34 copies/μL, which suggested that the sensitivity of Real-time qPCR was about 1000 times higher than that of the conventional PCR assay. The established method was applied to detect the samples in rainbow trout after artificial infection. Results showed that 15 of those samples were positive, which had complete agreement(100%) with bacteriological analysis by isolation and culture. In conclusion, the developed Real-time PCR assay for A.salmonicida is fast, highly specific, and sensitive. This method had a broad application for clinical diagnosis and disease surveillance in aquaculture.
In order to implement the early and quick quantitative determination of Aeromonas salmonicida, a SYBR Green Ⅰ Real-time PCR method of A. salmonicida was established based on the pathogen sequence information. Based on the vapA of virulence array protein gene sequence of A. salmonicida, a pair of primers was designed and used in a real time quantitative polymerase chain reaction(qPCR) assay. The specificity, sensitivity,repeatability and application of the system were also evaluated. The results showed that A. salmonicida and its subspecies can be clearly discriminated from the other 10 bacteria species by SYBR Green I Real-time qPCR,which indicated that the primer pair has good inter-species specificity. The standard curve established by recombinant plasmid showed a fine linear relationship between initial templates and threshold cycle, which can be described as y=–4.8345 x+42.535(R~2=0.998). The sensitivity analysis showed that the detection limit was34 copies/μL, which suggested that the sensitivity of Real-time qPCR was about 1000 times higher than that of the conventional PCR assay. The established method was applied to detect the samples in rainbow trout after artificial infection. Results showed that 15 of those samples were positive, which had complete agreement(100%) with bacteriological analysis by isolation and culture. In conclusion, the developed Real-time PCR assay for A.salmonicida is fast, highly specific, and sensitive. This method had a broad application for clinical diagnosis and disease surveillance in aquaculture.
引文
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[5]杨移斌,胥宁,董靖,等.斑点叉尾鲙源杀鲑气单胞菌无色亚种分离鉴定及药敏特性[J].水生生物学报,2017,41(4):787-792.Yang Y B,Xu N,Dong J,et al.Isolation and identification of Aeromonas salmonicida subsp.achromogenes from Ictalunes punctatus and its antimicrobial susceptibility[J].Acta Hydrobiologica Sinica,2017,41(4):787-792(in Chinese).
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[9]林如涛,周作勇,王小林,等.羊源杀鲑气单胞菌16Sr RNA基因的克隆测序及分析[J].西南大学学报(自然科学版),2011,33(8):46-51.Lin R T,Zhou Z Y,Wang X L,et al.Cloning and sequence analysis of the 16S r RNA gene of Aeromonas salmonicida in goat[J].Journal of Southwest University(Natural Science Edition),2011,33(8):46-51(in Chinese).
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[12]Gulla S,Lund V,Kristoffersen A B,et al.vap A(A-layer)typing differentiates Aeromonas salmonicida subspecies and identifies a number of previously undescribed subtypes[J].Journal of Fish Diseases,2016,39(3):329-342.
[13]刘宗晓,刘荭,史秀杰,等.杀鲑气单胞菌的实时定量PCR检测方法的建立和应用[J].海洋水产研究,2008,29(5):83-88.Liu Z X,Liu H,Shi X J,et al.Development and application of real-time PCR assay for detecion of Aeromonas salmonicida[J].Marine Fisheries Research,2008,29(5):83-88(in Chinese).
[14]张惟才,朱力,王玉飞.实时荧光定量PCR[M].北京:化学工业出版社,2013.Zhang W C,Zhu L,Wang Y F.Real-time QuantitativePCR[M].Beijing:Chemical Industry Press,2013(in Chinese).
[15]Bustin S A,Benes V,Nolan T,et al.Quantitative realtime RT-PCR-a perspective[J].Journal of Molecular Endocrinology,2005,34(3):597-601.
[16]石晓璐,扈庆华,张佳峰,等.多重实时PCR快速同时检测沙门菌和志贺菌[J].中华流行病学杂志,2006,27(12):1053-1056.Shi X L,Hu Q H,Zhang J F,et al.Rapid simultaneous detection of Salmonella and Shigella using modified molecular beacons and real-time PCR[J].Chinese Journal of Epidemiology,2006,27(12):1053-1056(in Chinese).
[17]李富祥,赵德宏,宋建领,等.猪丹毒杆菌Taq Man荧光定量PCR检测方法的建立[J].中国预防兽医学报,2016,38(5):385-389.Li F X,Zhao D H,Song J L,et al.Development of TaqMan real-time PCR assay for detection of Erysipelothrix rhusiopathiae[J].Chinese Journal of Preventive Veterinary Medicine,2016,38(5):385-389(in Chinese).
[18]荣小军,廖梅杰,张正,等.迟缓爱德华氏菌SYBRGreen I实时荧光定量PCR检测方法的建立及其应用[J].水产学报,2013,37(12):1829-1838.Rong X J,Liao M J,Zhang Z,et al.Development of an SYBR Green I real-time PCR assay for detection of Edwardsiella tarda and its application[J].Journal of Fisheries of China,2013,37(12):1829-1838(inChinese).
[19]王国良,刘璐,李思源.這鱼诺卡氏菌SYBR GreenⅠ实时荧光定量PCR检测方法的建立与应用[J].水产学报,2012,36(4):509-513.Wang G L,Liu L,Li S Y.Development of a SYBRGreenⅠreal-time PCR assay for detection of Nocardia seriolae and its application[J].Journal of Fisheries of China,2012,36(4):509-513(in Chinese).
[20]O’Regan E,Mc Cabe E,Burgess C,et al.Development of a real-time multiplex PCR assay for the detection of multiple Salmonella serotypes in chicken samples[J].BMC Microbiology,2008,8:156.
[21]Alperi A,Figueras M J,Inza I,et al.Analysis of 16Sr RNA gene mutations in a subset of Aeromonas strains and their impact in species delineation[J].International Microbiology,2008,11(3):185-194.
[22]Kulkarni A,Caipang C M A,Brinchmann M F,et al.Loop-mediated isothermal amplification-an assay for the detection of atypical furunculosis caused by Aeromonas salmonicida in Atlantic cod,Gadus morhua[J].Journal of Rapid Methods and Automation in Microbiology,2009,17(4):476-489.
[23]童裳亮,王作芸,周建玲.杀鲑气单胞菌的快速检测[J].动物检疫,1993,10(5):7-8.Tong S L,Wang Z Y,Zhou J L.Rapid detection of Aeromonas salmonicida using ELISA[J].Animal Quarantine,1993,10(5):7-8(in Chinese).
[2]Coscelli G A,Roberto B,Ana P L,et al.Acute Aeromonas salmonicida infection in turbot(Scophthalmus maximus L.).Histopathological and immunohistochemical studies[J].Aquaculture,2014,430:79-85.
[3]Erdal J I,Reitan L J.Immune response and protective immunity after vaccination of Atlantic salmon(Salmo salar L.)against furunculosis[J].Fish&Shellfish Immunology,1992,2(2):99-108.
[4]杨嘉龙,周丽,邢婧,等.养殖刺参溃疡病杀鲑气单胞菌的分离、致病性及胞外产物特性分析[J].中国水产科学,2007,14(6):981-989.Yang J L,Zhou L,Xing J,et al.Identification of Aeromonas salmonicida associated with skin ulceration of cultured sea cucumber Apostichopus japonicus and characterization of the extracellular products[J].Journal of Fishery Sciences of China,2007,14(6):981-989(in Chinese).
[5]杨移斌,胥宁,董靖,等.斑点叉尾鲙源杀鲑气单胞菌无色亚种分离鉴定及药敏特性[J].水生生物学报,2017,41(4):787-792.Yang Y B,Xu N,Dong J,et al.Isolation and identification of Aeromonas salmonicida subsp.achromogenes from Ictalunes punctatus and its antimicrobial susceptibility[J].Acta Hydrobiologica Sinica,2017,41(4):787-792(in Chinese).
[6]周冬仁,罗毅志,杭小英,等.1株乌鳢源杀鲑气单胞菌杀鲑亚种的分离与鉴定[J].海洋湖沼通报,2015(3):64-70.Zhou D R,Luo Y Z,Hang X Y,et al.Isolation and identification of pathogens Aeromonas Salmonicida Salmonicida subsp.from northern snakehead(Ophicephalus Argus)[J].Transactions of Oceanology and Limnology,2015(3):64-70(in Chinese).
[7]吕俊超,张晓华,王燕,等.养殖大菱鲆病原菌--杀鲑气单胞菌无色亚种的分离鉴定和组织病理学研究[J].中国海洋大学学报,2009,39(1):91-95.Lv J C,Zhang X H,Wang Y,et al.Isolation and identification of bacterial pathogen:Aeromonas salmonicida subsp.achromogenes in cultured turbot and histopathological study[J].Periodical of Ocean University of China,2009,39(1):91-95(in Chinese).
[8]Fontes M C,Saavedra M J,Martins S C,et al.Phylogenetic identification of Aeromonas from pigs slaughtered for consumption in slaughterhouses at the north of Portugal[J].International Journal of Food Microbiology,2011,146(2):118-122.
[9]林如涛,周作勇,王小林,等.羊源杀鲑气单胞菌16Sr RNA基因的克隆测序及分析[J].西南大学学报(自然科学版),2011,33(8):46-51.Lin R T,Zhou Z Y,Wang X L,et al.Cloning and sequence analysis of the 16S r RNA gene of Aeromonas salmonicida in goat[J].Journal of Southwest University(Natural Science Edition),2011,33(8):46-51(in Chinese).
[10]Austin B,Austin D A.Bacterial Fish Pathogens:Disease of Farmed and Wild Fish[M].4th ed.Netherlands:Springer,2007:162-164.
[11]曹成易,汪开毓,王玲,等.大西洋鲑杀鲑气单胞菌的分离鉴定[J].淡水渔业,2009,39(1):54-57.Cao C Y,Wang K Y,Wang L,et al.Isolation and identification of pathogenic bacteria causing ulcer disease of Atlantic salmon[J].Freshwater Fisheries,2009,39(1):54-57(in Chinese).
[12]Gulla S,Lund V,Kristoffersen A B,et al.vap A(A-layer)typing differentiates Aeromonas salmonicida subspecies and identifies a number of previously undescribed subtypes[J].Journal of Fish Diseases,2016,39(3):329-342.
[13]刘宗晓,刘荭,史秀杰,等.杀鲑气单胞菌的实时定量PCR检测方法的建立和应用[J].海洋水产研究,2008,29(5):83-88.Liu Z X,Liu H,Shi X J,et al.Development and application of real-time PCR assay for detecion of Aeromonas salmonicida[J].Marine Fisheries Research,2008,29(5):83-88(in Chinese).
[14]张惟才,朱力,王玉飞.实时荧光定量PCR[M].北京:化学工业出版社,2013.Zhang W C,Zhu L,Wang Y F.Real-time QuantitativePCR[M].Beijing:Chemical Industry Press,2013(in Chinese).
[15]Bustin S A,Benes V,Nolan T,et al.Quantitative realtime RT-PCR-a perspective[J].Journal of Molecular Endocrinology,2005,34(3):597-601.
[16]石晓璐,扈庆华,张佳峰,等.多重实时PCR快速同时检测沙门菌和志贺菌[J].中华流行病学杂志,2006,27(12):1053-1056.Shi X L,Hu Q H,Zhang J F,et al.Rapid simultaneous detection of Salmonella and Shigella using modified molecular beacons and real-time PCR[J].Chinese Journal of Epidemiology,2006,27(12):1053-1056(in Chinese).
[17]李富祥,赵德宏,宋建领,等.猪丹毒杆菌Taq Man荧光定量PCR检测方法的建立[J].中国预防兽医学报,2016,38(5):385-389.Li F X,Zhao D H,Song J L,et al.Development of TaqMan real-time PCR assay for detection of Erysipelothrix rhusiopathiae[J].Chinese Journal of Preventive Veterinary Medicine,2016,38(5):385-389(in Chinese).
[18]荣小军,廖梅杰,张正,等.迟缓爱德华氏菌SYBRGreen I实时荧光定量PCR检测方法的建立及其应用[J].水产学报,2013,37(12):1829-1838.Rong X J,Liao M J,Zhang Z,et al.Development of an SYBR Green I real-time PCR assay for detection of Edwardsiella tarda and its application[J].Journal of Fisheries of China,2013,37(12):1829-1838(inChinese).
[19]王国良,刘璐,李思源.這鱼诺卡氏菌SYBR GreenⅠ实时荧光定量PCR检测方法的建立与应用[J].水产学报,2012,36(4):509-513.Wang G L,Liu L,Li S Y.Development of a SYBRGreenⅠreal-time PCR assay for detection of Nocardia seriolae and its application[J].Journal of Fisheries of China,2012,36(4):509-513(in Chinese).
[20]O’Regan E,Mc Cabe E,Burgess C,et al.Development of a real-time multiplex PCR assay for the detection of multiple Salmonella serotypes in chicken samples[J].BMC Microbiology,2008,8:156.
[21]Alperi A,Figueras M J,Inza I,et al.Analysis of 16Sr RNA gene mutations in a subset of Aeromonas strains and their impact in species delineation[J].International Microbiology,2008,11(3):185-194.
[22]Kulkarni A,Caipang C M A,Brinchmann M F,et al.Loop-mediated isothermal amplification-an assay for the detection of atypical furunculosis caused by Aeromonas salmonicida in Atlantic cod,Gadus morhua[J].Journal of Rapid Methods and Automation in Microbiology,2009,17(4):476-489.
[23]童裳亮,王作芸,周建玲.杀鲑气单胞菌的快速检测[J].动物检疫,1993,10(5):7-8.Tong S L,Wang Z Y,Zhou J L.Rapid detection of Aeromonas salmonicida using ELISA[J].Animal Quarantine,1993,10(5):7-8(in Chinese).