[1]张兴民,张焕容*,汤 承.山羊4种腹泻病原菌多重PCR检测方法的建立[J].中国预防兽医学报,2018,(02):122-126.[doi:0.3969/j.issn.1008-0589.201704013]
 ZHANG Xing-min,ZHANG Huan-rong*,TANG Cheng.Establishment of multiplex PCR for detection of four kinds of diarrhea pathogenic bacteria in goats[J].Chinese journal of preventive veterinary medicine,2018,(02):122-126.[doi:0.3969/j.issn.1008-0589.201704013]
点击复制

山羊4种腹泻病原菌多重PCR检测方法的建立()
分享到:

《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2018年02
页码:
122-126
栏目:
诊断和检测技术
出版日期:
2018-02-15

文章信息/Info

Title:
Establishment of multiplex PCR for detection of  four kinds of diarrhea pathogenic bacteria in goats
文章编号:
1008-0589(2018)2-0122-05
作者:
 

张兴民张焕容*汤 承

 (西南民族大学 生命科学与技术学院,四川 成都 610041)
Author(s):
 

ZHANG Xing-min ZHANG Huan-rong* TANG Cheng

 (College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041, China)
关键词:
产肠毒素大肠杆菌沙门菌志贺氏菌奇异变形杆菌多重PCR
Keywords:
 Enterotixigenic Escherichia coli  Salmonella  Shigella  Proteus mirabilis  multiplex PCR
分类号:
S852.61
DOI:
0.3969/j.issn.1008-0589.201704013
文献标志码:
A
摘要:
为建立检测引起山羊腹泻的4种主要病原菌的多重PCR方法,本研究针对产肠毒素大肠杆菌(ETEC) K99基因,沙门菌invA基因,志贺氏菌侵袭性质粒H (ipaH)基因和奇异变形杆菌ureR基因设计4对特异性引物,通过对反应条件优化,建立快速检测引起山羊腹泻的4种主要病原菌的多重PCR方法,并应用该方法检测山羊腹泻样品。结果显示该多重PCR方法对巴氏杆菌等其它6种细菌无扩增;其敏感性分别为ETEC 103 cfu/mL、沙门菌102 cfu/mL、奇异变形杆菌102 cfu/mL、志贺氏菌103 cfu/mL;经检测该方法稳定性也好。应用该多重PCR方法对21份山羊临床腹泻样品的检测结果与细菌分离鉴定结果一致,无漏检。本研究建立的多重PCR方法可实现从山羊粪便样品中同时检测4种腹泻病原菌,对山羊腹泻病原菌的快速检测提供了技术支持。
  
Abstract:
This study was to establish a multiplex-PCR method for detection of four pathogens causing goat diarrhea. In this study, four pairs of primers were designed based on the k99 gene in ETEC, invA gene in Salmonella spp, ipaH gene in Shigella spp and ureR gene in Proteus mirabilis. The concentrations of the primes and the anneling temperatures were optimized to determine the reaction conditions, and the multiplex PCR was used to detect goat diarrhea samples. The results showed the multiplex PCR amplification bands were accordant with the expected bands, and those of 6 other pathogens were detected negatively. Under the optimized conditions, the detection sensitivity of the multiplex PCR for the four pathogens was 103 cfu/mL for Enterotixigenic Escherichia coli, 102 cfu/mL for Salmonella spp, 102 cfu/mL for Proteus mirabilis and 103 cfu/mL for shigella spp, and the stability of the  method was good. The multiplex PCR method was applied to detect 21 goat diarrhea samples, and the results were consistent with those of bacteria isolation and identifiction. In this study, the multiplex PCR method can detect four kinds of diarrheal pathogens from goat feces, which provides technical support for rapid detection of goat diarrhea pathogens.

参考文献/References:

[1] 妥鑫,刘永明,黄美州,等. 羔羊腹泻细菌和病毒病原的研究进展[J]. 中国畜牧兽医,2016,43(3):831-836.
[2]王晶晶,夏宾雁,岳华,等. 四川省部分地区山羊沙门氏菌健康带菌率及耐药性调查[J]. 中国畜牧兽医,2015,42(2):472-477.
[3]陈曦,王远微,王永,等. 山羊腹泻粪便中奇异变形杆菌的分离鉴定及系统发育分析[J]. 中国畜牧兽医,2014,41(8):215-218.
[4]王克恭. 幼畜腹泻与防治的研究进展[J]. 内蒙古农业大学学报(自然科学版),1985(2):71-75.
[5]汤继顺,惠文巧, 朱德建,等. 羔羊腹泻病原菌常规分离鉴定及药敏试验[J]. 中国草食动物科学,2016,36(4):42-45.
[6]余倩,黄梦娜. 三种食源性致病菌的多重PCR快速检测及应用[J]. 生物技术通报,2014(7):64-68.
[7]Jofre A, Martin B, Garriga M, et al. Simultaneous detection of Listeria monocytogenes and Salmonella by multiplex PCR in cooked ham [J]. Food Microbiol, 2005, 22(1): 109-115.
[8]曲泽慧,陈佩佩,张爱芹,等. 产肠毒素大肠杆菌双重PCR检测方法的建立[J]. 中国畜牧兽医,2013,40(7):65-68.
[9]郇娟,戈红雨,张成龙,等. 双重PCR检测沙门氏菌和变形杆菌方法的建立[J]. 天津医科大学学报,2015,21(1):84-86.
[10]姜宣鹏,张焕容. 产肠毒素大肠埃希菌 K99菌毛基因的克隆与原核表达[J]. 动物医学进展,2014(5):12-16.
[11]舒畅,姜琛璐,钟慈平,等. 三种食源性致病菌多重PCR检测方法的建立[J]. 食品工业科技,2014,35(12):49-54.
[12]林红乐,文岚,张如胜. PCR快速检测奇异变形杆菌的研究[J]. 实用预防医学,2009,16(2):560-562.
[13]Worrall L J, Vuckovic M, Strynadka N C J. Crystal structure of the C-terminal domain of the  Salmonella, type III secretion system export apparatus protein InvA [J]. Protein Sci, 2010, 19(5):1091-1096.
[14]Mccarthy N, Reen F J, Buckley J F, et al. Sensitive and rapid molecular detection assays for Salmonella enterica serovars Typhimurium and Heidelberg [J]. J Food Prot, 2009, 72(11):2350-2357.
[15]王琼,唐俊妮,汤承,等. 一种采用微波炉加热快速提取细菌DNA用于PCR扩增的方法[J]. 西南民族大学学报,2015,42(2):150-155.
[16]Yuste M, Orden J A, De L F R, et al. Polymerase chain reaction typing of genes of the locus of enterocyte effacement of ruminant attaching and effacing Escherichia coil [J].  Can J Vet Res, 2008, 72(5): 444.
[17]Jin J D, Lee D S, Shin E K, et al. Molecular Typing by Random Amplification of Polymorphic DNA (RAPD) and Detection of Virulence Genes of Salmonella enterica subspecies enterica serovar Gallinarum biovar Gallinarum [J]. J Vet Med Sci, 2006, 68(12): 1321-1326.
[18]Michelim L, Muller G, Zacaria J, et al. Comparison of PCR- based molecular markers for the characterization of Proteus mirabilis clinical isolates [J]. Braz J Infect Dis, 2008, 12(5): 423.
[19]Truong Q L, Yoon B I, Hahn T W. Development of a multiplex PCR to identify Salmonella,  Leptospira and Brucella species in tissue samples [J]. Korean J Vet Res, 2012, 52(2): 75-82.
(本文编辑:李   娜)

相似文献/References:

[1]刘淑杰,李永明,徐子伟*,等.ETEC粘附素蛋白亚基Fae G在乳酸乳球菌中的表达及免疫反应性分析 [J].中国预防兽医学报,2009,(09):688.
 LIU Shu-jie,LI Yong-ming,XU Zi-wei*,et al.Cloning and expression of the Fae G gene of enterotoxigenic Escherichia coli K88 fimbrial subunit in Lactococcus lactis [J].Chinese journal of preventive veterinary medicine,2009,(02):688.
[2]曲泽慧,陈佩佩,张爱芹,等.产肠毒素大肠杆菌多重PCR检测方法的建立[J].中国预防兽医学报,2012,(12):980.[doi:doi: 10.3969/j.issn.1008-0589.2012.12.12]
 QU Ze-hui,CHEN Pei-pei,ZHANG Ai-qin,et al.Multiplex PCR for detection of virulence geneof enterotoxigenic Escherichia coli[J].Chinese journal of preventive veterinary medicine,2012,(02):980.[doi:doi: 10.3969/j.issn.1008-0589.2012.12.12]
[3]郁 磊,吴 娟,朱 军,等.产肠毒素大肠杆菌K88ab/K88ad菌毛操纵子 fae全基因的克隆、表达及生物学活性的初步研究[J].中国预防兽医学报,2013,(01):23.
 YU Lei,WU Juan,ZHU Jun,et al.Cloning and expression of K88ab/K88ad fimbrial operons from enterotoxigenic Escherichia coli and their bioactivity[J].Chinese journal of preventive veterinary medicine,2013,(02):23.
[4]杨绪秋,方一臻,张艳艳,等.产肠毒素大肠杆菌F4菌毛抗体间接ELISA检测方法的建立[J].中国预防兽医学报,2014,(08):655.[doi:10.3969/j.issn.1008-0589.2014.08.17]
 YANG Xu-qiu,FANG Yi-zhen,ZHANG Yan-yan,et al.Development of an indirect ELISA for detecting specific antibodyto F4 pilus protein of enterotoxigenic Escherichia coli[J].Chinese journal of preventive veterinary medicine,2014,(02):655.[doi:10.3969/j.issn.1008-0589.2014.08.17]
[5]许 明,龚建森,林 勇,等.鸭源沙门菌江苏分离株的生物学特性研究[J].中国预防兽医学报,2014,(10):775.[doi:10.3969/j.issn.1008-0589.2014.10.08]
 XU Ming,GONG Jian-sen,LIN Yong,et al.Characterization of Salmonella spp. isolated from ducksin Jiangsu province[J].Chinese journal of preventive veterinary medicine,2014,(02):775.[doi:10.3969/j.issn.1008-0589.2014.10.08]
[6]赵 鹏,夏 爽,关乃瑜,等.产肠毒素大肠杆菌FaeG-mSTa-LTb-STb融合表达及其包涵体口服免疫小鼠诱导的免疫应答[J].中国预防兽医学报,2015,(10):790.[doi:10.3969/j.issn.1008-0589.2015.10.13]
 ZHAO Peng,XIA Shuang,GUAN Nai-yu,et al.Immune response of inclusion bodies from recombinant E.coli expressing FaeG-mSTa-LTb-STb fusion protein by the oral routein mouse model[J].Chinese journal of preventive veterinary medicine,2015,(02):790.[doi:10.3969/j.issn.1008-0589.2015.10.13]
[7]齐 宇,王 开,伊淑帅,等.产肠毒素大肠杆菌K88诱发BALB/c鼠肠炎模型的建立及评价[J].中国预防兽医学报,2016,(01):19.
 QI Yu,WANG Kai,YI Shu-shuai,et al.Establishment and evaluation of bacterial enteritis model inBALB/c mice induced by enterotoxigenic Escherichia coli K88[J].Chinese journal of preventive veterinary medicine,2016,(02):19.
[8]葛俊伟,宋满满,赵 鹏,等.产肠毒素大肠杆菌STa突变体、LTB和STb融合蛋白在乳酸菌中组成型分泌表达及其免疫原性分析[J].中国预防兽医学报,2017,(12):1003.[doi:10.3969/j.issn.1008-0589.201702025]
 GE Jun-wei,SONG Man-man,ZHAO Peng,et al.Secretory expression of mSTa, LTB and STb fusion protein of ETEC in Lactococcus lactis and the immunogenicity analysis[J].Chinese journal of preventive veterinary medicine,2017,(02):1003.[doi:10.3969/j.issn.1008-0589.201702025]
[9]杨 溢?,杨玮枫?,张 东,等.抗沙门菌PEG菌毛单克隆抗体的制备及初步临床应用[J].中国预防兽医学报,2018,(05):438.[doi:0.3969/j.issn.1008-0589.201708016]
 YANG Yi?,YANG Wei-feng?,ZHANG Dong,et al.Preparation and preliminary application of monoclonal antibodies against PEG fimbriae of Salmonella[J].Chinese journal of preventive veterinary medicine,2018,(02):438.[doi:0.3969/j.issn.1008-0589.201708016]
[10]徐耀辉?*,齐亚如?,邓同炜,等. 河南地区麻种鸡死胚中沙门菌的分离鉴定与耐药性分析[J].中国预防兽医学报,2018,(10):891.[doi:10.3969/j.issn.1008-0589.201805001]
 XU Yao-hui?*,QI Ya-ru?,DENG Tong-wei,et al. Isolation and antimicrobial resistance identification of Salmonella from dead chicken embryos inPartridge Shank breeding farms in Henan[J].Chinese journal of preventive veterinary medicine,2018,(02):891.[doi:10.3969/j.issn.1008-0589.201805001]

更新日期/Last Update: 2018-03-09