CN104152555A - 奶牛致病菌五重pcr反应试剂盒 - Google Patents
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Abstract
本发明涉及一种奶牛致病菌五重PCR反应试剂盒,包括检测产肠毒素大肠杆菌LT基因、产肠毒素性大肠杆菌ST基因、沙门氏菌invA基因、金黄色葡萄球菌nuc基因、单核细胞增生性李氏杆菌hlyA基因的五对引物。本发明以规模化牛场环境中常见的大肠杆菌、沙门氏菌、金黄色葡萄球菌、单核增生性李氏杆菌为研究对象,利用这些致病菌不同的靶基因建立特异性强、可靠性和灵敏度高的多重PCR反应试剂盒,这不仅可以提高检测的灵敏度和准确性,且能够检测到一些传统方法很难检测到的致病菌,还为快速、高效检测和控制牛场环境中多种致病菌提供思路和方法。
Description
技术领域
本发明属于动物分子生物学领域,涉及一种奶牛致病菌五重PCR反应试剂盒。
背景技术
随着规模化奶牛场的不断发展,国内外有关机构和研究者逐渐重视牛舍的环境质量问题。现在奶牛饲养密度大,每头牛的生活和运动空间都相对拥挤,牛舍环境质量下降,病原菌繁殖加快,致使牛舍受到污染。致病菌、条件性致病菌和非病原菌都可存在牛舍环境中,当这些细菌达到一定程度时,就会暴发系列疾病。牛舍环境质量的好坏不仅直接影响了牛的正常生长和生产性能,还决定了畜产品的质量和养牛业的生产效益。所以,环境中致病菌是影响人和动物健康的潜在因素,环境卫生管理一有疏忽,它们就会趁虚而入,给养殖业造成经济损失,严重者甚至影响到社会发展和政治稳定。因此检测和控制环境中病原菌的问题需亟待解决。
环境中的病原菌主要有大肠杆菌、沙门氏菌、金黄色葡萄球菌、单核增生性李氏杆菌等,它们在一定程度上均可导致动物的感染。致病型大肠杆菌有很多种,其中,肠毒素型大肠杆菌是引起腹泻最常见的大肠杆菌。在世界范围内,它可引起犊牛严重的水样腹泻。这种菌主要在小肠近端居留,通过产生肠毒素而致病。产肠毒素大肠杆菌有不耐热肠毒素(LT)和耐热肠毒素(ST)两种。沙门氏菌的主要致病物质有脂多糖、肠毒素、内毒素、侵袭力。沙门氏菌的侵袭蛋白(invasion protein,inv)决定细菌粘附和进入宿主上皮细胞的能力,与细菌致病性密切相关,它是由invA、invB、invC、invD和invE等一组基因编码,其中invA为沙门氏菌的主要毒力因子,也是毒力岛SPI基因之一。染色体上的侵袭基因invA是一段只存在于致病性沙门菌中独特的保守基因序列。在沙门氏菌属中invA基因序列有很高的同源性,与其它生物没有同源性,是目前沙门氏菌检测的主要靶点。金黄色葡萄球菌可以分泌多种毒力因子,如葡萄球菌溶血素、肠毒素、凝固酶、耐热核酸酶等,这些毒素和酶致病性强,可引起创伤性感染、脓肿、蜂窝织炎、乳腺炎、关节炎、败血症、毒素休克综合症、心内膜炎、中毒性呕吐、肠炎等症状。金黄色葡萄球菌nuc基因编码的耐热核酸酶是一种胞外酶,为金黄色葡萄球菌所特有,耐热核酸酶蛋白的nuc基因编码已全部定序,只要通过nuc基因的特异性扩增就可以快捷准确地检测出金黄色葡萄球菌。单核增生性李氏杆菌对人和动物均有侵袭力,可导致食物中毒,是一种人畜共患的食品病原菌。人、畜感染后主要表现为脑膜炎.败血症及孕妇流产,有的可出现单细胞增多,溶血素为李斯特菌的主要致病因子,由hly基因编码,单核细胞增多性李斯特菌hlyA基因具有较高的保守性和特异性。因此,hly基因可作为单核增生性李氏杆菌的靶基因。
传统的分离培养、生化鉴定等检测方法无法对难培养或不可培养的微生物进行检测,而且特异性不高、灵敏度低、操作繁琐耗时,难以满足飞速发展的集约化养殖生产对检测速度的要求,不能实现有效的监测、预防作用。因此,开发快速检测环境中致病菌技术,并对其进行控制成为保障畜舍环境公共卫生和畜产品安全的重要任务之一。
在国外,多年来,许多研究人员已创建了不少快速、特异、敏感、低耗且实用的细菌学检测方法。尤其是免疫酶联法、DNA探针、多聚酶联反应、生物感受器、基因芯片等技术的发展和应用,明显提高了人类对环境致病菌的认识及检测水平。但国内对致病菌的免疫学和分子生物学检测方法也有一定的研究,对其应用较少,相比传统方法所需仪器设备相比要求高,在很大程度上限制了这些方法的应用,大大降低了其实用价值。多重PCR技术简单、快速,实用性高,因此,可以考虑应用多重PCR技术为奶牛致病菌的快速检测提供一种新的思路。
发明内容
本发明的目的是提供一种奶牛致病菌五重PCR反应试剂盒,克服关于奶牛致病菌传统检测手段特异性不高、灵敏度低、操作繁琐耗时等问题,还能克服传统检测不能实现有效监测和预防作用的问题。
本发明通过以下技术方案来实现:奶牛致病菌五重PCR反应试剂盒,包括10×PCR反应缓冲液,Mg2+,dNTP,引物和Taq酶,所述的引物为:
(1)检测产肠毒素性大肠杆菌LT基因的引物:
F1:5′-CTGACTCTAGACCCCCAGATGAAAT-3′(SEQ ID No.1)
R1:5′-CTGTCCTGCTAAGTGAGCACTTCTC-3′(SEQ ID No.2)
(2)检测产肠毒素性大肠杆菌ST基因的引物:
F2:5′-AAAACCAGATAGCCAGACAATTAAC-3′(SEQ ID No.3)
R2:5′-AGGATTACAACAAAGTTCACAGCAG-3′(SEQ ID No.4)
(3)检测沙门氏菌invA基因的引物:
F3:5′-GATTTGTCCTCCGCTCTGTCTACTT-3′(SEQ ID No.5)
R3:5′-CAATACGATGCTGTTATCGTCCAGG-3′(SEQ ID No.6)
(4)检测金黄色葡萄球菌nuc基因的引物:
F4:5′-TAGGGATGGCTATCAGTAATGTTTC-3′(SEQ ID No.7)
R4:5′-CCATCAGCATAAATATACGCTAAGC-3′(SEQ ID No.8)
(5)检测单核细胞增生性李氏杆菌hlyA基因的引物:
F5:5′-TATGATGACGAAATGGCTTACAGTG-3′(SEQ ID No.9)
R5:5′-GAACAATTTCGTTACCTTCAGGATC-3′(SEQ ID No.10)。
进一步的,所述的试剂盒的反应体系为10×Buffer 5μL,25mmol·L-1的Mg2+6μL,5U·μL-1的Taq酶0.4μL,dNTP 3.0μL,模板DNA 1.0μL,10μmmol·L-1的上、下游引物各0.5μL,用灭菌去离子水补至50μL。
进一步的,所述的试剂盒的反应条件为94℃预变性5min,94℃变性45s,63℃退火45s,72℃延伸45s,共进行30个循环,最后72℃终延伸10min。
采用上述技术方案的积极效果:本发明以规模化牛场环境中常见的大肠杆菌、沙门氏菌、金黄色葡萄球菌、单核增生性李氏杆菌为研究对象,利用这些致病菌不同的靶基因建立特异性强、可靠性和灵敏度高的多重PCR反应试剂盒,这不仅可以提高检测的灵敏度和准确性,且能够检测到一些传统方法很难检测到的致病菌,还为快速、高效检测和控制牛场环境中多种致病菌提供思路和方法,而且对及时有效控制和预防环境中病原菌传播、降低病原菌对动物和人类带来的危害、加强牛舍环境公共卫生的监护与防疫、提高畜产品质量具有十分重大的意义。
附图说明
图1是本发明的试剂盒优化条件结果图;
M-DL2000DNA Marker;1~16-分别对应表1中试验1~16;17-阴性对照;
图2是本发明的试剂盒特异性实验检测结果图;
M-DL2000DNA Marker;1为金黄色葡萄球菌、沙门氏菌、产LT大肠杆菌;2为金黄色葡萄球菌、沙门氏菌、单核增生性李氏杆菌、产ST大肠杆菌;3为金黄色葡萄球菌、沙门氏菌、产LT大肠杆菌、产ST大肠杆菌;4为沙门氏菌、产ST大肠杆菌、产LT大肠杆菌;5为沙门氏菌、产LT大肠杆菌;6为金黄色葡萄球菌、产ST大肠杆菌、产LT大肠杆菌;7为单核增生性李氏杆菌、金黄色葡萄球菌、沙门氏菌、产LT大肠杆菌、产ST大肠杆菌;8为产ST大肠杆菌、产LT大肠杆菌;9为单核增生性李氏杆菌、产ST大肠杆菌;10为阴性对照;
图3是本发明的试剂盒敏感性检测结果图;
M-DL2000DNA Marker;1~7-五种菌浓度均依次为107~101cfu·mL-1。
具体实施方式
本发明中的生物材料来源的说明:
1、引物
检测产肠毒素大肠杆菌LT基因、产肠毒素性大肠杆菌ST基因、沙门氏菌invA基因、金黄色葡萄球菌nuc基因、单核细胞增生性李氏杆菌hlyA基因的引物均为自行设计并委托上海生工生物有限公司合成。
2、菌种
金黄色葡萄球菌(ATCC25923)、沙门氏菌(CVCC325)、单核细胞增生性李氏杆菌(ATCC19111)、产肠毒素大肠杆菌(ATCC83922)购自中国兽医微生物菌种保藏中心。
实施例1
将营养琼脂斜面活化的金黄色葡萄球菌(ATCC25923)、沙门氏菌(CVCC325)、单核细胞增生性李氏杆菌(ATCC19111)、产肠毒素大肠杆菌(ATCC83922)分别接种营养肉汤,37℃培养18h后进行DNA提取。用CTAB/NaCL2酚抽提法提取细菌DNA,取菌液1mL于1.5ml离心管,12000r/min离心2min,弃上清液,TE缓冲液(PH=8.0)洗涤,12000r/min离心2min;于沉淀中加入567μL TE缓冲液,重悬菌体,加入30μL10%SDS和3μL蛋白酶K(20mg/mL),混匀,37℃温浴1h;加入100μL 5mol/L NaCl,充分混匀,再加入80μL CTAB/NaCl溶液,混匀,于65℃温浴10min;加入等体积的氯仿/异戊醇(24:1),混匀,离心4~5min。将上清液转入一个新管中;加入等体积的酚/氯仿/异戊醇(24:24:1),离心5min,将上清转移到新管中;加入0.6体积异丙醇,轻轻混合至DNA沉淀下来,转移沉淀至1mL乙醇中洗涤;离心,弃上清,干燥,重溶于100μL TE缓冲液中,储存备用。
根据产肠毒素大肠杆菌LT基因(V00275)、产肠毒素性大肠杆菌ST基因(M25607)、沙门氏菌invA基因(V43272.1)、金黄色葡萄球菌nuc基因(V01281.1)、单核细胞增生性李氏杆菌hlyA基因的序列(AF253320.1),用Primer 5.0引物设计软件进行设计,5对引物由上海生工生物有限公司合成。引物序列如下:
(1)检测产肠毒素性大肠杆菌LT基因的引物:
F1:5′-CTGACTCTAGACCCCCAGATGAAAT-3′(SEQ ID No.1)
R1:5′-CTGTCCTGCTAAGTGAGCACTTCTC-3′(SEQ ID No.2)
(2)检测产肠毒素性大肠杆菌ST基因的引物:
F2:5′-AAAACCAGATAGCCAGACAATTAAC-3′(SEQ ID No.3)
R2:5′-AGGATTACAACAAAGTTCACAGCAG-3′(SEQ ID No.4)
(3)检测沙门氏菌invA基因的引物:
F3:5′-GATTTGTCCTCCGCTCTGTCTACTT-3′(SEQ ID No.5)
R3:5′-CAATACGATGCTGTTATCGTCCAGG-3′(SEQ ID No.6)
(4)检测金黄色葡萄球菌nuc基因的引物:
F4:5′-TAGGGATGGCTATCAGTAATGTTTC-3′(SEQ ID No.7)
R4:5′-CCATCAGCATAAATATACGCTAAGC-3′(SEQ ID No.8)
(5)检测单核细胞增生性李氏杆菌hlyA基因的引物:
F5:5′-TATGATGACGAAATGGCTTACAGTG-3′(SEQ ID No.9)
R5:5′-GAACAATTTCGTTACCTTCAGGATC-3′(SEQ ID No.10)。
构建50μL的反应体系,对退火温度、Mg2+、引物、Taq酶及dNTPs浓度进行正交试验L16(45),进而筛选最优的PCR反应体系。正交试验方案见表1。
表1PCR条件优化正交设计方案
注:以上均为50μL体系加入量。
取5μL PCR反应产物在1%琼脂糖凝胶上进行电泳,利用凝胶成像系统观察结果并成像。
如图1所示,第16组实验方案效果最佳,扩增条带清晰无杂带,试剂盒的反应体系最佳为10×Buffer 5μL,25mmol·L-1的Mg2+6μL,5U·μL-1的Taq酶0.4μL,dNTP 3.0μL,模板DNA 1.0μL,10μmmol·L-1的上、下游引物各0.5μL,用灭菌去离子水补至50μL。试剂盒的反应条件最佳为94℃预变性5min,94℃变性45s,63℃退火45s,72℃延伸45s,共进行30个循环,最后72℃终延伸10min。
实施例2
随机将金黄色葡萄球菌(ATCC25923)、沙门氏菌(CVCC325)、单核细胞增生性李氏杆菌(ATCC19111)、产肠毒素大肠杆菌(ATCC83922)进行组合,分为9组,1组为金黄色葡萄球菌、沙门氏菌、产LT大肠杆菌的组合,2组为金黄色葡萄球菌、沙门氏菌、单核增生性李氏杆菌、产ST大肠杆菌的组合,3组为金黄色葡萄球菌、沙门氏菌、产LT大肠杆菌、产ST大肠杆菌的组合,4组为沙门氏菌、产ST大肠杆菌、产LT大肠杆菌的组合,5组为沙门氏菌、产LT大肠杆菌的组合,6组为金黄色葡萄球菌、产ST大肠杆菌、产LT大肠杆菌的组合,7组为单核增生性李氏杆菌、金黄色葡萄球菌、沙门氏菌、产LT大肠杆菌、产ST大肠杆菌的组合,8组为产ST大肠杆菌、产LT大肠杆菌的组合,9为单核增生性李氏杆菌、产ST大肠杆菌的组合,利用试剂盒PCR扩增检测多重PCR反应的特异性。结果如图2所示,非目标菌及阴性对照均未出现扩增且引物间无交叉反应,说明引物及反应均具有较强特异性,不会扩增出非目的片段。
实施例3
等量混合5种菌液,使每种菌的含量达到107cfu/mL,以10倍稀释法进行梯度稀释,即每种菌的含量分别是107cfu/mL、106cfu/mL、105cfu/mL、104cfu/mL、103cfu/mL、102cfu/mL、101cfu/mL,分别提取DNA按已优化的多重PCR反应条件分别进行PCR扩增,测定多重PCR扩增的敏感性。结果如图3所示,试剂盒对金黄色葡萄球菌、产肠毒素大肠杆菌、沙门氏菌敏感性都为101cfu·mL-1,单增李斯特菌的敏感性为102cfu·mL-1,灵敏度高,可检测出样品中的痕量DNA。
本发明在传统检测方法基础上,建立起规模化牛场环境中常见致病菌的多重PCR检测技术,可广泛应用于牛场致病菌的检测及流行病学调查中,这种测定方法有望推广应用于环境监测、水源检测,食品卫生监督、商品检验检疫等领域。可为有效控制这几种菌的流行提供科学依据和预警资料,也为监测这几种致病菌提供理论基础。
Claims (3)
1.一种奶牛致病菌五重PCR反应试剂盒,包括10×PCR反应缓冲液,Mg2+,dNTP,引物和Taq酶,其特征在于:所述的引物为:
(1)检测产肠毒素性大肠杆菌LT基因的引物:
F1:5′-CTGACTCTAGACCCCCAGATGAAAT-3′(SEQ ID No.1)
R1:5′-CTGTCCTGCTAAGTGAGCACTTCTC-3′(SEQ ID No.2)
(2)检测产肠毒素性大肠杆菌ST基因的引物:
F2:5′-AAAACCAGATAGCCAGACAATTAAC-3′(SEQ ID No.3)
R2:5′-AGGATTACAACAAAGTTCACAGCAG-3′(SEQ ID No.4)
(3)检测沙门氏菌invA基因的引物:
F3:5′-GATTTGTCCTCCGCTCTGTCTACTT-3′(SEQ ID No.5)
R3:5′-CAATACGATGCTGTTATCGTCCAGG-3′(SEQ ID No.6)
(4)检测金黄色葡萄球菌nuc基因的引物:
F4:5′-TAGGGATGGCTATCAGTAATGTTTC-3′(SEQ ID No.7)
R4:5′-CCATCAGCATAAATATACGCTAAGC-3′(SEQ ID No.8)
(5)检测单核细胞增生性李氏杆菌hlyA基因的引物:
F5:5′-TATGATGACGAAATGGCTTACAGTG-3′(SEQ ID No.9)
R5:5′-GAACAATTTCGTTACCTTCAGGATC-3′(SEQ ID No.10)。
2.根据权利要求1所述的奶牛致病菌五重PCR反应试剂盒,其特征在于:所述的试剂盒的反应体系为10×Buffer 5μL,25mmol·L-1的Mg2+6μL,5U·μL-1的Taq酶0.4μL,dNTP 3.0μL,模板DNA 1.0μL,10μmmol·L-1的上、下游引物各0.5μL,用灭菌去离子水补至50μL。
3.根据权利要求1所述的奶牛致病菌五重PCR反应试剂盒,其特征在于:所述的试剂盒的反应条件为94℃预变性5min,94℃变性45s,63℃退火45s,72℃延伸45s,共进行30个循环,最后72℃终延伸10min。
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CN107699639A (zh) * | 2017-11-23 | 2018-02-16 | 广西壮族自治区兽医研究所 | 一种鉴别牛轮状病毒和产肠毒大肠杆菌的引物及方法 |
CN108179211A (zh) * | 2018-03-14 | 2018-06-19 | 贵州省畜牧兽医研究所 | 产肠毒素性大肠杆菌耐热肠毒素基因SYBR Green I荧光定量PCR诊断试剂盒 |
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Non-Patent Citations (3)
Title |
---|
牟恺等: "时检测四种病原茵的PCR方法研究", 《山东农业大学学报(自然科学版)》, vol. 41, no. 2, 31 December 2010 (2010-12-31), pages 253 - 257 * |
程晓艳: "几种食源性致病菌快速检测技术的建立", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》, no. 2, 15 February 2013 (2013-02-15), pages 024 - 48 * |
马广强等: "直接从粪便中检测致犊牛腹泻产肠毒素大肠杆菌的双重PCR方法的建立与应用", 《中国人兽共患病杂志》, vol. 22, no. 2, 31 December 2006 (2006-12-31), pages 153 - 156 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107699639A (zh) * | 2017-11-23 | 2018-02-16 | 广西壮族自治区兽医研究所 | 一种鉴别牛轮状病毒和产肠毒大肠杆菌的引物及方法 |
CN107699639B (zh) * | 2017-11-23 | 2019-12-20 | 广西壮族自治区兽医研究所 | 一种鉴别牛轮状病毒和产肠毒大肠杆菌的引物及方法 |
CN108179211A (zh) * | 2018-03-14 | 2018-06-19 | 贵州省畜牧兽医研究所 | 产肠毒素性大肠杆菌耐热肠毒素基因SYBR Green I荧光定量PCR诊断试剂盒 |
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