CN102653786B - 一种创伤弧菌pcr检测试剂盒及检测方法 - Google Patents
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Abstract
本发明提供了一种创伤弧菌PCR检测试剂盒，主要包括PCR特异性扩增引物、DNA聚合酶、脱氧三磷酸核苷混合物和PCR缓冲液，所述特异性扩增引物如下：上游引物v-toxR(+)：5′-TGATTCCAGCCTAACTCAAG-3′，下游引物vv-toxR(-)：5′-TTTGAACCGCGTC AGTACTG-3′。本发明通过提供一种对创伤弧菌特定基因片段具有特异性的引物及其利用上述引物检测是否存在创伤弧菌特定基因片段，进而确定标本中是否存在创伤弧菌。本发明检测试剂盒和检测方法具有敏感性高、特异性强、方便快捷、适用范围广等优点，可解决水产养殖过程中病原体的快速检测。
Description
(一)技术领域
本发明涉及一种创伤弧菌PCR检测试剂盒及检测方法。
(二)背景技术
创伤弧菌(Vibrio vulnificus)是一种革兰氏阴性嗜盐菌，主要分布于近海海洋环境中，因此又称海洋弧菌，是水产养殖虾、蟹和牡蛎等水生动物中最常见、流行最广、危害最严重的致病菌之一。与霍乱弧菌、肠炎弧菌并列为造成人类感染疾病之三大弧菌之一。人类通过食用被该菌污染的水产品或因创口接触带菌的海水和海洋生物而受传染，临床症状主要表现为胃肠道感染、严重的伤口感染、蜂窝组织炎、败血症及肢体坏死，过程相当迅速且死亡率高。一旦出现败血症，其病死率高达50～60％，在我国创伤弧菌感染多发于沿海地区。
由于创伤弧菌的高致病性危害，引起了世界各国的广泛关注，业已成为水产养殖和进出口水产重点检验检疫的致病菌之一。目前对该菌的检测仍以分离培养和理化鉴定为主，该方法虽然可靠，但整个过程耗时费力，灵敏度比较差，不能满足快速检测的需要。特别是由于创伤弧菌本身的表型不稳定，非典型菌株较多见，使得鉴定过程和结果复杂化。免疫学方法主要有免疫荧光、放射免疫法检测和酶联免疫吸附试验等，这些方法虽然具有一定的特异性和敏感性，但通常需要结合细菌分离才能进行，也难满足快速检测的需要。
随着分子生物学技术的发展，检测手段的日益改进，RT-PCR技术已被应用于细菌的诊断。虽然常规PCR法已具备快速、特异、灵敏度高的优点，但由于死菌中的DNA降解速度较慢，不能区分死菌和活菌，而对于某些感染性疾病来说，明确标本中的细菌死活可以使治疗更具针对性。RT-PCR技术是一种新的简便、快速、灵敏、特异并能区分细菌死活的检测手段。目前未有利用RT-PCR技术检测创伤弧菌的检测方法，以及对创伤弧菌toxR特定基因片段具有特异性的RT-PCR引物的报道。
(三)发明内容
本发明目的是提供一种能够特异性检测创伤弧菌的PCR检测试剂盒及检测方法。
本发明采用的技术方案是：
一种创伤弧菌PCR检测试剂盒，主要包括PCR特异性扩增引物、DNA聚合酶、脱氧三磷酸核苷混合物和PCR缓冲液，其特征在于：所述特异性扩增引物如下：
v-toxR(+)：5′-TGATTCCAGCCTAACTCAAG-3′
vv-toxR(-)：5′-TTTGAACCGCGTCAGTACTG-3′。
本发明试剂盒的关键在于引物的设计，试剂盒中的其他组分，如DNA聚合酶、脱氧三磷酸核苷混合物和PCR缓冲液等，对于本领域技术人员来说属于公知常识，可根据需要选用。
本发明试剂盒的靶基因为创伤弧菌的toxR(Vibrio vulnificustransmembrane regulatory protein ToxR)-GenBank登陆号：AF170883，本发明引物与所述靶基因的21位～250位核酸序列的一部分或其互补链互补。本发明扩增产物序列如下：tgattccagc ctaactcaag cgatttctac cttacgcaaaatgctcaaag actctacaaa gtcccctgag tttgtgaaaa cggttccaaa acgtggttat cagttgatctgttcggttga gcgcattaac ccgctcctgt cagattcaac caacaacgtg aatgacgcag cttctgaagcattagatcaa gaagaattag aaaacgaaat cagtactgac gcggttcaaa，全长230bp。
本发明还涉及一种利用所述试剂盒检测样品中创伤弧菌的方法，所述方法包括：
(1)按常规方法提取待测样品DNA；待测样品可以是鱼体的发病部位，如体表或相关组织，可直接提取DNA作为模板；待测样品也可以是细菌待检测标本，待测细菌用普通营养肉汤培养基37℃培养过夜，取1ml增菌液进行模板RNA的提取，并经反转录得cDNA作为扩增模板；
(2)以待测样品DNA为模板，加入特异性扩增引物、DNA聚合酶、脱氧三磷酸核苷混合物和PCR缓冲液配制PCR反应液，进行PCR扩增；所述特异性扩增引物如下：
v-toxR(+)：5′-TGATTCCAGCCTAACTCAAG-3′
vv-toxR(-)：5′-TTTGAACCGCGTCAGTACTG-3′；
(3)扩增产物进行琼脂糖凝胶电泳(1％琼脂糖凝胶，150V电泳约20min)，若电泳结果出现230bp特异性条带，则判断为阳性，否则为阴性。
所述PCR扩增条件如下：94℃5min一个循环；94℃30s，60℃30s，72℃30s，30个循环；72℃延伸反应7min。
本发明的有益效果主要体现在：本发明通过提供一种对创伤弧菌特定基因片段具有特异性的引物及其利用上述引物检测是否存在创伤弧菌特定基因片段，进而确定标本中是否存在创伤弧菌。本发明检测试剂盒和检测方法具有敏感性高、特异性强、方便快捷、适用范围广等优点，可解决水产养殖过程中病原体的快速检测。
(四)附图说明
图1为本发明所述引物进行RT-PCR扩增结果的电泳图；M：2kbDNA marker；1：创伤弧菌；2～8：分别为河流弧菌、鳗利斯顿氏菌、嗜水气单胞菌、温和气单胞菌、溶藻弧菌、哈维氏弧菌、副溶血弧菌。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
实施例1：引物设计
通过查阅文献和利用BLAST软件分析，筛选出创伤弧菌特异性基因-Vibrio vulnificus transmembrane regulatory protein ToxR(toxR)-GenBank登陆号：AF170883。设计的引物序列如下：
上游引物：vv-toxR(+)：5′-TGATTCCAGCCTAACTCAAG-3′；
下游引物：vv-toxR(-)：5′-TTTGAACCGCGTCAGTACTG-3′。
实施例2：toxR基因的扩增
1)菌种的准备
所获细菌均通过常规表型方法验证，增菌后确定为纯培养的细菌。取创伤弧菌单个菌落，溶于2ml营养肉汤中，37℃过夜培养(其它菌也接种于营养肉汤中)。
2)模板RNA提取
将培养好的增菌液12000r/min离心1min收集菌体，弃去上清后加入1mlTRIZOL振荡5min，静置5min。加入适量氯仿，静置5min，12000r/min离心15min。取上清，向上层水相中加入等量异丙醇，静置10min，12000r/min离心10min。弃上清，加入75％乙醇1mL洗涤沉淀，7500r/min离心5min，弃上清，室温干燥10min。将沉淀溶于20μLDEPC水，55～60℃水浴助溶。-20℃保存备用。(为去除基因组DNA的污染，可用DNase I加以处理)。
3)反转录
将处理后的RNA用Quantscript RT Kit进行反转录试验。反应体系及条件如下：10×RT mix 2μL，dNTP 2μL，Random 2μL，Quant 1μL，Rnase-free H2O 8μL，RNA 5μL，37℃孵育60min。
4)PCR扩增
将反转录后的cDNA进行PCR扩增。反应体系50μl，其中2×Mix(购自北京博凌科为生物科技有限公司，已含有2×Taq DNA Polymerase，2×PCR Buffer，2×dNTP及PCR反应的优化剂和稳定剂)25μL，H2O 19μl，上游引物2μl(浓度50μM)，下游引物2μl(浓度50M)，模板2μl(浓度约100ng/μL)，反应条件如下：94℃5min一个循环，以下流程30个循环(94℃30s，60℃30s，72℃30s)，72℃延伸反应7min。
5)产物检测
扩增产物用1％琼脂糖凝胶，150V电泳约20min。若电泳结果出现230bp特异性条带，判断为阳性。
实施例3：扩增产物的鉴定与测定
按照实施例2方法通过RT-PCR对1株创伤弧菌和7株非创伤弧菌的标准菌进行toxR基因扩增产物和特异性检测与鉴定，结果如图1和表1。
表1：本实施例实验的菌株与各菌株的扩增结果
| 序号 | 分子量标准品或供试菌株 | 有无扩增 |
| M | 2kb DNA marker |
| 1 | 创伤弧菌(ATCC27562) | 阳 |
| 2 | 河流弧菌(ATCC33816) | 阴 |
| 3 | 鳗利斯顿氏菌(ATCC43306) | 阴 |
| 4 | 嗜水气单胞菌(ATCC7966) | 阴 |
| 5 | 温和气单胞菌(ATCC43979) | 阴 |
| 6 | 溶藻弧菌(ATCC17749) | 阴 |
| 7 | 哈维氏弧菌(ATCC33866) | 阴 |
| 8 | 副溶血弧菌(ATCC33847) | 阴 |
可见，本发明试剂盒仅对创伤弧菌具有特异性，因此可用于创伤弧菌的检测。
SEQUENCE LISTING
<110> 杭州师范大学
<120> 一种创伤弧菌PCR检测试剂盒及检测方法
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attagatcaa gaagaattag aaaacgaaat cagtactgac gcggttcaaa 230
Claims (1)
1.一种创伤弧菌PCR检测试剂盒，主要包括PCR特异性扩增引物、DNA聚合酶、脱氧三磷酸核苷混合物和PCR缓冲液，其特征在于：所述特异性扩增引物如下：
v-toxR(+):5'-TGATTCCAGCCTAACTCAAG-3'
vv-toxR(-):5'-TTTGAACCGCGTCAGTACTG-3'。
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| CN1560274A (zh) * | 2004-02-26 | 2005-01-05 | 中山大学 | 海洋水产动物及人类病原菌－副溶血弧菌基因诊断试剂盒及检测方法 |
| CN101082063A (zh) * | 2007-04-17 | 2007-12-05 | 海南大学 | 创伤弧菌的一管法半巢式pcr检测试剂盒及检测方法 |
| CN101113473A (zh) * | 2007-06-07 | 2008-01-30 | 天津出入境检验检疫局动植物与食品检测中心 | 运用复合荧光pcr技术检测食源性致病弧菌的方法 |
| CN101348835A (zh) * | 2008-09-09 | 2009-01-21 | 南开大学 | 一种利用环介导等温扩增技术检测创伤弧菌的试剂盒 |
| CN102140515A (zh) * | 2011-01-04 | 2011-08-03 | 天津生物芯片技术有限责任公司 | 用于检测水产品中重要致病菌的核苷酸及其应用 |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1560274A (zh) * | 2004-02-26 | 2005-01-05 | 中山大学 | 海洋水产动物及人类病原菌－副溶血弧菌基因诊断试剂盒及检测方法 |
| CN101082063A (zh) * | 2007-04-17 | 2007-12-05 | 海南大学 | 创伤弧菌的一管法半巢式pcr检测试剂盒及检测方法 |
| CN101113473A (zh) * | 2007-06-07 | 2008-01-30 | 天津出入境检验检疫局动植物与食品检测中心 | 运用复合荧光pcr技术检测食源性致病弧菌的方法 |
| CN101348835A (zh) * | 2008-09-09 | 2009-01-21 | 南开大学 | 一种利用环介导等温扩增技术检测创伤弧菌的试剂盒 |
| CN102140515A (zh) * | 2011-01-04 | 2011-08-03 | 天津生物芯片技术有限责任公司 | 用于检测水产品中重要致病菌的核苷酸及其应用 |
Non-Patent Citations (2)
| Title |
|---|
| development of a quantitative real-time polmerase chain reaction targeted to the toxR for detection of Vibrio vulnificus;Hajime Takahashi et al;《journal of microbiological method》;20051231;第61卷;第77－85页 * |
| Hajime Takahashi et al.development of a quantitative real-time polmerase chain reaction targeted to the toxR for detection of Vibrio vulnificus.《journal of microbiological method》.2005,第61卷 |
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