CN104278102B - 一种沙门氏菌的四重pcr检测方法 - Google Patents
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Abstract
本发明公开了一种沙门氏菌的四重PCR检测方法。本发明中将hns基因(152bp)、fliM基因(418bp)、invA基因(681bp)和Hut基因(495bp)，作为PCR检测的四个基因靶点，将扩增这4个基因片段的4对引物放在一个PCR反应体系，通过各项参数调整优化，建立直接从样品中检测沙门氏菌的四重PCR检测方法。与传统PCR不同，本发明建立的快速检测沙门氏菌四重PCR体系，同时扩增沙门氏菌的四个毒力因子特异性序列，一方面具有四重保险的特点，提高检测的准确性和可靠性，另一方面还可以对其毒力因子编码基因结构变异进行分析，从而掌握其变异趋势，起到一箭双雕的效果。
Description
技术领域
本发明涉及一种生物技术领域的多重PCR检测方法，具体是一种能检测沙门氏菌的四重PCR方法。
背景技术
沙门氏菌是肠杆菌科的一类革兰氏阴性菌，目前已经发现有2500多个血清型。沙门氏菌除可感染人外，还可感染很多动物包括哺乳类、鸟、爬行类、鱼、两栖类及昆虫。沙门氏菌含有多个毒力因子，能够引起动物患肠胃炎、伤寒、菌血症、流产、脑膜炎、呼吸道疾病、心脏疾病、骨髓炎以及一些当地性疾病。沙门氏菌存在于人类的整个食物链中(动物饲料、饲养动物、屠宰场、动物产品零售、餐馆)。至今为止，沙门氏菌仍然威胁着养殖动物及其人类的健康，危害着养殖业的发展，沙门氏菌病是公共卫生学上具有重要意义的人畜共患病之一，包括食物中毒，胃肠炎、伤寒和副伤寒等。人畜感染后可呈无症状带菌状态，也可表现为有临床症状的致死疾，它可能加重病态或死亡率，或者降低动物的繁殖生产力。沙门氏菌的属的种类很多，其中最常见的引起食物中毒的沙门氏菌有鼠伤寒沙门氏菌、猪霍乱沙门氏菌、肠炎沙门氏菌等。沙门氏菌大多属于条件性致病菌，而且常与猪繁殖与呼吸综合征病毒、猪圆环病毒、猪胸膜肺炎放线杆菌、多杀性巴氏杆菌等猪呼吸道病原微生物混合或继发感染，所以区分出致病性沙门氏菌是确诊的关键所在。由此可见，建立有效、灵敏的检测方法无疑是防治沙门氏菌感染和沙门氏菌病的关键。
沙门氏菌是兼性胞内菌，在实验中较难培养，同时在采样过程中分离率也较低，传统的沙门氏菌检测方法(临床观察、培养镜检、玻片凝集试验、生化指标、和噬菌体分型)等耗时耗力，传统的血清型诊断方法有操作繁琐复杂，特异性和灵敏性低等多种不足。而已经报道的常规PCR检测方法，虽可以通过扩增沙门氏菌特异性基因片段，快速有效地检测沙门氏菌，但这些PCR检测方法不能很好地对所检出的沙门氏菌进行分型和致病性鉴定。
多重PCR(multiplexPCR)是在常规PCR的基础上加以改进，于一个PCR反应体系中加入多对特异性引物，针对多个DNA模板或同一模板的不同区域扩增多个目的片段的PCR技术。可用于同时检测多种病原体或鉴定出是那一型病原体感染。由于毒力因子是区分致病性的重要标志物。所以建立基于扩增毒力因子的沙门氏菌杆菌多重PCR检测方法对于及时准确掌握沙门氏菌感染分布状况、实时跟踪其流行趋势及遗传变异规律等都具有重要意义。
发明内容
鉴于沙门氏菌属的种类很多，且往往与其他常见病原体混合感染，传统的诊断技术如细菌分离、免疫学实验等费时费力，不适于临床快速诊断，也不适于大规模的流行病学调查的现状，本发明目的在于克服现有技术不足，提供一种直接从样品中检测沙门氏菌并对其进行分型的四重PCR检测方法。本发明的检测方法采用四重PCR体系检测，具有多重保险的特点，检测的准确性和可靠性高，检测速度快。
本发明针对实际生产中沙门氏菌常与多种病原体混合感染、且临床很难区分的情况，本着既能检测病原体又能分析其重要毒力因子编码基因变异特点的原则，从已发表的文献中筛选出沙门氏菌编码DNA结合蛋白的hns基因(152bp)、编码鞭毛开关蛋白的fliM基因(418bp)、与沙门氏菌入侵肠道上皮细胞有关的invA基因(681bp)以及编码组氨酸结合周质蛋白的Hut基因(495bp)，作为PCR检测的四个基因靶点，将扩增这4个基因片段的4对引物放在一个PCR反应体系，通过各项参数调整优化，建立直接从样品中检测沙门氏菌的四重PCR检测方法。
本发明是通过以下的技术方案实现的。
一种沙门氏菌的四重PCR检测方法，将沙门氏菌四个毒力因子hns基因(152bp)、fliM基因(418bp)、invA基因(681bp)和Hut基因(495bp)，作为PCR检测的四个基因靶点，采用四重PCR于PCR反应体系中一次性同时扩增上述4个基因片段，再通过琼脂糖凝胶电泳检测鉴定PCR扩增产物；其中：扩增hns基因的上游引物的序列如SEQIDNO.1所示，下游引物的序列如SEQIDNO.2所示；扩增fliM基因的上游引物的序列如SEQIDNO.3所示；下游引物的序列如SEQIDNO.4所示；扩增invA基因的上游引物的序列如SEQIDNO.5所示；下游引物的序列如SEQIDNO.6所示；扩增Hut基因的上游引物的序列如SEQIDNO.7所示；下游引物的序列如SEQIDNO.8所示(见表1)。
表1沙门氏菌四个毒力因子编码基因引物
上述检测方法中，PCR反应体系如下：cDNA模板1μl，4对上、下游引物各0.5μl，PCRMix12.5μl，超纯水补充至25μl。PCR反应条件：95℃预变性5min，进入95℃60s，58℃1min和72℃1min的循环，循环35次，最后72℃延伸5min。
与现有技术相比，本发明检测方法以沙门氏菌编码四个毒力因子基因片段作为PCR检测的四个基因靶点，较现有报道的PCR检测方法准确性高、特异性强，也提高了检测效率。
附图说明
图1为退火温度的优化结果；M：标准分子量；泳道1：54℃；泳道2：55℃；泳道3：56℃；泳道4：57℃.；泳道5：58℃；泳道6：59℃；泳道7：60℃；泳道8：61℃；泳道9：62℃.；泳道10：63℃。
图2为25ul多重PCR体系特异性实验结果；M：NDA2000Marker；泳道1-6：大肠杆菌、副猪嗜血杆菌、链球菌、巴氏杆菌、猪肺炎支原体、金黄色葡萄球菌；泳道7：阳性沙门氏菌。
图3为样品检测结果；M：标准分子量；泳道1-20：检测样品(部分)。
具体实施方式
本实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和过程，但本发明的保护范围不限于下述的实施例。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照试剂制造厂商所建议的条件。
将已知沙门氏菌菌株在LB培养基上划板，37℃培养12h后挑单菌落接种于2mLLB液体培养基中，12h后取沙门氏菌菌液作为阳性PCR模板。
实施例1检测沙门氏菌四重PCR方法退火温度的确定
1细菌DNA提取
本发明采用常规DNA提取方法，步骤如下：
1.1将上述菌液悬于1.5ml溶菌酶溶液(0.15mol/lNaCl，0.1mol/lNa2EDTA，15mg/ml溶菌酶，pH＝8)；
1.2.37℃温浴2h；
1.3.加入1.5ml10％SDS(0.1mol/lNaCl，0.5mol/lTris，10％SDS，pH＝8)反复颠倒混匀10min，10000r/min离心10min，取上清；
1.4用等体积苯酚：氯仿：异戊醇各抽提1次，水相中加入40μl的3mol/l乙酸铵和3ml无水乙醇，-20℃沉淀DNA1h，12000r/min离心10min；
1.5收集DNA沉淀，用100μlTE溶解。
2四重PCR扩增反应体系建立
在PCR反应体系，特别是多重PCR反应体系中，退火温度是一个关键的因素。本发明为了四重PCR体系中适合4对引物的退火温度，四重PCR反应体系如下：cDNA模板1μl，4对上、下游引物各0.5μl，PCRMix12.5μl，超纯水补充至25μl。PCR反应条件：95℃预变性5min，进入95℃60s,依次设定退火温度为54℃；55℃，56℃,57℃，58℃,59℃，60℃，61℃,62℃,63℃退火温度梯度1min和72℃1min的循环，循环35次，最后72℃延伸5min，PCR结束后，取8μlPCR产物于1℅的凝胶琼脂上电泳检测，确定体系退火温度，结果显示四重PCR检测体系在退火温度58℃时能更好的扩增出预期的条带，故选择58℃为本四重PCR体系的退火温度。
3结果
通过退火温度梯度实验，确定hns基因、flim基因、hut基因和fimH基因四重PCR的最佳退火温度，结果如图1所示当退火温度为57℃-60℃时(泳道4-7)，PCR结果显示出四个目的条带，取58℃为四重PCR检测方法的退火温度。
实施例2检测沙门氏菌四重PCR方法体系特异性
特异性是PCR反应体系又一个关键的因素。本发明为了确保所建立的四重PCR体系的特异性，利用大肠杆菌、沙门氏菌、嗜血杆菌、链球菌2型等作为对照，验证该体系的特异性，以体系只能扩增出沙门氏菌四个目的基因片段，而对照组不能扩增出片段为特异性合格。将实验室保存的沙门氏菌、大肠杆菌在LB培养基上划线，37℃培养过夜后挑单菌落接种于2mlLB液体培养基中，12h后取沙门氏菌、大肠杆菌菌液及金黄色葡萄球菌作为对照PCR模板；将实验室保存的猪肺炎支原体、多杀性巴氏杆菌、链球菌2型在TSA培养基上划线，37℃培养24h后挑取单菌落接种于TSB液体培养基中，12h后取菌液作为对照PCR模板。
1细菌DNA提取
同实施例1。
2四重PCR扩增反应体系建立
四重PCR反应体系如下：cDNA模板1μl，4对上、下游引物各0.5μl，PCRMix12.5μl，超纯水补充至25μl。PCR反应条件：95℃预变性5min，进入95℃60s，58℃1min和72℃1min的循环，循环35次，最后72℃延伸5min，PCR结束后，取8μl产物作琼脂糖凝胶电泳分析。
3结果
分别对大肠杆菌、副猪嗜血杆菌、链球菌、巴氏杆菌、猪肺炎支原体、金黄色葡萄球取1ul菌液作为四重PCR的模板进行检测，如图2所示，该PCR方法只有在沙门氏菌为模板时出现目标条带(泳道7)，证明该四重PCR方法具有良好的特异性。
实施例3四重PCR方法检测实际样品中沙门氏菌
为了进一步验证本发明提供的检测沙门氏菌四重PCR方法的实用性,本发明将养猪场送检的疑似沙门氏菌感染的组织样品在TSA培养基上划线，37℃培养24h后挑取单菌落接种于TSB培养液中，12h后取菌液作为提取沙门氏菌DNA样品。
1细菌DNA提取
同实施例1。
2四重PCR扩增反应体系扩增
四重PCR反应体系如下：cDNA模板1μl，4对上、下游引物各0.5μl，PCRMix12.5μl，超纯水补充至25μl。PCR反应条件：95℃预变性5min，进入95℃60s，58℃1min和72℃1min的循环，循环35次，最后72℃延伸5min，PCR结束后，取8μl产物作琼脂糖凝胶电泳分析。
应用四重PCR方法对猪场所采集的样品进行四重PCR，如图3所示共确定检测到沙门氏菌4株(泳道17-20)。
Claims (2)
1.一种非疾病诊断治疗目的的沙门氏菌的四重PCR检测方法，其特征在于，将沙门氏菌编码DNA结合蛋白的hns基因，编码鞭毛开关蛋白的fliM基因，沙门氏菌入侵肠道上皮细胞有关的invA基因和编码组氨酸结合周质蛋白的Hut基因作为4个基因靶点，采用四重PCR于PCR反应体系中一次性同时扩增上述4个基因片段，再通过琼脂糖凝胶电泳检测鉴定PCR扩增产物；其中：
扩增hns基因的上游引物的序列如SEQIDNO.1所示，下游引物的序列如SEQIDNO.2所示；
扩增fliM基因的上游引物的序列如SEQIDNO.3所示，下游引物的序列如SEQIDNO.4所示；
扩增invA基因的上游引物的序列如SEQIDNO.5所示，下游引物的序列如SEQIDNO.6所示；
扩增Hut基因的上游引物的序列如SEQIDNO.7所示，下游引物的序列如SEQIDNO.8所示。
2.根据权利要求1所述的非疾病诊断治疗目的的沙门氏菌的四重PCR检测方法，其特征在于，PCR反应体系如下：cDNA模板1μl，4对上、下游引物各0.5μl，PCRMix12.5μl，超纯水补充至25μl；PCR反应条件：95℃预变性5min，进入95℃60s，58℃1min和72℃1min的循环，循环35次，最后72℃延伸5min。
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