CN105755143B - 一种用于检测菌的试剂盒及检测方法 - Google Patents
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Abstract
本发明提供了一种用于检测猪丹毒杆菌的LAMP试剂盒，其包括引物、Bst DNA聚合酶、反应缓冲液和核酸染料。本发明还提供了一种检测猪丹毒杆菌的LAMP方法。使用本发明的LAMP试剂盒或方法能够快速、准确地对猪丹毒杆菌进行鉴定和检测，具有高特异性、耗时短、灵敏度高、鉴定简便等优点，适于实验室或田间检验使用。
Description
技术领域
本发明涉及微生物检测领域，具体地说，本发明涉及一种用于检测猪丹毒杆菌的LAMP试剂盒及检测方法。
背景技术
猪丹毒杆菌(Erysipelothrix rhusiopathiae)俗称猪丹毒丝菌或丹毒丝菌，属丹毒丝菌属(Erysipelothrix)，是一种纤细的小杆菌，革兰染色呈阳性。其能够广泛地、长时期地存在于环境当中，与种类繁多的野生和家养动物、鸟类和鱼类共生而发病。猪丹毒丝菌的形态学呈多变性，细长，呈直或稍弯的杆状，以单个或链状存在，有时可见到球状或棒状状态，但常见的是栅状和尖状，具有形成长丝状的倾向。本菌无运动性，不产生鞭毛，易被普通染料着色，但容易脱色。在人工培养基上传若干代后开始形成长丝，在液体培养基中容易形成团状的菌体丝。不同血清型的猪丹毒丝菌毒力差异很大，关于其致病机制了解较少。已知神经氨酸酶、透明质酸酶、粘附素和热不稳定性荚膜与疾病的致病性密切相关。
猪丹毒病在我国被认为是一种古老的疫病，上世纪80-90年代猪丹毒与猪肺疫、猪瘟被称为养猪业的三大疾病，曾给我国养猪业带来了严重的经济损失。近年来猪丹毒病时有发生，在我国南方的广西、广东、湖南、四川、福建、江西、湖南、安徽等地日趋活跃，频频爆发，造成不同生长阶段的猪群发病死亡。而且猪丹毒杆菌对不利的环境有相当的抵抗力，能耐干燥，动物组织中的细菌在各种条件下能够存活几个月，在冻肉、腐败的尸体、干燥的血液及鱼粉中长期存活，对盐腌、酸浸、烟熏有较强的抵抗力，在腌制烟熏的火腿中可以存活几个月。1909年，Rosenbach等从患者皮肤病灶中成功分离到了该菌，由此证明人也是本菌的宿主。人类食用了含有猪丹毒杆菌的食品或接触被病畜粪便污染的水源、土壤或饲养管理用具，均能够感染此菌而发病，产生腹泻、呕吐、败血症、皮肤疹块等症状，严重时甚至死亡。因此，对于猪丹毒杆菌的检测或鉴定需要引起食品安全部门以及畜牧业的关注与重视。
目前分子生物学检测以其快速、灵敏的特点，在微生物检测领域逐渐取代传统的形态学鉴定，成为热门研究方向。环介导等温扩增技术(loop-mediated isothermalamplification of DNA,LAMP)是日本学者Notomi于2000年建立的一种新型的核酸扩增技术。该技术通过肉眼观察颜色变化即可判定结果，无需电泳和紫外观察等步骤，与实验室常规检测的PCR方法相比具有操作简便、灵敏度高、特异性强等优势，在食品、动植物检验检疫中被广泛应用于各种病原检测。
发明内容
本发明的目的在于提供一种用于检测猪丹毒杆菌的LAMP试剂盒及LAMP方法。
为了实现本发明的目的，在一个方面中，本发明提供一种用于检测猪丹毒杆菌的LAMP试剂盒，其包括引物、反应缓冲液、Bst DNA聚合酶和核酸染料，其中所述引物如下所示:
外引物F3：ATGATGGAAAAAGAAATTCATCC(SEQ ID NO:1)
外引物B3：GAACATCTCCACTTCTTTGG(SEQ ID NO:2)
内引物FIP：ACGTTCCAAGTTTGGATATACATCT-TTGTATCT
TGAACTTTATGCTATGC(SEQ ID NO:3)
内引物BIP：GCGAACGCGGTTGTTGAATC-AGTTCCTGTAGT
TTCTTCTCTC(SEQ ID NO:4)。
优选地，所述反应缓冲液由2mM dNTP、10×ThermoPol反应缓冲液和6mM Mg2+组成。
优选地，所述核酸染料为1000×SYBR Green I。
优选地，本发明的试剂盒进一步包括DNA提取试剂以及阳性对照和阴性对照。
优选地，所述DNA提取试剂例如CTAB抽提缓冲液。
优选地，所述阳性对照是猪丹毒杆菌基因组DNA，并且所述阴性对照是100mMTris-HCl pH 8.0和50mM EDTA。
在另一个方面中，本发明提供了引物在制备用于检测猪丹毒杆菌的LAMP试剂盒中的应用，所述引物如序列SEQ ID NOs:1-4所示。
在又一个方面中，本发明还提供了一种检测猪丹毒杆菌的LAMP方法，其中使用本发明的试剂盒，所述方法包括以下步骤：
(1)向待测样品中加入CTAB抽提缓冲液，按照常规CTAB法提取DNA；
(2)在PCR管中制备LAMP反应体系，包括0.2μmol/μl的外引物F3 1μl、0.2μmol/μl的外引物B3 1μl、1.2μmol/μl的内引物FIP 1μl、1.2μmol/μl的内引物BIP 1μl、反应缓冲液2.5μl、8U/μl的Bst DNA聚合酶1μl、模板DNA 2μl，加水补充至25μl，并以猪丹毒杆菌基因组DNA作为阳性对照，以100mM Tris-HCl pH 8.0和50mM EDTA作为阴性对照；
(3)将步骤(2)中的PCR管于63℃恒温反应60min；
(4)分析判断反应产物结果：在(3)中所得反应产物中加入核酸染料SYBR GreenI，反应液为橙色表示结果为阴性，待测样品中不含猪丹毒杆菌，反应液为绿色表示结果为阳性，待测样品中含有猪丹毒杆菌。
本发明的发明人针对猪丹毒杆菌序列的6个区域设计出4条引物，灵敏度高、特异性好。利用具有链置换活性的DNA聚合酶在恒温条件下快速、高特异性地扩增靶基因，产物是两端带有茎环结构的哑铃状DNA分子。本发明用于检测猪丹毒杆菌的LAMP试剂盒及方法能够准确鉴定食品或动物病例中的猪丹毒杆菌，假阳性率低、快速、高效且操作简便，适于基础实验室和现场检测，值得推广应用。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。本领域技术人员应当领会的是，可以在不偏离本发明精神的情况下进行多种修改，这些修改将包含在本发明的范围内。
实施例1本发明的试剂盒的制备
1.1试剂
引物由天根生化科技(北京)有限公司合成；Bst DNA聚合酶和10×ThermoPol反应缓冲液购自Takara；SYBR Green I购自Invitrogen；其余PCR试剂和配制CTAB抽提缓冲液所需的试剂购自Sigma。
1.2试剂盒的制备：
本发明的试剂盒包括以下试剂：
CTAB抽提缓冲液：按照以下配方配制：100mM Tris-HCl pH 8.0、50mM EDTA、1MNaCl、1％(v/v)β-巯基乙醇、2％CTAB，调整pH值至7.2；
反应缓冲液：按照以下配方配制：2mM dNTP、10×ThermoPol反应缓冲液、6mMMgSO4；
引物：外引物F3，其核苷酸序列如SEQ ID NO:l所示；外引物B3，其核苷酸序列如SEQ ID NO:2所示；内引物FIP，其核苷酸序列如SEQ ID NO:3所示；内引物BIP，其核苷酸序列如SEQ ID NO:4所示。其中外引物F3和B3的浓度为0.2μmol/μl，内引物FIP和BIP的浓度为1.2μmol/μl。
浓度为8U/μl的Bst DNA聚合酶；
核酸染料：1000×SYBR Green I；
阳性对照：猪丹毒杆菌基因组DNA；
阴性对照：100mM Tris-HCl(pH 8.0)和50mM EDTA。
实施例2猪丹毒杆菌特异性检测
2.1LAMP特异性检测
2.1.1待测样品
待测样品采集自河北省任丘市长兴养殖服务专业合作社，包括2016年3月份发生疑似猪丹毒病疫情的病料，心、肝、脾组织共6份。模式菌株猪丹毒杆菌CVCC134、胸膜肺炎放线杆菌CVCC259、化脓链球菌CVCC594购自中国兽医微生物菌种保藏管理中心。
2.1.2样品预处理：
将少许病料组织用消毒剪刀切成小块，放入匀浆器中匀浆，700×g离心5min，随后弃去沉淀，取一部分上清接种于5％山羊血营养琼脂平板，于30℃培养，约48h后观察细菌生长情况。挑取具有典型猪丹毒杆菌形态特征的优势菌落进行分离纯化，并将纯化后的菌接种于5％山羊血琼脂平板，培养至菌落形成。挑取单菌落进行革兰氏染色镜检和凝集素试验，确认待测菌株为猪丹毒杆菌。
模式菌株接种于5％山羊血琼脂平板，30℃培养48h，观察菌落的形成和生长。挑取菌丝少量，在光学显微镜下观察其形态学特征。
2.1.3LAMP检测
使用实施例1制备的试剂盒按照以下步骤进行检测：
(1)收集山羊血琼脂平板上的菌落并离心，加入CTAB抽提缓冲液，按照常规CTAB法提取DNA；
(2)在PCR管中制备LAMP反应体系，其中四种引物各1μl、反应缓冲液2.5μl、8U/μl的Bst DNA聚合酶1μl、步骤(1)所得模板DNA2μl，加水补充至25μl，并以猪丹毒杆菌基因组DNA作为阳性对照，以100mM Tris-HCl pH 8.0和50mM EDTA作为阴性对照；
(3)将步骤(2)的PCR管置于63℃恒温反应60min；
(4)分析判断反应产物结果：在(3)中所得反应产物中加入1μl1000×SYBR GreenI，如反应液颜色为橙色，表示结果为阴性，如反应液颜色为绿色，表示结果为阳性。
2.2检测结果
6份病料组织以及模式菌株猪丹毒杆菌CVCC134的PCR管中均呈现绿色，而胸膜肺炎放线杆菌、化脓链球菌以及阴性对照的显色结果均为橙色，表明引物能够准确地鉴定出携带猪丹毒杆菌的样品，具有很强的特异性。
实施例3猪丹毒杆菌灵敏度检测
3.1LAMP灵敏度检测
按照实施例2的步骤(1)提取模式菌株猪丹毒杆菌CVCC134的DNA，紫外分光光度计测量其OD值，并以10倍浓度系列稀释法稀释成10ng、1ng、100pg、10pg、1pg、100fg共6个梯度。
按照实施例2第2.1.3节的步骤(2)-(4)进行LAMP检测。
3.2检测结果
猪丹毒杆菌DNA浓度为10ng、1ng、100pg、10pg、1pg的PCR管中均呈现绿色，表明本发明的检测方法的最低检测极限达到1pg DNA，灵敏度很高。
Claims (2)
1.一种用于检测猪丹毒杆菌的LAMP试剂盒，其特征在于，包括特异性引物组、反应缓冲液、Bst DNA聚合酶和核酸染料，其中所述引物如下所示:
外引物F3：ATGATGGAAAAAGAAATTCATCC(SEQ ID NO:1)外引物B3：GAACATCTCCACTTCTTTGG(SEQ ID NO:2)
内引物FIP：ACGTTCCAAGTTTGGATATACATCT-TTGTATCT TGAACTTTATGCTATGC(SEQ ID NO:3)
内引物BIP：GCGAACGCGGTTGTTGAATC-AGTTCCTGTAGT TTCTTCTCTC(SEQ ID NO:4)；
所述反应缓冲液由2mM dNTP、10×ThermoPol反应缓冲液和6mM Mg2+组成；
进一步包括DNA提取试剂以及阳性对照和阴性对照；
所述DNA提取试剂是CTAB抽提缓冲液。
2.一种检测猪丹毒杆菌的LAMP方法，所述方法用于对食品中的猪丹毒杆菌进行鉴定，包括以下步骤：
(1)向待测样品中加入CTAB抽提缓冲液，按照常规CTAB法提取DNA；
(2)在PCR管中制备LAMP反应体系，其包括0.2μmol/μl的外引物F3 1μl、0.2μmol/μl的外引物B3 1μl、1.2μmol/μl的内引物FIP 1μl、1.2μmol/μl的内引物BIP 1μl、反应缓冲液2.5μl、8U/μl的Bst DNA聚合酶1μl、模板DNA 2μl，加水补充至25μl，并以猪丹毒杆菌基因组DNA作为阳性对照，以100mM Tris-HCl pH 8.0和50mM EDTA作为阴性对照，
其中所述引物如下所示：
外引物F3：ATGATGGAAAAAGAAATTCATCC(SEQ ID NO:1)
外引物B3：GAACATCTCCACTTCTTTGG(SEQ ID NO:2)
内引物FIP：ACGTTCCAAGTTTGGATATACATCT-TTGTATCT TGAACTTTATGCTATGC(SEQ ID NO:3)
内引物BIP：GCGAACGCGGTTGTTGAATC-AGTTCCTGTAGT TTCTTCTCTC(SEQ ID NO:4)；
(3)将步骤(2)中的PCR管于63℃恒温反应60min；
(4)分析判断反应产物结果：在(3)中所得反应产物中加入核酸染料SYBR Green I，反应液为橙色表示结果为阴性，待测样品中不含猪丹毒杆菌，反应液为绿色表示结果为阳性，待测样品中含有猪丹毒杆菌。
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