CN105385685A - 用于快速预判禽源大肠杆菌分离株致病力的引物组合及试剂盒 - Google Patents

用于快速预判禽源大肠杆菌分离株致病力的引物组合及试剂盒 Download PDF

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CN105385685A
CN105385685A CN201510919559.9A CN201510919559A CN105385685A CN 105385685 A CN105385685 A CN 105385685A CN 201510919559 A CN201510919559 A CN 201510919559A CN 105385685 A CN105385685 A CN 105385685A
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高崧
董向磊
高清清
曹春光
张翠翠
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Abstract

本发明涉及用于快速预判禽源大肠杆菌分离株致病力的引物组合及试剂盒。所述引物组合是大肠杆菌iss、iroN、tsh、cvaC、iutA和irp2这6个基因的特异引物，它们的序列分别如SEQIDNo.1和2，SEQIDNo.3和4，SEQIDNo.5和6，SEQIDNo.7和8，SEQIDNo.9和10，SEQIDNo.11和12所示。包含上述引物组合的PCR试剂盒，对相应靶基因进行PCR扩增，通过扩增的靶基因片段数量来快速判定分离株的致病力。本发明可用于预判禽源大肠杆菌的致病力，与传统的动物试验相比，本发明不但减少了实验动物的用量，同时改善了动物福利，缩短了实验周期。

Description

用于快速预判禽源大肠杆菌分离株致病力的引物组合及试剂盒
技术领域
本发明涉及用于快速预判禽源大肠杆菌分离株致病力的引物组合及试剂盒。
背景技术
大肠杆菌病是造成世界各地家禽业高发病率和死亡率的一个主要病因。禽大肠杆菌病是由大肠埃希氏菌(Escherichiacoli)的某些致病性血清型引起的细菌性传染病。常引起继发性局部或全身性感染，可引起败血症、大肠杆菌性腹膜炎、心包炎、肝周炎、输卵管炎、脐炎、滑膜炎、气囊炎、肉芽肿、眼炎等多种病变。近年来报道大肠杆菌病还可引起脑型感染和肿头综合征。该病常与其他细菌病、病毒病、寄生虫病等混合感染造成死亡率升高，给养禽业带来巨大的损失。
由于大肠杆菌的血清型较多，一直以来在禽源大肠杆菌分离株的血清型方面研究较多，对于禽源大肠杆菌分离株的毒力基因与致病力之间关系的了解相对较少。而血清型鉴定只能初步判定有限血清型的禽致病性大肠杆菌(AvianpathogenicEscherichiacoli,APEC)分离株，因此，目前确定APEC分离株的致病力主要通过动物致死性试验加以判定，这种方法不仅消耗大量的实验动物，而且耗时、费力、费钱，有违动物福利，增加了实验成本。
发明内容
为了提高APEC分离株的致病力预判的效率，本发明提供了用于快速判定禽源大肠杆菌分离株致病力的引物组合及试剂盒。采用本发明的引物组合及试剂盒，通过一种双重PCR和单一PCR的检测方法可快速判定禽源大肠杆菌分离株的致病力。
本发明旨在建立禽致病性大肠杆菌(APEC)毒力基因与致病力之间的关系，根据已发表的大肠杆菌毒力基因的序列，对APEC分离株的32个毒力基因的检测结果表明，致病株比非致病株出现频率高(P<0.05)的有iucC、iucD、iutA、tsh、iroN、irp-2、iss和cvaC这8个毒力相关基因。气杆菌素合成基因iucC、iucD与编码气杆菌素受体的基因iutA处于一个操纵子上，iucC和iucD的检出率几乎一样，而iutA基因在致病株与非致病株的检出率间存在显著差异，我们选择保留iutA基因。因此，本发明建立iutA、iss、tsh、iroN、irp-2、cvaC这6个基因的检测模式来判定APEC。
再根据对89个禽源大肠杆菌分离株的致病性试验结果(表1)，结合这些分离株毒力基因的检测结果，得出：
(1)在受检的6个毒力基因中，若分离株毒力基因数≥2；分离株属致病株的概率为：63÷68＝92.64％≈93％
(2)在受检的6个毒力基因中，若分离株毒力基因数<2，分离株属非致病株的概率为：12÷21＝57.14％≈57％。
表189个APEC分离株毒力基因数量与致病性之间的关系
基于以上结果，建立基于毒力基因数量预测禽源大肠杆菌分离株致病性的方法。
本发明一方面提供了用于快速预判禽源大肠杆菌分离株致病力的引物组合，该引物组合是由6对引物组成，所述的6对引物分别是大肠杆菌iss、iroN、tsh、cvaC、iutA和irp2这6个基因的特异引物。
所述的大肠杆菌iss、iroN、tsh、cvaC、iutA和irp2基因的6对引物，其序列分别如SEQIDNo.1和2，SEQIDNo.3和4，SEQIDNo.5和6，SEQIDNo.7和8，SEQIDNo.9和10，SEQIDNo.11和12所示。
本发明还提供一种用于快速预判禽源大肠杆菌分离株致病力的PCR试剂盒，该试剂盒包括上述引物组合及常规PCR试剂。所述PCR试剂为10×buffer、MgCl2(25mmol/L)、dNTPs(2.5mmol/L)、TaqDNA聚合酶(500U)及超纯水。
采用本发明引物组合或试剂盒进行靶基因的特异性扩增，完成PCR扩增产物的鉴定，确定大肠杆菌分离株所含毒力基因的数量，从而快速鉴定APEC分离株的致病力。在受检的6个毒力基因中，若分离株毒力基因数≥2，判定该分离株为禽致病性大肠杆菌(APEC)。
与现有技术相比，本发明利用合成的6对引物对相应靶基因进行PCR扩增，通过扩增的靶基因片段数量来快速判定分离株的致病力。本研究建立的方法可用于预判禽源大肠杆菌的致病力，只有需确证上述分离株致病力的情形下，才需考虑动物试验。因此，与传统的动物试验相比，本发明不但减少了实验动物的用量，同时改善了动物福利，缩短了实验周期。
具体实施方式
本发明中致病菌株和非致病菌株的定义：
致病菌株：以1×107菌落形成单位(colonyformingunits,CFU)的禽源大肠杆菌分离株气管或气囊接种1日龄易感鸡5-6羽，连续观察1周，能引起接种鸡死亡的分离株为致病菌株。
非致病菌株：以1×107菌落形成单位(colonyformingunits,CFU)的禽源大肠杆菌分离株气管或气囊接种1日龄易感鸡5-6羽，连续观察1周，不能引起接种鸡死亡的分离株为非致病菌株。
采用本发明引物组合进行预测禽源大肠杆菌分离株致病力的方法如下：
(1)引物的设计
引物序列如表2：
表2发明所涉引物序列与预期扩增片段大小
(2)DNA模版的制备
通过全菌裂解法进行细菌DNA模板的制备。在LB固体培养基上挑取单个菌落接种于LB液体培养基中，37℃，220r/min摇振培养过夜，取适量于指形管中，12000rpm×5min离心，弃去残存培养基，瞬离，吸干，用去离子水悬浮沉淀，置100℃10min，冰浴10min，12000rpm×10min离心，取上清即为DNA模板。
(3)PCR扩增毒力基因片段
6个毒力基因PCR扩增体系及反应程序如下：
iss、iroN毒力基因的双重PCR扩增体系：10×buffer2.5μL、MgCl21.5μL、dNTPs0.5μL、TaqDNA聚合酶0.5μL、超纯水16μL引物iss-F、iss-R各0.5μL，iroN-F、iroN-R各0.5μL(浓度为20μmol/ml)，2μLDNA模板。反应程序：首先94℃预变性4min；然后进行30个循环，条件为94℃1min，55℃1min、72℃1min；最后72℃延伸10min。同时以标准菌株作阳性对照，以灭菌超纯水作空白对照。
tsh、cvaC毒力基因的双重PCR扩增体系：10×buffer2.5μL、MgCl21.5μL、dNTPs0.5μL、TaqDNA聚合酶0.5μL、超纯水16μL，引物tsh-F、tsh-R各0.5μL，cvaC-F、cvaC-R各0.5μL(浓度为20μmol/ml)，2μLDNA模板。反应程序：首先94℃预变性4min；然后进行30个循环，条件为94℃1min，55℃1min、72℃1min；最后72℃延伸10min。同时以标准菌株作阳性对照，以灭菌超纯水作空白对照。
iutA毒力基因的PCR扩增体系：10×buffer2.5μL、MgCl21.5μL、dNTPs0.5μL、TaqDNA聚合酶0.5μL、超纯水17μL，引物iutA-F、iutA-R各0.5μL(浓度为20μmol/ml)，2μLDNA模板。反应程序：首先94℃预变性4min；然后进行30个循环，条件为94℃30s，63℃30s，72℃1min；最后72℃延伸10min。同时以标准菌株作阳性对照，以灭菌超纯水作空白对照。
irP2毒力基因的PCR扩增体系：10×buffer2.5μL、MgCl21.5μL、dNTPs0.5μL、TaqDNA聚合酶0.5μL、超纯水17μL，引物irP2-F、irP2-R各0.5μL(浓度为20μmol/ml)，2μLDNA模板。反应程序：首先94℃预变性4min；然后进行30个循环，条件为94℃30s，59℃30s，72℃1min；最后72℃延伸10min。同时以标准菌株作阳性对照，以灭菌超纯水作空白对照。
(4)PCR扩增产物的鉴定
取PCR扩增产物8μl，点样于1％琼脂糖凝胶(含0.5μg/mL溴化乙锭)，以5V/cm的电场强度于0.5×TBE缓冲液中电泳，以100bpLaddermarker作为标准参照，凝胶成像系统拍照，确定待检分离株拥有6个受检毒力基因的数量，如果分离株的上述毒力基因数不小于2个，即可判定该分离株具有致病性。

Claims (4)

1.用于快速预判禽源大肠杆菌分离株致病力的引物组合，其特征在于，是由6对引物组成，所述的6对引物分别是大肠杆菌iss、iroN、tsh、cvaC、iutA和irp2这6个基因的特异引物。
2.根据权利要求1所述的引物组合，其特征在于，所述的6对引物，其序列如SEQIDNo.1和2，SEQIDNo.3和4，SEQIDNo.5和6，SEQIDNo.7和8，SEQIDNo.9和10，SEQIDNo.11和12所示。
3.一种用于快速预判定禽源大肠杆菌分离株致病力的PCR试剂盒，其特征在于包括权利要求1所述的引物组合以及PCR试剂。
4.根据权利要求3所的试剂盒，其特征在于，所述的PCR试剂是10×buffer、25mmol/L的MgCl2、2.5mmol/LdNTPs、500U的TaqDNA聚合酶及超纯水。
CN201510919559.9A 2015-12-11 2015-12-11 用于快速预判禽源大肠杆菌分离株致病力的引物组合及试剂盒 Pending CN105385685A (zh)

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