CN110423828A - 一种检测胎儿弯曲菌龟亚种的pcr试剂盒 - Google Patents

一种检测胎儿弯曲菌龟亚种的pcr试剂盒 Download PDF

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CN110423828A
CN110423828A CN201910752523.4A CN201910752523A CN110423828A CN 110423828 A CN110423828 A CN 110423828A CN 201910752523 A CN201910752523 A CN 201910752523A CN 110423828 A CN110423828 A CN 110423828A
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campylobacter fetus
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侯水平
吴新伟
张周斌
杨智聪
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Abstract

本发明公开了一种检测胎儿弯曲菌龟亚种的PCR试剂盒。本发明所述的检测胎儿弯曲菌龟亚种的PCR试剂盒包括：以胎儿弯曲菌龟亚种的COA基因作为靶基因设计的正向引物和反向引物。本发明所述的试剂盒不仅可以检测龟亚种，还可用来区分龟亚种和胎儿亚种、性病亚种。

Description

一种检测胎儿弯曲菌龟亚种的PCR试剂盒
技术领域
本发明属于生物医药技术领域，涉及一种PCR检测试剂盒，特别是涉及一种检测胎儿弯曲菌龟亚种的PCR试剂盒。
背景技术
弯曲菌属是一类革兰氏阴性，氧化酶触酶阳性，呈S形、或螺旋状弯曲，单极或双极鞭毛、嗜微需氧生长的人兽共患传染病病原体。弯曲菌常定居于温血动物(如家禽、野鸟)的肠道内，以“粪-口传播”的方式传染给人类，主要引起一种“痢疾样”急性腹泻性疾病。而胎儿弯曲菌(Campylobacter fetus)可导致人体肠道外感染如早产、菌血症、腹膜炎、脑膜炎、关节炎等。胎儿弯曲菌按照宿主的不同及所致疾病的差异可分为三个亚种：胎儿亚种、性病亚种和龟亚种。胎儿亚种来源于牛羊及鸟类，可导致牛羊早产，但症状较轻；性病亚种主要来源于牛，可导致牛羊流产、生殖道炎症；龟亚种来源于爬行动物并具有独特的分子特征，因此对亚种的准确鉴定可以推测其感染的来源，为流行病学调查提供依据。
胎儿弯曲菌三个亚种之间的生化反应差异较小：1％甘氨酸生长实验性病亚种为阳性，而胎儿亚种和龟亚种为阴性，目前不能根据生化试验将其三个亚种之间进行区分。文献报道使用多重PCR鉴别胎儿亚种和性病亚种的原理为扩增针对胎儿弯曲菌CstA基因和针对性病亚种的Par基因，如果扩增出现960bp片段和140bp片段则为性病亚种，如果仅有960bp为胎儿亚种。但是对龟亚种目前仅能通过对16S RNA测序然后在Genbank数据库中比对，以及通过MALDI-TOF MS取得峰图跟标准菌株比对后确定是否为龟亚种，但目前尚没有对龟亚种的普通PCR鉴定方法。
本申请人在2014年8月29日已申请发明专利“一种快速鉴别胎儿弯曲菌和性病亚种的方法和试剂盒”(专利号：CN201410437158.5)。该专利涉及的试剂盒仅能鉴别胎儿弯曲菌和性病亚种，检测的是胎儿弯曲菌的sap保守基因和性病亚种的par基因，而不能对龟亚种进行检测。当时尚未有国际权威期刊对龟亚种的定义。目前对胎儿弯曲菌龟亚种没有通过核酸的快速检测方法。
发明内容
本发明的目的在于提供一种检测胎儿弯曲菌龟亚种的PCR试剂盒。
本发明所述的检测胎儿弯曲菌龟亚种的PCR试剂盒包括：以胎儿弯曲菌龟亚种的COA基因作为靶基因设计的正向引物和反向引物。
根据本发明所述的检测胎儿弯曲菌龟亚种的PCR试剂盒的进一步特征，所述以COA基因为靶基因设计的正向引物的序列为SEQ ID NO.1，反向引物的序列为SEQ ID NO.2。
本发明所述的检测胎儿弯曲菌龟亚种的PCR试剂盒是用于检测龟亚种的COA基因，此基因在胎儿亚种和性病亚种均不存在，因此本发明所述的试剂盒不仅可以检测龟亚种，还可用来区分龟亚种和胎儿亚种、性病亚种。
附图说明
图1是本发明所述的PCR试剂盒的PCR产物电泳图，泳道1-7分别为1:10-1:106稀释度的PCR产物。
图2是本发明所述的PCR试剂盒的特异性实验PCR产物电泳图。
具体实施方式
以下通过具体实施例并结合附图对本发明进行详细说明。
1.引物设计
选择胎儿弯曲菌龟亚种的COA基因作为靶基因，设计PCT扩增引物(见表1)。
表1：扩增COA基因的引物序列
引物 序列(5’-3’) 序列编号
正向引物 ACATAGCCGTAGTTGGATTTAGTA SEQ ID NO:1
反向引物 CCTCGGCTTTTTGCTTCA SEQ ID NO:2
注：COA基因的扩增片段长度为294bp。
2.PCR反应体系和PCR扩增程序
反应体系：50μL反应体系(PCR mix 25μL，正向引物(10μM/L)1μL，反向引物(10μM/L)1μL，ddH2O 21μL，DNA 2μL)。
反应条件：预变性95℃5min；95℃30s，55℃30s，72℃45s，35个循环；72℃5min。
琼脂糖凝胶电泳：1％琼脂糖凝胶，120V电泳60min，染色后紫外成像在294bp处片段即为阳性。
3.灵敏度实验
将标准菌株(ATCC BAA-2539)新鲜菌落用10mlPBS稀释成均匀的菌悬液，并梯度稀释成1:10、1:102、1:103、1:104、1:105、1:106等6个稀释度的菌悬液。
3.1每个稀释度菌落计数。
分别取每个稀释度的菌悬液100μl均匀涂抹在血平板上(每个稀释度同时做一个平行样)，然后将血平板在37℃微需氧培养箱培养3天后，对血平板上的弯曲菌菌落分别计数。并参照《GB4789.2-2006》计算出菌悬液的浓度。
3.2每个稀释度提取核酸及PCR。
取每个稀释度100μl，95℃加热10min，12000rpm离心2min，上清液即为待用模板。每个稀释度的模板按照前述反应体系PCR。
3.3取5μlPCR反应产物在1％琼脂糖凝胶100V电泳70min后照相。
3.4结果：
PCR产物电泳图见图1；经计算该PCR方法检测灵敏度为9.7CFU/μl(见表2)。
表2：胎儿弯曲菌龟亚种(ATCC BAA-2539)多个稀释度的PCR结果
稀释度 菌落数量(CFU/100μl) PCR结果
1:10 多不可计 +
1:10<sup>2</sup> 多不可计 +
1:10<sup>3</sup> 多不可计 +
1:10<sup>4</sup> 97 -
1:10<sup>5</sup> 8 -
1:10<sup>6</sup> 0 -
4.特异性实验
4.1将表3的菌株用脑心浸液肉汤复苏增菌培养，取菌液1ml，12000rpm离心2min，去掉上清液，加入50μl DNA提取液，95℃10min，12000rpm离心2min，上清液即为待检用模板。
4.2取上述模板2μl按照本试剂盒的反应体系及反应条件进行PCR扩增，扩增结束后取5μlPCR产物在1％琼脂糖凝胶100V电泳70min后照相(见图2)。
4.38株胎儿弯曲菌龟亚种PCR产物电泳后在300bp位置出现明显条带，结果均为阳性。而其他27株其他细菌和阴性对照在300bp位置未出现条带，结果均为阴性，特异性为100％。
表3：特异性实验用的菌株及PCR检测结果
胎儿弯曲菌龟亚种检测方法的特异性实验PCR产物电泳结果见图2。
5.结论
本发明建立了一种检测胎儿弯曲菌龟亚种的PCR试剂盒，检测灵敏度为9.7CFU/μl，特异性为100％，可应用于临床对胎儿弯曲菌龟亚种菌株进行快速准确的鉴定。
SEQUENCE LISTING
<110> 广州市疾病预防控制中心
<120> 一种检测胎儿弯曲菌龟亚种的PCR试剂盒
<130>
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 24
<212> DNA
<213> 人工合成
<400> 1
acatagccgt agttggattt agta 24
<210> 2
<211> 18
<212> DNA
<213> 人工合成
<400> 2
cctcggcttt ttgcttca 18

Claims (2)

1.一种检测胎儿弯曲菌龟亚种的PCR试剂盒，其特征在于，包括：
以胎儿弯曲菌龟亚种的COA基因作为靶基因设计的正向引物和反向引物。
2.根据权利要求1所述的检测胎儿弯曲菌龟亚种的PCR试剂盒，其特征在于：所述以COA基因为靶基因设计的正向引物的序列为SEQ ID NO.1，反向引物的序列为SEQ ID NO.2。
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