CN103725764A - 高特异高敏感检测副溶血性弧菌trh毒素基因的方法 - Google Patents

高特异高敏感检测副溶血性弧菌trh毒素基因的方法 Download PDF

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CN103725764A
CN103725764A CN201210385670.0A CN201210385670A CN103725764A CN 103725764 A CN103725764 A CN 103725764A CN 201210385670 A CN201210385670 A CN 201210385670A CN 103725764 A CN103725764 A CN 103725764A
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trh
vibrio parahemolyticus
vptrh
staphylococcus
nucleic acid
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金京勋
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STONE LAKE PHARMA TECH Co Ltd
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Abstract

本发明公开了一种高特异高敏感检测副溶血性弧菌TRH毒素基因的方法，包括以下步骤：步骤1）制备反应液；步骤2）使用副溶血性弧菌TRH+菌株的核酸、TDH-菌株的核酸，以及若干临床分离金黄色葡萄球菌、头皮葡萄球菌、人型葡萄球菌、表皮葡萄球菌、草绿色链球菌、大肠杆菌、克雷伯菌的核酸，多次进行反应，观察它们的阴阳性。本发明通过配制反应液，设计引物序列，能快速、准确的检测副溶血性弧菌TRH毒素基因，并及时采取相应的措施，减少了疾病的发生率。

Description

高特异高敏感检测副溶血性弧菌TRH毒素基因的方法
技术领域
 本发明涉及疾病检测技术领域，具体的说，是一种高特异高敏感检测副溶血性弧菌TRH毒素基因的方法。
背景技术
副溶血性弧菌（Vibrio Parahemolyticus）是一种嗜盐性细菌。副溶血性弧菌食物中毒，是进食含有该菌的食物所致，主要来自海产品，如墨鱼、海鱼、海虾、海蟹、海蜇，以及含盐分较高的腌制食品，如咸菜、腌肉等。本菌存活能力强，在抹布和砧板上能生存1个月以上，海水中可存活47天。临床上以急性起病、腹痛、呕吐、腹泻及水样便为主要症状。本病多在夏秋季发生于沿海地区，常造成集体发病。和近年来由于海鲜空运，内地城市病例也渐增多。副溶血弧菌有分泌TRH毒素的菌株和分泌TRH毒素的菌株，TRH毒素与直接溶血关系密切。快速检测副溶血性弧菌的TRH毒素基因，对提高副溶血性弧菌引起的食物中毒确定有效治疗方案具有很大意义。
环介导等温扩增技术是由日本荣研（EIKEN）株式会社开发的高特异性敏感度的核酸检测系统，无须昂贵的设备，已经成为核酸快速检测法的良好手段，本发明结合环介导等温扩增技术，设计针对副溶血性弧菌TRH基因基因序列的一对外引物（VPtrh-F3和VPtrh-B3）和一对内引物（VPtrh-FIP和VPtrh-BIP）进行环介导等温扩增。
发明内容
本发明公开了一种高特异高敏感检测副溶血性弧菌TRH毒素基因的方法，提高了它的检测和诊断效率。
为实现上述技术目的，达到上述技术效果，本发明通过以下技术方案实现：
高特异高敏感检测副溶血性弧菌TRH毒素基因的方法，包括以下步骤：
步骤1）制备反应液
反应液组成及含量为
8u/ul Bst核酸聚合酶                         8u
10X耐热聚合酶反应缓冲液                 1X,MgSO4 2mM
4M 甜菜碱                                    1M
10mM d-NTP混合液                           400μM
内引物
   VPtrh-FIP                                40umol
   VPtrh-BIP                                40umol
外引物
     VPtrh-F3                               5umol
     VPtrh-B3                               5umol
标本核酸                                     20ng
添加焦碳酸二乙酯处理水至总量达到25ul;
步骤2）使用副溶血性弧菌TRH+菌株的核酸、TDH-菌株的核酸，以及若干临床分离金黄色葡萄球菌、头皮葡萄球菌、人型葡萄球菌、表皮葡萄球菌、草绿色链球菌、大肠杆菌、克雷伯菌的核酸，多次进行反应，观察它们的阴阳性。
进一步的，所述外引物VPtrh-F3的序列为TTCCATTAATGTTTACCGTCAT。
进一步的，所述外引物VPtrh-B3的序列为ATGAGCTACTATTTGTCGTTAG。  
进一步的，所述内引物VPtrh-FIP的序列为
ACCATATAAAAGCGTTCACGGT-CGCTTAACCATTTTGAGCC。
进一步的，所述内引物VPtrh-BIP的序列为TCGTTTTATGTTTCGGTTTGTCCA-ACAATAAAAACTGAATCACCAGT。
进一步的，所述反应液中加入荧光显示物质溴化乙锭、钙黄绿素进行实时检查。
本发明的有益效果是：
    本发明通过配制反应液，设计引物序列，能快速、准确的检测副溶血性弧菌TRH毒素基因，并及时采取相应的措施，减少了疾病的发生率。
具体实施方式
下面将结合实施例，来详细说明本发明。
 高特异高敏感检测副溶血性弧菌TRH毒素基因的方法，包括以下步骤：
步骤1）制备反应液
反应液组成及含量为
8u/ul Bst核酸聚合酶                         8u
10X耐热聚合酶反应缓冲液                 1X,MgSO4 2mM
4M 甜菜碱                                    1M
10mM d-NTP混合液                           400μM
内引物
   VPtrh-FIP                                40umol
   VPtrh-BIP                                40umol
外引物
     VPtrh-F3                               5umol
     VPtrh-B3                               5umol
标本核酸                                     20ng
添加焦碳酸二乙酯处理水至总量达到25ul。
参考副溶血性弧菌的基因trh（基因序列来源美国国立生物技术信息中心-NCBI）设计引物。所述外引物VPtrh-F3的序列为TTCCATTAATGTTTACCGTCAT，所述外引物VPtrh-B3的序列为ATGAGCTACTATTTGTCGTTAG，所述内引物VPtrh-FIP的序列为
ACCATATAAAAGCGTTCACGGT-CGCTTAACCATTTTGAGCC，所述内引物VPtrh-BIP的序列为TCGTTTTATGTTTCGGTTTGTCCA-ACAATAAAAACTGAATCACCAGT。
进一步的，所述反应液中加入荧光显示物质溴化乙锭、钙黄绿素进行实时检查。
步骤2）使用副溶血性弧菌TRH+菌株2株的核酸、TDH-菌株3株的核酸，以及若干临床分离金黄色葡萄球菌、头皮葡萄球菌、人型葡萄球菌、表皮葡萄球菌、草绿色链球菌、大肠杆菌、克雷伯菌的核酸，多次进行反应，观察它们的阴阳性。结果显示：副溶血性弧菌TRH+菌株2株的核酸每次均为阳性，其余菌种为阴性。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
<110>  苏州四同医药科技有限公司
 
<120>  高特异高敏感检测副溶血性弧菌TRH毒素基因的方法
 
 
<160>  4   
 
 
<210>  1
<211>  22
<212>  DNA
<213>  人工序列
 
<400>  1
1 TTCCATTAATGTTTACCGTCAT
                              
 
<210>  2
<211>  22
<212>  DNA
<213>  人工序列
 
<400>  2 
1 ATGAGCTACTATTTGTCGTTAG
 
      
<210>  3
<211>  41
<212>  DNA
<213>  人工序列
 
<400>  3 
1 ACCATATAAAAGCGTTCACGGT-CGCTTAACCATTTTGAGCC
 
 
<210>  4
<211>  47
<212>  DNA
<213>  人工序列
 
<400>  4 
1 TCGTTTTATGTTTCGGTTTGTCCA-ACAATAAAAACTGAATCACCAGT    

Claims (6)

1.高特异高敏感检测副溶血性弧菌TRH毒素基因的方法,其特征在于，包括以下步骤：
步骤1）制备反应液
反应液组成及含量为
8u/ul Bst核酸聚合酶                         8u
10X耐热聚合酶反应缓冲液                 1X,MgSO4 2mM
4M 甜菜碱                                    1M
10mM d-NTP混合液                           400μM
内引物
   VPtrh-FIP                                40umol
   VPtrh-BIP                                40umol
外引物
     VPtrh-F3                               5umol
     VPtrh-B3                               5umol
标本核酸                                     20ng
添加焦碳酸二乙酯处理水至总量达到25ul;
步骤2）使用副溶血性弧菌TRH+菌株的核酸、TDH-菌株的核酸，以及若干临床分离金黄色葡萄球菌、头皮葡萄球菌、人型葡萄球菌、表皮葡萄球菌、草绿色链球菌、大肠杆菌、克雷伯菌的核酸，多次进行反应，观察它们的阴阳性。
2.根据权利要求1所述的高特异高敏感检测副溶血性弧菌TRH毒素基因的方法，其特征在于：所述外引物VPtrh-F3的序列为TTCCATTAATGTTTACCGTCAT。
3.根据权利要求2所述的高特异高敏感检测副溶血性弧菌TRH毒素基因的方法，其特征在于：所述外引物VPtrh-B3的序列为ATGAGCTACTATTTGTCGTTAG。
4.根据权利要求3所述的高特异高敏感检测副溶血性弧菌TRH毒素基因的方法，其特征在于：所述内引物VPtrh-FIP的序列为
ACCATATAAAAGCGTTCACGGT-CGCTTAACCATTTTGAGCC。
5.根据权利要求4所述的高特异高敏感检测副溶血性弧菌TRH毒素基因的方法，其特征在于：所述内引物VPtrh-BIP的序列为TCGTTTTATGTTTCGGTTTGTCCA-ACAATAAAAACTGAATCACCAGT。
6.根据权利要求5所述的高特异高敏感检测副溶血性弧菌TRH毒素基因的方法，其特征在于：所述反应液中加入荧光显示物质溴化乙锭、钙黄绿素进行实时检查。
CN201210385670.0A 2012-10-12 2012-10-12 高特异高敏感检测副溶血性弧菌trh毒素基因的方法 Pending CN103725764A (zh)

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Application publication date: 20140416