CN103725757A - 一种高特异高敏感检测分泌肠毒素蜡样芽孢杆菌的方法 - Google Patents

一种高特异高敏感检测分泌肠毒素蜡样芽孢杆菌的方法 Download PDF

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CN103725757A
CN103725757A CN201210380671.6A CN201210380671A CN103725757A CN 103725757 A CN103725757 A CN 103725757A CN 201210380671 A CN201210380671 A CN 201210380671A CN 103725757 A CN103725757 A CN 103725757A
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bacillus cereus
enterotoxin
bce
secretion
staphylococcus
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金京勋
吕少波
李苏杨
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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分泌肠毒素蜡样芽孢杆菌的方法，包括以下步骤：步骤1）制备反应液；步骤2）提取临床分离分泌肠毒素蜡样芽胞杆菌，分泌呕吐毒素蜡样芽胞杆菌、环境分离蜡样芽胞杆菌，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、准确的检测出蜡样芽胞杆菌，提高了致病性蜡样芽胞杆菌的检测和诊断效率，对确定有效的治疗方案具有很大的指引作用。

Description

一种高特异高敏感检测分泌肠毒素蜡样芽孢杆菌的方法
技术领域
 本发明涉及生物技术领域，具体的说，是一种高特异高敏感检测分泌肠毒素蜡样芽孢杆菌的方法。
背景技术
蜡样芽孢杆菌是芽孢杆菌属中的一种，1950年首次在挪威报告，会引起食物中毒，中毒者症状为呕吐、腹痛、腹泻，腹痛腹泻是由蜡样芽胞杆菌肠毒素引起，并且该产物不是蜡样芽胞杆菌的标准产物之一，蜡样芽胞杆菌产生的肠毒素与其它细菌所产生的肠毒素不相同。快速检测分泌肠毒素蜡样芽胞杆菌，对提高致病性蜡样芽胞杆菌的检测和诊断效率，确定有效治疗方案具有很大意义。LAMP-PCR系统是由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。
发明内容
本发明公开了一种高特异高敏感检测分泌肠毒素蜡样芽孢杆菌的方法，能快速检测分泌肠毒素蜡样芽胞杆菌，提高了致病性蜡样芽胞杆菌的检测和诊断效率。
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：
一种高特异高敏感检测分泌肠毒素蜡样芽孢杆菌的方法,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
   BCE-FIP                                 40umol
   BCE-BIP                                 40umol
外引物
    BCE-F3                                 5umol
    BCE-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）提取临床分离分泌肠毒素蜡样芽胞杆菌，分泌呕吐毒素蜡样芽胞杆菌、环境分离蜡样芽胞杆菌，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BCE-F3的序列为TGCGAAAGATAGCTGGAA。
进一步的，所述外引物BCE-B3的序列为GAATTAGCCGACTGGATGA。
进一步的，所述内引物BCE-FIP的序列为GCCCTTATGCTTTTTTTGTGTCT-ACTATGCAGAAAAGATTTATGCTG。
进一步的，所述内引物BCE-BIP的序列为AGAGGGGAATACTAAGTACATGTCA-TAAGTGACCGAAACTGGC。
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、准确的检测出蜡样芽胞杆菌，提高了致病性蜡样芽胞杆菌的检测和诊断效率，对确定有效的治疗方案具有很大的指引作用。
具体实施方式
下面将结合实施例，来详细说明本发明。
一种高特异高敏感检测分泌肠毒素蜡样芽孢杆菌的方法,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
   BCE-FIP                                 40umol
   BCE-BIP                                 40umol
外引物
    BCE-F3                               5umol
    BCE-B3                               5umol
标本核酸                                    20ng
添加焦碳酸二乙酯处理水至总量达到25ul。
参考控制分泌蜡样芽胞杆菌肠毒素的基因nheB（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BCE-F3的序列为TGCGAAAGATAGCTGGAA，所述外引物BCE-B3的序列为GAATTAGCCGACTGGATGA，所述内引物BCE-FIP的序列为GCCCTTATGCTTTTTTTGTGTCT-ACTATGCAGAAAAGATTTATGCTG，所述内引物BCE-BIP的序列为AGAGGGGAATACTAAGTACATGTCA-TAAGTGACCGAAACTGGC。
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）提取临床分离分泌肠毒素蜡样芽胞杆菌3株，分泌呕吐毒素蜡样芽胞杆菌2株、环境分离蜡样芽胞杆菌2株，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：3株分泌呕吐毒素蜡样芽胞杆菌每次均为阳性，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。

Claims (6)

1.一种高特异高敏感检测分泌肠毒素蜡样芽孢杆菌的方法,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
   BCE-FIP                                 40umol
   BCE-BIP                                 40umol
外引物
    BCE-F3                                 5umol
    BCE-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）提取临床分离分泌肠毒素蜡样芽胞杆菌，分泌呕吐毒素蜡样芽胞杆菌、环境分离蜡样芽胞杆菌，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分泌肠毒素蜡样芽孢杆菌的方法，其特征在于：所述外引物BCE-F3的序列为TGCGAAAGATAGCTGGAA。
3.根据权利要求2所述的高特异高敏感检测分泌肠毒素蜡样芽孢杆菌的方法，其特征在于：所述外引物BCE-B3的序列为GAATTAGCCGACTGGATGA。
4.根据权利要求3所述的高特异高敏感检测分泌肠毒素蜡样芽孢杆菌的方法，其特征在于：所述内引物BCE-FIP的序列为GCCCTTATGCTTTTTTTGTGTCT-ACTATGCAGAAAAGATTTATGCTG。
5.根据权利要求4所述的高特异高敏感检测分泌肠毒素蜡样芽孢杆菌的方法，其特征在于：所述内引物BCE-BIP的序列为AGAGGGGAATACTAAGTACATGTCA-TAAGTGACCGAAACTGGC。
6.根据权利要求5所述的高特异高敏感检测分泌肠毒素蜡样芽孢杆菌的方法，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。
CN201210380671.6A 2012-10-10 2012-10-10 一种高特异高敏感检测分泌肠毒素蜡样芽孢杆菌的方法 Pending CN103725757A (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111690757A (zh) * 2020-05-19 2020-09-22 广东岭南职业技术学院 用于快速鉴定产呕吐毒素蜡样芽胞杆菌的引物及检测方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111690757A (zh) * 2020-05-19 2020-09-22 广东岭南职业技术学院 用于快速鉴定产呕吐毒素蜡样芽胞杆菌的引物及检测方法

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