CN103725751A - 一种高特异高敏感检测分泌呕吐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呕吐毒素蜡样芽孢杆菌的方法 Download PDF

Info

Publication number
CN103725751A
CN103725751A CN201210380431.6A CN201210380431A CN103725751A CN 103725751 A CN103725751 A CN 103725751A CN 201210380431 A CN201210380431 A CN 201210380431A CN 103725751 A CN103725751 A CN 103725751A
Authority
CN
China
Prior art keywords
bacillus cereus
vomitoxin
staphylococcus
reaction solution
secretion
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201210380431.6A
Other languages
English (en)
Inventor
金京勋
吕少波
李苏杨
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
STONE LAKE PHARMA TECH Co Ltd
Original Assignee
STONE LAKE PHARMA TECH Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by STONE LAKE PHARMA TECH Co Ltd filed Critical STONE LAKE PHARMA TECH Co Ltd
Priority to CN201210380431.6A priority Critical patent/CN103725751A/zh
Publication of CN103725751A publication Critical patent/CN103725751A/zh
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Toxicology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明公开了一种高特异高敏感检测分泌呕吐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）将上述反应液放入63℃烘箱进行反应30-60min，浑浊为阳性；步骤3）提取临床分离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、准确的检测分泌呕吐毒素蜡样芽孢杆菌，并及时采取相应的措施，减少了疾病的发生率。

Description

一种高特异高敏感检测分泌呕吐毒素蜡样芽孢杆菌的方法
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分泌呕吐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，其中呕吐症状是由蜡样芽孢杆菌致吐（cereulide）毒素造成，并且该产物不是蜡样芽胞杆菌的标准产物之一，蜡样芽孢杆菌致吐（cereulide）毒素是一种由非核糖调控缩氨酸综合(NRPS)产生的即使在菌体内也是不常见的多肽。快速检测分泌呕吐毒素蜡样芽胞杆菌，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。
发明内容
本发明公开了一种高特异高敏感检测分泌呕吐毒素蜡样芽孢杆菌的方法，提高了分泌呕吐毒素蜡样芽孢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呕吐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
   BC-FIP                                   40umol
   BC-BIP                                   40umol
外引物
    BC-F3                                   5umol
    BC-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）将上述反应液放入63℃烘箱进行反应30-60min，浑浊为阳性;
步骤3）提取临床分离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BC-F3的序列为CTCTTGCATGCTTGGAATT。
进一步的，所述外引物BC-B3的序列为CCTTCGCTTCTAATCCAACA。  
进一步的，所述内引物BC-FIP的序列为
ACACACCAAGATATTGCGCAT-GATACAGTTCCAACTTTTAGATCG。
 进一步的，所述内引物BC-BIP的序列为TTAGTGTGATGTTCGCACTTCAA-CTATTTGTCGCAATCACCGT。
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、准确的检测分泌呕吐毒素蜡样芽孢杆菌，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。
 一种高特异高敏感检测分泌呕吐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
   BC-FIP                                   40umol
   BC-BIP                                   40umol
外引物
    BC-F3                                   5umol
    BC-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孢杆菌致吐毒素的基因ces（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BC-F3的序列为CTCTTGCATGCTTGGAATT，所述BC-B3的序列为CCTTCGCTTCTAATCCAACA，所述BC-FIP的序列为ACACACCAAGATATTGCGCAT-GATACAGTTCCAACTTTTAGATCG，所述BC-BIP的序列为TTAGTGTGATGTTCGCACTTCAA-CTATTTGTCGCAATCACCGT。
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）将上述反应液放入63℃烘箱进行反应30-60min，浑浊为阳性;
步骤3）提取临床分离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：2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呕吐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
   BC-FIP                                   40umol
   BC-BIP                                   40umol
外引物
    BC-F3                                   5umol
    BC-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）将上述反应液放入63℃烘箱进行反应30-60min，浑浊为阳性;
步骤3）提取临床分离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呕吐毒素蜡样芽孢杆菌的方法，其特征在于：所述外引物BC-F3的序列为CTCTTGCATGCTTGGAATT。
3.根据权利要求2所述的高特异高敏感检测分泌呕吐毒素蜡样芽孢杆菌的方法，其特征在于：所述外引物BC-B3的序列为CCTTCGCTTCTAATCCAACA。
4.根据权利要求3所述的高特异高敏感检测分泌呕吐毒素蜡样芽孢杆菌的方法，其特征在于：所述内引物BC-FIP的序列为
ACACACCAAGATATTGCGCAT-GATACAGTTCCAACTTTTAGATCG。
5.根据权利要求4所述的高特异高敏感检测分泌呕吐毒素蜡样芽孢杆菌的方法，其特征在于：所述内引物BC-BIP的序列为TTAGTGTGATGTTCGCACTTCAA-CTATTTGTCGCAATCACCGT。
6.根据权利要求5所述的高特异高敏感检测分泌呕吐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。
CN201210380431.6A 2012-10-10 2012-10-10 一种高特异高敏感检测分泌呕吐毒素蜡样芽孢杆菌的方法 Pending CN103725751A (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201210380431.6A CN103725751A (zh) 2012-10-10 2012-10-10 一种高特异高敏感检测分泌呕吐毒素蜡样芽孢杆菌的方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201210380431.6A CN103725751A (zh) 2012-10-10 2012-10-10 一种高特异高敏感检测分泌呕吐毒素蜡样芽孢杆菌的方法

Publications (1)

Publication Number Publication Date
CN103725751A true CN103725751A (zh) 2014-04-16

Family

ID=50450042

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201210380431.6A Pending CN103725751A (zh) 2012-10-10 2012-10-10 一种高特异高敏感检测分泌呕吐毒素蜡样芽孢杆菌的方法

Country Status (1)

Country Link
CN (1) CN103725751A (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107475401A (zh) * 2017-09-08 2017-12-15 江苏农林职业技术学院 利用环介导等温扩增技术检测食源性蜡样芽孢杆菌的方法及引物
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

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101386884A (zh) * 2008-10-31 2009-03-18 东北农业大学 一种同时检测原料乳中多种致病菌的方法及检测试剂盒
CN101831493A (zh) * 2009-11-06 2010-09-15 武汉工业学院 致病性蜡样芽孢杆菌的环介导等温扩增引物对与检测方法
CN102181545A (zh) * 2011-04-11 2011-09-14 深圳国际旅行卫生保健中心 食源性致病菌检测试剂盒及检测方法
CN102311993A (zh) * 2010-07-08 2012-01-11 天津生物芯片技术有限责任公司 用于检测水产品中重要致病菌的基因芯片及其试剂盒

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101386884A (zh) * 2008-10-31 2009-03-18 东北农业大学 一种同时检测原料乳中多种致病菌的方法及检测试剂盒
CN101831493A (zh) * 2009-11-06 2010-09-15 武汉工业学院 致病性蜡样芽孢杆菌的环介导等温扩增引物对与检测方法
CN102311993A (zh) * 2010-07-08 2012-01-11 天津生物芯片技术有限责任公司 用于检测水产品中重要致病菌的基因芯片及其试剂盒
CN102181545A (zh) * 2011-04-11 2011-09-14 深圳国际旅行卫生保健中心 食源性致病菌检测试剂盒及检测方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107475401A (zh) * 2017-09-08 2017-12-15 江苏农林职业技术学院 利用环介导等温扩增技术检测食源性蜡样芽孢杆菌的方法及引物
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

Similar Documents

Publication Publication Date Title
US9951341B2 (en) Lactococcus CRISPR-Cas sequences
Siragusa et al. Synthesis of γ-aminobutyric acid by lactic acid bacteria isolated from a variety of Italian cheeses
Lhomme et al. The predominance of Lactobacillus sanfranciscensis in French organic sourdoughs and its impact on related bread characteristics
Yousif et al. Phylogenetic Characterization of Staphylococcus aureus isolated from the women breast abscess in Al-Qadisiyah Governorate, Iraq
Shi et al. Evaluation of safety and probiotic properties of a strain of Enterococcus faecium isolated from chicken bile
CN103725751A (zh) 一种高特异高敏感检测分泌呕吐毒素蜡样芽孢杆菌的方法
Shen et al. Evaluation of the potential probiotic yeast characteristics with anti-MRSA abilities
Su et al. Insights into complex infection by two Pectobacterium species causing potato blackleg and soft rot
Ferreira et al. Increased expression of clp genes in Lactobacillus delbrueckii UFV H2b20 exposed to acid stress and bile salts
Mostafa et al. Characterization of Bacillus cereus isolated from contaminated foods with sequencing of virulence genes in Egypt
CN101033471B (zh) 可表达和分泌β-半乳糖苷酶的大肠杆菌-乳酸菌穿梭质粒及其构建方法与应用
Zhu et al. BOX-PCR and PCR-DGGE analysis for bacterial diversity of a naturally fermented functional food (Enzyme®)
WO2015052665A4 (en) Process for the preparation of semi-finished flour based food products comprising an element with trans-glutaminas activity and a source of lysine
CN105646680B (zh) 布鲁氏菌s2疫苗gl_0002181蛋白的应用
Burke et al. Epidemiology of invasive Klebsiella pneumoniae with hypermucoviscosity phenotype in a research colony of nonhuman primates
Gao et al. The effects of elimination of fungi on microbial population and fiber degradation in sheep rumen
CN106011154A (zh) 一种致肝脓肿肺炎克雷伯菌分子检测试剂盒及其应用
CN110669707A (zh) 一种麦康凯琼脂平板及其制备方法
CN103205491A (zh) 一种高特异高敏感检测金黄色葡萄球菌的方法
CN102242092A (zh) 一种家蚕肠道益生菌的分离鉴定及其酶基因的克隆与表达
CN103725757A (zh) 一种高特异高敏感检测分泌肠毒素蜡样芽孢杆菌的方法
CN104611411A (zh) 产高效抗菌肽butyrisin的丁酸梭菌的筛选法
CN103725752A (zh) 一种高特异高敏感检测食源性致病性耶尔森氏菌的方法
CN105063077B (zh) 一种提高乳酸菌乙醇胁迫抗性的方法
CN103725756A (zh) 一种高特异高敏感检测肠聚集性大肠杆菌的方法

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C05 Deemed withdrawal (patent law before 1993)
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20140416