CN103725767A - High-specificity high-sensitivity vibrio cholerae detection method - Google Patents
High-specificity high-sensitivity vibrio cholerae detection method Download PDFInfo
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- CN103725767A CN103725767A CN201210385900.3A CN201210385900A CN103725767A CN 103725767 A CN103725767 A CN 103725767A CN 201210385900 A CN201210385900 A CN 201210385900A CN 103725767 A CN103725767 A CN 103725767A
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- vibrio cholerae
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- C12Q1/6844—Nucleic acid amplification reactions
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Abstract
The invention discloses a high-specificity high-sensitivity vibrio cholerae detection method. The method comprises the following steps of 1, preparing a reaction solution, 2, putting the prepared reaction solution into an oven having a temperature of 63 DEG C and carrying out a reaction process for 30-60min, wherein if the reaction solution becomes turbid, the detection result is positive, and 3, taking nucleic acids of vibrio cholera and nucleic acids of clinical isolates of staphylococcus aureus, scalp staphylococcus, human staphylococcus, staphylococcus epidermidis, viridans streptococcus, escherichia coli and Klebsiella, carrying out multiple reaction processes and carrying out positive and negative result observation. Through reaction solution preparation and primer sequence design, vibrio cholerae can be fast and accurately detected. According to the detection result, corresponding measures are adopted timely so that disease incidence is reduced.
Description
Technical field
The present invention relates to disease detection technical field, specifically, is a kind of high special high responsive method that detects vibrio cholerae.
Background technology
Cholera (cholera) is a kind of severe intestinal transmissible disease, and one of two kinds of category A infectious diseases, by vibrio cholerae (Vibrio cholerae) polluted water and food and cause propagation.Clinically hurried take onset, acutely rush down that to tell, drain a large amount of water in which rice has been washed sample intestinal contentses, dehydration, myospasm oliguresis and anuria be feature.It is main that the source of infection of vibrio cholerae is mainly contaminated diet, and the vibrio cholerae in rapid detection diet has very large meaning to prevention food poisoning and definite effective treatment plan.
Loop-mediated isothermal amplification technique is the nucleic acid detection system by the high specific susceptibility of Japanese Rong Yan (EIKEN) Co., Ltd. exploitation, equipment that need not be expensive, become the excellent means of nucleic acid fast detection method, coupling collar mediated isothermal amplification technology of the present invention, design is carried out ring mediated isothermal amplification for a pair of outer primer (V-F3 and V-B3) and a pair of inner primer (V-FIP and V-BIP) of vibrio cholerae specific gene sequence.
Summary of the invention
The invention discloses a kind of high special high responsive method that detects vibrio cholerae, improved its diagnosis and detection efficiency.
For realizing above-mentioned technical purpose, reach above-mentioned technique effect, the present invention is achieved through the following technical solutions:
A high special high responsive method that detects vibrio cholerae, comprises the following steps:
Step 1) preparation feedback liquid
Reaction solution composition and content are
8u/ul Bst nucleic acid polymerase 8u
10X heat-resisting polymerase reaction buffer 1X, MgSO4 2mM
4M trimethyl-glycine 1M
10mM d-NTP mixed solution 400 μ M
Inner primer
V-FIP 40umol
V-BIP 40umol
Outer primer
V-F3 5umol
V-B3 5umol
Sample nucleic acid 20ng
Add diethylpyrocarbonate processing water to total amount and reach 25ul;
Step 2) above-mentioned reaction solution is put into 63 ℃ of baking ovens react 30-60min, muddy positive;
Step 3) is used the nucleic acid of vibrio cholerae, and the nucleic acid of some clinical SEPARATION OF GOLD staphylococcus aureus, scalp staphylococcus, human-like staphylococcus, staphylococcus epidermidis, Streptococcus viridans, intestinal bacteria, klebsiella, repeatedly react, observe their yin and yang attribute.
Further, the sequence of described outer primer V-F3 is CTGCTAACGTTGGCTTTG.
Further, the sequence of described outer primer V-B3 is GGCTTTATTTTCGAAATAGGGC.
Further, the sequence of described inner primer V-FIP is
CCGTTGTTTCAATATTGGCATACC-TTATATGCTCAATGATAGCTGGT。
Further, the sequence of described inner primer V-BIP is AGCAGGTGCAGATGCCAAAT-GTTAGAACTTATAACCACCCG.
Further, in described reaction solution, add fluorescence display material ethidium bromide, fluorexon to carry out real-time inspection.
The invention has the beneficial effects as follows:
The present invention is by preparation reaction solution, and design primer sequence, can detect fast and accurately vibrio cholerae, and take appropriate measures in time, has reduced the incidence of disease.
Embodiment
Below in conjunction with embodiment, describe the present invention in detail.
A high special high responsive method that detects vibrio cholerae, comprises the following steps:
Step 1) preparation feedback liquid
Reaction solution composition and content are
8u/ul Bst nucleic acid polymerase 8u
10X heat-resisting polymerase reaction buffer 1X, MgSO4 2mM
4M trimethyl-glycine 1M
10mM d-NTP mixed solution 400 μ M
Inner primer
V-FIP 40umol
V-BIP 40umol
Outer primer
V-F3 5umol
V-B3 5umol
Sample nucleic acid 20ng
Add diethylpyrocarbonate processing water to total amount and reach 25ul.
Choose the specific gene astA(gene order source state-run biotechnology information center-NCBI of the U.S. of vibrio cholerae), design primer sequence.The sequence of described outer primer V-F3 is CTGCTAACGTTGGCTTTG, and the sequence of described outer primer V-B3 is GGCTTTATTTTCGAAATAGGGC, and the sequence of described inner primer V-FIP is
CCGTTGTTTCAATATTGGCATACC-TTATATGCTCAATGATAGCTGGT, the sequence of described inner primer V-BIP is AGCAGGTGCAGATGCCAAAT-GTTAGAACTTATAACCACCCG.
Further, in described reaction solution, add fluorescence display material ethidium bromide, fluorexon to carry out real-time inspection.
Step 2) above-mentioned reaction solution is put into 63 ℃ of baking ovens react 30-60min, muddy positive;
Step 3) is used the nucleic acid of vibrio cholerae 3 strains, and the nucleic acid of some clinical SEPARATION OF GOLD staphylococcus aureus, scalp staphylococcus, human-like staphylococcus, staphylococcus epidermidis, Streptococcus viridans, intestinal bacteria, klebsiella, repeatedly react, observe their yin and yang attribute.Result shows: the nucleic acid of 3 strain vibrio cholerae is at every turn all positive, and all the other bacterial classifications are negative.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
<110> Suzhou Si Tong Pharmaceutical Technology Co., Ltd
Mono-kind high special high responsive method that detects vibrio cholerae of <120>
<160> 4
<210> 1
<211> 18
<212> DNA
<213> artificial sequence
<400> 1
1 CTGCTAACGTTGGCTTTG
<210> 2
<211> 22
<212> DNA
<213> artificial sequence
<400> 2
1 GGCTTTATTTTCGAAATAGGGC
<210> 3
<211> 47
<212> DNA
<213> artificial sequence
<400> 3
1 CCGTTGTTTCAATATTGGCATACC-TTATATGCTCAATGATAGCTGGT
<210> 4
<211> 41
<212> DNA
<213> artificial sequence
<400> 4
1 AGCAGGTGCAGATGCCAAAT-GTTAGAACTTATAACCACCCG
Claims (6)
1. a high special high responsive method that detects vibrio cholerae, is characterized in that, comprises the following steps:
Step 1) preparation feedback liquid
Reaction solution composition and content are
8u/ul Bst nucleic acid polymerase 8u
10X heat-resisting polymerase reaction buffer 1X, MgSO4 2mM
4M trimethyl-glycine 1M
10mM d-NTP mixed solution 400 μ M
Inner primer
V-FIP 40umol
V-BIP 40umol
Outer primer
V-F3 5umol
V-B3 5umol
Sample nucleic acid 20ng
Add diethylpyrocarbonate processing water to total amount and reach 25ul;
Step 2) above-mentioned reaction solution is put into 63 ℃ of baking ovens react 30-60min, muddy positive;
Step 3) is used the nucleic acid of vibrio cholerae, and the nucleic acid of some clinical SEPARATION OF GOLD staphylococcus aureus, scalp staphylococcus, human-like staphylococcus, staphylococcus epidermidis, Streptococcus viridans, intestinal bacteria, klebsiella, repeatedly react, observe their yin and yang attribute.
2. the special high responsive method that detects vibrio cholerae of height according to claim 1, is characterized in that: the sequence of described outer primer V-F3 is CTGCTAACGTTGGCTTTG.
3. the special high responsive method that detects vibrio cholerae of height according to claim 2, is characterized in that: the sequence of described outer primer V-B3 is GGCTTTATTTTCGAAATAGGGC.
4. the special high responsive method that detects vibrio cholerae of height according to claim 3, is characterized in that: the sequence of described inner primer V-FIP is
CCGTTGTTTCAATATTGGCATACC-TTATATGCTCAATGATAGCTGGT。
5. the special high responsive method that detects vibrio cholerae of height according to claim 4, is characterized in that: the sequence of described inner primer V-BIP is AGCAGGTGCAGATGCCAAAT-GTTAGAACTTATAACCACCCG.
6. the special high responsive method that detects vibrio cholerae of height according to claim 5, is characterized in that: in described reaction solution, add fluorescence display material ethidium bromide, fluorexon to carry out real-time inspection.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101020926A (en) * | 2007-03-09 | 2007-08-22 | 中国科学院南海海洋研究所 | Reagent kit and process for detecting cholera vibrio in circular mediated constant temperature amplification method |
| CN101153332A (en) * | 2007-09-21 | 2008-04-02 | 珠海市疾病预防控制中心 | Primer, detection method and detection reagent kit for detecting cholera vibrio |
| CN101386885A (en) * | 2008-10-15 | 2009-03-18 | 山东出入境检验检疫局检验检疫技术中心 | Toxigenous commabacillus cholera vibrio ring mediated isothermal amplification rapid detection method |
| CN102242216A (en) * | 2011-07-14 | 2011-11-16 | 浙江省疾病预防控制中心 | Fluorescent PCR (polymerase chain reaction) detection kit for vibrio cholerae and systematic identification method thereof |
-
2012
- 2012-10-12 CN CN201210385900.3A patent/CN103725767A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101020926A (en) * | 2007-03-09 | 2007-08-22 | 中国科学院南海海洋研究所 | Reagent kit and process for detecting cholera vibrio in circular mediated constant temperature amplification method |
| CN101153332A (en) * | 2007-09-21 | 2008-04-02 | 珠海市疾病预防控制中心 | Primer, detection method and detection reagent kit for detecting cholera vibrio |
| CN101386885A (en) * | 2008-10-15 | 2009-03-18 | 山东出入境检验检疫局检验检疫技术中心 | Toxigenous commabacillus cholera vibrio ring mediated isothermal amplification rapid detection method |
| CN102242216A (en) * | 2011-07-14 | 2011-11-16 | 浙江省疾病预防控制中心 | Fluorescent PCR (polymerase chain reaction) detection kit for vibrio cholerae and systematic identification method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 柯雪梅等: "霍乱弧菌LAMP快速检测方法的建立及应用", 《南方医科大学学报》, vol. 29, no. 10, 31 December 2009 (2009-12-31), pages 2059 - 2063 * |
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Application publication date: 20140416 |