CN101178363B - Assistant hemolysis vibrion constant temperature gene amplification fast detecting reagent box and methods - Google Patents
Assistant hemolysis vibrion constant temperature gene amplification fast detecting reagent box and methods Download PDFInfo
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- CN101178363B CN101178363B CN2007100309481A CN200710030948A CN101178363B CN 101178363 B CN101178363 B CN 101178363B CN 2007100309481 A CN2007100309481 A CN 2007100309481A CN 200710030948 A CN200710030948 A CN 200710030948A CN 101178363 B CN101178363 B CN 101178363B
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Abstract
The invention discloses a vibrio parahaemolyticus constant temperature gene amplification fast-detection kit and a method thereof, the kit includes primer mixed liquid with a sequence of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and so on, Bst DNA polymerase, reaction solution, sample pretreatment solution, chromogenic agent, positive control and negative control. The invention can make use of the kit to realize the high specific, rapid, high sensitive and simple detection of the vibrio parahaemolyticus by the extraction of the vibrio parahaemolyticus gene group DNA, constant temperature gene amplification reaction and the judgement of the results of the reaction products.
Description
Technical field
The present invention relates to microorganism detection reagent and method, be specifically related to a kind of assistant hemolysis vibrion constant temperature gene amplification fast detecting reagent box and method.
Background technology
Vibrio parahaemolytious (Vibrio parahaemolyticus is called for short VP) is a kind of halophagia bacterium that is distributed widely in ocean and the salt lake, and this bacterium is often arranged in the marine product.Eaten when polluting marine product life or that do not boil that vibrio parahaemolytious is arranged and can cause serious acute enterogastritis, in the formation of food posioning, the acute gastroenteritis that causes with vibrio parahaemolytious accounts for the first place again.Therefore, the detection means of rapid sensitive is the key that the prevention and control vibrio parahaemolytious is propagated.Common traditional authentication method is separation and Culture, microscopy observation, biochemical identification, halophagia test etc., and the not only consuming time but also complicated operation of these detection meanss, sensitivity are low.In recent years, there is a large amount of reports to confirm that the method for PCR-based successfully has been used to detect vibrio parahaemolytious, PCR method improves a lot than conventional method on the running time and aspect specificity that detects and the sensitivity, but, PCR method must have accurate temperature cycles device, its detection time is still long (4～8 hours) also, and course of reaction is easy to the influence of contaminated thing, thereby make this method can not satisfy the needs that detect on the spot.Except PCR method, also have other nucleic acid amplification methods, such as amplification of nucleic acid sequences method (NASBA), from sequence replica method (3SR) and strand displacement replica method (SDA).The detection level of these three kinds of methods all is no less than 10 copies, in the similar scope that just target nucleic acid can be increased in about 1 hour consuming time, however, they are not strong to the specific amplification of target sequence, also need detailed experimental implementation means to detect amplified production after the feasible amplification.The immunology detection technology is fast and convenient with low cost, but requires the monoclonal antibody of high-quality high stability, otherwise because of accuracy is not enough, can only be the auxiliary detection means at present.So, the newest fruits of biotech development is applied to the pathogenic microorganism detection, significant.Also have some detection methods in addition, such as immunochromatography, DNA hybrid method, though the detection sensitivity height, equipment needed thereby and operating process more complicated can not satisfy the demand of fast detecting.
Dna circle mediated constant temperature nucleic acid amplification technology (loop-mediated isothermal amplification ofDNA, LAMP) overcome the deficiency of gene amplification method in the past, can specificity under isothermy, carry out the amplification of nucleic acid efficiently, apace, have a lot of superiority.This method is normally carried out as follows: step 1, tested sample is carried out pre-service, the DNA of the tested sample of rapid extraction or RNA; The preparation of step 2, Auele Specific Primer: after determining the target gene of sample to be measured, obtain 2 pairs of Auele Specific Primers, each is a pair of for inner primer and outer primer; Step 3, carry out loop-mediated isothermal amplification technique (LAMP) reaction: will mix with the BstDNA polymerase through sample, primer, the reaction buffer of pre-treatment, be incubated 0.5 to 1.5 hour at 63～65 ℃ and carry out the endless-chain displacement reaction; Step 4, analysis and judgement reaction product result.Though disclosed LAMP technology has high sensitivity, can be under isothermy amplification of nucleic acid efficiently, prior art also has weak point, mainly is: 1. its special primer requires the very harsh widespread use that limits this method; 2. the milky white precipitate thing judged result that only generates with reaction is accurately clear inadequately, also is difficult for the realization response miniaturization, limits it and further develops.Be necessary existing LAMP technology is improved in order to overcome these shortcomings.
Summary of the invention
The objective of the invention is to overcome above-mentioned technological deficiency, a kind of assistant hemolysis vibrion constant temperature gene amplification fast detecting reagent box and method are provided.
To achieve the object of the present invention, by the following technical solutions:
A kind of assistant hemolysis vibrion constant temperature gene amplification fast detecting reagent box of the present invention, its reagent comprises:
---the primer mixed liquor: four primer concentrations are 10pmol/ μ l, primer sequence such as SEQ ID NO.1, SEQ ID NO.2, SEQ IDNO.3, SEQ ID NO.4;
---the Bst archaeal dna polymerase: concentration is 8U/ μ l;
---reactant liquor: 10mM dNTP: 10 * ThermoPol reaction buffer: 150mM MgSO
4=8: 5: 2;
---sample pretreatment liquid: 20mM Tris-HCl (pH 8.0), 2mMEDTA, 1.2%TritonX-100;
---developer: fluorescent dye 1 * SYBR Green I;
---positive control is vibrio parahaemolyticus gene group DNA, and negative control is not for containing the reaction mixture of template.
The method that detects vibrio parahaemolytious with above-mentioned kit may further comprise the steps:
(1) vibrio parahaemolytious DNA extraction process to be checked: the enrichment liquid of getting incubated overnight is in centrifuge tube, and centrifugal 2 minutes of 1000rpm removes supernatant; Add sample pretreatment liquid in centrifuge tube, mix with precipitation gained thalline; Boil in the boiling water after 10 minutes and placed cooled on ice immediately 10 minutes; Centrifugal 2 minutes of 10000-12000rpm, supernatant promptly can be used as vibrio parahaemolytious DNA to be checked; Wherein the amount ratio of enrichment liquid and sample pretreatment liquid is 5: 8;
(2) constant temperature gene amplification reaction: prepare reaction solution at the 200ulPCR pipe: primer mixture 1.5 μ l, reactant liquor 5.5 μ l, Bst archaeal dna polymerase 0.5 μ l, template DNA 2 μ l add water to 11.5 μ l; When the positive control reaction is set, substitute the vibrio parahaemolytious DNA to be checked that obtains in (1), when the negative control reaction is set, substitute the vibrio parahaemolytious DNA to be checked that obtains in (1) with the reaction mixture that does not contain template with vibrio parahaemolyticus gene group DNA; The PCR pipe that will contain the reaction solution for preparing was in 65 ℃ of reactions 1.5 hours;
(3) analysis and judgement reaction product result: obtain adding in the product 2.5 μ l fluorescent dyes, 1 * SYBR Green I in (2), mixing leaves standstill 5min; If shows green is then positive, orange then negative.
A kind of assistant hemolysis vibrion constant temperature gene amplification fast detecting reagent box of the present invention and method have following beneficial effect:
1, high specific: the present invention has designed four Auele Specific Primers according to vibrio parahaemolytious tlh virulence gene, primer sequence such as SEQ IDNO1, SEQ ID NO2, SEQ ID NO3, SEQ ID NO4;
Use above-mentioned four primers, whether the existence that just can judge target substance according to whether increasing, and positive rate can reach greater than 99%, and false positive rate is less than 1%;
2, fast efficient amplification: amplification can be finished less than 1 hour, and the productive rate height;
3, highly sensitive: the lowest detection limit reaches 10CFU/ml, and the recall rate of sample reaches 99%;
4, evaluation is easy: identify by visual inspection, need not other any analytical procedures such as electrophoresis.
Embodiment
Press following formulation vibrio parahaemolytious isothermal gene amplification fast detecting kit:
---the primer mixed liquor: four primer concentrations are 10pmol/ μ l, primer sequence such as SEQ ID NO.1, SEQ ID NO.2, SEQ IDNO.3, SEQ ID NO.4;
---the Bst archaeal dna polymerase: concentration is 8U/ μ l;
---reactant liquor: 80 μ l 10mM dNTP, 50 μ l, 10 * ThermoPol reaction buffer, 20 μ l 150mM MgSO
4
---sample pretreatment liquid: 20mM Tris-HCl (pH 8.0), 2mM EDTA, 1.2%Triton X-100;
---developer: fluorescent dye 1 * SYBR Green I;
---positive control is vibrio parahaemolyticus gene group DNA, and negative control is not for containing the reaction mixture of template.
By the following method vibrio parahaemolytious is detected with above-mentioned kit:
1, the simple and easy leaching process of vibrio parahaemolytious DNA to be checked:
(1), the enrichment liquid of getting 50 μ l incubated overnight in centrifuge tube, centrifugal 2 minutes of 1000rpm removes supernatant;
(2), add 80 μ l sample pretreatment liquid in centrifuge tube, mix with precipitation gained thalline;
(3), boil in the boiling water after 10 minutes and placed cooled on ice immediately 10 minutes;
(4), centrifugal 2 minutes of 10000rpm, supernatant promptly can be used as vibrio parahaemolytious DNA to be checked.
2, loop-mediated isothermal amplification (LAMP) reaction:
(1), at 200ulPCR pipe preparation reaction mixture: primer mixture 1.5 μ l, reactant liquor 5.5 μ l, Bst archaeal dna polymerase 0.5 μ l, template DNA 2 μ l add water to 11.5 μ l; When the positive control reaction is set, substitute vibrio parahaemolytious DNA to be checked, when the negative control reaction is set, substitute vibrio parahaemolytious DNA to be checked with the reaction mixture that does not contain template with vibrio parahaemolyticus gene group DNA;
(2), will contain the PCR pipe of the reaction mixture for preparing in 65 ℃ of reactions 1.5 hours.
3, analysis and judgement reaction product result:
Add 2.5 μ l fluorescent dyes (1 * SYBR Green I) in reaction product, mixing leaves standstill 5min.If shows green is then positive, orange then negative.
The primer sequence table:
SEQUENCE?LISTING
SEQ?ID?NO.1
＜110〉South China Science ﹠ Engineering University
Sunlight, the Shandong
Beautiful, the king
Beautiful jade, Lee
＜120〉assistant hemolysis vibrion constant temperature gene amplification fast detecting reagent box and method
<130>Notomi?T，Okayama?H，Masubuchi?H，YonekawaT，Watanabe?K，AminoN，Hase?T.Loop-mediated?isothermal?amplification?ofDNA，NucleicAcids?Res.2000?Jun?15；28(12)：E63.
<160>1
<170>PatentIn?version?3.4
<210>1
<211>19
<212>DNA
<213>Vibrio?parahaemolyticus
<400>1
cgcaccagct?actcgaaag 19
SEQUENCE?LISTING
SEQ?ID?NO.2
＜110〉South China Science ﹠ Engineering University
Sunlight, the Shandong
Beautiful, the king
Beautiful jade, Lee
＜120〉assistant hemolysis vibrion constant temperature gene amplification fast detecting reagent box and method
<130>Notomi?T，Okayama?H，Masubuchi?H，Yonekawa?T，Watanabe?K，AminoN，Hase?T.Loop-mediated?isothermal?amplification?ofDNA，NucleicAcids?Res.2000?Jun?15；28(12)：E63.
<160>1
<170>PatentIn?version?3.4
<210>1
<211>20
<212>DNA
<213>Vibrio?parahaemolyticus
<400>1
cggcgaagaa?cgtaatgtct 20
SEQUENCE?LISTING
SEQ?ID?NO.3
＜110〉South China Science ﹠ Engineering University
Sunlight, the Shandong
Beautiful, the king
Beautiful jade, Lee
＜120〉assistant hemolysis vibrion constant temperature gene amplification fast detecting reagent box and method
<130>Notomi?T，Okayama?H，Masubuchi?H，Yonekawa?T，Watanabe?K，AminoN，Hase?T.Loop-mediated?isothermal?amplification?ofDNA，NucleicAcids?Res.2000?Jun?15；28(12)：E63.
<160>1
<170>PatentIn?version?3.4
<210>1
<211>40
<212>DNA
<213>Vibrio?parahaemolyticus
<400>1
ccaccagtag?ccgtcaatgg?tggcgaccga?ttgggaatgg 40
SEQUENCE?LISTING
SEQ?ID?NO.4
＜110〉South China Science ﹠ Engineering University
Sunlight, the Shandong
Beautiful, the king
Beautiful jade, Lee
＜120〉assistant hemolysis vibrion constant temperature gene amplification fast detecting reagent box and method
<130>Notomi?T，Okayama?H，Masubuchi?H，Yonekawa?T，Watanabe?K，AminoN，Hase?T.Loop-mediatedisothermal?amplification?ofDNA，NucleicAcids?Res.2000?Jun?15；28(12)：E63.
<160>1
<170>PatentIn?version?3.4
<210>1
<211>41
<212>DNA
<213>Vibrio?parahaemolyticus
<400>1
acaccaacac?gtcgcaaaac?gtgcgttctc?gttcgccaaa?t 41
Claims (2)
1. an assistant hemolysis vibrion constant temperature gene amplification detection kit is characterized in that, its reagent comprises:
---the primer mixed liquor: four primer concentrations are 10pmol/ μ l, and primer sequence is SEQ ID NO.1, SEQ ID NO.2, SEQ IDNO.3, SEQ ID NO.4;
---the Bst archaeal dna polymerase: concentration is 8U/ μ l;
---reactant liquor: 10mMd NTP: 10 * ThermoPol reaction buffer: 150mM MgSO
4=8: 5: 2;
---sample pretreatment liquid: 20mM Tris-HCl, pH8.0,2mMEDTA, 1.2% TritonX-100;
---developer: fluorescent dye 1 * SYBR Green I;
---positive control is vibrio parahaemolyticus gene group DNA, and negative control is not for containing the reaction mixture of template.
2. the method with the described kit detection of claim 1 vibrio parahaemolytious is characterized in that, may further comprise the steps:
(1) vibrio parahaemolytious DNA extraction process to be checked: the enrichment liquid of getting incubated overnight is in centrifuge tube, and centrifugal 2 minutes of 1000rpm removes supernatant; Add sample pretreatment liquid in centrifuge tube, mix with precipitation gained thalline; Boil in the boiling water after 10 minutes and placed cooled on ice immediately 10 minutes; Centrifugal 2 minutes of 10000-12000rpm, supernatant promptly can be used as vibrio parahaemolytious DNA to be checked; Wherein the amount ratio of enrichment liquid and sample pretreatment liquid is 5: 8;
(2) constant temperature gene amplification reaction: in the PCR pipe, prepare reaction solution: primer mixed liquor 1.5 μ l, reactant liquor 5.5 μ l, Bst archaeal dna polymerase 0.5 μ l, the DNA to be checked 2 μ l that obtain in (1) add water to 11.5 μ l; When the positive control reaction is set, substitute the vibrio parahaemolytious DNA to be checked that obtains in (1), when the negative control reaction is set, substitute the vibrio parahaemolytious DNA to be checked that obtains in (1) with the reaction mixture that does not contain template with vibrio parahaemolyticus gene group DNA; The PCR pipe that will contain the reaction solution for preparing was in 65 ℃ of reactions 1.5 hours;
(3) analysis and judgement reaction product result: add 2.5 μ l developers in the product that obtains in (2), mixing leaves standstill 5min; If shows green is then positive, orange then negative.
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| CN101178363B true CN101178363B (en) | 2010-10-27 |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060230A (en) * | 2012-12-19 | 2013-04-24 | 华南农业大学 | Culture medium for promoting vibrio parahemolyticus to enter non-culturable state and method |
| CN105331679A (en) * | 2014-08-15 | 2016-02-17 | 中山鼎晟生物科技有限公司 | Kit for detecting vibrio parahaemolyticus in food and detection method |
| CN104561275A (en) * | 2014-12-18 | 2015-04-29 | 浙江省疾病预防控制中心 | Vibrio parahaemolyticus isothermal amplification detection kit and detection method |
| CN109517913B (en) * | 2018-12-25 | 2022-08-16 | 华南理工大学 | Primer, kit and method for PSR (phosphoenolpyruvate carboxylase) detection of heat-resistant direct hemolysin and heat-resistant related hemolysin |
| CN112239789A (en) * | 2020-11-13 | 2021-01-19 | 宁波大学 | Primer and kit capable of simultaneously detecting 4 virulence genes of vibrio parahaemolyticus |
| CN113337627B (en) * | 2021-06-03 | 2022-08-05 | 浙江省农业科学院 | Label-free visual detection method for vibrio parahaemolyticus gene based on CRISPR/Cas12a |
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