CN101487057B - Loop-mediated isothermal amplification fast detecting reagent kit for O157:H7 coliform - Google Patents
Loop-mediated isothermal amplification fast detecting reagent kit for O157:H7 coliform Download PDFInfo
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- CN101487057B CN101487057B CN2009100960306A CN200910096030A CN101487057B CN 101487057 B CN101487057 B CN 101487057B CN 2009100960306 A CN2009100960306 A CN 2009100960306A CN 200910096030 A CN200910096030 A CN 200910096030A CN 101487057 B CN101487057 B CN 101487057B
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Abstract
The invention provides a quick detecting kit that is used for loop-mediated isothermal augmentation of O157:H7 coliform, designs LAMP primers respectively for O157:H7 O-antigen encoding rfbE, Shiga-like toxin stx2 and H flagellar antigen encoding flic gene, provides a quick detecting kit and a detecting method with strong specificity and high sensitivity that are used for loop-mediated isothermal augmentation of O157:H7 coliform, and has following essential advantages: (1) expensive and precise instruments and equipments are unnecessary, and only a metal pyrolyzer/temperature-constant water bathing device is required; (2) the detecting kit and the detecting method consume less time, and can finish reaction in about 1 hour at constant temperature; (3) a result can be determined by observing color changes of a reaction system with naked eyes under white light, thus having simple and convenient operation; and (4) the method is simple, easy, quick, specific and sensitive, and has wider application prospect in quick detection aspects.
Description
(1) technical field
The present invention relates to loop-mediated isothermal amplification fast detecting reagent kit and the detection method of O157:H7 colibacillus.
(2) background technology
New discovery and the pathogenic micro-organism that comes back again are the new problems that our times medical science faces, and enterohemorrhagic colibacillus O157:H7 is exactly wherein a kind of.O157:H7 is that the enterohemorrhagic Escherichia coli, E coli (found in the U.S. first in nineteen eighty-two, is the New Development pathogenic bacteria by Enterohemorrhagic Escherichia coli, EHEC) topmost serotype.It is pathogenic strong, and infective dose is extremely low, eats 10 bacteriums of less than and can cause disease.This bacterium mainly cause bleeding enteritis (HC), hemolytic uremic syndrome (HCS) and thrombotic thrombocytopenic purpura (TTP), case fatality rate are the worldwide public health problems that the whole world is paid close attention to up to more than 30%.China from 1988 after the Xuzhou detects first strain EHEC O157:H7, the food inspection to enteron aisle outpatient service, livestock and poultry and meat product etc. has been strengthened in various places, repeatedly detects the O157:H7 colibacillus in succession from diarrhoea patient, domestic animal, poultry and food.By our province O157 strain isolated virulence gene detection (PCR method) is in recent years found that our province is mainly based on Stx2, the bacterial strain that carries Stx2 and Stx1 gene simultaneously is then fewer, this is similar to domestic other provinces and cities isolated most of bacterial strains, but then different with report such as Fagan, be that target gene detects so the stx2 gene is mainly chosen in this research.RfbE and fliC gene encode respectively O157:H7 O antigen O antigen and flagellar H antigens will help the further checking of O157:H7 serotype diagnosis at the detection of these two kinds of genes.
At present, the most of laboratory qualification O157:H7 of basic unit colibacilluss still mainly depend on traditional cultural method, biochemistry and serological identification method, and the method for inspection is loaded down with trivial details, waste time and energy, and the report assay time is long, is difficult to adapt to the requirement of rapid detection.In recent years, the PCR method detects the O157:H7 colibacillus and has obtained developing rapidly, mainly contains conventional PCR, multiplex PCR, Real-time PCR etc., but these methods all need special instrument, are not suitable for promoting the use of in basic unit or small test chamber.Notomi etc. are a kind of new dna circle mediated isothermal amplification method (the loop-mediated isothermal amplification that develops in 2000, LAMP), because of it has characteristics such as easy to operation, that interpretation as a result is simple, can promote the use of in the basic unit laboratory, this method is widely used in some countries and regions, domestic then at the early-stage to this technical study, use LAMP technology for detection O157:H7 colibacillus and do not see bibliographical information at present.
Loop-mediated isothermal amplification technique principle: ring mediated isothermal amplification (loop-mediated isothermalamplification, LAMP) technology is the constant-temperature amplification method of a kind of new dna circle mediation of developing in 2000 such as Notomi, promptly utilize Bst large fragment DNA polysaccharase and the 4-6 bar primer that designs according to different target sequences, discern 6 specific regions on the target sequence specifically, the purpose target gene that efficiently increases specifically under 60～65 ℃ of isothermal conditions, end product are the stem circular DNAs that has with the anti-phase tumor-necrosis factor glycoproteins of purpose target gene.If design 1-2 bar ring primer again, then can improve amplified reaction speed greatly.One big characteristics of this method are, can whether judge the existence of target gene by byproduct of reaction-sedimentary generation of white magnesium pyrophosphate, or add fluorescent dye reagent in system, observe under ultraviolet lamp, and reaction product is monitored in real time.
(3) summary of the invention
The present invention is exactly according to above-mentioned principle, design the LAMP primer respectively at O antigen encoding rfbE, the shiga-like toxin stx2 of O157:H7, H flagellar antigen coding flic gene, loop-mediated isothermal amplification fast detecting reagent kit and the detection method of a kind of high specificity, highly sensitive O157:H7 colibacillus is provided.
The technical solution used in the present invention is:
The loop-mediated isothermal amplification fast detecting reagent kit of O157:H7 colibacillus mainly comprises specificity amplification primer, Bst archaeal dna polymerase, deoxyribonucleotide mixture, PCR damping fluid, and described specificity amplification primer sequence is as follows:
RfbE FIP (O antigen encoding rfbE specific amplification inner primer FIP):
CTCTCTTTCCTCTGCGGTCCGATGTTTTTCACACTTATTGGAT；
RfbE BIP (O antigen encoding rfbE specific amplification inner primer BIP):
TAAGGAATCACCTTGCAGATAAACTAGTACATTGGCATCGTGT；
RfbE F3 (O antigen encoding rfbE specific amplification outer primer F3):
AACAGTCTTGTACAAGTCCA；
RfbE B3 (O antigen encoding rfbE specific amplification outer primer B3):
GGTGCTTTTGATATTTTTCCG；
RfbE LB (O antigen encoding rfbE specific amplification ring primer LB):
CGAAACAAGGCCAGTTTTTTACC；
Stx2FIP (shiga-like toxin stx2 specific amplification inner primer FIP):
GGTTAATAACAGACACCGATGTGG-ACAGAGATATCGACCCCT；
Stx2BIP (shiga-like toxin stx2 specific amplification inner primer BIP):
TAAGGAATCACCTTGCAGATAAACTAGTACATTGGCATCGTGT；
Stx2F3 (shiga-like toxin stx2 specific amplification outer primer F3):
GTCTCTTCGTTAAATAGTATACGG；
Stx2B3 (shiga-like toxin stx2 specific amplification outer primer B3):
GGCCACATAAATTATTTTGCTC；
Flic FIP (H flagellar antigen coding flic gene specific amplification inner primer FIP):
GCCGGATACGCGGTCAATTTCACTCCGATTCTGACCTGGACT；
Flic BIP (H flagellar antigen coding flic gene specific amplification inner primer BIP):
TGAACGTGCTGGCGAAAGACGTTTCAGGTCGATCGTGATGG；
Flic F3 (H flagellar antigen coding flic gene specific amplification outer primer F3):
GAACTGACGGTTCAGGCC；
Flic B3 (H flagellar antigen coding flic gene specific amplification outer primer B3):
AGCCATTCAGACCCAGAGT。
Above-mentionedly want composition for the group of test kit, other ancillary components can be selected by this area ordinary method.Wherein deoxidation nucleoside triphosphate mixture (dNTP) is the mixing of dATP, dTTP, dCTP, dGTP, is generally dATP, dTTP, dCTP, dGTP amount of substance than 1: 1: 1: 1 mixture.
Also can comprise in the described test kit: trimethyl-glycine (Betaine), MgSO
4, MnCl
2And fluorexon (Calcein).The inventive method is main with reference to reports such as Tomita in 2008, can add an amount of MgSO in the LAMP reaction system
4, MnCl
2, fluorexon, whether reaction result generates by the visual inspection green under white light is judged, realization response and product detect a step and finish.This method is an improved fresh approach in addition on the method basis of Notomi etc., whether the result can produce green calcium manganese mixture by visual inspection under white light is judged that whether target gene exists, and it did not produce white magnesium pyrophosphate precipitation and be easier to judge than have by visual inspection under white light in the past.
The invention still further relates to the loop-mediated isothermal amplification fast detection method of O157:H7 colibacillus, described method comprises:
(1) design specificity amplification primer sequence is as follows:
rfbE?FIP：
CTCTCTTTCCTCTGCGGTCCGATGTTTTTCACACTTATTGGAT；
rfbE?BIP：
TAAGGAATCACCTTGCAGATAAACTAGTACATTGGCATCGTGT；
rfbE?F3：AACAGTCTTGTACAAGTCCA；
rfbE?B3：GGTGCTTTTGATATTTTTCCG；
rfbE?LB：CGAAACAAGGCCAGTTTTTTACC；
stx2FIP：
GGTTAATAACAGACACCGATGTGG-ACAGAGATATCGACCCCT；
stx2BIP：
TAAGGAATCACCTTGCAGATAAACTAGTACATTGGCATCGTGT；
stx2F3：GTCTCTTCGTTAAATAGTATACGG；
stx2B?3：GGCCACATATAAATTATTTTGCTC；
flic?FIP：
GCCGGATACGCGGTCAATTTCACTCCGATTCTGACCTGGACT；
flic?BIP：
TGAACGTGCTGGCGAAAGACGTTTCAGGTCGATCGTGATGG；
flic?F3：GAACTGACGGTTCAGGCC；
flic?B3：AGCCATTCAGACCCAGAGT；
(2) extracting testing sample DNA, is being that sequence rfbE FIP, rfbE BIP, rfbE B3, rfbE LB, stx2FIP, stx2BIP, stx2F3, stx2B3, flic FIP, flic BIP, flic F3 and flic B3 carry out LAMP in the reaction system of primer to react in template, step (1) with testing sample DNA;
(3) after the LAMP reaction finishes, analyze,, then contain the O157:H7 colibacillus in the testing sample if the LAMP reaction result is positive.
The main component final concentration is composed as follows in the described reaction system:
Bst dna polymerase buffer liquid final concentration is 1 *
Primer rfbE FIP, stx2FIP, each 1.2～2.0 μ M of flic FIP
Primer rfbE BIP, stx2BIP, each 1.2～2.0 μ M of flic BIP
Primer rfbE F3, stx2F3, each 0.1～0.5 μ M of flic F3
Primer rfbE B3, stx2B3, each 0.1～0.5 μ M of flic B3
Primer rfbE LB, 0.1～0.5 μ M
dNTP 0.4mM～1.0mM
Bst archaeal dna polymerase 5～10U/ reaction
Solvent is a DEPC water.Described DEPC water refers to that diethypyrocarbonate (diethylpyrocarbonate) handled and through the MiliQ of high temperature, autoclave sterilization level pure water.
Described damping fluid final concentration is 1 *, be meant that each component is same at reaction system final concentration and 1 * Bst dna polymerase buffer liquid phase in the damping fluid, represent that promptly each component final concentration in reaction solution is as follows in the damping fluid: 20mM Tris-HCl (pH8.8), 10mM KCl, 10mM (NH
4)
2SO
4, 4mMMgSO
4, 0.1%TritonX-100.
Also comprise the trimethyl-glycine of the component that final concentration is following: 0.5～1.0M, the MgSO of 2～6mM in the described reaction system
4, 0.3～0.8mM MnCl
2Fluorexon with 10～30 μ M.
Described step (2) LAMP reaction conditions is set to: 60 ℃ of 1h, 80 ℃ of 2min.
Concrete, described method is as follows:
(1) design specificity amplification primer sequence rfbE FIP, rfbE BIP, rfbE F3, rfbE B3, rfbELB, stx2FIP, stx2BIP, stx2F3, stx2B3, flic FIP, flic BIP, flic F3 and flic B3;
(2) extract testing sample DNA, preparation LAMP reaction system final concentration is composed as follows:
10 * Bst dna polymerase buffer liquid final concentration is 1 *
Primer rfbE FIP, stx2FIP, each 1.6 μ M of flic FIP
Primer rfbE BIP, stx2BIP, each 1.6 μ M of flic BIP
Primer rfbE F3, stx2F3, each 0.2 μ M of flic F3
Primer rfbE B3, stx2B3, each 0.2 μ M of flic B3
Primer rfbE LB 0.4 μ M
DNTP mixture 0.6mM
Trimethyl-glycine 0.8M
Fluorexon 25 μ M
MnCl
2 0.5mM
MgSO
4 4mM
Bst archaeal dna polymerase 8U/ reaction
Sample DNA is an amount of
Solvent is a DEPC water;
The LAMP reaction conditions is set to: 60 ℃ of 1h, 80 ℃ of 2min;
(3) after the LAMP reaction finishes, visual inspection product colour-change, present obvious green and be judged to be the positive, be that sample contains the O157:H7 colibacillus, whether present the orange feminine gender that is judged as, present limegreen and then carry out gel electrophoresis by 2% agarose and observe band and occur, the obvious person of electrophoretic band is judged to be the positive, not obvious person is judged to be feminine gender (or getting corresponding LAMP product 2ul LAMP once more, the observation colour-change).The yin and yang attribute contrast is all done in each test.
Beneficial effect of the present invention is mainly reflected in:
1. present method does not need to use the plant and instrument of costliness, precision, only needs a metal cracking instrument/constant temperature water bath apparatus;
2. weak point consuming time reacts under constant temperature, can finish about 1 hour, has shortened the sense cycle (detect to finish only need about 1.5h from being extracted into of bacterial strain nucleic acid) of this bacterium greatly;
3. can carry out the result by naked eyes range estimation reaction system colour-change under the white light and judge that realization response and product detect a step and finish, and be easy and simple to handle;
4. this method detects the another new method means that provide for rapid gene;
5. this method is simple and easy, quick, special, responsive, its characteristics and advantage are, it not only is suitable for scientific effort, can also be as uses such as basic unit's inspection department and the disease emergent diagnosis of small test chamber, as prevailing disease scholar person is trained a little, can be voluntarily use on emergency car or when carrying out on-the-spot epidemiology survey, comparatively application prospects is arranged aspect rapid detection.
(4) description of drawings
Fig. 1 is 16 strain O157 type strains and Local Isolates rfbE gene LAMP amplification;
Fig. 2 is 16 strain O157 type strains and Local Isolates Stx2 gene LAMP amplification;
Fig. 3 is 16 strain O157 type strains and Local Isolates fliC gene LAMP amplification.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
Utilize LAMP amplification detection method of the present invention that 56 strain O157 bacterial strains (comprising type strain O157:H7, local O157 strain isolated and other enteron aisle experiment strain) are detected, this result and regular-PCR, quantitative PCR method result are compared, with checking present method specificity.Its fliC gene of colibacillus O28ac84-1373, LT-85-44 and 84-135 LAMP result is positive in the experiment, its regular-PCR and quantitative PCR result are also all positive as a result, infer and to think that this may all to carry the fliC gene of coding O157:H7 flagellar H antigens relevant with this three strains bacterium.
Reaction system is as follows:
10 * damping fluid, 2.5 μ L
Primer rfbE FIP, stx2FIP, each 1.6 μ M of flic FIP
Primer rfbE BIP, stx2BIP, each 1.6 μ M of flic BIP
Primer rfbE F3, stx2F3, each 0.2 μ M of flic F3
Primer rfbE B3, stx2B3, each 0.2 μ M of flic B3
Primer rfbE LB 0.4 μ M
DNTP mixture (1: 1: 1: 1) be total to 0.6mM
Trimethyl-glycine 0.8M
Fluorexon 25 μ M
MnCl
2 0.5mM
MgSO
4 4mM
Bst polysaccharase 8U enzyme/reaction
Sample bacterial strain DNA 4 μ L
DEPC water complements to 25 μ L.
The LAMP amplification condition is set to 60 ℃ of 1h, 80 ℃ of 2min.
Wherein 16 strain O157 type strains and Local Isolates rfbE, Stx2, fliC gene LAMP amplification are seen Fig. 1, Fig. 2 and Fig. 3 respectively; (1～16 bacterial strain is followed successively by 882364 type strains, W933 type strain, 97-1 (O157:H7), 99-1 (O157:H7), 00-2 (O157:H-), 00-4 (O157:H7), 00-5 (O157:H7), 05-1 (O157:H7), 00-6 (O157:H7), 00-7 (O157:H7), 06-12 (O157:H7), Hangzhoupro 3-325 (O157:H-), Hangzhoupro 3-189 (O157:H-), HZ1-68 (O157:H-), Hangzhoupro 4-257 (O157:H-), Hangzhoupro 3-307 (O157:H-) among the figure).
The LAMP amplification of part bacterial strain as can be seen, uses the inventive method to detect by experiment, can detect evaluation at aimed strain, and specificity is good.
Sensitivity detects:
Getting the bacterium amount is 5 * 10
7The O157:H7 colibacillus of cfu/mL carries out 10 times of dilutions with distilled water, respectively from 10
-1～10
-8Draw 100uL in the diluent, again behind the high speed centrifugation, respectively make the dna profiling of 100uL, carry out the LAMP sensitivity test according to above-mentioned condition through 100 ℃ of 15min cracking.
With the minimum extent of dilution template amount meter sensitivity that detects, the minimum detectability that body series detects three kinds of target genes is the 26cfu/ reaction, and is highly sensitive.
SEQUENCE?LISTING
＜110〉Zhejiang Center For Disease Control and Prevention
＜120〉loop-mediated isothermal amplification fast detecting reagent kit of O157:H7 colibacillus and method
<130>
<160>13
<170>PatentIn?version?3.4
<210>1
<211>43
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>1
ctctctttcc?tctgcggtcc?gatgtttttc?acacttattg?gat 43
<210>2
<211>43
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>2
taaggaatca?ccttgcagat?aaactagtac?attggcatcg?tgt 43
<210>3
<211>20
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>3
aacagtcttg?tacaagtcca 20
<210>4
<211>21
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>4
ggtgcttttg?atatttttcc?g 21
<210>5
<211>23
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>5
cgaaacaagg?ccagtttttt?acc 23
<210>6
<211>42
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>6
ggttaataac?agacaccgat?gtggacagag?atatcgaccc?ct 42
<210>7
<211>43
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>7
taaggaatca?ccttgcagat?aaactagtac?attggcatcg?tgt 43
<210>8
<211>24
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>8
gtctcttcgt?taaatagtat?acgg 24
<210>9
<211>24
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>9
ggccacatat?aaattatttt?gctc 24
<210>10
<211>42
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>10
gccggatacg?cggtcaattt?cactccgatt?ctgacctgga?ct 42
<210>11
<211>41
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>11
tgaacgtgct?ggcgaaagac?gtttcaggtc?gatcgtgatg?g 41
<210>12
<211>18
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>12
gaactgacgg?ttcaggcc 18
<210>13
<211>19
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>13
agccattcag?acccagagt 19
Claims (2)
1.O157:H7 the loop-mediated isothermal amplification detection kit of colibacillus mainly comprises specificity amplification primer, Bst archaeal dna polymerase, deoxyribonucleotide mixture and PCR damping fluid, it is characterized in that: described specificity amplification primer sequence is as follows:
rfbE?FIP：CTCTCTTTCCTCTGCGGTCCGATGTTTTTCACACTTATTGGAT；
rfbE?BIP：TAAGGAATCACCTTGCAGATAAAC?TAGTACATTGGCATCGTGT；
rfbE?F3：AACAGTCTTGTACAAGTCCA；
rfbE?B3：GGTGCTTTTGATATTTTTCCG；
rfbE?LB：CGAAACAAGGCCAGTTTTTTACC；
stx2FIP：GGTTAATAACAGACACCGATGTGGACAGAGATATCGACCCCT；
stx2BIP：TAAGGAATCACCTTGCAGATAAACTAGTACATTGGCATCGTGT；
stx2F3：GTCTCTTCGTTAAATAGTATACGG；
stx2B3：GGCCACATATAAATTATTTTGCTC；
flic?FIP：GCCGGATACGCGGTCAATTTCACTCCGATTCTGACCTGGACT；
flic?BIP：TGAACGTGCTGGCGAAAGACGTTTCAGGTCGATCGTGATGG；
flic?F3：GAACTGACGGTTCAGGCC；
flic?B3：AGCCATTCAGACCCAGAGT。
2. test kit as claimed in claim 1 is characterized in that also comprising in the described test kit: trimethyl-glycine, MgSO
4, MnCl
2And fluorexon.
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| CN106676173A (en) * | 2017-01-03 | 2017-05-17 | 宁夏出入境检验检疫局检验检疫综合技术中心 | Primer group for detecting EHEC (enterohaemorrhagic escherichia coli) O157:H7 with LAMP (loop-mediated isothermal amplification) and detection method |
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| CN102363816B (en) * | 2011-11-25 | 2013-04-17 | 杭州优思达生物技术有限公司 | Reagent for detecting enterohemorrhagic escherichia coli by using cross primer nucleic acid isothermal amplification technology, amplification method and detection method for enterohemorrhagic escherichia coli |
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| CN110951896A (en) * | 2019-12-26 | 2020-04-03 | 华南理工大学 | CPA detection primer, kit and method for Escherichia coli Shiga toxin II |
| CN112280833A (en) * | 2020-11-11 | 2021-01-29 | 上海市嘉定区中心医院(上海健康医学院附属嘉定区中心医院、上海交通大学医学院附属仁济医院嘉定分院) | Primer group for quantitative detection of Escherichia coli DNA on surface of urinary catheter |
| CN113684289A (en) * | 2021-09-08 | 2021-11-23 | 武汉海关技术中心 | Primer group, detection method, kit and application for rapidly detecting enterohemorrhagic escherichia coli by LAMP technology |
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| CN106676173A (en) * | 2017-01-03 | 2017-05-17 | 宁夏出入境检验检疫局检验检疫综合技术中心 | Primer group for detecting EHEC (enterohaemorrhagic escherichia coli) O157:H7 with LAMP (loop-mediated isothermal amplification) and detection method |
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| CN101487057A (en) | 2009-07-22 |
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