CN108531631A - One kind is for detecting enteropathogenic E. Coli O157:The RPA-LFD primer pairs and probe of H7 and its application - Google Patents
One kind is for detecting enteropathogenic E. Coli O157:The RPA-LFD primer pairs and probe of H7 and its application Download PDFInfo
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Abstract
The invention discloses one kind for detecting enteropathogenic E. Coli O157:The RPA LFD primer pairs and probe of H7 and its application, primer pair and probe sequence are as follows：The nucleotides sequence of sense primer EOF4 is classified as SEQ ID NO.6；The nucleotides sequence of downstream primer EOR3 is classified as SEQ ID NO.7；The nucleotides sequence of probe EOProb is classified as SEQ ID NO.9.Method safety, special, quick, sensitive, the easy Site Detection of the present invention, compensates for the deficiency of existing detection technique.It is demonstrated experimentally that the minimal reaction volume of RPA LFD technologies of the present invention is 10 μ L, optimal reactive temperature is 40 DEG C, and most short detection time 15min has not only greatly saved reaction cost, but also shortens detection required time.Best primer specificity by RPA reaction screenings is strong, and RPA LFD sensitivity is 100 times of PCR, has very high sensitivity.
Description
Technical field
The present invention relates to one kind for detecting enteropathogenic E. Coli O157:The RPA-LFD primer pairs and probe of H7 and its
Using belonging to technical field of food safety detection.
Background technology
In recent years, International Food Safety Control event emerges one after another, and food-borne pathogens are widely distributed and annual in the whole world
The human diseases for causing thousands of examples is the No.1 food-safety problem in the world.According to the World Health Organization (WHO) newest publication
Report display, the annual food origin disease that occurs in the whole world reach number 1,000,000,000, and even if developed country, at least 1/3 people suffers from food-borne disease
Disease.If there are about 4,8,000,000 food origin disease patients every year for U.S.'s estimation, wherein 12.8 ten thousand people are hospitalized for treatment, 3000 people are dead.
In China, it is equally food pollution caused by invasive organism to threaten the greatest problem of food security.It is logical that planning commission is defended according to country
Report, 2015 years, China's microbes food poisoning number are up to 3181 people, account for the 53.7% of annual food poisoning total number of persons,
For food poisoning number most.Adequately statistics indicate that, food-borne pathogens are to lead to the micro- life of the principal causative of food origin disease
Object causes food origin disease, to the public's often with contaminated food products in the production of raw-food material, processing, packaging and transporting procedures
Physical and mental health causes serious harm, it has also become global public health problem causes government department, food enterprise, food and drink
Unit, researcher and the highest attention of consumer.In entire food posioning case, food-borne pathogens large intestine
Bacillus O157:The infection rate of H7 occupy always forefront with incidence, which can be caused by contaminated food products, drinking water and environment
Human infection causes hemorrhagic diarrhea, the clinical symptoms such as hemolytic uremic syndrome (HUS), severe patient lethal can be died.Therefore,
It is how quick, sensitive, efficiently to the enteropathogenic E. Coli O157 in agri-food supply chains:H7 carries out effectively monitoring to cope with food
The occurrence and development of source property disease are the significant problems that current food-borne pathogens prevention and control field intends to solve.
However, for enteropathogenic E. Coli O157 in food:H7 is detected, and mostly uses traditional (improvement) culture identification at present
Technology, PCR and its deriving technology, immuno analytical method, metabolism analytical technology, fluorescence resonance energy transfer technology, biology pass
The means such as sensor technology, biochip technology, flow cytometer showed technology and molecular logic gate technique.However, these technological means
That there are sample treatments is cumbersome, detection cycle is long, expensive equipment, reagent is costly, sensitivity is low, easy generation false retrieval and missing inspection and
The pathogenic bacteria for being manually difficult to cultivate can not be detected etc. with many limitations, therefore, scene when being unfavorable for epidemic outbreaks is fast
Speed is diagnosed and is accurately traced to the source, and does not also meet required quick, special, the sensitive principle of food-borne pathogens detection.Therefore, for
Enteropathogenic E. Coli O157 in food:Easy quick, sensitive efficient, the economic and practical visualization of presence of exploitation is badly in need of in H7 pollutions
Detection technique.Recombinase polymerase nucleic acid amplification (Recombinase polymerase amplification, RPA) is a kind of
Nucleic acid-templated amplification can be arrived detectable level in a short time at 37-42 DEG C, be tried with Sidestream chromatography by isothermal amplification
Paper slip (Lateral flow dipsticks, LFD) combination (RPA-LFD) is with sensitive, special, easy, visualization and now
Many advantages, such as field is quick, is highly suitable for food-borne pathogenic Escherichia coli O 157:The detection of H7.
Invention content
In view of the deficiencies of the prior art, the object of the present invention is to provide one kind for detecting enteropathogenic E. Coli O157:
The RPA-LFD primer pairs and probe of H7 and its application, to realize to Escherichia coli O 157:H7's is safe and reliable, simple and efficient, clever
Quick efficient live quick visualization detection.
To achieve the goals above, the technical solution adopted in the present invention is as follows：
One kind is for detecting enteropathogenic E. Coli O157:The RPA-LFD primer pairs and probe of H7, primer pair and probe sequence
Row are as follows：
The nucleotides sequence of sense primer EOF4 is classified as SEQ ID NO.6；
The nucleotides sequence of downstream primer EOR3 is classified as SEQ ID NO.7；
The nucleotides sequence of probe EOProb is classified as SEQ ID NO.9.
5 ' the end applicating biotin Biotin labels of downstream primer EOR3.
5 ' the ends of probe EOProb are using Fluoresceincarboxylic acid FAM labels.
3 ' the ends of probe EOProb are closed using C3-Spacer, and the base A from 5 ' the 33rd, ends is replaced by tetrahydrofuran
Generation.
A kind of RPA-LFD amplimers pair and probe are preparing detection enteropathogenic E. Coli O157:Reagent, the reagent of H7
Application in box.
A kind of RPA-LFD amplimers pair and probe diagnose or the enteropathogenic E. Coli of therapy field in non-disease
O157:Application in H7 detections.
Non-disease diagnoses or therapy field is field of detection of food safety.
Enteropathogenic E. Coli O157 in a kind of detection food:The method of H7, includes the following steps：
(1) RPA reaction systems：
(2) RPA reactions amplification：
The above-mentioned reagent in addition to magnesium acetate solution is sequentially added in sterile centrifugation tube, is mixed well, by magnesium acetate solution
It is added in inside sterile centrifugation tube pipe lid, covers tightly rear brief centrifugation, mixing is put into rapidly the PCR instrument or constant temperature for closing heat lid after centrifugation
It is incubated in water-bath, carries out RPA reactions；
(3) the LFD detections of RPA products：2 μ L are taken to be added to the RPA amplified productions that dual anti-original FAM and Biotin is marked
Then LFD test strips are dipped vertically into mixed by mixing in the 96 hole microwell plates of the Tris-Cl Buffer containing 100 μ L pH 8.0
It closes in solution and carries out colour developing 5min, pass through test strips colour developing situation judging result.
RPA reaction conditions are：40℃、10min.
The detection line and control line of test strips take on a red color, and result judgement is the positive；Only control line takes on a red color, detection line
It does not develop the color, result judgement is feminine gender；Control line does not develop the color, and result judgement is invalid.
Advantageous effect of the present invention：
The present invention provides a kind of quick detection enteropathogenic E. Coli O157 based on molecular biology:The method of H7, with
It realizes to Escherichia coli O 157:Safe, special, quick, sensitive, the easy Site Detection of H7, compensates for existing detection technique
Deficiency.It is demonstrated experimentally that the minimal reaction volume of RPA-LFD technologies of the present invention is 10 μ L, optimal reactive temperature is 40 DEG C, most short
Detection time 15min.Best primer specificity by RPA reaction screenings is strong, PCR and RPA-LFD can detect that respectively 100fg with
The Escherichia coli O 157 of 1fg:H7 genomic DNAs, RPA-LFD sensitivity are 100 times of PCR, have very high sensitivity.This hair
Bright reaction system is only 10 μ L, and it is only 15min to complete total time-consuming to detection since extracting bacterial genomes, not only greatly
Reaction cost has been saved on ground, and shortens detection required time, is conducive to be promoted and applied on a large scale.Finally, of the invention
Using test strips can realize visualized presence detect, can in time, effectively monitor Escherichia coli O 157:H7 epidemic situations are sent out
Exhibition has important public health meaning.
Description of the drawings
The specific implementation mode of the present invention is described in further detail below in conjunction with attached drawing.
Fig. 1：The RPA primer PCRs of the method for the present invention and the double screening electrophoresis result figures of RPA.In figure, swimming lane M is
DNAMarker, swimming lane 1,2：1st group of primer EO1 (EOF1/EOR1), swimming lane 3,4：2nd group of primer EO2 (EOF2/EOR2), swimming
Road 5,6：3rd group of primer EO3 (EOF3/EOR2), swimming lane 7,8：4th group of primer EO4 (EOF4/EOR2), swimming lane 9,10：5th group
Primer EO5 (EOF3/EOR3), swimming lane 11,12：6th group of primer EO6 (EOF4/EOR3), swimming lane 13,14：7th group of primer EO7
(EOF4/EOR4)；Swimming lane 1,3,5,7,9,11,13 is respectively the PCR the selection results of 1-7 group primers；Swimming lane 2,4,6,8,10,
12,14 be respectively 1-7 group primers RPA the selection results.
Fig. 2：The best primer pairs of Escherichia coli RPA-LFD and probe design diagram.In figure, sense primer EOF4 and downstream
Primer EOR3 sequences are irised out by box；Probe EOProb is indicated by overstriking underscore, wherein the base " A " not underlined is being visited
It is substituted by tetrahydrofuran (THF) in needle.
Fig. 3：The Escherichia coli O 157 of the method for the present invention:H7RPA reaction volume optimum results figures.In figure, swimming lane M is
DNAMarker, swimming lane 1-10 correspond to respectively 5 μ L of RPA reaction volumes, 10 μ L, 15 μ L, 20 μ L, 25 μ L, 30 μ L, 35 μ L, 40 μ L,
45 μ L and 50 μ L.
Fig. 4：The Escherichia coli O 157 of the method for the present invention:H7RPA reaction time optimum results figures.In figure, swimming lane M is
DNAMarker, swimming lane 1-6 are corresponding in turn to reaction time 5min, 10min, 15min, 20min, 25min and 30min；Fig. 4 A are
The RPA reaction time optimizes RPA-AGE result figures；Fig. 4 B are to optimize RPA-LFD result figures in the RPA reaction time, and C is control line, and T is
Detection line.
Fig. 5：The Escherichia coli O 157 of the method for the present invention:H7RPA reaction temperature optimum results figures.In figure, swimming lane M is
DNAMarker, swimming lane 1-6 are corresponding in turn to 37 DEG C of RPA reaction temperatures, 38 DEG C, 39 DEG C, 40 DEG C, 41 DEG C and 42 DEG C of optimum results figures；
Fig. 5 A are the RPA-AGE result figures of RPA reaction temperatures optimization；Fig. 5 B are the RPA-LFD result figures of RPA reaction temperatures optimization, and C is
Control line, T are detection line.
Fig. 6：The Escherichia coli O 157 of the method for the present invention:The RPA atopic analysis result figures of H7.In figure, swimming lane M is
DNA Marker, swimming lane 1 are positive control plasmid rpUCm-rfbE；Swimming lane 2-12 is corresponding in turn to Escherichia coli O 157:H7, large intestine
Bacillus, salmonella typhimurium, Listeria monocytogenes, staphylococcus aureus, vibrio parahemolyticus, Enterobacter sakazakii, Fu Shi
Shigella, pseudomonas aeruginosa, bacillus cereus and Campylobacter jejuni gene group DNA；Fig. 6 A are RPA atopics
RPA-AGE result figures；Fig. 6 B are the RPA-LFD result figures of RPA atopics, and C is control line, and T is detection line.
Fig. 7：The Escherichia coli O 157 of the method for the present invention:The sensitivity analysis result figure of H7 genomic DNAs.In figure, swimming lane M
For DNA Marker；Swimming lane 1-8 is corresponding in turn to 105fg、104fg、103fg、102fg、101Fg, 5fg, 1fg and 0.1fg；Fig. 7 A are
Escherichia coli O 157:The PCR-AGE result figures of H7 genomic DNA sensitivity analyses；Fig. 7 B are Escherichia coli O 157:H7 genomes
The RPA-AGE result figures of DNA sensitivity analyses；Fig. 7 C are Escherichia coli O 157:The RPA- of H7 genomic DNA sensitivity analyses
LFD result figures, C are control line, and T is detection line.
Fig. 8：The detection of artificial contaminated bacteria samples milk limits result figure in the method for the present invention.In figure, swimming lane M is DNA Marker, swimming
Road 1-8 is corresponding in turn to 4.4 × 107CFU/mL、4.4×106CFU/mL、4.4×105CFU/mL、4.4×104CFU/mL、4.4×
103CFU/mL、4.4×102CFU/mL、4.4×101CFU/mL and 4.4 × 100CFU/mL, swimming lane 9 are pollution-free raw milk pair
According to；Fig. 8 A are the PCR-AGE detection limit result figures of artificial contaminated bacteria samples milk；Fig. 8 B are the RPA-AGE detection limits of artificial contaminated bacteria samples milk
Result figure；Fig. 8 C are the RPA-LFD detection limit result figures of artificial contaminated bacteria samples milk, and C is control line, and T is detection line.
Specific implementation mode
Recombinase polymeric enzymatic amplification technology (RPA) is that one kind is participated in by a variety of enzymes and albumen, realizes core under constant temperature
The isothermal duplication new technology of acid index amplification.RPA is expanded by simulating in DNA bodies, can be under 37-42 DEG C of isothermy
The DNA copy of detection level can be generated in 30min.Lateral flow test strips (LFD), have merged immunochromatography and molecular biology
Technology can form coloured detection line on paper slip and be quickly detected from target product to realize.Recombinase polymerase expands
Increasing technology (RPA) is combined (RPA-LFD), it can be achieved that RPA amplified production Site Detections are visual with Sidestream chromatography test strips (LFD)
Change, testing result is obviously intuitive, and without equipment and technology restriction, is very suitable for food-borne pathogenic Escherichia coli O 157:H7's
Field quick detection can make emergency situations rapid accurate judgement.However, being had been found that in practical applications without effective
Primer and probe combines, and can often lead to occur in detection process false positive or the low problem of detection sensitivity.
The specific implementation mode of the present invention is described in further detail with reference to embodiments.
Embodiment 1, primer screening and label
1, RPA design of primers
According to Escherichia coli O 157:H7rfbE genes (Genbank no：S83460.1), conserved region is analyzed by Blast
RPA primers are designed in domain, and upstream and downstream primer separately designs 4, and it is bis- to carry out PCR and RPA to 7 kinds of different upstream and downstream primer combinations
It screens again, primer sets and its sequence information are as follows：
1st group (EO1)
Sense primer EOF1：
5’-GCCCAGTTAGAACAAGCTGATGATTTTATATCACG-3’(SEQ ID NO.1)
Downstream primer EOR1：
5’-CCTTGTTTCGATGAGTTTATCTGCAAGGTGATTCC-3’(SEQ ID NO.2)
Product length：207bp
2nd group (EO2)
Sense primer EOF2：
5’-CATCCATGTGATATGGAACAAATTGTAGAACTGGC-3’(SEQ ID NO.3)
Downstream primer EOR2：
5’-CCCACATATTTACCTTTATATTTAGAACCAAAGGC-3’(SEQ ID NO.4)
Product length：110bp
3rd group (EO3)
Sense primer EOF3：
5’-CCCCATTTTCGTTGATTCAGATAATGAAACTTGGC-3’(SEQ ID NO.5)
Downstream primer EOR2：
5’-CCCACATATTTACCTTTATATTTAGAACCAAAGGC-3’(SEQ ID NO.4)
Product length：218bp
4th group (EO4)
Sense primer EOF4：
5’-TCTTCATTTAGCTTTGTTAGCGTTAGGTATATCGG-3’(SEQ ID NO.6)
Downstream primer EOR2：
5’-CCCACATATTTACCTTTATATTTAGAACCAAAGGC-3’(SEQ ID NO.4)
Product length：327bp
5th group (EO5)
Sense primer EOF3：
5’-CCCCATTTTCGTTGATTCAGATAATGAAACTTGGC-3’(SEQ ID NO.5)
Downstream primer EOR3：
5’-CCATATCACATGGATGTCCGTATAAATGGACACAC-3’(SEQ ID NO.7)
Product length：125bp
6th group (EO6)
Sense primer EOF4：
5’-TCTTCATTTAGCTTTGTTAGCGTTAGGTATATCGG-3’(SEQ ID NO.6)
Downstream primer EOR3：
5’-CCATATCACATGGATGTCCGTATAAATGGACACAC-3’(SEQ ID NO.7)
Product length：233bp
7th group (EO7)
Sense primer EOF4：
5’-TCTTCATTTAGCTTTGTTAGCGTTAGGTATATCGG-3’(SEQ ID NO.6)
Downstream primer EOR4：
5’-CCAAGTTTCATTATCTGAATCAACGAAAATGGGGG-3’(SEQ ID NO.8)
Product length：142bp
2, the PCR of primer
PCR reaction systems (25 μ L)
Remarks：EOF, EOR are respectively the upstream and downstream primer of 7 groups of primers, and template is Escherichia coli O 157:H7 genomic DNAs.
PCR reactions carry out in nucleic acid augmentative instrument, PCR response procedures：First, 95 DEG C of pre-degeneration 5min；Then, into 30
A cycle, each cycle 95 DEG C of denaturation 25s, 55 DEG C of annealing 25s, 72 DEG C of extension 45s；Finally, 10min is re-extended for 72 DEG C.
3, the RPA of primer
(1) RPA reaction systems (10 μ L)
(2) RPA reacts
The above-mentioned reagent in addition to magnesium acetate solution is sequentially added in sterile centrifugation tube, is mixed well, then, by magnesium acetate
Solution is added in inside sterile centrifugation tube pipe lid, covers tightly rear brief centrifugation, after centrifugation mixing be put into rapidly close heat lid PCR instrument or
It is incubated 10min in 40 DEG C of waters bath with thermostatic control.
4, the electrophoresis detection of PCR and RPA products
PCR with after PCR product Purification Kit RPA products into row agarose gel electrophoresis (AGE) analyze.Such as figure
Shown in 1, the corresponding RPA electrophoretic bands of the 6th group of primer are brighter and without non-specific amplification, illustrate under the same reaction conditions, the 6th
The RPA expanding effects of group primer are substantially better than other 6 groups of primers.
5, primer mark
The best primer pair that 6th group of primer EO6 (EOF4/EOR3) is reacted as RPA of the present invention, to downstream primer EOR3
5 ' end applicating biotin Biotin labels.
Embodiment 2, RPA-LFD are established
1, probe designs
For best primer pair EOF4/EOR3 design probes EOProb：5’-FAM-TGTCTGTTAGTGACATAGAACAAA
AAATCACT-THF-ATAAAACTAAAGCTATT-C3-Spacer-3 ' (SEQ ID NO.9) (Fig. 2).
Probe length：49bp
Corresponding RPA product lengths：233bp
2, RPA reaction systems (10 μ L)
3, RPA reaction systems expand
The above-mentioned reagent in addition to magnesium acetate is added in sterile centrifugation tube, is mixed well.Then, magnesium acetate solution is added in
Inside sterile centrifugation tube pipe lid, rear brief centrifugation is covered tightly, mixing is put into rapidly the PCR instrument or 40 DEG C of constant temperature for closing heat lid after centrifugation
10min is incubated in water-bath.
4, the LFD detections of RPA products
Take 2 μ L RPA products that mixing in the 96 hole microwell plates containing 100 μ L Tris-Cl Buffer (pH 8.0) is added,
Then the test strips sample pipetting volume areas LFD one end is dipped vertically into above-mentioned buffer solution the 5min that develops the color, is developed the color feelings by test strips
Condition judges result：(1) RPA products containing the bis- antigenic marks of FAM and Biotin with after gold marks anti-FAM antibody to be combined on LFD,
Anti- gold on coated anti-Biotin antibody and control line (C) can be detected on survey line (T) and mark anti-FAM antibody secondary antibody capture, T lines
It takes on a red color with C lines, result judgement is the positive；(2) downstream primer only containing Biotin labels or the spy only containing FAM labels
Needle or other unrelated products without containing FAM/Biotin double labellings then can only mark anti-FAM antibody by coated anti-gold on C lines
Secondary antibody captures, and only C lines take on a red color, and result judgement is feminine gender；(3) gold marks anti-FAM antibody not marked with the anti-gold on C lines
Anti- two anti-binding of FAM antibody, C lines do not develop the color, and result judgement is invalid.
Embodiment 3, RPA reaction condition optimizations
To Escherichia coli O 157:RPA optimum responses volume, optimum reacting time and the optimal reaction temperature of H7RPA carries out
Grope.RPA reaction volumes set 10 gradients：5 μ L, 10 μ L, 15 μ L, 20 μ L, 25 μ L, 30 μ L, 35 μ L, 40 μ L, 45 μ L and 50 μ
L, each corresponding RPA reacted constituents content of reaction volume and its volume are equal proportion composition.RPA is reacted in constant temperature electric heating water-bath
It carries out in pot, is analyzed by 2%AGE and LFD after the corresponding RPA product purifications of differential responses volume.As a result such as Fig. 3 institutes
Show, RPA amplified production amounts are minimum when RPA reaction volumes are 5 μ L, are held essentially constant in 9 gradients of 10 μ L to 30 μ L.
The RPA reaction time sets 6 gradients：5min, 10min, 15min, 20min, 25min and 30min, RPA reaction systems in addition to when
Between different outer, remaining all same.RPA reactions carry out in constant temperature electric heating water-bath, and the differential responses time, corresponding RPA products were pure
It is analyzed by 2%AGE and LFD after change.The results are shown in Figure 4, and RPA amplified production amounts are when the RPA reaction time is 5min
It is minimum, it is held essentially constant in 5 gradients of 10min to 30min.RPA reaction temperatures set 6 gradients：37℃、38℃、39
DEG C, 40 DEG C, 41 DEG C with 42 DEG C, RPA reaction systems are other than temperature is different, remaining all same.RPA reactions are in the temperature for closing heat lid
It is carried out in degree grads PCR instrument, the corresponding RPA products of differential responses temperature are analyzed by 2%AGE and LFD.As a result such as Fig. 5
Shown, RPA amplified production amounts reach maximum value when RPA reaction temperatures are 40 DEG C.In summary, Escherichia coli O 157:H7's
RPA is 10 μ L, reaction time 10min in reaction volume, in the case of reaction temperature is 40 DEG C, RPA product amounts up to maximum value,
Reaction effect is best.Illustrate that RPA reactions can be completed in 10 μ L micro volumes in 10min, economical in reaction, rapid, this advantage
It is highly suitable for quickly detecting.
Embodiment 4, specificity analysis
To contain Escherichia coli O 157:The recombinant plasmid rpUCm-rfbE (positive control) of H7 specific genes rfbE and big
Enterobacteria O157:H7, Escherichia coli, salmonella typhimurium, Listeria monocytogenes, staphylococcus aureus, parahemolyticas arc
Bacterium, Enterobacter sakazakii, shigella flexneri, pseudomonas aeruginosa, bacillus cereus and campylobacter jejuni genomic DNA
As RPA reaction templates, template quantity 0.1ng carries out specific analysis to the RPA-LFD detection methods of foundation, while with
ddH2O is as blank control.For RPA reaction systems as previously mentioned, RPA systems react 10min at 40 DEG C, RPA amplified productions are logical
It crosses AGE and LFD and carries out double analysis assessment (Fig. 6).The results show that RPA is with positive control plasmid and Escherichia coli O 157:H7
Genomic DNA, which is template, effectively to be expanded, and is negative knot by the amplification that template carries out of other bacterial genomes DNA
Fruit.It can be seen that RPA-LFD of the present invention can be realized to Escherichia coli O 157:The specific detection of H7, not to other related food sources
Property pathogenic bacteria occur cross reaction, illustrate the present invention have very strong specificity.
Embodiment 5, sensitivity analysis
Under best RPA reaction conditions, with Escherichia coli O 157:H7 not same amount genomic DNAs (105fg、104fg、
103fg、102fg、101Fg, 5fg, 1fg, 0.1fg) be template, carry out RPA-LFD sensitivity analyses, at the same with RPA-AGE and
PCR-AGE is as parallel control.The results are shown in Figure 7, RPA-LFD, and RPA-AGE and PCR-AGE can detect 1fg respectively, 5fg with
The Escherichia coli O 157 of 100fg:H7 genomic DNAs.It is not difficult to find out, the sensibility of RPA-LFD is RPA-AGE and PCR- respectively
5 times of AGE methods with 100 times.Therefore, for Escherichia coli O 157:H7 genomic DNAs, RPA-LFD of the present invention have high
Sensitivity.
Embodiment 6, practical application
1, raw milk artificial contaminated bacteria samples
Before carrying out artificial contamination to raw milk, examined in raw milk without big according to National Standard Method (GB4789.3-2016)
Enterobacteria O157:H7.Take 200 Escherichia coli O 157s of the μ L through plate count:The stoste 4.4 × 10 of H78CFU/mL is added to
Mixing in the raw milk of 1.8mL then makees 10 times of doubling dilutions until Escherichia coli O 157 backward successively:H7 bacterium solution final concentrations reach
To 4.4 × 100CFU/mL finally makes in raw milk bacteria containing amount between 4.4 × 107CFU/mL-4.4×100CFU/mL (totally 8 ladders
Degree).The Escherichia coli O 157 of corresponding each dilution:H7 genomic DNA application commercialization genome DNA extraction kit into
Row extracting.
2, artificial contaminated bacteria samples milk RPA-LFD detections limit
With different dilutions (4.4 × 107CFU/mL-4.4×100CFU/mL) Escherichia coli O 157:H7 genomic DNAs are
Template analyzes the detection limit of PCR-AGE, RPA-AGE and RPA-LFD.The results are shown in Figure 8, RPA-LFD and RPA-
AGE is for Escherichia coli O 157 in artificial contamination's raw milk:H7 detection limit be 4.4CFU/mL, than PCR-AGE (4.4 ×
102CFU/mL) 100 times high.It can be seen that RPA-LFD is for Escherichia coli O 157 in microbiological contamination milk:H7 detections have high
Sensitivity illustrates that RPA-LFD technologies have a good application prospect in food-borne pathogens detection field.
The foregoing is merely preferred embodiments, and for those skilled in the art, the present invention can have respectively
Kind change and variation.All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should all wrap
Containing within protection scope of the present invention.
Sequence table
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<120>One kind is for detecting enteropathogenic E. Coli O157:The RPA-LFD primer pairs and probe of H7 and its application
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Claims (10)
1. one kind is for detecting enteropathogenic E. Coli O157:The RPA-LFD primer pairs and probe of H7, which is characterized in that primer
Pair and probe sequence it is as follows：
The nucleotides sequence of sense primer EOF4 is classified as SEQ ID NO.6；
The nucleotides sequence of downstream primer EOR3 is classified as SEQ ID NO.7；
The nucleotides sequence of probe EOProb is classified as SEQ ID NO.9.
2. amplimer pair according to claim 1 and probe, which is characterized in that answer at the 5 ' ends of the downstream primer EOR3
It is marked with biotin Biotin.
3. amplimer pair according to claim 1 and probe, which is characterized in that 5 ' the end applications of the probe EOProb
Fluoresceincarboxylic acid FAM labels.
4. amplimer pair according to claim 1 and probe, which is characterized in that 3 ' the end applications of the probe EOProb
C3-Spacer is closed, and the base A from 5 ' the 33rd, ends is substituted by tetrahydrofuran.
5. a kind of amplimer pair as described in claim 1 and probe are preparing detection enteropathogenic E. Coli O157:H7's
Application in reagent, kit.
6. a kind of amplimer pair as described in claim 1 and probe are in non-disease diagnosis or the pathogenic large intestine of therapy field
Bacillus O157:Application in H7 detections.
7. application according to claim 6, which is characterized in that the non-disease diagnosis or therapy field are examined for food security
Survey field.
8. enteropathogenic E. Coli O157 in a kind of detection food:The method of H7, which is characterized in that include the following steps：
(1) RPA reaction systems：
(2) RPA reactions amplification：
The above-mentioned reagent in addition to magnesium acetate solution is sequentially added in sterile centrifugation tube, mixes well, magnesium acetate solution is added in
Inside sterile centrifugation tube pipe lid, rear brief centrifugation is covered tightly, mixing is put into rapidly the PCR instrument for closing heat lid or water bath with thermostatic control after centrifugation
Middle incubation carries out RPA reactions；
(3) the LFD detections of RPA products：Take the RPA amplified productions that 2 μ L are marked with dual anti-original FAM and Biotin be added to containing
Then it is molten to be dipped vertically into mixing by mixing in the 96 hole microwell plates of the Tris-Cl Buffer of 100 μ L pH 8.0 for LFD test strips
Colour developing 5min is carried out in liquid, passes through test strips colour developing situation judging result.
9. according to the method described in claim 8, it is characterized in that, RPA reaction conditions are：40℃、10min.
10. according to the method described in claim 8, it is characterized in that, the detection line and control line of test strips take on a red color, as a result
It is determined as the positive；Only control line takes on a red color, and detection line does not develop the color, and result judgement is feminine gender；Control line does not develop the color, result judgement
It is invalid.
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| CN109988823A (en) * | 2019-05-15 | 2019-07-09 | 南京林业大学 | Recombinase-mediated isothermal duplication-Sidestream chromatography technology detection Oak Tree phytophthora primer and probe combination and its application |
| CN110066885A (en) * | 2019-04-30 | 2019-07-30 | 南京林业大学 | A kind of primer and probe combination and its application based on RPA- Sidestream chromatography technology detection chestnut Heisui River phytophthora |
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| CN109957622A (en) * | 2019-03-27 | 2019-07-02 | 华南农业大学 | It is a kind of detect duck infectious serositis RPA-LFD visualizing agent box and its application |
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| KR102557448B1 (en) | 2021-11-19 | 2023-07-19 | 주식회사 풀무원 | Composition for detecting E.coli O157:H7 based on CRISPR-Cas, and E.coli O157:H7 detection method using the same |
| CN114606330A (en) * | 2022-04-20 | 2022-06-10 | 青岛国际旅行卫生保健中心(青岛海关口岸门诊部) | Detection kit for rapidly detecting Escherichia coli O157H 7 through RPA visualization |
| CN116200509A (en) * | 2022-08-17 | 2023-06-02 | 最高人民检察院检察技术信息研究中心 | Food-borne pathogenic bacteria RPA nucleic acid isothermal amplification primer probe group, kit, detection method and application thereof |
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