CN106701991A - Detection primer group of escherichia coli O111 and escherichia coli O157, kit and dual-PCR (Polymerase Chain Reaction) detection method - Google Patents
Detection primer group of escherichia coli O111 and escherichia coli O157, kit and dual-PCR (Polymerase Chain Reaction) detection method Download PDFInfo
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Abstract
The invention discloses a detection primer group of escherichia coli O111 and escherichia coli O157, a kit and a dual-PCR (Polymerase Chain Reaction) detection method. The nucleotide sequence of a primer pair for detecting the escherichia coli O111 is as follows: F1: 3'-TTAGTTTGTTTGATAGGCTC-5'; R1: 3'-AAAAAGAATAACGCAAGACA-5'; and the nucleotide sequence of a primer pair for detecting the escherichia coli O157 is as follows: F2: 3'-CCCAGCCTATCGAATGAGCA-5'; and R2: 3'-CACCGCCCCACTCGTAAAA-5'. The detection method disclosed by the invention has the advantages of high specificity, high sensitivity, rapidness and reliability in detection and simplicity and convenience for operation; and an effective means is provided for synchronous detection of the escherichia coli O111 and the escherichia coli O157.
Description
Technical field
The invention belongs to field of food safety, it is related to double PCR technology, and in particular to Escherichia coli O111 and Escherichia coli
O157 detections primer sets, kit and detection method.
Background technology
Escherichia coli (Escherichia coli) are a kind of common bacteriums in people and warm-blooded animal enteron aisle.It is most of
Coli strain is harmless.However, some Escherichia coli can cause food origin disease.The antigenic component of Escherichia coli is complicated, can
It is divided into somatic antigen (O), flagellar antigen (H) and surface antigen (K), according to the difference of somatic antigen, Escherichia coli can be divided into
More than 200 type, some of which serotype has pathogenic, can cause diarrhoea, is referred to as diarrheagenic E. coli.According to biology
Diarrheagenic E. coli can be divided into 5 major classes by the difference of characteristic：Enteropathogenic E.Coli, intestines enterotoxigenic E.coli, intestines
Enteroinvasive E. Coli, EAEC and EHEC (EHEC).EHEC can be produced
Raw shiga-like toxin is thus also called (STEC) by shiga toxin producing escherichia coli.EHEC is once in multiple countries
Outbreak of epidemic, becomes global public health problem.Enterohemorrhagic Escherichia coli (EHEC) infection mainly results in diarrhoea, can cause tight
The food origin disease of weight, it is mainly transmitted to the mankind by eating contaminated food, comminuted meat product that is such as raw or not being cooked,
Raw milk and contaminated raw vegetables and shoot vegetable.Escherichia coli O 157:H7 is that the most important intestines relevant with public health go out
The serum type of courageous and upright Escherichia coli；Other Escherichia coli O111 serotypes are also more typical EHEC serum
One of type, uncommon EHEC serotype has about 40 kinds.
The conventional inspection method of Escherichia coli O111 and Escherichia coli O 157 is needed as the detection of other pathogenic microorganisms
Serological Identification is carried out on the basis of microbial growth, biochemical identification, although reliable but very time-consuming, laborious, it is necessary to 4
Could complete within~7 days, therefore, need in time, the security of Fast Evaluation microorganism in food when, be not used generally.Cause
This be highly desirable to set up it is a kind of it is quick, sensitive, can be while the method for detecting Escherichia coli O111 and Escherichia coli O 157.
The content of the invention
It is an object of the invention to provide a kind of Escherichia coli O111 and detection primer group, the kit of Escherichia coli O 157
And dual PCR detection method.
The technical solution used in the present invention is：
The double PCR detection primer group of Escherichia coli O111 and Escherichia coli O 157, wherein, for detecting Escherichia coli
The nucleotide sequence of the primer pair of O111 is as follows：
F1：3’-TTAGTTTGTTTGATAGGCTC-5’(SEQ ID NO.1)；
R1：3’-AAAAAGAATAACGCAAGACA-5’(SEQ ID NO.2).
Nucleotide sequence for detecting the primer pair of Escherichia coli O 157 is as follows：
F2：3’-CCCAGCCTATCGAATGAGCA-5’(SEQ ID NO.3)；
R2：3’-CACCGCCCCACTCGTAAAA-5’(SEQ ID NO.4).
The double PCR detection kit of Escherichia coli O111 and Escherichia coli O 157, it includes above-mentioned primer sets.
The dual PCR detection method of Escherichia coli O111 and Escherichia coli O 157, comprises the following steps：
(1) measuring samples DNA is extracted；
(2) double PCR amplified reaction is carried out using above-mentioned primer sets；
(3) judge whether contain Escherichia coli O111 and/or Escherichia coli O 157 in measuring samples according to PCR product.
The composition of double PCR amplification reaction system is as follows：
The program of double PCR amplified reaction is：94 DEG C of predegeneration 5min；94 DEG C of denaturation 20s, 60 DEG C of anneal 1min, 45
Circulation；4 DEG C of preservation products.
The beneficial effects of the invention are as follows：Detection method of the invention have high specificity, sensitivity it is high, detection quickly may be used
By, advantage easy to operate, for the synchronous detection of Escherichia coli O111 and Escherichia coli O 157 provides a kind of effective means.
Brief description of the drawings
Fig. 1-Fig. 3 is the amplification electrophoretogram of the Escherichia coli O111 of embodiment one and Escherichia coli O 157 double PCR；
Fig. 4 is the specificity experiments result electrophoretogram of the Escherichia coli O111 of embodiment two and Escherichia coli O 157 double PCR；
Fig. 5 is the Escherichia coli O111 sensitivity experiment result electrophoretograms of embodiment three；
Fig. 6 is the Escherichia coli O 157 sensitivity experiment result electrophoretogram of embodiment three.
Specific embodiment
With reference to embodiment, the present invention is further illustrated, but is not limited thereto.
Molecular biology experiment technology employed in following examples include PCR amplifications, plasmid extraction, plasmid convert,
DNA fragmentation connection, digestion, gel electrophoresis etc., unless otherwise specified, generally conventionally operate, and for details, reference can be made to《Molecule
Cloning experimentation guide》(third edition) (Sambrook J, Russell DW, Janssen K, Argentine J. Huang Peitangs etc. are translated,
2002, Beijing：Science Press), or according to the condition proposed by manufacturer.
The foundation of the dual PCR detection method of the Escherichia coli O111 of embodiment 1 and Escherichia coli O 157
1st, design of primers
It is designed for detecting the specific primer pair of Escherichia coli O111 and Escherichia coli O 157, wherein, it is big for detecting
The nucleotide sequence of the primer pair of enterobacteria O111 is as follows：
F1：3’-TTAGTTTGTTTGATAGGCTC-5’(SEQ ID NO.1)；
R1：3’-AAAAAGAATAACGCAAGACA-5’(SEQ ID NO.2).
Nucleotide sequence for detecting the primer pair of Escherichia coli O 157 is as follows：
F2：3’-CCCAGCCTATCGAATGAGCA-5’(SEQ ID NO.3)；
R2：3’-CACCGCCCCACTCGTAAAA-5’(SEQ ID NO.4).
2nd, double PCR amplified reaction
(1) measuring samples DNA is extracted；
(2) double PCR amplification reaction system and program：
Configuration cumulative volume is the reaction system of the double PCR of 50 μ L, and concrete composition is as shown in table 1：
Table 1
| Component | Working solution concentration | Sample-adding amount (μ L) |
| F1 | 10μmol/L | 2 |
| R1 | 10μmol/L | 2 |
| F2 | 10μmol/L | 2 |
| R2 | 10μmol/L | 2 |
| dNTPs | 10mmol/L | 2 |
| PCR buffer solutions | 10× | 5 |
| Taq polymerase | 5U/μL | 1 |
| Template DNA to be checked | / | 2 |
| Deionized water | / | 32 |
Response procedures：Double PCR reaction system is placed in PCR instrument carries out double PCR reaction by following reaction conditions：Instead
Answer condition：94 DEG C of predegeneration 5min；94 DEG C of denaturation 20s, 60 DEG C of annealing 1min, 45 circulations；4 DEG C of preservation products.
3rd, amplified production gel electrophoresis
4th, result judges：Enterobacteria O111 and Escherichia coli O 157 amplified production length are respectively 238bp, 139bp, according to
Amplified production electrophoresis whether there is the band of expected size, judges PCR amplifications.There is the band of 238bp then in amplified production electrophoresis
Can determine that the sample is positive for Escherichia coli O111；The band that 139bp occurs in amplified production electrophoresis can determine that sample Escherichia coli
O157 results are positive；Two band that 238bp and 139bp occurs in amplified production electrophoresis can determine that sample Escherichia coli O111 and big
Enterobacteria O157 is the positive；Otherwise can determine that sample Escherichia coli O111 and Escherichia coli O 157 are feminine gender.
Selecting above-mentioned primer sets carries out double PCR detection, molten with the standard DNA of Escherichia coli O111 and Escherichia coli O 157
Liquid replaces template DNA to be checked, replaces template DNA to be checked as negative control using deionized water, is additionally carried out above-mentioned steps；It is dual
PCR testing results are shown in Fig. 1-Fig. 3.M is that DNA Marker DL2000, B correspondence deionized waters are made instead of template DNA to be checked in Fig. 1
It is negative control, the standard DNA solution of 1 and 2 corresponding Escherichia coli ATCC 43887 replaces template DNA to be checked.M is DNA in Fig. 2
Marker DL2000, B correspondence deionized waters replace template DNA to be checked as negative control, 1 and 2 corresponding Escherichia coli ATCC
35150 standard DNA solution replaces template DNA to be checked.M is that DNA Marker DL2000, B correspondence deionized waters replace in Fig. 3
Used as negative control, the standard DNA solution of 1 corresponding Escherichia coli ATCC 43887 replaces template DNA to be checked, 2 to template DNA to be checked
The standard DNA solution of correspondence Escherichia coli ATCC 35150 replaces template DNA to be checked.3 corresponding Escherichia coli ATCC's 43887
Each 1 μ L of standard DNA solution of standard DNA solution and Escherichia coli ATCC 35150 replace template DNA to be checked.
The specificity experiments of embodiment 2
In this example, specificity experiments mainly are carried out to the primer sets that embodiment one is provided, is concretely comprised the following steps：With table 2
In the DNA of coli strain replace template DNA to be checked respectively, using deionized water as negative control.
Table 2
Experimental result is：Three plants of Escherichia coli O111 bacterial strains and seven plants of Escherichia coli O 157 bacterial strains are amplified respectively in upper table
Length is respectively the product fragment of 238bp, 139bp, completely the same with positive control and expected results, and other bacterial strains are special nothing but
Specific amplification is feminine gender, and the amplification electrophoretogram of part bacterial strain is shown in Fig. 4.M is DNA Marker DL2000 in Fig. 4, B correspondences go from
Sub- water replaces template DNA to be checked as negative control, the standard DNA solution and large intestine bar of 1 corresponding Escherichia coli ATCC 43887
Each 1 μ L of standard DNA solution of bacterium ATCC 35150 replace template DNA to be checked, the standard of 2 corresponding Escherichia coli ATCC 43887
DNA solution replaces the standard DNA solution of template DNA to be checked, 3 corresponding Escherichia coli ATCC 29552 to replace template DNA to be checked, 4
The standard DNA solution of correspondence Escherichia coli ATCC 33780 replaces template DNA to be checked, 5 corresponding Escherichia coli ATCC's 35150
Standard DNA solution replaces the standard DNA solution of template DNA to be checked, 6 corresponding Escherichia coli CCTCC AB 200051 to replace to be checked
Template DNA, the standard DNA solution of 7 corresponding Escherichia coli EHEC Stx1 replaces template DNA to be checked, 8 corresponding Escherichia coli EHEC
The standard DNA solution of Stx2 replaces the standard DNA solution of template DNA to be checked, 9 corresponding Escherichia coli NCTC 12900 to replace to be checked
Template DNA, the standard DNA solution of 10 corresponding Escherichia coli ECO-13-01 replaces template DNA to be checked, 11 corresponding Escherichia coli
The standard DNA solution of ECO-14-01 replaces template DNA to be checked, the standard DNA solution generation of 12 corresponding Escherichia coli ATCC 12795
For template DNA to be checked, the standard DNA solution of 13 corresponding Escherichia coli ATCC BAA-2469 replaces template DNA to be checked,；14 correspondences
The standard DNA solution of Escherichia coli ATCC 23982 replaces template DNA to be checked, the standard DNA of 15 corresponding Escherichia coli EC O104
Solution replaces the standard DNA solution of template DNA to be checked, 16 corresponding Escherichia coli ATCC BAA-2190 to replace template DNA to be checked,
The standard DNA solution of 17 corresponding Escherichia coli ATCC BAA-2216 replaces template DNA to be checked, 18 corresponding Escherichia coli EC O4's
Standard DNA solution replaces the standard DNA solution of template DNA to be checked, 19 corresponding Escherichia coli ETEC O8 to replace template DNA to be checked,
The standard DNA solution of 20 correspondence Escherichia coli replaces template DNA to be checked, and the standard DNA of 21 corresponding Escherichia coli ETEC O78 is molten
Liquid replaces template DNA to be checked.
As seen from the figure, above-mentioned primer sets have specificity to Escherichia coli O111 and Escherichia coli O 157, therefore, it is above-mentioned to draw
The high specificity of thing group, therefore, herein under the premise of, the present invention provide detection method it is very reliable.
The sensitivity experiment of embodiment 3
Sensitivity experiment mainly is carried out to the primer sets that embodiment one is provided in this example, is concretely comprised the following steps：
By the μ L of standard DNA solution 2 of Escherichia coli ATCC 43887, acquisition DNA is measured using nucleic acid-protein analyzer molten
Liquid concentration is 35ng/ μ L.10 times of solution deionized water is incremental to be diluted to concentration 3.5ng/ μ L, 350pg/ μ L, 35pg/ μ respectively
L, 3.5pg/ μ L, 0.35pg/ μ L, 0.035pg/ μ L, 0.0035pg/ μ L, replace template DNA to be checked to operate embodiment one in addition
Described in the step of 2.Experimental result is shown in Fig. 5, and M is DNA Marker DL2000 in figure, and 1 to 9 is corresponding dense respectively successively in figure
Spend is 35ng/ μ L, 3.5ng/ μ L, 350pg/ μ L, 35pg/ μ L, 3.5pg/ μ L, 0.35pg/ μ L, 0.035pg/ μ L, 0.0035pg/
The amplification curve of μ L, the standard DNA solution of the Escherichia coli ATCC 43887 of 0.00035pg/ μ L, therefore, can determine whether that this is dual
PCR detection Escherichia coli O111 sensitivity is high, and its detection sensitivity is reacted up to 0.7pg/, and genome of E.coli size is
0.004pg, sensitivity is converted to copy number and is 175 copies/reaction.
By the μ L of standard DNA solution 2 of Escherichia coli ATCC 35150, acquisition DNA is measured using nucleic acid-protein analyzer molten
Liquid concentration is 30ng/ μ L；10 times of solution deionized water is incremental to be diluted to 3ng/ μ L, 300pg/ μ L, 30pg/ μ L, 3pg/ μ respectively
L, 0.3pg/ μ L, 0.03pg/ μ L, 0.003pg/ μ L and 0.0003pg/ μ L, replace template DNA to be checked to operate embodiment in addition
The step of described in one 2.
Experimental result is shown in Fig. 5, and M is DNA Marker DL2000 in figure, and 1 to 9 corresponding concentration is respectively successively in figure
30ng/ μ L, 3ng/ μ L, 300pg/ μ L, 30pg/ μ L, 3pg/ μ L, 0.3pg/ μ L, 0.03pg/ μ L, 0.003pg/ μ L and
The amplification curve of the standard DNA solution of 0.0003pg/ μ L；Therefore, can determine whether that the double PCR detects Escherichia coli O 157 sensitivity
Height, its detection sensitivity is reacted up to 6pg/, and genome of E.coli size is 0.004pg, and sensitivity is converted to copy number i.e.
It is 1500 copies/reaction.
The food samples mark-on of embodiment 4 is tested
The object bacteria of fresh cultured is made the bacteria suspension of about 0.35McF concentration, ten times are incremented by and are diluted to 10- 7Dilution factor,
Now sample bacteria suspension concentration is 101The bacteria suspension of CFU/mL is tested for sample mark-on.
25g food samples to 225mL EC meat soups are taken according to table 3 is aseptic respectively, that distinguishes will correspond to aimed strain in table
101The bacteria suspension 1mL of CFU/mL add to food samples meat soup be prepared into it is high by 101The mark-on sample of CFU/25g, slap type homogenizer
Homogeneous 2min.
Above-mentioned EC meat soups are put into 36 DEG C of increasing bacterium 24h, dual regular-PCR is respectively adopted and is detected corresponding Escherichia coli O111 and big
Enterobacteria O157.Each concentration addition Coupon Testing Results are shown in Table 7 and table 8, and this method is used for sample detection result as shown in Table 3
Consistent with expection, its detection sensitivity is up to 101CFU/25g。
The food mark-on sample multiplexed PCR amplification result of table 3
Embodiment 5
Sample contamination investigation mainly is carried out to the primer sets that embodiment one is provided in this example, is concretely comprised the following steps：
Raw pork, totally 196 parts of raw beef sample, daily fresh, the jelly for accepting inspection of our unit are purchased and collected from the market
43 parts of poultry product, fresh, 46 parts of frozen aquatic products, can 54 parts of salad vegetable, altogether check 339 parts of samples.
All samples are aseptic to weigh 25g to 225mL EC meat soups, slap type homogenizer homogeneous 2min, by above-mentioned EC meat soups
36 DEG C of increasing bacterium 24h are put, pyrolysis method extracts nucleic acid, dual regular-PCR method detection is respectively adopted, sample detection the results are shown in Table 4.
Escherichia coli O111 and Escherichia coli O 157 double PCR testing result in the food samples of table 4
As shown in Table 4,339 parts of totally 16 parts of food samples detection Escherichia coli O111, account for 5.3%, detect 5 Escherichia coli
Totally 7 parts of O157, accounts for 2.1%, shows that the detection method can be efficiently applied to Escherichia coli O111 and the Escherichia coli of food samples
The detection of O157.
The present invention is not limited to above-described embodiment, simple replacement based on above-described embodiment, not making creative work,
Should belong to the invention discloses scope.
SEQUENCE LISTING
<110>Inspection ＆ Quarantine Technology Center of Zhuhai Entry-Exit Inspection ＆ Quarantine Bureau
<120>The detection primer group of Escherichia coli O111 and Escherichia coli O 157, kit and dual PCR detection method
<130>
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
ttagtttgtt tgataggctc 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
aaaaagaata acgcaagaca 20
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
cccagcctat cgaatgagca 20
<210> 4
<211> 19
<212> DNA
<213>Artificial sequence
<400> 4
caccgcccca ctcgtaaaa 19
Claims (5)
1. the double PCR detection primer group of Escherichia coli O111 and Escherichia coli O 157, it is included for detecting Escherichia coli
The primer pair of O111 and the primer pair for detecting Escherichia coli O 157, it is characterised in that described for detecting Escherichia coli
The nucleotide sequence of the primer pair of O111 is as follows:
F1：3 '-TTAGTTTGTTTGATAGGCTC-5 ',
R1：3’-AAAAAGAATAACGCAAGACA-5’；
It is described as follows for detecting the nucleotide sequence of the primer pair of Escherichia coli O 157：
F2：3 '-CCCAGCCTATCGAATGAGCA-5 ',
R2：3’-CACCGCCCCACTCGTAAAA-5’.
2. the double PCR detection kit of Escherichia coli O111 and Escherichia coli O 157, it includes the primer described in claim 1
Group.
3. the dual PCR detection method of Escherichia coli O111 and Escherichia coli O 157, comprises the following steps：
(1) measuring samples DNA is extracted；
(2) double PCR amplified reaction is carried out using the primer sets described in claim 1；
(3) judge whether contain Escherichia coli O111 and/or Escherichia coli O 157 in measuring samples according to PCR product.
4. dual PCR detection method according to claim 3, it is characterised in that the double PCR amplification reaction system
Composition is as follows：
5. dual PCR detection method according to claim 3, it is characterised in that the program of the double PCR amplified reaction
For：94 DEG C of predegeneration 5min；94 DEG C of denaturation 20s, 60 DEG C of annealing 1min, 45 circulations；4 DEG C of preservation products.
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| CN201710076815.1A CN106701991A (en) | 2017-02-13 | 2017-02-13 | Detection primer group of escherichia coli O111 and escherichia coli O157, kit and dual-PCR (Polymerase Chain Reaction) detection method |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1749414A (en) * | 2004-08-20 | 2006-03-22 | 深圳太太基因工程有限公司 | Primer for detecting E. coli 0157:H7 nucleotide segment and probe sequence |
| EP1466011B1 (en) * | 2001-12-19 | 2006-10-11 | Angles D'Auriac, Marc B. | New primers for the detection and identification of bacterial indicator groups and virulence factors |
-
2017
- 2017-02-13 CN CN201710076815.1A patent/CN106701991A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1466011B1 (en) * | 2001-12-19 | 2006-10-11 | Angles D'Auriac, Marc B. | New primers for the detection and identification of bacterial indicator groups and virulence factors |
| CN1749414A (en) * | 2004-08-20 | 2006-03-22 | 深圳太太基因工程有限公司 | Primer for detecting E. coli 0157:H7 nucleotide segment and probe sequence |
Non-Patent Citations (2)
| Title |
|---|
| BASTIN,D.A.等: "Accession ID:AF078736.1,Escherichia coli O111 O antigen gene cluster,partial sequence", 《GENBANK DATABASE》 * |
| 晚观生: "多重PCR-DHPLC快速检测食品中产志贺毒素大肠杆菌", 《工业微生物》 * |
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Application publication date: 20170524 |