CN103866031A - PCR (polymerase chain reaction) detection primer and detection kit for escherichia coli 0157: H7 - Google Patents
PCR (polymerase chain reaction) detection primer and detection kit for escherichia coli 0157: H7 Download PDFInfo
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Abstract
The invention provides a PCR (polymerase chain reaction) detection primer and detection kit for escherichia coli 0157: H7, belonging to the field of biotechnology. The kit is a rapid technique for detecting escherichia coli 0157: H7 in a sample by using specific primer pairs, which is implemented through the following steps: 1, extracting nucleotides in a sample to be detected; 2, detecting nucleotides in the sample by using the PCR kit, and judging whether the sample contains escherichia coli 0157: H7. The kit provided by the invention has strong pertinence, and is simple and convenient to operate. The primer pairs include a primer pair consisting of an upstream primer SEQ ID NO.1 with a sequence of 5-atgaataagaatctcatc-3 and a downstream primer SEQ ID NO.2 with a sequence of 5-tttcttctcgcagtttcg-3. The set of primer sequences is designed according to the unique conservative sequence of escherichia coli 0157: H7 on GenBank, and has extremely high specificity and sensitivity.
Description
one, technical field
The PCR that the invention provides a kind of Escherichia coli O 157: H7 detects primer and detection kit, belongs to biological technical field.
two, background technology
Intestinal bacteria (
escherichia coli,
e.coli) O157:H7 is important foodborne bacterial pathogens, can in worldwide, cause that animal, poultry and the mankind break out infection.China is by Escherichia coli O 157: H7 classifies 21 century as may to compatriots' health one of 12 kinds of pathogenic micro-organisms of great effect, and strong to people's virulence, infective dose is low, and infection carrier bacteria containing amount reaches l0/g can cause infection above.After germs have invaded the human body, can causing bleeding property or non hemorrhagic diarrhoea, hemorrhagic colitis and hemolytic uremic syndrome.Escherichia coli O 157: H7 not only concerns food safety, and closely related with medical treatment and public health problem.Infect in order to monitor timely and effectively following possible outburst, setting up responsive, special and reliable technology is extremely necessary for detecting fast and optionally Escherichia coli O 157: H7.
Current, many reports are attempted method detection Escherichia coli O 157: the H7 that development is more suitable for.Traditional culture identification utilization selective medium or colour developing are dull and stereotyped detects Escherichia coli O 157: H7, but this method is time-consuming, effort, poor accuracy.Round pcr each detection field that has been widely used, is bringing into play very important effect.But, use round pcr to detect Escherichia coli O 157: the obstacle of H7 maximum is the unique special label gene establishment method of How to choose.Many scholars select shiga toxin gene
stx1 He
stx2,
uidA,
eae,
fimA,
rfbEwith
fliCidentification of escherichia coli O157:H7, although these genes provide some authentication information to a certain extent, most gene is not the exclusive gene of Escherichia coli O 157: H7, can not directly be judged to be Escherichia coli O 157: H7.Therefore, select more special and stable gene identification Escherichia coli O 157: H7, set up Fast Detection Technique, more meaningful and necessity.
In previous research work, by existing Escherichia coli O 157: H7 whole genome sequence information biology, compare of analysis, find new reading frame unique be present in Escherichia coli O 157: in H7 genome.We select Escherichia coli O 157: H7 reference strain EDL933, non-Escherichia coli O 157 bacterial strain and similar enteron aisle bacterial strain Salmonellas, Shigellae etc., set up PCR and detect and distinguish these pathogenic bacterias, result shows that this new reading frame is Escherichia coli O 157: the label gene that H7 is special and stable.These results of study impel us further to develop the novel PC R test kit for detection of Escherichia coli O 157: H7.
three, find content
technical problemthe object of the invention is to provide a kind of specific detection Escherichia coli O 157: H7 primer sequence and test kit.
technical schemespecific embodiment of the invention scheme is as follows:
1, design of primers: BLAST compares Escherichia coli O 157: H7 genome sequence, find its peculiar gene, and use DNAStar software design primer pair, formed by sequence SEQ ID NO.1 and SEQ ID NO.2.Amplified production is 354bp.
2, pcr amplification, comprises the following steps:
(1) Escherichia coli O 157: H7 microbial culture and template preparation: transfering loop dips-70 DEG C and preserves bacterial classification, streak inoculation is in sorbyl alcohol Mai Kangkai flat board, cultivate 20h for 37 DEG C, in single colony inoculation 5mL heart and brain leach liquor substratum (Hangzhou microorganism reagent goods company); If sample to be checked is liquid, directly getting 1mL joins in 9mL heart and brain leach liquor substratum, in substratum, adding 100 uL concentration is that 50mg/mL Vulkamycin. PA-93 (Ameresco import packing) is cultivated 20h again, next day, get inoculum 1mL, the centrifugal 10min of 12000r, the resuspended bacterium mud of 100 μ L pure water, boil 10min, the centrifugal 10min of 12000r, supernatant is template to be checked;
(2) the primer pZF of pcr amplification: MgCl2 1.5 uL, 20pmol/uL base sequence that add respectively dNTPs 2.0uL, the 25mM of 10 × PCR damping fluid 2.5uL, 2.5mM in the PCR of 0.2mL reaction tubes as shown in SEQ ID NO.1 and SEQ ID NO.2 and each 0.2 uL of pZR, archaeal dna polymerase 1, DNA profiling 2uL, add ultrapure water 15.6 uL to cumulative volume 25uL.95 DEG C of denaturation 5min, 94 DEG C of 30s, 50 DEG C of-60 DEG C of Gradient annealing 30s, 72 DEG C of 40s, 29 circulations, 72 DEG C are extended 5min again, amplification PCR product, 1.2% agarose gel electrophoresis observations, only has the highest annealing temperature of object band, and optimum annealing temperature is 60 DEG C.
(3) sensitivity test: first select Escherichia coli O 157: H7 pure cultures of bacteria, then select the Escherichia coli O 157 of simulation preparation: H7 contaminated samples is analyzed the susceptibility of PCR.
(4) specific test: select very easily the bacterium of obscuring with Escherichia coli O 157: H7 to carry out specific detection.
(5) test kit assembling: PCR premixed liquid, archaeal dna polymerase, positive control, negative control and pure water.
beneficial effect
(1) the present invention is based on the discovery of early-stage Study, Escherichia coli O 157: there is exclusive gene fragment in H7 genome, be different from other intestinal bacteria.According to this fragment gene, design primer increases, and specificity is good.
(2) due to the selected peculiar gene design primer of Escherichia coli O 157: H7 of the present invention, the PCR method of setting up to the e. coli serotype O55 very easily obscuring and all negative amplifications of O26, there is very good specificity.
(3) primer provided by the invention, without secondary structure, avoids the non-specific binding of primer self, causes false negative amplification.
(4) primer detection sensitivity provided by the invention is high, to Escherichia coli O 157: H7 bacterial cultures lowest detectable limit 10
4cFU/mL, to simulating positive sewage sample and meat sample, detectability 10CFU/mL (g) after increasing bacterium.
four, brief description of the drawings
The PCR sensitivity test figure of Fig. 1: pZF and pZR primer pair
1-6: Escherichia coli O 157: H7 pollutes after beef sample increases bacterium and detects, and 10
4cFU/g, 10
3cFU/g, 10
2cFU/g, 10CFU/g, 1CFU/g and negative control.M:DL-2000 standard molecular weight; 8-15: Escherichia coli O 157: H7 pollutes beef sample direct-detection, positive control, 10
8cFU/g, 10
7cFU/g, 10
6cFU/g, 10
5cFU/g, 10
4cFU/g, 10
3cFU/g and 10
2cFU/g.
five, embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should illustrate, these embodiment are only not used in and limit the scope of the invention for the present invention.The test method of unreceipted specific experiment condition in embodiment below, conventionally according to normal condition, for example Sambrook equimolecular clone; Condition described in laboratory manual (New York:Cold Spring Harber Laboratory Press, 1989), or the condition of advising according to manufacturer.
embodiment 1
Design of primers
Escherichia coli O 157 according to delivering on GenBank: H7 EDL933(accession number: AE005174) exclusive conservative property gene order design primer, pZF and pZR primer sequence are SEQ ID NO.1 and SEQ ID NO.2.Amplification conservative gene fragment is 354bp, and its sequence is SEQ ID NO.3.SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 are as follows:
SEQ ID NO.1：5- atgaataagaatctcatc-3
SEQ ID NO.2：5- tttcttctcgcagtttcg-3
SEQ ID NO.3：atgaataagaatctcatcctcgcatttgcattattttccttacctgtatttgcagaagaagatctggggccaggcaaata
tgtttgtgacattaggatttccagcctggataccgctactcaaattctgtcaaaatccgcaacagttctcgataatggaa
acaatttcatcgttcaaatgccaaatggagatcaattgtactctccagatctggaaaatgttgatgatggtataaaacaa
aaagcaacaataggtggagtgacatttattcgtcgtccaacttttaacgatcgcttcatcgtggaagatggaaatacagg
attcttctataagatgcgaaactgcgagaagaaa
embodiment 2
The sensitivity test of test kit, detects simulation preparation O157:H7 positive by above-mentioned primer.
It is all through immunomagnetic beads enrichment (Dynabeads anti-that O157:H7 positive is prepared in all simulations below
e. colio157 immunomagnetic beads, purchased from Invitrogen company), color developing culture medium bacterium separates (method reference: the separation of ox source O157:H7 and virulence factor analysis, Chinese animal doctor's journal, 2012,32 (8): 1148-1153.) turn out to be O157:H7 feminine gender and just can select.
2.1 sewage sample preparations: gather sewage from plant, 10mL/ pipe, adds Escherichia coli O 157: H7 culture, bacterium final concentration is respectively 10 successively
6cFU/mL, 10
5cFU/mL, 10
4cFU/mL, 10
3cFU/mL, 10
2cFU/mL, 10 CFU/mL and 1CFU/mL.
2.2 beef sample preparations: beef mud 200g is bought in supermarket, and every 10g joins in 90mL phosphate buffered saline buffer, then adds Escherichia coli O 157: H7 culture, bacterium final concentration is respectively 10
8cFU/g, 10
7cFU/g, 10
6cFU/g, 10
5cFU/g, 10
4cFU/g, 10
3cFU/g, 10
2cFU/g, 10 CFU/g and 1CFU/g.
The preparation of 2.3 templates to be checked:
By the positive beef mud sample of above-mentioned preparation, getting respectively 1mL joins in 9mL heart and brain leach liquor substratum (Hangzhou microorganism reagent goods company), in substratum, adding 100 uL concentration is 50mg/mL Vulkamycin. PA-93 (Ameresco import packing) again, 37 DEG C are continued to cultivate 18h, draw 1mL culture, the centrifugal 15min of 500 r, abandon precipitation, draw supernatant to new centrifuge tube, the centrifugal 10min of 12000r, abandon supernatant, get precipitation and be resuspended in 1/10 volume distilled water, boiling water bath 10min, 4 DEG C 12000 leaves heart 10min, draw supernatant, i.e. detection template DNA.
Get the positive sewage sample of above-mentioned preparation, getting respectively 1mL joins in 9mL heart and brain leach liquor substratum (Hangzhou microorganism reagent goods company), in substratum, adding 100 uL concentration is 50mg/mL Vulkamycin. PA-93 (Ameresco import packing) again, 37 DEG C are continued to cultivate 18h, draw 1mL culture, the centrifugal 15min of 500 r, abandon precipitation, draw supernatant to new centrifuge tube, the centrifugal 10min of 12000r, abandon supernatant, get precipitation and be resuspended in 1/10 volume distilled water, boiling water bath 10min, 4 DEG C of centrifugal 10min of 12000r, draw supernatant, i.e. detection template DNA.
2.4pcr amplification condition: add respectively 21 uL PCR premixed liquids in the PCR of 0.2mL reaction tubes, archaeal dna polymerase 1uL, DNA profiling 3 uL.
2.5by PCR pipe as for increasing in PCR instrument; Reaction conditions is: 95 DEG C of denaturation 5min, and 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 40s, 29 circulations, 72 DEG C are extended 5min again.Electrophoresis on the sepharose that is 1.2% in mass ratio by PCR product.
2.6result is judged: if amplified production has the band of 354bp, indicate the existence of Escherichia coli O 157: H7; If amplified production does not have the band of 354bp, indicate without Escherichia coli O 157: the existence of H7.PCR direct-detection Escherichia coli O 157: H7 pure growth template, lowest detection is limited to 10
4cFU/mL.PCR detects respectively each extent of dilution 1 CFU/mL-10 of the positive sewage sample of simulation
6cFU/mL, lowest detectable limit 10
4cFU/mL, lowest detectable limit 10CFU/mL after increasing bacterium; PCR detects respectively each extent of dilution 1 CFU/g-10 of the positive meat sample of simulation
8cFU/g, lowest detectable limit 10
5cFU/g, lowest detectable limit 10CFU/g after increasing bacterium.Fig. 1.
embodiment 3
The specific test of test kit, comprises the following steps:
Detect Escherichia coli O 157 according to test kit pcr amplification method in embodiment 2: H7 EDL933, intestinal bacteria O26, intestinal bacteria O55 and non-intestinal bacteria cause of disease (suis, Salmonella, pasteurellosis bacillus, Shigella dysenteriae, shigella flexneri 2a) (Li Li, etc.Foundation and the application of the double PCR of EHEC O157H7.North China agronomy report, 2011,26 (6): 61.) result all do not amplify any band, only has Escherichia coli O 157: H7 EDL933 amplifies the band of 354bp.
embodiment 4
Above-mentioned primer sequence and test kit detect clinical sample, comprise the following steps:
4.1sample collection: gather 20 parts of cow manures, 20 parts of Feces of Beef Cattles, 25-30g/ part; Different stands beef is bought in country fair, each 250g, totally 6 samples; Vegetables and fruit wholesale market, buy spinach, bean sprouts, leek, water chestnut and romaine lettuce, each 250g, totally 30 parts.All collections and purchase sample are stored in sample collection bag (Nanjing assistant research fellow company), sealing stored refrigerated.
4.2the preparation of template to be checked: take out 10 grams of ight soil or foods from sample collection bag, join in 90mL phosphate buffered saline buffer, after 37 DEG C of concussion 2h, getting 1mL joins in 9mL heart and brain leach liquor substratum (Hangzhou microorganism reagent goods company), in substratum, adding 100 uL concentration is 50mg/mL Vulkamycin. PA-93 (Ameresco import packing) again, 37 DEG C are continued to cultivate 18h, draw 1mL culture, the centrifugal 15min of 500 r, abandon precipitation, draw supernatant to new centrifuge tube, the centrifugal 10min of 12000r, abandon supernatant, getting precipitation is resuspended in 1/10 volume distilled water, boiling water bath 10min, 4 DEG C of centrifugal 10min of 12000r, draw supernatant, i.e. detection template DNA.
4.3pcr amplification condition: add respectively 22uL PCR premixed liquid in the PCR of 0.2mL reaction tubes, archaeal dna polymerase 1uL, DNA profiling 2uL.
4.4by PCR pipe as for increasing in PCR instrument; Reaction conditions is: 95 DEG C of denaturation 5min, and 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 40s, 29 circulations, 72 DEG C are extended 5min again.
4.5electrophoresis on the sepharose that is 1.2% in mass ratio by PCR product.
4.6result is judged: if amplified production has the band of 354bp, indicate the existence of Escherichia coli O 157: H7; If amplified production does not have the band of 354bp, indicate without Escherichia coli O 157: the existence of H7.
Result shows, 76 parts altogether of above-mentioned all samples, positive 12 parts of Escherichia coli O 157: H7, wherein positive 9 parts of ight soil, positive 1 part of water chestnut, positive 2 parts of spinach.
SEQUENCE LISTING
<110> Jiangsu Province Agriculture Science Institute
The PCR of <120> Escherichia coli O 157: H7 detects primer and detection kit
<130> 0
<160> 3
<170> PatentIn version 3.1
<210> 1
<211> 18
<212> DNA
<213> is artificial
<220>
<221> upstream primer pZF
<222> (1)..(18)
<223>
<400> 1
atgaataaga atctcatc 18
<210> 2
<211> 18
<212> DNA
<213> is artificial
<220>
<221> downstream primer pZR
<222> (1)..(18)
<223>
<400> 2
tttcttctcg cagtttcg 18
<210> 3
<211> 354
<212> DNA
<213> is artificial
<220>
<221> conservative gene fragment
<222> (1)..(354)
<223>
<400> 3
atgaataaga atctcatcct cgcatttgca ttattttcct tacctgtatt tgcagaagaa 60
gatctggggc caggcaaata tgtttgtgac attaggattt ccagcctgga taccgctact 120
caaattctgt caaaatccgc aacagttctc gataatggaa acaatttcat cgttcaaatg 180
ccaaatggag atcaattgta ctctccagat ctggaaaatg ttgatgatgg tataaaacaa 240
aaagcaacaa taggtggagt gacatttatt cgtcgtccaa cttttaacga tcgcttcatc 300
gtggaagatg gaaatacagg attcttctat aagatgcgaa actgcgagaa gaaa 354
Claims (3)
1. the PCR of Escherichia coli O 157: H7 detects a primer, it is characterized in that: be made up of sequence SEQ ID NO.1 and SEQ ID NO.2, amplified production is 354bp:
SEQ ID NO.1：5- atgaataagaatctcatc-3
SEQ ID NO.2：5- tttcttctcgcagtttcg-3。
2. contain the detection Escherichia coli O 157 of primer described in claim 1: the test kit of H7, it is characterized in that, test kit comprises PCR premixed liquid and archaeal dna polymerase, described PCR premixed liquid comprises:
The reaction system of every 22uL comprises the dNTPs 2.0uL of 10 × PCR damping fluid 2.5uL, 2.5mM, the MgCl of 25mM
21.5 uL, 20pmol/uL base sequence each 0.2 uL of primer, the ultrapure water 15.6uL as shown in SEQ ID NO.1 and SEQ ID NO.2;
Each detection reaction cumulative volume 25 uL, contain 22uLPCR premixed liquid, archaeal dna polymerase 1 uL, DNA profiling 2uL to be checked.
3. test kit according to claim 2, for detection of the PCR method of Escherichia coli O 157: H7, comprises the following steps:
(1) preparation of template to be checked: get 10 grams of ight soil or foods, join in 90mL phosphate buffered saline buffer, after 37 DEG C of concussion 2h, get 1mL and join in 9mL heart and brain leach liquor substratum; If sample to be checked is liquid, directly to get 1mL liquid sample and join in 9mL heart and brain leach liquor substratum, in substratum, adding 100 uL concentration is 50mg/mL Vulkamycin. PA-93 again, 37 DEG C are continued to cultivate 18h, draw 1mL culture, the centrifugal 15min of 500 r, abandon precipitation, draw supernatant to new centrifuge tube, the centrifugal 10min of 12000r, abandon supernatant, get precipitation and be resuspended in 1/10 volume distilled water, boiling water bath 10min, 4 DEG C 12000 leaves heart 10min, draw supernatant, i.e. detection template DNA;
(2) pcr amplification condition: add respectively 22uLPCR premixed liquid in the PCR of 0.2mL reaction tubes, archaeal dna polymerase 1uL, DNA profiling 2uL;
(3) by PCR pipe as for increasing in PCR instrument; Reaction conditions is: 95 DEG C of denaturation 5min, and 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 40s, 29 circulations, 72 DEG C are extended 5min again;
(4) electrophoresis on the sepharose that is 1.2% by PCR product in mass ratio;
(5) result is judged: if amplified production has the band of 354bp, indicate the existence of Escherichia coli O 157: H7; If amplified production does not have the band of 354bp, indicate without Escherichia coli O 157: the existence of H7.
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| CN109988852A (en) * | 2019-04-17 | 2019-07-09 | 江苏省农业科学院 | A kind of enterohemorrhagic escherichia coli O157:H7 detection kit and its application |
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| CN104862394A (en) * | 2015-05-11 | 2015-08-26 | 昆明理工大学 | Primer for detecting Escherichia coli, and method and application of primer |
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| CN109988852A (en) * | 2019-04-17 | 2019-07-09 | 江苏省农业科学院 | A kind of enterohemorrhagic escherichia coli O157:H7 detection kit and its application |
| CN110923348A (en) * | 2019-12-23 | 2020-03-27 | 河北农业大学 | Primer and method for identifying five pilus genes of escherichia coli at one time |
| CN110923348B (en) * | 2019-12-23 | 2022-09-16 | 河北农业大学 | Primer and method for identifying five pilus genes of escherichia coli at one time |
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