CN105087576A - Detection kit for Yersinia rohdei causing enterocolitis and detection method - Google Patents
Detection kit for Yersinia rohdei causing enterocolitis and detection method Download PDFInfo
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- CN105087576A CN105087576A CN201510634138.1A CN201510634138A CN105087576A CN 105087576 A CN105087576 A CN 105087576A CN 201510634138 A CN201510634138 A CN 201510634138A CN 105087576 A CN105087576 A CN 105087576A
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Abstract
The invention discloses a detection kit for Yersinia rohdei causing enterocolitis. The detection kit comprises primer pairs shown as SEQ ID NO: (1 to 2), SEQ ID NO: (3 to 4) and SEQ ID NO: (5 to 6). The invention further provides a detection method of Yersinia rohdei, the three primer pairs, and application of the primer pairs. The detection kit and the detection method has the advantages that Yersinia rohdei can be detected accurately and effectively; the specificity and the sensitivity are high; the time consumption is low; the detection is fast; fast detection and forecast of water sources and foods can be realized; diseases can be prevented; the economic benefit is improved.
Description
Technical field
The present invention relates to detection kit and the detection method of the detection technique of a kind of bacterium, particularly yersinia entero-colitica.
Background technology
Yersinia entero-colitica (Y.entrocolitica) is a kind of gram negative bacillus or coccus, is under the jurisdiction of enterobacteriaceae yersinia's genus, is the another pathogenic bacterium after Yersinia pestis and artificial tuberculosis yersinia genus.This bacterium is just subject to international concern as far back as the eighties in 20th century, from environment, water body, crowd, animal and food, detects this pathogenic bacterium, and propagates by approach such as people-people, people-animal, animal-animal, food, water sources.Be the complication such as stomach and intestine phenomenon and erythema nodosum, sacroiliitis, osteomyelitis, hepatitis, septicemia such as acute abdominal pain, diarrhoea, vomiting by this microbial clinical symptom main manifestations.Based on above characteristic, yersinia entero-colitica has become China's import and export food must examine one of microorganism.
The method of traditional detection yersinia entero-colitica need by series of complex processes such as middle temperature Zengjing Granule, physics and chemistry qualification, serological analysis, existence is wasted time and energy, the features such as complex operation, and recall rate is not high, be unfavorable for promptly and accurately diagnosing yersinia entero-colitica, be therefore necessary to set up a kind of easy, detection technique fast and efficiently.The yersinia entero-colitica detection method of current report mainly contains fluorescent quantitation and Enzyme-linked Immunosorbent Assay technology, and these methods not only need expensive plant and instrument, and requires also higher to the operating skill of experimenter, is unfavorable for universal utilization.Develop rapidly along with molecular biological, relevant scholar has had more intensive understanding to the virulence gene of yersinia entero-colitica and mechanism of causing a disease, and this is identify that the virulence of yersinia entero-colitica and assessment bacterial strain provides theoretical basis and technical support from molecular level.
Notification number be 103468811B patent discloses a kind of test kit detecting yersinia entero-colitica, it needs to increase with 6 pairs of primer pairs simultaneously, and amplification is complicated, and cost is high, sensitivity only 10
5cFU/mL.
Summary of the invention
In order to solve the problem, the invention provides a kind of detection kit and detection method of yersinia entero-colitica.
The present invention provide firstly primer pair shown in SEQIDNO:1 ~ 2, SEQIDNO:3 ~ 4 and SEQIDNO:5 ~ 6.
Present invention also offers the purposes of primer pair shown in SEQIDNO:1 ~ 2, SEQIDNO:3 ~ 4 and SEQIDNO:5 ~ 6 in the reagent of preparation detection yersinia entero-colitica.
Present invention also offers a kind of detection kit of yersinia entero-colitica, it comprises primer pair shown in SEQIDNO:1 ~ 2, SEQIDNO:3 ~ 4 and SEQIDNO:5 ~ 6.
Present invention also offers a kind of detection method of yersinia entero-colitica, comprise the steps:
A, extracts sample DNA: extract the DNA in sample to be checked;
B, gene amplification: increase with the DNA that aforesaid test kit is treated in sample basis;
C, result detects: detect DNA cloning result.
Wherein, the sample described in step a is aquaculture water.
Test kit provided by the invention and method can specific amplified yersinia entero-coliticas, and high specificity, sensitivity reaches 17.04 × 10
-3ng/ μ L, consuming time short, for rapid detection water source, food, can the generation of preventing intestinal colitis Yersinia infectious diseases timely and effectively, have a good application prospect.
The embodiment of form by the following examples, is described in further detail foregoing of the present invention.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following embodiment.The technology that all contents recorded based on claims of the present invention realize all belongs to scope of the present invention.
Accompanying drawing explanation
3 heavy pcr amplification product electrophorogram and substance pcr amplification product, the wherein M:DL2000Marker of ail, inv, intB of Fig. 1 yersinia entero-colitica; The 3 heavy pcr amplification products of 1:ail, inv, intB; The amplified production of 2:ail gene, 351bp; The amplified production of 3:inv gene, 520bp; The amplified production of 4:intB gene, 722bp;
Fig. 2 detects the specific electrophorogram of amplification system, wherein M:DL2000Marker; A1, A2: be crucian small intestine Yersinia; 2: fourth salmon Aeromonas veronii; 3: trigger fish Vibrio harveyi; 4: tiger fur catfish pseudomonas; 5: elderly person for whom a birthday celebration is being held fish vibrio mimicus; 6: globe fish Aeromonas hydrophila: 7: African kind madai citric acid bacillus; 8: mullet Aeromonas media.
Fig. 3 is the electrophorogram detecting amplification system sensitivity, wherein M:DL2000Marker; 1: yersinia entero-colitica 17.04ng/ μ L; 2:8.52ng/ μ L; 3:3.41ng/ μ L; 4:17.04 × 10
-1ng/ μ L; 5:17.04 × 10
-2ng/ μ L; 6:17.04 × 10
-3ng/ μ L; 7:17.04 × 10
-4ng/ μ L; 8:17.04 × 10
-5ng/ μ L; 9:17.04 × 10
-6ng/ μ L.
Embodiment
One, experiment material and instrument
1, test strain
Table 1 test strain table
| Strain name | Source | Numbering |
| Yersinia entero-colitica | Chinese medicine DSMZ | CMCC52204 |
| Aeromonas veronii | Be separated from fourth salmon | JY-01 |
| Vibrio harveyi | Be separated from trigger fish | YD-09 |
| Pseudomonas | Be separated from tiger fur catfish | LF-11 |
| Vibrio mimicus | Be separated from elderly person for whom a birthday celebration is being held fish | CICE1457 |
| Aeromonas hydrophila | Be separated from globe fish | GM311 |
| Citric acid bacillus | The kind madai in Africa | JY-12 |
| Aeromonas media | Be separated from mullet | HI-10 |
2, main agents and equipment
CIN-1 substratum and matched reagent, improvement Y substratum-Hangzhou microorganism reagent company limited; PremixTaq (TaKaRaTaqVer.2.0plusdye), the raw biological company limited of nucleic acid molecular weight standard substance (DL2000Marker) 100 ~ 2000bp-Dalian treasured; GoldenView-; The biological company limited of bacterial genomes DNA extraction kit-sky root; Tris-Amresco; Ethylenediamine tetraacetic acid (EDTA), glacial acetic acid-Chengdu Ke Long chemical reagent factory; Agar-GENECOMPANYLTD; PCR instrument-Eppendorf, electrophoresis apparatus, gel imaging system-BioRAD.
Embodiment 1 design of primers
One, experimental technique
1, PCR primer Design and synthesis
Upstream and downstream primer (SEQIDNO.1 ~ 2) for ail gene: ail-F:5 ˊ-taatgtgtacgctgcgag-3 ˊ, ail-R:5 ˊ-gacgtcttacttgcactg-3 ˊ, expection clip size 351bp; Inv gene upstream and downstream primer (SEQIDNO.3 ~ 4): intB-Forword:5 ˊ-tgcgccatgcggtccatc-3 ˊ, intB-Reverse:5 ˊ-ggtgcataagattctcgg-3 ˊ, expection clip size 520bp; IntB gene upstream and downstream primer (SEQIDNO.5 ~ 6): intB-F:5 ˊ-tgcgccatgcggtccatc-3 ˊ, intB-R:5 ˊ-ggtgcataagattctcgg-3 ˊ, expection clip size 722bp.Primer all has Shanghai Ying Weijie base trade Co., Ltd to synthesize.
2, Template preparation
Be placed in by yersinia entero-colitica and be inoculated in LB broth culture 28 DEG C after rapid fluid resuscitation respectively under 37 DEG C of conditions and cultivate 18h, daily root bacterial genomes DNA extraction kit operation steps extracts bacterial genomes DNA, is used for pcr amplification as masterplate.
3, pcr amplification
Substance pcr amplification:
Reaction is totally 25 μ L, PremixTaq12.5 μ L, each 1 μ L of upstream and downstream primer, DNA profiling 1.0 μ L, distilled water 8.5 μ L.
Response procedures is 94 DEG C of denaturation 4min, and 94 DEG C of sex change 45s, 50 DEG C of annealing 45s, turn 72 DEG C of extension 40s, 30 circulations, last 72 DEG C of incubation 5min, 4 DEG C of preservations are to be checked.
Multiplexed PCR amplification of the present invention:
Detection primer ratio for ail:inv:intB is 3:2:1.
Reaction is totally 25 μ L, in 200 μ L centrifuge tubes, once add PremixTaq12.5 μ L, and ail upstream and downstream is because each 1.5 μ L, each 1.0 μ L of inv upstream and downstream primer, each 0.5 μ L of intB upstream and downstream primer, DNA profiling 2.0 μ L, distilled water 4.5 μ L.
Response procedures is 94 DEG C of denaturation 4min, and 94 DEG C of sex change 45s, 50 DEG C of annealing 45s, turn 72 DEG C of extension 40s, 30 circulations, last 72 DEG C of incubation 5min, 4 DEG C of preservations are to be checked.
4, result detects
Get 5 μ LPCR amplified productions and nucleic acid molecular weight 1% agarose carries out horizontal strip electrophoresis, under being placed in gel imaging system, observe pcr amplification result.
Two, result
As shown in Figure 1, the substance PCR for ail:inv:intB all can amplify target stripe to experimental result respectively, and multiplex PCR of the present invention can increase three objective bands simultaneously.
Experiment proves, primer pair of the present invention can accurately increase the gene fragment of ail:inv:intB gene of yersinia entero-colitica.
Embodiment 2 specific test
One, test method
1, PCR primer
With the primer pair (shown in SEQIDNO.1 ~ 2, SEQIDNO.3 ~ 4, SEQIDNO.5 ~ 6 primer pair) of embodiment 1.
2, Template preparation:
Yersinia entero-colitica and Aeromonas veronii, Vibrio harveyi, pseudomonas, vibrio mimicus, drowsiness Aeromonas, citric acid bacillus, Aeromonas media are placed in and are inoculated in LB broth culture 28 DEG C respectively after rapid fluid resuscitation under 37 DEG C of conditions and cultivate 18h, daily root bacterial genomes DNA extraction kit operation steps extracts bacterial genomes DNA, is used for pcr amplification as masterplate.
With embodiment 1.
3, pcr amplification
With embodiment 1 multiplexed PCR amplification.
4, result detects
With embodiment 1.
Two, result
As shown in Figure 2, only have yersinia entero-colitica can to increase three bands, other bacteriums are then all without amplified band for result.
Experiment proves, detection method accurately can distinguish yersinia entero-colitica and other bacteriums, high specificity.
Embodiment 3 sensitivity test
One, test method
1, PCR primer
With the primer pair (shown in SEQIDNO.1 ~ 2, SEQIDNO.3 ~ 4, SEQIDNO.5 ~ 6 primer pair) of embodiment 1.
2, Template preparation:
Yersinia entero-colitica is inoculated in LB broth culture 28 DEG C respectively under 37 DEG C of conditions cultivates 18h with being placed in after rapid fluid resuscitation, daily root bacterial genomes DNA extraction kit operation steps extracts bacterial genomes DNA.
Measure the concentration of DNA sample, do 10 times, 20 times, 50 times, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7dilute.After measured, the concentration of DNA sample is 170.4ng/ μ L, and obtaining concentration after dilution is 17.04ng/ μ L, 8.52ng/ μ L, 3.41ng/ μ L, 17.04 × 10
-1ng/ μ L, 17.04 × 10
-2ng/ μ L, 17.04 × 10
-3ng/ μ L, 17.04 × 10
-4ng/ μ L, 17.04 × 10
-5ng/ μ L, 17.04 × 10
-6the DNA sample liquid of ng/ μ L.
3, pcr amplification
With embodiment 1 multiplexed PCR amplification.
4, result detects
With embodiment 1.
Two, result
As shown in Figure 3, can occur clear band when sample liquid DNA concentration is 17.04ng/ μ L, sample liquid DNA concentration is less than 17.04 × 10
-3occur ambiguous band during ng/ μ L, the minimum template concentrations that therefore really can measure yersinia entero-colitica is 17.04 × 10
-3ng/ μ L.
Description of test, in measuring samples, the DNA concentration of yersinia entero-colitica is not less than 17.04 × 10
-3ng/ μ L, the inventive method can realize accurate detection, highly sensitive.
To sum up, detection kit provided by the invention and detection method can specific amplified yersinia entero-coliticas, high specificity, highly sensitive, consuming time short, detect fast, have a good application prospect.
Claims (5)
- Primer pair shown in 1.SEQIDNO:1 ~ 2, SEQIDNO:3 ~ 4 and SEQIDNO:5 ~ 6.
- Primer pair shown in 2.SEQIDNO:1 ~ 2, SEQIDNO:3 ~ 4 and SEQIDNO:5 ~ 6 detects the purposes in the reagent of yersinia entero-colitica in preparation.
- 3. a detection kit for yersinia entero-colitica, is characterized in that: it comprises primer pair shown in SEQIDNO:1 ~ 2, SEQIDNO:3 ~ 4 and SEQIDNO:5 ~ 6.
- 4. a detection method for yersinia entero-colitica, is characterized in that: comprise the steps:A, extracts sample DNA: extract the DNA in sample to be checked;B, gene amplification: increase with the DNA that test kit according to claim 3 is treated in sample basis;C, result detects: detect DNA cloning result.
- 5. method according to claim 4, is characterized in that: the sample described in step a is water source, food.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110923349A (en) * | 2019-12-30 | 2020-03-27 | 广东省微生物研究所（广东省微生物分析检测中心） | Species-specific detection molecular tags 3283 and 3316 of yersinia enterocolitica and rapid detection method thereof |
| CN112795669A (en) * | 2020-12-30 | 2021-05-14 | 广东省微生物研究所（广东省微生物分析检测中心） | Yersinia enterocolitica standard strain containing specific molecular target and detection and application thereof |
Citations (3)
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| CN102329864A (en) * | 2011-09-16 | 2012-01-25 | 广西出入境检验检疫局检验检疫技术中心 | Fluorescence PCR (polymerase chain reaction) kit for detecting yersinia enterocolitica |
| CN103468811A (en) * | 2013-09-17 | 2013-12-25 | 北京卓诚惠生生物科技有限公司 | Yersinia enterocolitica virulence gene multiplex-PCR (Polymerase Chain Reaction) detection primer group and kit |
| CN104388574A (en) * | 2014-12-09 | 2015-03-04 | 合肥工业大学 | Dual-PCR detecting method for pathogenic enterocolitis yersinia |
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2015
- 2015-09-29 CN CN201510634138.1A patent/CN105087576A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102329864A (en) * | 2011-09-16 | 2012-01-25 | 广西出入境检验检疫局检验检疫技术中心 | Fluorescence PCR (polymerase chain reaction) kit for detecting yersinia enterocolitica |
| CN103468811A (en) * | 2013-09-17 | 2013-12-25 | 北京卓诚惠生生物科技有限公司 | Yersinia enterocolitica virulence gene multiplex-PCR (Polymerase Chain Reaction) detection primer group and kit |
| CN104388574A (en) * | 2014-12-09 | 2015-03-04 | 合肥工业大学 | Dual-PCR detecting method for pathogenic enterocolitis yersinia |
Non-Patent Citations (1)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110923349A (en) * | 2019-12-30 | 2020-03-27 | 广东省微生物研究所（广东省微生物分析检测中心） | Species-specific detection molecular tags 3283 and 3316 of yersinia enterocolitica and rapid detection method thereof |
| CN112795669A (en) * | 2020-12-30 | 2021-05-14 | 广东省微生物研究所（广东省微生物分析检测中心） | Yersinia enterocolitica standard strain containing specific molecular target and detection and application thereof |
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