CN102329865A - PCR (polymerase chain reaction) detection kit of campylobacter jejunii and applications thereof - Google Patents
PCR (polymerase chain reaction) detection kit of campylobacter jejunii and applications thereof Download PDFInfo
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- CN102329865A CN102329865A CN201110276557A CN201110276557A CN102329865A CN 102329865 A CN102329865 A CN 102329865A CN 201110276557 A CN201110276557 A CN 201110276557A CN 201110276557 A CN201110276557 A CN 201110276557A CN 102329865 A CN102329865 A CN 102329865A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a PCR (polymerase chain reaction) detection kit of campylobacter jejunii, comprising a pair of specific primers designed for conserved areas of hippuricase (Hip0) gene of the campylobacter jejunii; and the primers comprise an upstream primer H1 5'-ctgaacttgccgattcggata-3'(SEQID NO:1) and a downstream primer H2 5'-aggcaattcccgactggaca-3'(SEQ ID NO:2). The invention also provides applications of the kit in detection of the campylobacter jejunii existing in clinic samples. In the PCR detection kit, the component is simple, the detection operation is convenient, the specificity is good, the campylobacter jejunii and the common similar strains can be effectively distinguished, and the detection sensitivity is higher.
Description
Technical field
The present invention relates to the molecular Biological Detection test kit of bacterium in the biological technical field, particularly relate to campylobacter jejuni PCR detection kit and application thereof.
Background technology
Campylobacter jejuni (campylobacter jejuni) is a kind of of campylobacter; Be to receive the infecting both domestic animals and human cause of disease bacterium of extensively paying attention in recent ten years both at home and abroad; Mainly can cause human body acute enteritis and food poisoning, and immunity injury diseases such as the reactive arthritis that occurs together, hepatitis, auspicious Te Shi disease and Green-barre syndrome.Veterinarily, can cause that the domestic animal miscarriage is infertile, mastitis and cub fowl diarrhoea and poultry hepatitis.This bacterium gets in the environment through animal carrier or trouble patient's ight soil; " live non-the cultivation " (the viable but nonculturerable that in water surrounding, can get into a kind of being called; VBNC) resting stage of state; The bacterium that is in the VBNC state can recover and have pathogenic (Vanniasinkam etc., 1999) under given conditions.The contact fowl poultry kind, or eat the main path that the poultry product, water source and the milk that are polluted are this bacterium infection human bodies.Epidemiology survey shows that this bacterium is only second to Salmonellas and Shigellae as the cause of disease of enteritis, in a lot of areas even the trend (Sahin etc., 2003) that ranks first above Salmonellas is arranged.Setting up quick, specific separation of campylobacter jejuni and detection method, is the key point of effectively controlling and preventing such food source sexually transmitted disease.Campylobacter jejuni is a microaerobe, and culture condition is harsh, does not grow at 25 ℃, and 42 ℃ of well-growns get into the VBNC state easily.These characteristics make conventional separation and Culture and biochemical identification campylobacter jejuni time and effort consuming, sensitivity not high, can't fully satisfy the requirement of Clinical Laboratory, when occurring breaking out epidemic situation, more can't satisfy the needs of quick diagnosis.And existing campylobacter jejuni molecular Biological Detection method is used loop-mediated isothermal amplification technique or real-time fluorescence quantitative PCR technology (CN1598544A, CN101886120A, CN101886133A etc.) more, more or less also has complex equipments, operation steps problem how.Therefore provide a kind of fast, simple, reliably can qualitative detection campylobacter jejuni test kit still very necessary.
Summary of the invention
To the problem of enumerating in the background technology, one aspect of the present invention provides a kind of test kit that detects campylobacter jejuni, and it comprises a pair of Auele Specific Primer to campylobacter jejuni histozyme (Hip0) gene conservative zone design:
Upstream primer H1 5 '-ctgaacttgccgattcggata-3 ' (SEQ ID NO:1)
Downstream primer H2 5 '-aggcaattcccgactggaca-3 ' (SEQ ID NO:2)
Further, test kit of the present invention also comprises the dNTP of 10 * PCR damping fluid, 2.5mmol/L, the Taq enzyme of 2.5U/ μ L, the Mg of 25mmol/L
2+Deng reagent commonly used in the PCR reaction.
On the other hand, the present invention also provides the application in the campylobacter jejuni that test kit of the present invention exists in detecting clinical sample.
The use step of test kit of the present invention is:
With according to needing direct test sample or using the Bolton substratum to increase bacterium in sample, use the DNA extraction test kit to extract testing sample DNA then, preparation sample DNA template.
Use test kit provided by the invention to make up the pcr amplification system, pcr amplification system (25 μ L) is: the Taq enzyme 0.4 μ L of the dNTP1.0 μ L of 10 * PCR damping fluid, 2.5 μ L, 2.5mmol/L, 2.5U/ μ L, the Mg of 25mmol/L
2+2.0 the primer of μ L, 1.5 μ M is to 1.0 μ L, sample DNA template solution 2 μ L, sterilized water 16.1 μ L.
The PCR loop parameter is: 94 ℃ of preparatory sex change 5 minutes; 35 circulations subsequently, each cycling program are that 94 ℃ of sex change 30 seconds, 65 ℃ of annealing were extended 30 seconds for 30 seconds, 72 ℃; 72 ℃ were extended 10 minutes after the loop ends, were cooled to 12 ℃ at last, finished the PCR program.
Carry out electrophoresis and use gel imaging appearance observations,, then show and contain campylobacter jejuni in the sample if can amplify the fragment about 276bp.
Description of drawings
Accompanying drawing has shown the result who uses method disclosed by the invention and test kit to detect campylobacter jejuni ATCC33560 (swimming lane 1), campylobacter jejuni ATCC29428 (swimming lane 2), dust Xi Shi intestinal bacteria ATCC8739 (swimming lane 3), campylobacter coli ATCC43478 (swimming lane 4), Shigella flexneri ATCC12022 (swimming lane 5), aseptic ultrapure water (swimming lane 6).
Embodiment
The campylobacter jejuni genome sequence of including with reference to GeneBank; With itself and the similar campylobacter coli sequence alignment of genome structure with it; Select histozyme (Hip0) gene conservative zone, use software Primer Premier to design a pair of primer, sequence is following:
Upstream primer H1 5 '-ctgaacttgccgattcggata-3 ' (SEQ ID NO:1)
Downstream primer H2 5 '-aggcaattcccgactggaca-3 ' (SEQ ID NO:2).
The foundation of embodiment 2 campylobacter jejuni PCR detection methods
Use designed probe H1, H2 among the synthetic embodiment 1 of this area ordinary method.
Direct test sample or use the Bolton substratum to increase bacterium the sample uses the DNA extraction test kit to extract testing sample DNA, preparation sample DNA template then as required.
Pcr amplification system (25 μ L) is: the Taq enzyme 0.4 μ L of the dNTP1.0 μ L of 10 * PCR damping fluid, 2.5 μ L, 2.5mmol/L, 2.5U/ μ L, the Mg of 25mmol/L
2+2.0 the primer of μ L, 1.5 μ M is to 1.0 μ L, sample DNA template solution 2 μ L, sterilized water 16.1 μ L.
The PCR loop parameter is: 94 ℃ of preparatory sex change 5 minutes; 35 circulations subsequently, each cycling program are that 94 ℃ of sex change 30 seconds, 65 ℃ of annealing were extended 30 seconds for 30 seconds, 72 ℃; 72 ℃ were extended 10 minutes after the loop ends, were cooled to 12 ℃ at last, finished the PCR program.
Electrophoresis also uses gel imaging appearance observations, if can amplify the fragment about 276bp, then shows and contains campylobacter jejuni in the sample.
The specificity of embodiment 3 campylobacter jejuni PCR detection kit
The DNA that extracts campylobacter jejuni ATCC33560, campylobacter jejuni ATCC29428, dust Xi Shi intestinal bacteria ATCC8739, campylobacter coli ATCC43478, Shigella flexneri ATCC12022 (above-mentioned bacterial classification is all bought the ATCC from USS type culture collection institute) is as template; Detect according to the PCR detection method among the embodiment 2, to verify the specificity of this detection method.Detected result is shown in Figure of description; Only campylobacter jejuni ATCC33560 (swimming lane 1), campylobacter jejuni ATCC29428 (swimming lane 2) amplify the fragment (276bp) of expection size; Several kinds of proximate bacterial strain dust Xi Shi intestinal bacteria ATCC8739, campylobacter coli ATCC43478, Shigella flexneri ATCC12022 and aseptic ultrapure waters (swimming lane 6) all do not amplify any fragment, explain that detection method provided by the invention possesses good specificity.
The sensitivity of embodiment 4 campylobacter jejuni PCR detection methods
With increasing bacterium 48 hours in the campylobacter jejuni ATCC33560 adding Bolton substratum.Use enrichment liquid to prepare the dna profiling of concentration, respectively these templates are detected according to the detection method of embodiment 2 as 18.7ng, 1.87ng, 187pg, 18.7pg, 1.87pg, 187fg, 18.7fg, 1.87fg.The template of 18.7ng, 1.87ng, 187pg, 18.7pg, 1.87pg, 187fg all can amplify the clear band of 276bp as a result, and the template of 18.7fg, 1.87fg is then failed to amplify and can be known the band of distinguishing (electrophorogram of sensitivity test does not show).Evidence test kit detection sensitivity of the present invention can reach more than the 187fg/DNA template, can satisfy the needs of clinical detection basically.
Can find out from the foregoing description, compare existing molecular Biological Detection method, campylobacter jejuni PCR detection kit composition provided by the invention is simple, detecting operation convenient (only needing a simple PCR amplification can carry out electrophoresis judges); And possess good specificity, can effectively distinguish campylobacter jejuni and common similar bacterial classification; Also possesses the above detection sensitivity of 187fg/DNA template.
Claims (3)
1. test kit that detects campylobacter jejuni comprises a pair of campylobacter jejuni Auele Specific Primer:
H1?5’-ctgaacttgccgattcggata-3’(SEQ?ID?NO：1)，
H2?5’-aggcaattcccgactggaca-3’(SEQ?ID?NO：2)。
2. the said test kit of claim 1 further comprises the dNTP of 10 * PCR damping fluid, 2.5mmol/L, the Taq enzyme of 2.5U/ μ L, the Mg of 25mmol/L
2+
3. the application in the campylobacter jejuni that in detecting clinical sample, exists of the described test kit of claim 2.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111020039A (en) * | 2019-12-30 | 2020-04-17 | 广东省微生物研究所（广东省微生物分析检测中心） | Campylobacter jejuni species specific molecular target and rapid detection method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111020039A (en) * | 2019-12-30 | 2020-04-17 | 广东省微生物研究所（广东省微生物分析检测中心） | Campylobacter jejuni species specific molecular target and rapid detection method thereof |
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Application publication date: 20120125 |