CN101624625B - Detection kit for common campylobacter in foods and detection method thereof - Google Patents
Detection kit for common campylobacter in foods and detection method thereof Download PDFInfo
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- CN101624625B CN101624625B CN2009100107949A CN200910010794A CN101624625B CN 101624625 B CN101624625 B CN 101624625B CN 2009100107949 A CN2009100107949 A CN 2009100107949A CN 200910010794 A CN200910010794 A CN 200910010794A CN 101624625 B CN101624625 B CN 101624625B
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Abstract
The invention relates to a detection kit for common campylobacter in foods and a detection method thereof, which is used for detecting and monitoring the microbial contamination of the foods. In the kit, 1mL of detection solution contains 10mM of Tris.Cl, 50mM of KCl, 25mM of MgCl2, 2.5mM of dNTP (dATP, dGTP, dCTP and dTTP) respectively, 5U/mu L of Taq DNA polymerase and 10muM of four kinds of common campylobacter primer pairs; the method adopts the virulence factor genes of the campylobacter to carry out detection, and can simultaneously differentiate four types when detecting the campylobacter; the detection time is only 1.5 days and is greatly shortened in comparison with the detection time of a traditional microbiology method; and the detection limit is below 15cfu/mL, and the campylobacter can be detected so long as the number of the campylobacter in bacterium liquid per milliliter is not less than 15. Moreover, the method has shorter detection time in comparison with the traditional method, is simpler and quicker, can save a large number of labour force and resources and is suitable for the requirement of quick detection.
Description
Technical field
The present invention relates to the detection method of pathogenic micro-organism, relate in particular to the mPCR-DHPLC detection method of pathogenic Campylobacter mikrobe in the food, also relate to detection compositions for use, i.e. test kit simultaneously.
Background technology
Campylobacter infection is a kind of zoonosis, and generation is all arranged all over the world, can cause multiple diseases such as human acute enteritis, Guillain Barre syndrome, reactive arthritis, Reiter ' s syndromes.Two heat-resisting kinds (thermophilic campylobacter) of this genus: the crooked bacterium (Campylobacter coli) of campylobacter jejuni (Campylobacter jejuni) and colon, almost can separate from the case of all Campylobacter clinical infections.Thermophilic Campylobacter bacterium, especially campylobacter jejuni are considered to the The main pathogenic fungi of human bacterial diarrhea.In the nz and the U.S., the food borne bacteria infection that is caused by campylobacter jejuni has surpassed Salmonellas and Shigellae within the specific limits.Animal (particularly poultry) is the main host plants of this bacterium, and raw milk, crude meat product and raw water also are main carriers in addition.Crooked bacterium is difficult in food, growing, but causes that the infective dose of morbidity is but very low, approximately takes in 400～500 thalline and just can cause intestinal tract infections, and this has caused serious threat to bird product safety and human health.The domestic molecular Biological Detection technical study that to Salmonellas in the food microorganisms, singly increases Lee Salmonella, vibrio cholerae, streptococcus aureus etc. comparatively deeply and extensively, but to crooked bacterium fast, high-throughput, somatotype detection technique study that then the finding report is very few.Present many developed countries set up the comparatively perfect monitoring and the hierarchy of control to the crooked bacterium in the food, and China's correlation study report is less.
At present, be exactly that selective enrichment is cultivated to the topmost method of the detection of common campylobacter in the food, but because the quantity of this bacterium in sample is few, and the growth conditions requirement is harsh, therefore is difficult to from sample, detect.Recent discovers, the state that under some certain conditions, that crooked bacterium also exists is a kind of " exist but can not cultivate ", the difficult problem that this traditional especially separation and Culture detection method can't overcome.Therefore, setting up a kind of efficient, quick, high-throughout Lapactic colon bacillus somatotype authenticate technology is the urgent problem that food safety faces.
Sex change performance liquid chromatography (DHPLC) adopts the principle of pair ion RPLC; Be through using special high temperature resistant liquid phase chromatography column to adopt the mode of temperature adjusting simultaneously; The nucleotide fragments molecule is carried out the method for analytical separation; Therefore, person also being arranged should technology be temperature adjusting pair ion RPLC.The invention of this technology is that the foranalysis of nucleic acids in the life science provides convenient and practical technology platform.Its fast high-flux, full-automatic operation, accuracy height, good reproducibility and the high advantage of susceptibility make it in foranalysis of nucleic acids, have the advantage that can not be substituted.DHPLC technology for detection mikrobe is an analysis platform of well cultivating ind mixed micro organism sample, can not only identify common mikrobe, can also identify uncommon, harsh and anaerobic bacterium.This technology is used widely in known detection and fields such as screening, gene type, gene quantification and length polymorphism analysis with the unknown gene sudden change.
Based on this, the inventor attempts to set up efficient, quick, easy the to be suitable clinical detection of utilizing DHPLC uses special and detection method common campylobacter in the sensitive food.
Summary of the invention
The object of the present invention is to provide a kind of be used for sensitivity quickly test sample, especially detect the food common campylobacter, with test kit and the detection method thereof of judging whether sample to be checked is polluted.
At first, the common campylobacter detection kit is in the food of the present invention:
1mL detects solution and contains 10mM TrisCl, 50mM KCl, 25mM MgCl
2, each 2.5mM of dNTP (dATP, dGTP, dCTP and dTTP), Taq archaeal dna polymerase 5000U be 5U/ μ L and four kinds of common campylobacter primers to each 10 μ M.
Wherein, the specific primer sequence of Campylobacter mikrobe to be detected is following:
The present invention also provides the detection method of utilizing the mentioned reagent box to detect common campylobacter in the food, comprises the steps:
1. get 1ul testing sample dna solution, add 10ul test kit solution and 14ul sterilization ultrapure water, TV 25ul; The centrifugal 10s of 5000r/min, carry out pcr amplification by following parameter then:
Sex change in advance: 94 ℃, 3min;
Get into circulation: 94 ℃ of sex change 60s, 56 ℃ of annealing 60s, 72 ℃ are extended 60s, 35 circulations;
Stop extending: 72 ℃, 7min;
2. DHPLC analyzes: chromatographic column: PS-DVB & C18 DNASep chromatographic column (4.6mm * 50mm, granularity 3 μ m); Column temperature: 50 ℃;
Moving phase (volume ratio): 0.0min, 55.0% buffered soln A, 45.0% buffered soln B;
0.5min, 50.2% buffered soln A, 49.8% buffered soln B;
2.4min, 44.2% buffered soln A, 55.8% buffered soln B;
4.3min, 40.9% buffered soln A, 59.1% buffered soln B;
6.1min, 38.8% buffered soln A, 61.2% buffered soln B;
8mm, 37.3% buffered soln A, 62.7% buffered soln B;
Wherein, buffered soln A is the TEAA aqueous solution of concentration 0.1mM; Buffered soln B is that concentration is the 0.1M TEAA aqueous solution and 3: 1 by volume mixing solutions of acetonitrile;
Flow velocity: 0.9mL/min;
Detector: fluorimetric detector (light source: 150W Xenon lamp; PLE bandwidth: 15nm; Emission spectrum bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s);
Applied sample amount: PCR product 5 μ L.
After detect finishing, according to DHPLC detect characteristic chromatographic peak in the collection of illustrative plates peak shape, RT and with the contrast of positive control spectrogram, confirm the microbiological contamination situation of sample.
Can establish positive control and negative control respectively in the testing process.Positive control is the positive colony molecular dna or the positive strain DNA of amplified fragments, and negative control is non-target DNA of pathogenic.
Negative control: under above-mentioned DHPLC analysis condition, no absorption peak occurs;
Positive control: under above-mentioned DHPLC analysis condition, campylobacter jejuni PCR product absorption peak occurs at about 3.8min; The crooked bacterium of colon PCR product absorption peak occurs at about 4.2min; The crooked bacterium of red mouth gull PCR product absorption peak occurs at about 4.5min; The crooked bacterium of embryo PCR product absorption peak occurs at about 8.7min.And above typical PCR product absorption peak is greater than 2mV;
Under above-mentioned DHPLC analysis condition, PCR product absorption peak appears in about 3.8min, is campylobacter jejuni; PCR product absorption peak appears in about 4.2min, is the crooked bacterium of colon; PCR product absorption peak appears in about 4.5min, is the crooked bacterium of red mouth gull; PCR product absorption peak appears in about 8.7min, is the crooked bacterium of embryo.If at the appearance time identical with positive control, test sample does not have the amplification absorption peak and occurs, and the decidable sample result is negative, and directly report does not detect corresponding pathogenic bacterium.
The present invention adopts multiplex PCR to combine sex change performance liquid chromatography (PCR-DHPLC) technology, sets up sensitivity detection method easily to 4 types of common crooked bacterium in the food, and on this basis, sets up common campylobacter quick detection kit in the food.Present method adopts the virulence factor gene of crooked bacterium to detect, and can when detecting crooked bacterium, carry out four types differentiation; Only 1.5 days detection time, shorten dramatically detection time than traditional micro-biological process; Detectability is lower than 15cfu/mL, as long as each crooked bacterium number is not less than 15 and can detects in every milliliter of bacterium liquid.And present method inspection side time ratio traditional method is short, and method is simpler and more direct, can save great amount of labor and financial resources, is fit to the requirement of rapid detection.
Description of drawings
Accompanying drawing 5 width of cloth of the present invention are specificity test-results figure between the kind of embodiment, wherein:
Fig. 1 is the detection collection of illustrative plates of campylobacter jejuni in the specificity test between the kind of the inventive method;
Fig. 2 is the detection collection of illustrative plates of the crooked bacterium of colon in the specificity test between the kind of the inventive method;
Fig. 3 is the detection collection of illustrative plates of the crooked bacterium of red mouth gull in the specificity test between the kind of the inventive method;
Fig. 4 is the detection collection of illustrative plates of the crooked bacterium of embryo in the specificity test between the kind of the inventive method;
Fig. 5 is the mPCR-DHPLC detected result of four kinds of crooked bacterium;
In the above-mentioned accompanying drawing, X-coordinate is RT, and (unit: minute min), ordinate zou is represented absorption peak strength of signal (unit: millivolt mV).
Wherein, 1. campylobacter jejuni gene group DNA ATCC 33560D-5;
2. campylobacter jejuni ATCC 33291;
3. the crooked bacterium genomic dna of colon ATCC BAA-1061D;
4. colon bending bacterium ATCC 43478;
5. the crooked bacterium genomic dna of red mouth gull ATCC BAA-1060D;
6. the crooked bacterium ATCC of red mouth gull BAA-1060;
7. embryo's bending bacterium ATCC 27374;
8. embryo's bending bacterium ATCC 43478;
9. shigella flexneri ATCC 12022;
10. enteropathogenic Escherichia coli ATCC 43887;
11. labor ground citric acid bacillus ATCC8090 not;
12. Salmonella enteritidis ATCC 13076;
13. yersinia entero-colitica ATCC 9610
In the above-mentioned accompanying drawing, X-coordinate is RT, and (unit: minute min), ordinate zou is represented absorption peak strength of signal (unit: millivolt mV).
Embodiment
Be specific embodiment of the present invention below, its foundation and application thereof to present method is further described, but does not limit content of the present invention in any form.
If no specified otherwise, the employed main agents in this part, instrument and merchandise resources thereof are: reagent such as bacterial genomes DNA a small amount of purification kit (TakaRa MiniBEST Bacterial Genomic DNA Extractionkit), Taq enzyme and PCR damping fluid are all available from precious biotechnology (Dalian) ltd; Crooked bacterium culture medium (Campylobacter Agar Base) is available from U.S. company BD; Brucella broth is available from Beijing overpass Technical responsibilities ltd; Automatic bacterial culture systems MART-II (Mart Microbiology B.V. company, Holland); Regular-PCR appearance PE 24000 (PerkinElmer company, the U.S.); Sex change high performance liquid chromatograph NAV-99-4500 (Transgenomic company, the U.S.); Supercentrifuge centrifuge 5804 (Eppendorf company, Germany).
Reference culture that this part content relates to and reference culture genomic dna available from U.S. ATCC standard biological article collecting center and Chinese medicine microbial strains preservation administrative center (CMCC), see table 1 for details respectively.
Before the use, get listed test strain in the table 1, after cultivating, extract genomic dna respectively, set up ATL and supply this part embodiment to test use.The bacterial strain of Campylobacter is on crooked bacterium selective medium, and (5% oxygen, 10% carbonic acid gas and 85% nitrogen) is cultivated 24h under 42 ℃ of little aerobic environments.Other bacterial strain is cultivated 24h in 37 ℃ on Trypsin soy agar (TSA).
Table 1
The foundation of the detection kit of common campylobacter and detection method thereof in embodiment 1, the food
Goal gene that present embodiment is confirmed to be used to detect and primer thereof are shown in following table (table 2):
Table 2
On this basis, being designed in the food of pcr amplification common campylobacter detection kit: 1mL detects solution and contains 10mM TrisCl, 50mM KCl, 25mM MgCl
2, in each 2.5mM of dNTP (dATP, dGTP, dCTP and dTTP), Taq archaeal dna polymerase 5U/ μ L and the above-mentioned table 2 four kinds of common campylobacter primers to each 10 μ M;
Use the PCR-DHPLC method of the crooked bacterium in this test kit test sample to comprise the steps:
1. the preparation of sample to be checked:
A. food samples: the 10g collected specimens is added in the 90mL brucella broth, and (5% oxygen, 10% carbonic acid gas and 85% nitrogen) is cultivated 24h under 42 ℃ of little aerobic conditions.Use bacterial genomes to extract test kit and extract its genomic dna, produce pcr template.Mark is directly as pcr template or-20 ℃ of preservations.
B. purifying bacterial strain sample: the single bacterium colony of picking is in brucella broth, and (5% oxygen, 10% carbonic acid gas and 85% nitrogen) is cultivated 24h under 42 ℃ of little aerobic conditions.Use bacterial genomes to extract test kit and extract its genomic dna, produce pcr template.Mark is directly as pcr template or-20 ℃ of preservations.
2. get 1ul testing sample dna solution, add 10ul test kit solution and 14ul sterilization ultrapure water, TV 25ul; The centrifugal 10s of 5000r/min, carry out pcr amplification by following parameter then:
Sex change in advance: 94 ℃, 3min;
Get into circulation: 94 ℃ of sex change 60s, 56 ℃ of annealing 60s, 72 ℃ are extended 60s, 35 circulations;
Stop extending: 72 ℃, 7min;
3. DHPLC analyzes: chromatographic column: PS-DVB & C18 DNASep chromatographic column (4.6mm * 50mm, granularity 3 μ m); Column temperature: 50 ℃;
Moving phase (volume ratio): 0.0min, 55.0% buffered soln A, 45.0% buffered soln B;
0.5min, 50.2% buffered soln A, 49.8% buffered soln B;
2.4min, 44.2% buffered soln A, 55.8% buffered soln B;
4.3min, 40.9% buffered soln A, 59.1% buffered soln B;
6.1mm, 38.8% buffered soln A, 61.2% buffered soln B;
8mm, 37.3% buffered soln A, 62.7% buffered soln B;
Wherein, buffered soln A is the TEAA aqueous solution of concentration 0.1mM; Buffered soln B is that concentration is the 0.1M TEAA aqueous solution and 3: 1 by volume mixing solutions of acetonitrile;
Flow velocity: 0.9mL/min;
Detector: fluorimetric detector (light source: 150W Xenon lamp; PLE bandwidth: 15nm; Emission spectrum bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s);
Applied sample amount: PCR product 5 μ L.
After detect finishing, according to DHPLC detect characteristic chromatographic peak in the collection of illustrative plates peak shape, RT and with the contrast of positive control spectrogram, confirm to measure the result.
Need to prove: the judgement for mPCR-DHPLC result need combine positive control and negative control to carry out.The mPCR-DHPLC detection spectrogram of testing sample and the mPCR-DHPLC of negative control and positive control are detected spectrogram relatively; When having any specificity absorption peak, negative control do not occur; And positive control is when the specificity absorption peak of correct position occurring; If test sample with the same position of positive amplification on the specificity absorption peak appears, during and peak height＞2mV, be judged to be the positive; If test sample does not have the specificity absorption peak in this position, then be judged to be feminine gender; If test sample with the same position of positive amplification on the specificity absorption peak appears, during but peak height＜2mV, then be suspicious specimen, need confirm through national standard method or additive method.
Get listed test strain in the table 1, after cultivating, extract genomic dna respectively, set up an ATL.Be template with these genomic dnas then, carry out the mPCR-DHPLC analyzing and testing according to above-mentioned condition and method.4 kinds of dissimilar crooked bacterium DHPLC specific detection collection of illustrative plates are shown in accompanying drawing 1～4, and the peak type of arrow indication is the specificity absorption peak of each crooked bacterium to be detected.The result can find out from diagram:
A.2 a campylobacter jejuni has all amplified the gryA target gene fragment, the then whole negative results of other non-jejunum campylobacter bacterial strains (accompanying drawing 1);
B.2 the crooked bacterium of a colon has all amplified the cdt target gene fragment, the crooked then whole negative results of bacterium (accompanying drawing 2) of other non-colons;
C.1 the crooked bacterium of the red mouth gull of strain has amplified the cdt target gene fragment, the crooked then whole negative results of bacterium (accompanying drawing 3) of other non-red mouth gulls;
D.1 the crooked bacterium amplification of strain embryo obtains the sap target gene fragment, the crooked whole negative results of bacterium (accompanying drawing 4) of all the other non-embryos.
The above results shows: 4 kinds of primers of this test design are applicable to that PCR-DHPLC detects crooked bacterium, has specificity between good kind.
1. campylobacter jejuni gene group DNA (ATCC 33560D-5) the 5 μ g lyophilized powders that ATCC is buied, 2. crooked bacterium genomic dna (ATCC BAA-1061D) the 10 μ g of colon, 3. crooked bacterium genomic dna (ATCC BAA-1060D) the 10 μ g of red mouth gull dissolve with the TE damping fluid and carry out 10 times of gradient dilutions; Concentration is respectively: 10ng/ μ L; 1ng/ μ L; 100pg/ μ L, 10pg/ μ L and 1pg/ μ L.4. with crooked bacterium (ATCC 27374) genomic dna of embryo with spectrophotometric determination concentration after, be adjusted into 10ng/ μ L, 1ng/ μ L, 100pg/ μ L, 10pg/ μ L and 1pg/ μ L.The template of different concns detects with DHPLC through multi-PRC reaction., template DNA concentration still can detect the specificity absorption peak when being respectively the crooked bacterium 1pg/ of campylobacter jejuni 10pg/ μ L, colon μ L, the crooked bacterium 1pg/ μ L of red mouth gull and the crooked bacterium 100pg/ of embryo μ L.Table 3 is the sensitivity that on genomic level, detects each crooked bacterium.
Table 3
The DHPLC spectrogram of measuring is shown in accompanying drawing 5.It can also be seen that by this result: the mPCR-DHPLC behavior of four kinds of common campylobacters has evident difference, and test kit and the detection method of using embodiment 1 to be set up can be distinguished identification with these the four kinds of bacterium in the microbiological contamination sample very effectively in one-time detection.
Bacteria containing amount is respectively 1.5 * 10
5, 1.5 * 10
4, 1.5 * 10
3, 1.5 * 10
2, 1.5 * 10
1, 1.5 * 10
0The simulating pollution sample put in the 90mL brucella broth, 42 ℃ of little aerobic conditions are cultivated 24h down.Each gradient nutrient solution adopts test kit extraction method (TakaRa MiniBEST Bacterial Genomic DNA Extractionkit) preparation template DNA, carries out PCR-DHPLC and detects.Detectability such as the table 4 of each crooked bacterium.Promptly when containing the crooked bacterium of 1.5 campylobacter jejunis, the crooked bacterium of 1.5 colons, 1.5 crooked bacterium of red mouth gull or 15 embryos in the initial analog sample, cultivating 24h down through 42 ℃ of little aerobic conditions can be detected.
Table 4
Adopt described mPCR-DHPLC method of present embodiment and national standard method (GB/T4789.9-2003) and industry standard method (SN/T 0175-92) that 172 parts of frozen chicken meat samples of random acquisition are detected simultaneously.Wherein mPCR-DHPLC of the present invention before increase the bacterium method and be: the 10g collected specimens is added in the crooked bacteria liquid enrichment medium of 90mL, and (5% oxygen, 10% carbonic acid gas and 85% nitrogen) is cultivated 24h under 42 ℃ of little aerobic conditions.
Through statistics, detect 18 strain campylobacter jejunis with mPCR-DHPLC method of the present invention, the crooked bacterium of 7 strain colons.Adopt country and industry standard method to detect 4 strain campylobacter jejunis, the crooked bacterium of 1 strain colon.Detect positive to all the other mPCR-DHPLC of the present invention; And standard method detects negative sample; Adopting increases the suspicious bacterial strain quantity of screening, utilizes immunological analysis reagent bar (VIDAS) and quick biochemical identification multiple confirmation methods such as (API), all turns out to be positive findings.
What crooked bacterium was adopted PCR-DHPLC and two kinds of method detected results of GB/SN relatively sees table 5:
Table 5
In this test, mPCR-DHPLC method recall rate of the present invention is higher than the detected result of traditional national standard method.Possibly be because traditional detection method for be in can not cultivation conditions crooked bacterium do not have detectivity, so fail to reflect strictly according to the facts the actual pollution situation of crooked bacterium in the sample.And mPCR-DHPLC method of the present invention for be in can not cultivation conditions crooked bacterium have the ability of detection equally, this will help the pollution situation of crooked bacterium in the frozen product is detected monitoring.
SEQUENCE?LISTING
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< 120>common campylobacter detection kit and detection method thereof in the food
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Claims (2)
1. common campylobacter detection kit in the food is characterized in that:
1mL detects solution and contains 10mM TrisCl, 50mM KCl, 25mM MgCl
2, each 2.5mM of dNTP, Taq archaeal dna polymerase 5U/ μ L and four kinds of common campylobacter primers are to each 10 μ M;
Wherein, the specific primer sequence of Campylobacter mikrobe to be detected is following:
2. the detection method of common campylobacter in the food is characterized in that using the described test kit of claim 1, comprises the steps:
1. get 1ul testing sample dna solution, add 10ul test kit solution and 14ul sterilization ultrapure water, TV 25ul; The centrifugal 10s of 5000r/min, carry out pcr amplification by following parameter then:
Sex change in advance: 94 ℃, 3min;
Get into circulation: 94 ℃ of sex change 60s, 56 ℃ of annealing 60s, 72 ℃ are extended 60s, 35 circulations;
Stop extending: 72 ℃, 7min;
2. DHPLC analyzes: chromatographic column: PS-DVB&C18DNASep chromatographic column, 4.6mm * 50mm, granularity 3 μ m; Column temperature: 50 ℃;
Moving phase, by volume: 0.0min, 55.0% buffered soln A, 45.0% buffered soln B;
0.5min, 50.2% buffered soln A, 49.8% buffered soln B;
2.4min, 44.2% buffered soln A, 55.8% buffered soln B;
4.3min, 40.9% buffered soln A, 59.1% buffered soln B;
6.1min, 38.8% buffered soln A, 61.2% buffered soln B;
8min, 37.3% buffered soln A, 62.7% buffered soln B;
Wherein, buffered soln A is the TEAA aqueous solution of concentration 0.1mM; Buffered soln B is that concentration is the 0.1M TEAA aqueous solution and 3: 1 by volume mixing solutions of acetonitrile;
Flow velocity: 0.9mL/min;
Detector: fluorimetric detector, light source: 150W Xenon lamp; PLE bandwidth: 15nm; Emission spectrum bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;
Applied sample amount: PCR product 5 μ L.
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| CN105695585A (en) * | 2016-03-16 | 2016-06-22 | 中国疾病预防控制中心传染病预防控制所 | Campylobacteria sixtuple PCR detection kit |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1536090A (en) * | 2003-04-07 | 2004-10-13 | 中国人民解放军军事医学科学院卫生学 | Food-originated pathogenic bactenium quick detection gene chip and its application |
| CN100342033C (en) * | 2004-08-13 | 2007-10-10 | 汕头大学 | Method for detecting jejunum arcuation bacterial |
| CN100395345C (en) * | 2004-08-20 | 2008-06-18 | 深圳太太基因工程有限公司 | Primer for detecting nucleotide segment of jejunum campylobacter and probe sequence |
| CN101255459A (en) * | 2008-03-25 | 2008-09-03 | 山东出入境检验检疫局检验检疫技术中心 | Preparation and detection method of jejunum vibrio coli rapid detection reagent kit |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1536090A (en) * | 2003-04-07 | 2004-10-13 | 中国人民解放军军事医学科学院卫生学 | Food-originated pathogenic bactenium quick detection gene chip and its application |
| CN100342033C (en) * | 2004-08-13 | 2007-10-10 | 汕头大学 | Method for detecting jejunum arcuation bacterial |
| CN100395345C (en) * | 2004-08-20 | 2008-06-18 | 深圳太太基因工程有限公司 | Primer for detecting nucleotide segment of jejunum campylobacter and probe sequence |
| CN101255459A (en) * | 2008-03-25 | 2008-09-03 | 山东出入境检验检疫局检验检疫技术中心 | Preparation and detection method of jejunum vibrio coli rapid detection reagent kit |
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