CN101624624B - Detection kit for salmonella and shigella in feeds and detection method thereof - Google Patents
Detection kit for salmonella and shigella in feeds and detection method thereof Download PDFInfo
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- CN101624624B CN101624624B CN2009100107915A CN200910010791A CN101624624B CN 101624624 B CN101624624 B CN 101624624B CN 2009100107915 A CN2009100107915 A CN 2009100107915A CN 200910010791 A CN200910010791 A CN 200910010791A CN 101624624 B CN101624624 B CN 101624624B
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Abstract
The invention relates to a detection kit for salmonella and shigella in feeds and a detection method thereof. The kit comprises a detection solution which contains 10mM of Tris.Cl, 50mM of KCl, 25mM of MgCl2, 2.5mM of dNTP, 5U/mu L of Taq DNA polymerase, 10mu M of salmonella primer pair and 10mu M of shigella primer pair. By using the detection kit and the detection method, the salmonella and theshigella in the feeds can be detected with high sensitivity, the detection time consumption is short, the operation is simple and quick, and a large number of labour force and resources can be saved;the detection kit and the detection method are suitable for the requirement of quick detection.
Description
Technical field
The present invention relates to Salmonellas and Shigellae detection kit and detection method thereof in the feed, relate in particular to the detection method and the employed reagent that utilize the PCR-DHPLC technology to detect.
Background technology
Animal derived feeds product very easily is subjected to microbial contamination, and with the after stain animal products, causes human diseases by food chain again.If animal derived feeds product from the epidemic-stricken area carry disease germs, dead livestock and poultry or without the byproduct raw material of strict sterilization processing, regular meeting causes the animal epidemic diffusion and causes human ill by food chain.Some pathogenic and relative pathogenic bacteria also can cause the fowl bacterial forage poisoning.The microbial contamination feed is the important channel that infectious diseases common to human beings and animals is propagated.The evaluation index of microbial contamination harm mainly contains total plate count, coliform, pathogenic bacterium and mould usually.Common pathogenic bacterium mainly are meant pathogen enterobacteria (as Salmonellas, Shigellae etc.).Generally pathogenic bacterium are all made the regulation of " must not detect " in the feed safety hygienic standard, to guarantee the safety and sanitation of feed.
In the traditional detection method, microbial culture and identify it is a not only consuming time but also complicated job is because factor such as quality instabilities such as some biochemical reagents, serum, specificity be bad brings difference often for the result of evaluation.A lot of at the method for quick of Salmonellas and Shigellae both at home and abroad at present, as PCR-gel electrophoresis, real-time fluorescence PCR method, gene chip method of testing, automatic bacterial biochemical identification instrument etc., also have immunofluorescence assay (VIDAS), colloid gold immune test strip etc. at Salmonellas in addition.Although method is numerous, all there be advantage and shortcoming separately in the whole bag of tricks: cultivate the biochemical identification method be subjected to artificial and the agents influence factor bigger; The automatic biochemical identification equipment circulation ratio of bacterium is better, but owing to just be confined to several biochemical reactions, differentiates difficulty especially between similar bacterial strain; It is high that immunofluorescence assay (VIDAS) detects cost, and exist the problem of spurious results; Though it is low that PCR-detected through gel electrophoresis technology detects cost, exists the shortcoming that the reaction product aftertreatment very easily pollutes; The real-time fluorescence PCR technology has high specificity, advantages such as sensitivity height, but it is higher to exist the detection cost, and the fluorescent probe shelf time is than deficiencies such as weak points; Method for gene chip then lacks practicality.
In the actual detected, to Salmonellas, when Shigellae detects, because these two kinds of used enrichment liquid differences of bacterium check, even to also increasing bacterium respectively with a sample, line separates respectively, identify respectively, breadboard workload is multiplied, and the while is inequality owing to both increase the bacterium time, Salmonellas 18～24h, Shigellae 6～8h brings quite big difficulty to the inspection routine arrangement.Given this, the present inventor thinks that the utmost point is necessary to develop a kind of new detection method, with the Salmonellas that detects testing sample efficiently, fast, delicately, the microbiological contamination situation of Shigellae.
Summary of the invention
The present invention at first is provided for the test kit that detects based on mPCR-DHPLC of Salmonellas and Shigellae in the rapid detection feed simultaneously.
Comprise a kind of detection solution in the test kit of the present invention, contain 10mM TrisCl, 50mM KCl, 25mM MgCl in this detection solution
2, each 2.5mM of dNTP, Taq archaeal dna polymerase 5U/ μ L and Salmonellas and Shigellae primer be to each 10 μ M;
Wherein, the specific primer sequence of Salmonellas and Shigellae is as follows:
The present invention also provides the mPCR-DHPLC method of utilizing the mentioned reagent box to detect Salmonellas and Shigellae in the feed, comprises the steps:
1. get 1ul testing sample dna solution, add detection solution and 14ul sterilization ultrapure water in the 10ul test kit, cumulative volume 25ul; The centrifugal 10s of 5000r/min, carry out pcr amplification by following parameter then:
Pre-sex change: 94 ℃, 3min;
Enter circulation: 94 ℃ of sex change 60s, 60 ℃ of annealing 60s, 72 ℃ are extended 60s, 35 circulations;
Stop extending: 72 ℃, 7min;
2. pcr amplification product being carried out DHPLC analyzes:
Chromatographic column: PS-DVB ﹠amp; C18 DNASep chromatographic column, 4.6mm * 50mm, granularity 3 μ m;
Column temperature: 50 ℃;
Moving phase (volume ratio): 0.0min, 48.0% buffered soln A, 52.0% buffered soln B;
0.5min, 42.9% buffered soln A, 57.1% buffered soln B;
3.6min, 38.8% buffered soln A, 61.2% buffered soln B;
6.8min, 36.6% buffered soln A, 63.4% buffered soln B;
9.9min, 35.2% buffered soln A, 64.8% buffered soln B;
13min, 34.3% buffered soln A, 65.7% 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; Excitation spectrum 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 feature chromatographic peak in the collection of illustrative plates peak shape, retention time and with the contrast of positive control spectrogram, determine the microbiological contamination situation of sample.
The present invention adopts the mPCR-DHPLC technology, sets up sensitivity detection method easily at Salmonellas in the feed and Shigellae, and sets up the quick detection kit that is applied to this method.Use detection kit of the present invention and detection method, can detect Salmonellas and Shigellae in the feed in high sensitivity, and detect weak point consuming time, simple to operation, can save a large amount of labours and financial resources, be fit to the requirement of rapid detection.
Description of drawings
Accompanying drawing 4 width of cloth of the present invention,
Fig. 1 .a is that PCR-DHPLC detects the positive absorption peak spectrogram of Salmonellas;
Fig. 1 .b is that PCR-DHPLC detects the positive absorption peak spectrogram of Shigellae;
Fig. 2 .a is that multiplex PCR-DHPLC detects the compound positive absorption peak spectrogram of Salmonellas, Shigellae that increases bacterium;
Fig. 2 .b is that multiplex PCR-DHPLC detects blended Salmonellas, the positive absorption peak spectrogram of Shigellae.
Embodiment
Be specific embodiments of the 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.
Test used main medium and reagent source in this part is: nutrient broth medium, SC enrichment liquid, GN enrichment liquid be respectively available from U.S. company BD, and by its explanation preparation; Reagent such as bacterial genomes DNA extraction reagent (TakaRa MiniBEST Bacterial Genomic DNA Extraction kit) and Ex Taq are available from precious biotechnology (Dalian) company limited; Triethylamine acetyl salt (TEAA, chromatographically pure) is available from Transgenomic company; Acetonitrile (chromatographically pure) is available from Fisher company.
Key instrument that this part test is used and equipment comprise gene-amplificative instrament (American AB I company) and sex change high performance liquid chromatography (being called for short DHPLC, U.S. Transgenomic company).
The used reference culture in this part is all available from U.S. typical case DSMZ (ATCC) or Chinese medicine microbial strains preservation administrative center (CMCC).
The foundation of Salmonellas and Shigellae detection kit and detection method thereof in embodiment 1, the feed
(1) assembling of primer design, synthetic and test kit:
Above-mentioned primer entrusts precious biotechnology (Dalian) company limited synthetic.
On this basis, be designed for the test kit that PCR-DHPLC detects: comprise a kind of detection solution in this test kit, contain 10mM TrisCl, 50mM KCl, 25mM MgCl in this detection solution
2, each 2.5mM of dNTP (dATP, dGTP, dCTP and dTTP), Taq archaeal dna polymerase 5U/ μ L and Salmonellas and Shigellae primer be to each 10 μ M;
(2) PCR-DHPLC detection method
The test kit that the PCR-DHPLC that this detection method uses present embodiment to set up detects comprises the steps:
1. the preparation of sample to be checked: adopt the test kit extraction method to prepare testing sample DNA genome
A. take by weighing animal derived feed sample 25g, be added in 225mL Salmonellas, the general enrichment liquid of Shigellae, after 36 ℃, 24h increase bacterium and cultivate, get general enrichment culture medium 1.5mL, the centrifugal 1min of 12000r/min inhales and abandons supernatant liquor, gets precipitation, adopt DNA of bacteria to extract test kit (TakaRa MiniBESTBacterial Genomic DNA Extraction kit) and extract DNA of bacteria, be stored in-20 ℃ standby with to be detected.
Wherein, the preparation method of general enrichment liquid is: peptone 20.0g, sodium-chlor 10.0g, disodium hydrogen phosphate 18.0g and potassium primary phosphate 3.0g are added in the 1000mL distilled water, mix evenly, leave standstill about 10min, heated and boiled is to dissolving fully, transfer to pH7.2 ± 0.1,121 ℃ autoclaving 15min.
B. reference culture: the single bacterium colony of picking is cultivated 18～24h in nutrient broth, uses bacterial genomes to extract test kit and extracts its genomic dna, produces pcr template.Mark is directly as pcr template or-20 ℃ of preservations.；
2. pcr amplification:
Get 1ul testing sample dna solution, add detection solution and 14ul sterilization ultrapure water in the 10ul test kit, cumulative volume 25ul; The centrifugal 10s of 5000r/min, carry out pcr amplification by following parameter then:
Pre-sex change: 94 ℃, 3min;
Enter circulation: 94 ℃ of sex change 60s, 60 ℃ of annealing 60s, 72 ℃ are extended 60s, 35 circulations;
Stop extending: 72 ℃, 7min;
3. DHPLC analyzes:
Chromatographic column: PS-DVB ﹠amp; C18 DNASep chromatographic column, 4.6mm * 50mm, granularity 3 μ m;
Column temperature: 50 ℃;
Moving phase (volume ratio): 0.0min, 48.0% buffered soln A, 52.0% buffered soln B;
0.5min, 42.9% buffered soln A, 57.1% buffered soln B;
3.6min, 38.8% buffered soln A, 61.2% buffered soln B;
6.8min, 36.6% buffered soln A, 63.4% buffered soln B;
9.9min, 35.2% buffered soln A, 64.8% buffered soln B;
13min, 34.3% buffered soln A, 65.7% 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; Excitation spectrum bandwidth: 15nm; Emission spectrum bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;
Applied sample amount: PCR product 5 μ L.
Get listed test strain in the table 1, after cultivating, extract genomic dna respectively, set up template base.。Be template with these genomic dnas then, adopt the method that embodiment set up to carry out the PCR-DHPLC analyzing and testing.Detected result shows that all Salmonellass are used the positive absorption peak (accompanying drawing 1.a) of the Salmonellas that has all detected DHPLC, the about 6.3min of appearance time, but not the Salmonellas detected result is all negative; Shigellae has all detected the positive absorption peak (accompanying drawing 1.b) of Shigellae of DHPLC, and appearance time is-10.4min, but not the Shigellae detected result is all negative.
The above results shows: Salmonellas that this test is adopted and Shigellae detect primer and all have good evaluation specificity.
Table 1
Get the composite general enrichment culture medium of Salmonella enteritidis and shigella flexneri, promptly the mixed culture enrichment liquid of two kinds of reference cultures extracts DNA, carries out the PCR-DHPLC detection according to the method that embodiment 1 is set up.The result detects the peak that goes out of Salmonellas and Shigellae respectively at 6.3min and 10.4min shown in accompanying drawing 2.a.
As a comparison, embodiment simultaneously with Salmonella enteritidis and shigella flexneri separately the product of single pcr amplification respectively get 2.5 μ L and mix the back and under identical conditions, carry out DHPLC and detect, the result is shown in accompanying drawing 2.b, Salmonellas and Shigellae have also detected positive absorption peak separately respectively, and appearance time is same as described above.
In order to confirm that the positive absorption peak that composite PCR-DHPLC detects is the gene fragment of Salmonella enteritidis and shigella flexneri, clone after we reclaim two positive absorption peak products of DHPLC respectively separately and check order, the result respectively with genbank in the comparison of Salmonella enteritidis and shigella flexneri gene order, proved that two positive absorption peaks of composite PCR-DHPLC are respectively the gene fragment of Salmonella enteritidis and shigella flexneri.
Get the composite general enrichment culture medium of Salmonella enteritidis and shigella flexneri, with the composite bacteria liquid concentration of quantitative Salmonellas of cultivating of turbidimetry and Shigellae.At first make OD with Maxwell colorimetric cylinder test kit
550Value---the typical curve of the bacterium number in every mL bacterium liquid, the OD of the mensuration bacterium liquid of cultivating again
550Value is with OD
550Be worth the substitution typical curve, obtain the concentration of the bacterium liquid of cultivating, unit is CFU/mL.Bacterium liquid is diluted to 1 * 10
1CFU/mL, 1 * 10
2CFU/mL, 1 * 10
3CFU/mL, 1 * 10
4CFU/mL, 1 * 10
5CFU/mL, 1 * 10
6The CFU/mL constant gradient.Extract DNA according to the method that embodiment 1 sets up, each gradient is respectively got 2 μ L as template, and the sensitivity of carrying out PCR-DHPLC detects.
When Salmonella enteritidis and the template that shigella flexneri is compound after increasing bacterium be 1 * 10
2CFU/mL and 1 * 10
3During DNA that CFU/mL extracted, the template DNA that adds 2 μ L still can detect typical positive absorption peak through PCR-DHPLC, and masterplate is 1 * 10
1Then do not detect positive absorption peak during CFU/mL.
The result shows that the sensitivity of present method is very high, can reach 1 * 10
2About CFU/mL.
Embodiment 5, be applied to the detection test of actual sample
The Salmonellas and the Shigellae actual survey that the PCR-DHPLC method of embodiment 1 foundation are used for animal derived feed samples such as digested tankage, meat meal tankage, fish meal, plasma powder, gelatin, shrimp shell meal and whey powder, adopt national standard method (GB/T 4789.5-2003 simultaneously,) and inspection and quarantine industry standard (SN/T0184.1-2005,), to compare.In 476 parts of samples of checking successively, detecting 9 duplicate samples successively through present method is the Salmonellas positive, and 1 duplicate samples is that Salmonellas and Shigellae are all positive.In full accord with the result who adopts national standard method (GB/T 4789.5-2003) and inspection and quarantine industry standard (SN/T 0184.1-2005) to detect.The result shows: composite PCR-DHPLC high throughput testing method accuracy is 100%, shows that this method has accuracy and suitability preferably.
In the present embodiment, adopt the method for national standard method (GB/T 4789.5-2003) and inspection and quarantine industry standard (SN/T 0184.1-2005), detect each sample average (comprising specimen preparation) always consuming time 96h, detect (not comprising specimen preparation) consuming time 94h; Use the mPCR-DHPLC method, detect each sample average (comprising specimen preparation) always consuming time 25h, detect (not comprising specimen preparation) consuming time 0.5h.
SEQUENCE?LISTING
＜110〉Cao Jijuan
＜120〉Salmonellas and Shigellae detection kit and detection method thereof in the feed
<130>N/A
<160>4
<170>PatentIn?version?3.3
<210>1
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＜213〉artificial sequence
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gtgaaattat?cgccacgttc?gggcaa 26
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＜213〉artificial sequence
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tcatcgcacc?gtcaaaggaa?cc 22
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gccggtcagc?caccctctga?gagtac 26
Claims (1)
1. the detection method of Salmonellas and Shigellae in the feed is characterized in that using test kit, comprises a kind of detection solution in the described test kit, contains 10mM TrisCl, 50mM KCl, 25mM MgCl in this detection solution
2, dATP, dGTP, each 2.5mM of dCTP and dTTP, Taq archaeal dna polymerase 5U/ μ L and Salmonellas and Shigellae primer are to each 10 μ M; Wherein, the specific primer sequence of Salmonellas and Shigellae is as follows:
Described detection method comprises the steps:
1. get 1 μ L testing sample dna solution, add detection solution and 14 μ L sterilization ultrapure water in the 10ul test kit, cumulative volume 25 μ L; The centrifugal 10s of 5000r/min, carry out pcr amplification by following parameter then:
Pre-sex change: 94 ℃, 3min;
Enter circulation: 94 ℃ of sex change 60s, 60 ℃ of annealing 60s, 72 ℃ are extended 60s, 35 circulations;
Stop extending: 72 ℃, 7min;
2. pcr amplification product being carried out DHPLC analyzes:
Chromatographic column: PS-DVB﹠amp; The C18DNASep chromatographic column, 4.6mm * 50mm, granularity 3 μ m;
Column temperature: 50 ℃;
Moving phase (volume ratio): 0.0min, 48.0% buffered soln A, 52.0% buffered soln B;
0.5min, 42.9% buffered soln A, 57.1% buffered soln B;
3.6min, 38.8% buffered soln A, 61.2% buffered soln B;
6.8min, 36.6% buffered soln A, 63.4% buffered soln B;
9.9min, 35.2% buffered soln A, 64.8% buffered soln B;
13min, 34.3% buffered soln A, 65.7% 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; Excitation spectrum 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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| CN102260740B (en) * | 2011-07-05 | 2013-04-10 | 江苏硕世生物科技有限公司 | Salmonella/shigella dual-fluorescence PCR detection kit |
| CN102643926A (en) * | 2012-05-21 | 2012-08-22 | 河南省兽药监察所 | Method and kit for rapidly detecting salmonella living cells in feed by combining ethidium monoazide (EMA) and polymerase chain reaction (PCR) |
| CN102719424A (en) * | 2012-05-30 | 2012-10-10 | 曹际娟 | Salmonella enteritidis nucleic acid standard sample as well as building method and application thereof |
| CN104894245A (en) * | 2015-05-12 | 2015-09-09 | 王秋艳 | Detection primer, kit and method for identifying components of cashmere and components of sheep wool at the same time |
| CN110643691A (en) * | 2019-11-06 | 2020-01-03 | 甘肃农业大学 | Method for monitoring salmonella |
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| CN1673391A (en) * | 2004-10-11 | 2005-09-28 | 复旦大学附属华山医院 | Method for detecting drug resistant gene polymorphism of polydrug |
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| CN1673391A (en) * | 2004-10-11 | 2005-09-28 | 复旦大学附属华山医院 | Method for detecting drug resistant gene polymorphism of polydrug |
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| J.萨姆布鲁克等.PCR反应体系.《分子克隆实验指南》.科学出版社,1992,(第二版),680-681. * |
| 曹际娟等.变性高效液相色谱高通量检测动物源性饲料中沙门氏菌和志贺氏菌.《饲料工业》.2008,第29卷(第14期),50-53. * |
| 闫平平等.食品中沙门菌变性高效液相色谱检测技术的研究与方法建立.《中国卫生检验杂志》.2008,第18卷(第9期),1739－1741,1750. * |
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