CN103484546A - Fourteen-food-borne pathogenic bacterium multiplex PCR detection primer set and kit - Google Patents
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Abstract
The invention discloses a fourteen-food-borne pathogenic bacterium multiplex PCR detection primer set and a kit. The detection primer set is composed of primer pairs for detecting salmonella, Shigella, Vibrio parahemolyticus, campylobacter jejuni, campylobacter coli, staphylococcus aureus, bacillus cereus, Listeria monocytogenes, yersinia enterocolitica, enterobacter sakazakii, Escherichia coli, vibrio cholerae, Escherichia coli O157, aeromonas hydrophila and internal positive control. A multiplex PCR detection method based on an ordinary PCR platform is built, multiplex PCR reactions are carried out on genomic DNA, extracted from a sample to be tested, of bacteria in the same reaction system through the primer pairs acquired through analysis and design, and whether the food-borne pathogenic bacteria are contained in the sample or not is judged through the electrophoretic analysis of reaction products.
Description
Technical field
The invention belongs to the microorganism detection field, relate to 14 kinds of multiple fast PCRs of food-borne pathogens and detect primer sets and test kit.
Background technology
Food-borne pathogenic microorganism is that the important biomolecule that causes food poisoning is learned factor.Along with the attention of country to food safety work, rapid screening food poisoning reason becomes the task of top priority.The design of test kit of the present invention can be used for the common food source property microorganism that causes diarrhoea of examination, they comprise Salmonellas, Shigellae, Vibrio parahemolyticus, campylobacter jejuni, Campylobacter Coli, streptococcus aureus, bacillus cereus, Listeria monocytogenes, yersinia entero-colitica, Enterobacter sakazakii, colon bacillus, vibrio cholerae, colon bacillus O157 and Aeromonas hydrophila, totally ten four kinds of food-borne pathogenic microorganisms, detect the enrichment liquid that sample comes from clinical samples or contaminated food sample.
The present invention is based on the multiplex PCR principle, can in 1-2 PCR reaction, complete the rapid screening to above-mentioned 14 kinds of food-borne pathogens, saved operating time and operating manpower, greatly improved the detectivity in laboratory in the food poisoning.
At present, do not have similar test kit can accomplish 14 kinds of common food-borne pathogenic microorganisms of examination simultaneously, can only adopt real-time fluorescence PCR, regular-PCR method, immunological method (as enzyme-linked immunosorbent assay (ELISA), Colloidal Gold) to detect successively 14 kinds of pathogenic microorganism single targets.The common problem of these substance detection methods is to lack a complete set of operational detection reagent to realize the examination to 14 kinds of food-borne pathogenic microorganisms; Use the substance detection method to operate one by one, configuration reaction system number is many, consume a large amount of reagent, human and material resources and time.Immune colloid gold method detection sensitivity and specificity are lower.ELISA method operating process complexity, detection means underaction, the rapid screening of the scenes such as very difficult reply emergency disposal.
With the similar detection method of the present invention, multiple PCR technique based on the liquid-phase chip detection platform, multifinder multiple PCR technique based on two probe hybridizations and the multiple PCR technique based on the arm-PCR principle etc. are arranged.These technology and something in common of the present invention have all adopted multiple PCR technique, and a plurality of target fragment that simultaneously increase in same reaction system are saved time to reach, the target of manpower, reagent and sample.
Multiple PCR technique based on the liquid-phase chip detection platform, the multifinder multiple PCR technique based on two probe hybridizations all can be realized the rapid screening to a plurality of detection targets, but the test kit that at present based on these technology, does not all detect 14 kinds of food-borne pathogenic microorganisms is researched and developed successfully.And the multiple PCR technique based on the liquid-phase chip detection platform carries out in two steps the PCR reaction in a PCR reaction tubes, the design of first round PCR lower concentration Auele Specific Primer causes the sensitivity of PCR reaction significantly to reduce, thereby affects the sensitivity of whole detection reagent; The poor problem of polychrome microballoon marker detection PCR product technology existence and stability.And the acquisition expenses of instrument is high, reagent consumptive material costliness.
Multifinder multiple PCR technique based on two probe hybridizations is used two adjacent specific probe hybridization, has affected the sensitivity of examination when improving detection specificity, easily produces false negative result; This technology adopts generation order-checking detection of platform PCR product, as ABI3130, and ABI3500 etc., reagent consumptive material costliness; Testing process is through steps such as nucleic acid extraction, two probe hybridization, probe connection, pcr amplification, product analysis, operate more loaded down with trivial details, very high to operator's technical requirements, need to be grasped the check order use of platform of a generation.
Multiple PCR technique based on the arm-PCR principle, although solved the problem of operation automation and product pollution, this technology drops into larger, the consumptive material costliness.
Summary of the invention
In order to solve problems of the prior art, the purpose of this invention is to provide primer sets and test kit that a kind of multiplex PCR detects 14 kinds of food-borne pathogens, but the above-mentioned 14 kinds of food-borne pathogens of rapid screening, set up a kind of multi-PCR detection method based on the regular-PCR platform.
In order to realize the object of the invention, at first the present invention provides the technical scheme of design and screening Auele Specific Primer and the primer sets that a kind of multiplex PCR detects food-borne pathogens:
1, build positive internal reference (IAC) amplimer and template
Take the pET28a plasmid as the stencil design pair of primers, and the fragment of amplification 1176bp size wherein, as the IAC detected.IAC upstream primer sequence is 5 '-TGCAGGTCGACTCTAGAGGA-3 ', and the sequence of IAC downstream primer is 5 '-TTCGAGCTCGGTACCCGGGGA-3 '.The base sequence of pET28a is as shown in SEQ ID No.31.
2, the screening specific gene of different bacterium or DNA fragmentation are for design of primers
The present invention is by document analysis and detect demand analysis, has obtained gene or the DNA fragmentation (in Table 1) of each pathogenic bacterium candidate amplification.
14 kinds of common food-borne pathogens multiplex PCRs of table 1 detect the candidate target gene
| Target microorganism | Candidate's amplification gene |
| Salmonellas | invA |
| Shigellae | ipaH |
| Vibrio parahemolyticus | toxR+toxS |
| Campylobacter jejuni | hipO |
| Campylobacter Coli | cdtA |
| Streptococcus aureus | nuc |
| Bacillus cereus | groB |
| Listeria monocytogenes | hly |
| Yersinia entero-colitica | 16S?rRNA |
| Enterobacter sakazakii | zpx |
| Colon bacillus | uidA |
| Vibrio cholerae | hlyA |
| Colon bacillus O157 | rfbE O157 |
| Aeromonas hydrophila | aha1 |
Annotate: the uidA gene all has amplification in colon bacillus and Shigellae.
3, primer candidate scheme design
The present invention uses primer premier6 software for all amplification target candidate genes or fragment design 1-2 cover alterant.
4, the primer candidate scheme of amplification target gene carried out to the Blast analysis
In Genbank, all primer candidate schemes are carried out to the Blast analysis, obtain primer scheme that specificity is higher as the experimental verification scheme, each pathogenic bacterium may be selected a plurality of amplification targets, and each target that increases has 1-2 experimental verification scheme.
5, each available experimental verification scheme is carried out to substance PCR checking, determine the operability of primer, comprise specificity assessment and sensitivity assessment.
6, the primer of all amplification targets is mixed and carries out multiplex PCR, the operability when checking primer increases jointly, need to be replaced according to the new primer of step 2-4 design iterations inoperable primer.The final a set of special primer sequence provided by the invention (referring to table 2) that obtains.
14 kinds of common food-borne pathogens multiple PCR detection primer groups of table 2
Annotate: although the gene of selected detection target or DNA fragmentation are conservative region, the possible type that still has indivedual bases to insert or lack, the size of PCR product also can increase thereupon or reduce several bp, does not affect the judgement of result.
Based on technique scheme, the invention provides the primer sets that a kind of multiplex PCR detects food-borne pathogens, it is comprised of the primer pair that detects Salmonellas, Shigellae, Vibrio parahemolyticus, campylobacter jejuni, Campylobacter Coli, streptococcus aureus, bacillus cereus, Listeria monocytogenes, yersinia entero-colitica, Enterobacter sakazakii, colon bacillus, vibrio cholerae, colon bacillus O157 and Aeromonas hydrophila;
The nucleotide sequence of the upstream and downstream primer of described detection streptococcus aureus primer pair is respectively by shown in SEQ ID NO.1, SEQ ID NO.2;
The nucleotide sequence of the upstream and downstream primer of described detection colon bacillus O157 primer pair is respectively by shown in SEQ ID NO.3, SEQ ID NO.4;
The nucleotide sequence of the upstream and downstream primer of described detection Salmonellas primer pair is respectively by shown in SEQ ID NO.5, SEQ ID NO.6;
The nucleotide sequence of the upstream and downstream primer of described detection Shigellae primer pair is respectively by shown in SEQ ID NO.7, SEQ ID NO.8;
The nucleotide sequence of the upstream and downstream primer of described detection Enterobacter sakazakii primer pair is respectively by shown in SEQ ID NO.9, SEQ ID NO.10;
The nucleotide sequence of the upstream and downstream primer of described detection vibrio cholerae primer pair is respectively by shown in SEQ ID NO.11, SEQ ID NO.12;
The nucleotide sequence of the upstream and downstream primer of described detection yersinia entero-colitica primer pair is respectively by shown in SEQ ID NO.13, SEQ ID NO.14;
The nucleotide sequence of the upstream and downstream primer of described detection Campylobacter Coli primer pair is respectively by shown in SEQ ID NO.15, SEQ ID NO.16;
The nucleotide sequence of the upstream and downstream primer of described detection Aeromonas hydrophila primer pair is respectively by shown in SEQ ID NO.17, SEQ ID NO.18;
The nucleotide sequence of the upstream and downstream primer of described detection Vibrio parahemolyticus primer pair is respectively by shown in SEQ ID NO.19, SEQ ID NO.20;
The nucleotide sequence of the upstream and downstream primer of described detection bacillus cereus primer pair is respectively by shown in SEQ ID NO.21, SEQ ID NO.22;
The nucleotide sequence of the upstream and downstream primer of described detection colon bacillus primer pair is respectively by shown in SEQ ID NO.23, SEQ ID NO.24;
The nucleotide sequence of the upstream and downstream primer of described detection Listeria monocytogenes primer pair is respectively by shown in SEQ ID NO.25, SEQ ID NO.26;
The nucleotide sequence of the upstream and downstream primer of described detection campylobacter jejuni primer pair is respectively by shown in SEQ ID NO.27, SEQ ID NO.28.
The present invention also provides a kind of method that 14 kinds of food-borne pathogens are carried out to the multiplex PCR detection, the DNA of described primer sets and testing sample is carried out to multi-PRC reaction, and reaction is carried out electrophoretic analysis to determine in sample whether contain above-mentioned 14 kinds of food-borne pathogens to the PCR reaction product after finishing.
Further, when carrying out multi-PRC reaction, also add positive internal reference primer pair and corresponding template in reaction system.
The nucleotide sequence of the upstream and downstream primer of described positive internal reference primer pair is respectively by shown in SEQID NO.29 and SEQ ID NO.30.
The method is totally ten four kinds of Rapid Screening Salmonellass, Shigellae, Vibrio parahemolyticus, campylobacter jejuni, Campylobacter Coli, streptococcus aureus, bacillus cereus, Listeria monocytogenes, yersinia entero-colitica, Enterobacter sakazakii, colon bacillus, vibrio cholerae, colon bacillus O157, Aeromonas hydrophila comprehensively, and its technical scheme is specific as follows:
1, primer is synthetic
According to the primer sequence in table 2, in the oligonucleotide sequence of the synthetic all primers of gene Synesis Company.
2, DNA profiling extracts
Select Salmonellas, Shigellae, Vibrio parahemolyticus, campylobacter jejuni, Campylobacter Coli, streptococcus aureus, bacillus cereus, Listeria monocytogenes, yersinia entero-colitica, Enterobacter sakazakii, colon bacillus, vibrio cholerae, colon bacillus O157, Aeromonas hydrophila as the target microorganism of setting up detection method, according to GB or industry standard method, 14 kinds of microorganisms are increased to bacterium respectively and cultivated, the bacterial concentration of 14 kinds of templates is adjusted to 10
8cFU/mL, adopt bacterial genomes to extract test kit and extract DNA profiling.
3, use regular-PCR platform construction multiplex PCR detection system
In definite multiple reaction system, after primer sequence, also need to be optimized respectively from aspects such as primer concentration, annealing temperature, reaction system configuration, reaction conditionss.The configuration of reaction system is as follows:
Taq archaeal dna polymerase 1-2U, 5 * PCR buffer(TrisHCl 100mM (PH8.3), KCl 250mM, tween-200.2%) 5ul, the dNTP0.5-1.5ul of 10mm, the MgCl of 25mM
21-5ul, primer mixture 2.5ul (comprising Auele Specific Primer and IAC primer, the primer concentration 0.4um-4um of each target to be measured), pET28a0.0001-0.01ng(is as the IAC template), DNA profiling 2-5ul, ultrapure water is mended to 25ul.
The mixture that described primer mixture is 15 kinds of primer pairs in table 2.
The reaction conditions of multiple system is as follows:
a：95℃4min；
b：95℃15-60S，
c：55-65℃15-60s，
D:72 ℃ of 30-120s, 30-40 reaction of b-d circulation;
e：72℃5-10min。
4, multiple PCR products analysis and result are judged
The 5ul multiple PCR products is carried out to electrophoretic analysis PCR product clip size, judge the situation of amplification target gene according to clip size synopsis 2.
5, the checking of detection system
Multi-PCR detection method for 14 kinds of common food-borne pathogenic microorganisms having set up carries out specificity checking and susceptibility checking.
Specificity checking: select Vibrio flurialis, Bacillus anthracis, Webster genus bacillus, sheep listeria spp, bordetella pertussis, legionella, suis etc. as specificity assessment bacterial strain, carry out nucleic acid extraction, adopt the reaction conditions of setting up and optimizing early stage, apply detection reagent of the present invention and detected.
Result shows the positive internal reference of IAC() all positive.Except IAC, negative control and the specific band amplification nothing but of specificity checking bacterial strain, the duplicate detection result is all consistent, shows that this detection method has specificity preferably, non-object bacteria effectively can be distinguished.
Susceptibility checking: be 10 to starting point concentration
814 kinds of object bacteria DNA of CFU/mL are diluted to respectively 10
7cFU/mL, 10
6cFU/mL, 10
5cFU/mL, 10
4cFU/mL.Adopt the reaction conditions of setting up early stage and optimizing to be detected, the detectability of every kind of object bacteria all can reach 10
5cFU/mL, the duplicate detection result is all consistent, shows that this detection method has the susceptibility of height and good repeatability.
The present invention also provides a kind of quick detection kit, and described test kit comprises aforementioned primer sets, positive internal reference primer pair, positive internal reference template, archaeal dna polymerase, 5 * PCR damping fluid, Mg2+, dNTP, positive control and deionized water.
Beneficial effect of the present invention is:
The multiple food-borne pathogenic microorganism detection method that the present invention sets up, can overcome the weak point of other technologies means, reaches following detection effect:
(1) Multiple detection
The detection method that the present invention sets up can examination simultaneously comprise Salmonellas in 1-2 PCR reaction, Shigellae, Vibrio parahemolyticus, campylobacter jejuni, Campylobacter Coli, streptococcus aureus, bacillus cereus, Listeria monocytogenes, yersinia entero-colitica, Enterobacter sakazakii, colon bacillus, vibrio cholerae, colon bacillus O157, 14 kinds of common food-borne pathogenic microorganisms of Aeromonas hydrophila, contained the common and important pathogenic microorganism of food safety examination comprehensively, obtain fast detected result, save time, man power and material's cost.
(2) specificity is high
The detection method specificity that the present invention sets up is mainly reflected in the specificity of a whole set of primer, and all primers all pass through the Blast compare of analysis, has conservative property and the specificity of height; Simultaneously through the specificity checking, can be good at distinguishing and detect the bacterium that target species symbolic animal of the birth year is near, living environment is identical, comprise Vibrio flurialis, Bacillus anthracis, Webster genus bacillus, sheep listeria spp, bordetella pertussis, legionella, suis etc., the proof detection method has the specificity of height, non-target bacteria accurately can be distinguished.
(3) highly sensitive
The screening when detection method that the present invention sets up can realize 14 object bacteria, in each reaction system, the detection sensitivity of each object bacteria can reach 10
5cFU/mL.
(4) cost is lower
The multi-PCR detection method that the present invention sets up has reduced human cost and time cost on operability, and originally substance detects needs artificial and 14 times of times 14 times, uses now this method only to need 1 time manually and the time of 1 secondary response; This multiple detection method has been saved the reagent consumption of the same sample of duplicate detection simultaneously, and maximum can be saved the reagent cost more than 50%.
(5) prevention false negative result
The IAC primer added in system and corresponding template, can effectively point out the false negative detected result.
The rapid detection that the present invention is multiple common food-borne pathogenic microorganism provides complete solution, can realize the quick Emergent detection after food poisoning occurs, and for disposing as early as possible epidemic situation, saves the valuable time.
In sum, the invention provides the primer sets that a kind of multiplex PCR detects 14 kinds of common food-borne pathogens, and set up a kind of multi-PCR detection method based on the regular-PCR platform.The present invention adopts highly sensitive and specific primer sequence, guarantees the quality of detected result; This detection method is simple to operate, time saving and energy saving; The detection flux is high, and the reagent consumables cost is low; Can the enrichment liquid of sample be detected, be compared to traditional detection method, can shift to an earlier date 24-48 hour and lock suspicious pathogenic bacterium; To detection platform and testing staff require lowly, can extensively promote at the inspection and quarantine First Line.
The accompanying drawing explanation
The pcr amplification product agarose gel electrophoresis result that Fig. 1 is specificity analyses in the embodiment of the present invention 2;
Wherein: M:100bp DNA marker; 1.invA-1; 2.invA-2; 3.invA-3; 4. positive control.
The pcr amplification product agarose gel electrophoresis result that Fig. 2 is sensitivity assessment in the embodiment of the present invention 2;
Wherein: M:100bp DNA marker; 1.invA-1; 2.invA-2; 3.invA-3.
Fig. 3 is that 14 kinds of food-borne pathogenic microorganism multiplex PCRs of the present invention detect the qiaxcel electrophoresis result;
Wherein: 1. Staphylococcus aureus; 2. colon bacillus O157; 3. Salmonellas; 4. Shigellae; 5. Enterobacter sakazakii; 6. vibrio cholerae; 7. yersinia entero-colitica; 8. Campylobacter Coli; 9. have a liking for the aqueous vapor unit cell; 10. Vibrio parahemolyticus; 11. bacillus cereus; 12. colon bacillus; 13. Listeria monocytogenes; 14. campylobacter jejuni; 15. positive control (14 kinds of templates are mixed); 16. blank.
Fig. 4 is that 14 kinds of food-borne pathogenic microorganism multiplex PCRs of the present invention detect the agarose gel electrophoresis result;
Wherein: M:100bp marker; A1. streptococcus aureus; A2. Salmonellas; A3. Enterobacter sakazakii; A4. yersinia entero-colitica; A5. have a liking for the aqueous vapor unit cell; A6. bacillus cereus; A7. Listeria monocytogenes; B1. colon bacillus O157; B2. Shigellae; B3. vibrio cholerae; B4. Campylobacter Coli; B5. Vibrio parahemolyticus; B6. colon bacillus; B7. campylobacter jejuni; Blank. blank.
Embodiment
Following examples are used for the present invention is described, but are not used for limiting the scope of the invention.
The design of primers of embodiment 1 Salmonellas is with synthetic
Download 65 of the full length sequences of Salmonellas invA gene in Genbank, 65 invA full length gene sequences are imported in Mega4 software, use the conserved sequence of alignment function compare of analysis invA gene, obtain the conserved sequence section, base sequence is as shown in sequence table SEQ ID No.32.
In the conserved sequence of above-mentioned invA gene input primer premier6 software, automatic analysis obtains multiple design of primers scheme.Manual regulation primer location and sequence length, be set as 55 ± 5 ℃ by the Tm value on this basis, and GC content 35%～60% does not produce hairpin structure and primer dimer as far as possible, and inerrancy causes generation.The design of primers scheme that above-mentioned manual regulation is obtained is carried out the Blast analysis on the NCBI website, if wherein there are cross reaction in any primer pair and non-object bacteria, need to readjust position and the sequence length of primer, until obtain the invA gene primer sequence of high specific.Final three kinds of invA gene primer alternativess that obtain as table 3.
Table 3invA gene primer alternatives
| The primer numbering | Upstream sequence (5 '-3 ') | Downstream sequence (5 '-3 ') | Amplification length (bp) |
| invA-1 | TTATTGGCGATAGCCTGGC | AATACCGGCCTTCAAATC | 173 |
| invA-2 | ATGGGGTCGTTCTACATTG | GCTATCGCCAATAACGAATT | 174 |
| invA-3 | CCGGKGAAATTATCGCCA | GTTTACCGGGCATACCAT | 171 |
The primer design method of other genes is identical with the invA gene.The primer alternatives obtained is all synthetic in invitrogen company.
The bacterium group of specificity assessment: comprise Shigellae, Vibrio parahemolyticus, campylobacter jejuni, Campylobacter Coli, streptococcus aureus, bacillus cereus, Listeria monocytogenes, yersinia entero-colitica, Enterobacter sakazakii, colon bacillus, vibrio cholerae, colon bacillus O157, detection object bacteria in 14 kinds of test kits such as Aeromonas hydrophila, also comprise with detection target species symbolic animal of the birth year near, the bacterium that living environment is identical, as Vibrio flurialis, Bacillus anthracis, the Webster genus bacillus, the sheep listeria spp, bordetella pertussis, legionella, 7 kinds, suis etc.
To above-mentioned bacterium, adopt bacterial genomes to extract test kit acquisition DNA profiling standby.The template of extraction is respectively got to 5ul and mix, as the template of specificity checking.
The specificity analyses test of Salmonellas: use the primer pair designed in embodiment 1, according to following project configuration PCR reaction system: Taq archaeal dna polymerase 0.4ul(5U/ul), 5 * PCR buffer(TrisHCl 100mM (PH8.3), KCl 250mM, tween-200.2%) 5ul, dNTP0.75ul(10mM), MgCl
24ul(25mM), every couple of primer 1ul(20uM); Specificity validating DNA template 5ul, deionized water is mended to 25ul.Carry out pcr amplification according to following reaction conditions:
a：95℃4min；
b：95℃30s，
c：62℃30s，
D:72 ℃ of 45s, b-d 30 reactions that circulate.
Pcr amplification product 5ul is used 2% agarose gel electrophoresis analytical results as shown in Figure 1.As shown in Figure 1, under the prerequisite of setting up at positive control (primer and the template that add the sramana), three Salmonellas primer invA-1 of design, invA-2 and invA-3 and Shigellae, Vibrio parahemolyticus, campylobacter jejuni, Campylobacter Coli, streptococcus aureus, bacillus cereus, Listeria monocytogenes, yersinia entero-colitica, Enterobacter sakazakii, colon bacillus, vibrio cholerae, colon bacillus O157, Aeromonas hydrophila, Vibrio flurialis, Bacillus anthracis, the Webster genus bacillus, the sheep listeria spp, bordetella pertussis, legionella, the habitats such as suis are similar, high frequency the equal no cross reaction of the bacterium of depositing.Therefore can think, three primer invA-1, invA-2 and invA-3 all have specificity preferably.
Sensitivity assessment: select the template of 1 strain Salmonellas, its starting point concentration is approximately 10
7cFU/mL, gradient dilution to 10
5cFU/mL, as the template of invA gene amplification primer sensitivity assessment.System configurations is as follows: Taq archaeal dna polymerase 0.4ul(5U/ul), and 5 * PCRbuffer(TrisHCl 100mM (PH8.3), KCl250mM, tween-200.2%) 5ul, dNTP0.75ul(10mM), MgCl24ul(25mM), every couple of primer 1ul(20uM); Salmonellas template 2ul, deionized water is mended to 25ul.The experiment of PCR reaction conditions homospecificity.
Pcr amplification product 5ul is used 2% agarose gel electrophoresis analytical results as shown in Figure 2.As seen from Figure 2, three primer pairs all can reach 10
5cFU/mL, wherein the band brightness of primer pair invA-3 is the highest, is secondly invA-1, invA-2.
Overall sensitivity and specific assessment result, select the primer of invA-3 as invA gene amplification.
The appraisal procedure of other genes is identical with Salmonellas invA gene.
The establishment of 314 kinds of common food-borne pathogenic microorganism multiple PCR detection kits of embodiment
This test kit consists of 2 * reaction system damping fluid, archaeal dna polymerase, 10 * primer mixed solution, positive control, deionized water, its concrete component is as follows: 2 * PCR Buffer(TrisHCl 40mM (PH8.3), KCl 100mM, tween-200.08%, 0.0006ng/ulpET28a, 1mm dNTP, 8mm MgCl2); 25 * archaeal dna polymerase: 2U/ul; 10 * primer mixed solution (comprise Auele Specific Primer and IAC primer, determine kind and the number of tubes of primer mixed solution according to testing goal and electrophoresis platform), positive control (14 kinds of food-borne pathogens hybrid templates, every kind 10
6cFU/ml).
The reaction system that test kit detects is 25ul, and its configuration is as follows: 2 * PCR Buffer12.5ul; 25 * archaeal dna polymerase 1ul; 10 * primer mixed solution (determining kind and the number of tubes of primer mixed solution according to testing goal and electrophoresis platform) 2.5ul; Template 2ul, deionized water 7ul.
The operation of embodiment 4 test kits and result judgement (according to the full-automatic nucleic acid-protein electrophoresis platform of qiaxcel)
1, genomic extraction
1-2mL bacterium enrichment liquid or suspension liquid, extract the test kit specification sheets according to bacterial genomes and extract template DNA.
2, the preparation of reaction system
Test kit is set up: this test kit consists of 2 * reaction system damping fluid, archaeal dna polymerase, 10 * primer mixed solution, positive control, deionized water, its concrete component is as follows: the reaction system damping fluid: 2 * PCR Buffer(TrisHCl 40mM (PH8.3), KCl100mM, tween-200.08%, 0.0006ng/ul pET28a, 1mm dNTP, 8mm MgCl2); 25 * archaeal dna polymerase: 2U/ul; 10 * primer mixed solution (14 kinds of food-borne pathogens primers and IAC primer mixed solution, every kind of final concentration 2um); Positive control (hybrid template of 14 kinds of food-borne pathogens, every kind 10
6cFU/ml).
Get the reaction system of the PCR pipe configuration 25ul of 200ul, its configuration is as follows: 2 * PCRBuffer12.5ul; 25 * archaeal dna polymerase 1ul; 10 * primer mixed solution (14 kinds of food-borne pathogens primers and IAC primer mixed solution, every kind of final concentration 2um) 2.5ul, template 2ul, deionized water 7ul.
3, PCR reaction
The PCR pipe is put into to Bio-Rad C1000 type PCR instrument, after opening the heat lid, carry out the PCR reaction according to following program: 95 ℃ of 4min; 95 ℃ of 30s, 62 ℃ of 30s, 72 ℃ of 90s, 30 circulations; 72 ℃ of 5min.
4, the full-automatic nucleic acid-protein electrophoresis platform of qiaxcel is analyzed the PCR product
5, result judgement
The pcr amplification result as shown in Figure 3, as shown in Figure 3, the all swimming lanes that comprise blank all have a band amplification at least, blank (16 swimming lane) has the amplification of IAC, other detections have target stripe and (or) amplification of IAC, show all establishments of all PCR reactions, get rid of false-negative result.Otherwise invalid depending on experiment, need repetition.
On the basis of IAC amplification, the 117bp amplified band also appears in the 1st swimming lane, shows that this bacterial strain is streptococcus aureus, and the amplified band of 142bp and 601bp also appears in the 2nd swimming lane, shows that this bacterial strain is colon bacillus O157 serotype; The 171bp amplified band also appears in the 3rd swimming lane, shows that this bacterial strain is Salmonellas; The amplified band of 210bp and 601bp also appears in the 4th swimming lane, shows that this bacterial strain is Shigellae; The 253bp amplified band also appears in the 5th swimming lane, shows that this bacterial strain is Enterobacter sakazakii; The 262bp amplified band also appears in the 6th swimming lane, shows that this bacterial strain is vibrio cholerae; The 315bp amplified band also appears in the 7th swimming lane, shows that this bacterial strain is yersinia entero-colitica; The 385bp amplified band also appears in the 8th swimming lane, shows that this bacterial strain is Campylobacter Coli; The 407bp amplified band also appears in the 9th swimming lane, shows that this bacterial strain is to have a liking for the aqueous vapor unit cell; The 478bp amplified band also appears in the 10th swimming lane, shows that this bacterial strain is Vibrio parahemolyticus; The 515bp amplified band also appears in the 11st swimming lane, shows that this bacterial strain is bacillus cereus; The 601bp amplified band also appears in the 12nd swimming lane, shows that this bacterial strain is colon bacillus; The 700bp amplified band also appears in the 13rd swimming lane, shows that this bacterial strain is Listeria monocytogenes.The 980bp amplified band also appears in the 14th swimming lane, shows that this bacterial strain is campylobacter jejuni; The amplification of 15 specific bands appears in the 15th swimming lane, corresponding 14 amplifications that detect targets and an IAC; Low 16 swimming lanes are blank, only have the IAC amplification.
Detect sample: totally ten four kinds of pathogen gene group DNA are as the template of testing to select Salmonellas, Shigellae, Vibrio parahemolyticus, campylobacter jejuni, Campylobacter Coli, streptococcus aureus, bacillus cereus, Listeria monocytogenes, yersinia entero-colitica, Enterobacter sakazakii, colon bacillus, vibrio cholerae, colon bacillus O157, Aeromonas hydrophila, and the concentration of every kind of template is approximately 10
5cFU/ml.Test kit is set up: this test kit consists of 2 * reaction system damping fluid, archaeal dna polymerase, 10 * primer mixed liquor A, 10 * primer mixed liquid B, positive control, deionized water, its concrete component is as follows: the reaction system damping fluid: 2 * PCR Buffer(TrisHCl 40mM (PH8.3), KCl 100mM, tween-200.08%, 0.0006ng/ul pET28a, 1mm dNTP, 8mmMgCl2); 25 * archaeal dna polymerase: 2U/ul; 10 * primer mixed liquor A (contains streptococcus aureus, Salmonellas, E.sakazakii, yersinia entero-colitica, has a liking for aqueous vapor unit cell, Bacillus cereus, Listeria monocytogenes and IAC primer, every kind of final concentration is 0.2um) and 10 * primer mixed liquid B (containing colon bacillus O157, Shigellae, vibrio cholerae, Campylobacter Coli, Vibrio parahemolyticus, colon bacillus, campylobacter jejuni and IAC primer, every kind of final concentration is 0.2um); Positive control (hybrid template of 14 kinds of food-borne pathogens, every kind 10
6cFU/ml).
Test kit operation: according to embodiment 4 operation test kits.
The Multiple detection experimental result as shown in Figure 4.
As shown in Figure 4: all swimming lanes that comprise blank all have a band amplification at least, and blank has the amplification of IAC, and other detections have the amplification of target stripe or IAC.Show that all PCR reactive systems all set up, get rid of false-negative result.On the basis of IAC amplification, the 117bp amplified band also appears in the A1 swimming lane, shows that this bacterial strain is streptococcus aureus; The 171bp amplified band also appears in A2, shows that this bacterial strain is Salmonellas; The 253bp amplified band also appears in the A3 swimming lane, shows that this bacterial strain is Enterobacter sakazakii; The 315bp amplified band also appears in the A4 swimming lane, shows that this bacterial strain is yersinia entero-colitica; The 407bp amplified band also appears in the A5 swimming lane, shows that this bacterial strain is to have a liking for the aqueous vapor unit cell; The 515bp amplified band also appears in the A6 swimming lane, shows that this bacterial strain is bacillus cereus; The 700bp amplified band also appears in the A7 swimming lane, shows that this bacterial strain is Listeria monocytogenes.
The amplified band of 142bp and 601bp also appears in the B1 swimming lane, shows that this bacterial strain is colon bacillus O157 serotype; The amplified band of 210bp and 601bp also appears in the B2 swimming lane, shows that this bacterial strain is Shigellae; The 262bp amplified band also appears in the B3 swimming lane, shows that this bacterial strain is vibrio cholerae; The 385bp amplified band also appears in the B4 swimming lane, shows that this bacterial strain is Campylobacter Coli; The 478bp amplified band also appears in the B5 swimming lane, shows that this bacterial strain is Vibrio parahemolyticus; The 601bp amplified band also appears in the B6 swimming lane, shows that this bacterial strain is colon bacillus; The 980bp amplified band also appears in the B7 swimming lane, shows that this bacterial strain is campylobacter jejuni.
The preservation period test of embodiment 6 test kits
With 10
5the Salmonellas of CFU/ml as assessment with detecting sample, in the time of the 0th day, be distributed into 9 parts frozen in-70 ℃ of refrigerators.Be positioned over-20 ℃ of preservations by setting up complete test kit, the test kit of getting respectively 0,10,20,30,60,90,120,150 and 180 day carries out the preservation period test.The preservation period detected result is as shown in table 4:
Table 4 preservation period test-results
| Preservation period | The IAC specific band | The Salmonellas specific band |
| The 0th day | + | + |
| The 10th day | + | + |
| The 20th day | + | + |
| The 30th day | + | + |
| The 60th day | + | + |
| The 90th day | + | + |
| The 120th day | + | + |
| The 150th day | + | + |
| The 180th day | + | + |
As shown in Table 4, test kit is kept at-20 ℃ of refrigerators, and the result that detects Salmonellas in different preservation perives is all positive, shows that the preservation period of this test kit is at least 6 months.
The specific test of embodiment 7 test kits
Select Vibrio flurialis, Bacillus anthracis, Webster genus bacillus, sheep listeria spp, bordetella pertussis, legionella, suis etc. close with detection object bacteria kind, exist like environmental facies bacterium as bacterium to be checked, all only have after testing an IAC band amplification, produce without assorted band.And, when object bacteria is detected, except specific band, without assorted band, produce.Show that test kit of the present invention can effectively distinguish non-target bacteria, there is specificity preferably.
The sensitivity test of embodiment 8 test kits
Assessment is with detecting sample: the concentration of 14 kinds of object bacteria is adjusted to 10
8the CFU/mL order of magnitude, adopt bacterial genomes to extract test kit and extract 14 kinds of DNA that detect object bacteria.Each template gradient dilution becomes 10
7cFU/mL, 10
6cFU/mL, 10
5cFU/mL, 10
4the detection sample of CFU/mL.
Use test kit of the present invention to detect respectively the different dilution templates of 14 object bacteria.According to example 4 determination methods as a result, it is as shown in table 5 that test kit detects the sensitivity test result of 14 kinds of object bacteria:
Table 5 test kit detects 14 kinds of object bacteria sensitivity test results
As seen from Table 5, the susceptibility of 14 kinds of food pathogenic microorganisms of test kit detection is 10
5cFU/mL.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (6)
1. a multiplex PCR detects the primer sets of food-borne pathogens, it is characterized in that, it is comprised of the primer pair that detects Salmonellas, Shigellae, Vibrio parahemolyticus, campylobacter jejuni, Campylobacter Coli, streptococcus aureus, bacillus cereus, Listeria monocytogenes, yersinia entero-colitica, Enterobacter sakazakii, colon bacillus, vibrio cholerae, colon bacillus O157 and Aeromonas hydrophila;
The nucleotide sequence of the upstream and downstream primer of described detection streptococcus aureus primer pair is respectively by shown in SEQ ID NO.1, SEQ ID NO.2;
The nucleotide sequence of the upstream and downstream primer of described detection colon bacillus O157 primer pair is respectively by shown in SEQ ID NO.3, SEQ ID NO.4;
The nucleotide sequence of the upstream and downstream primer of described detection Salmonellas primer pair is respectively by shown in SEQ ID NO.5, SEQ ID NO.6;
The nucleotide sequence of the upstream and downstream primer of described detection Shigellae primer pair is respectively by shown in SEQ ID NO.7, SEQ ID NO.8;
The nucleotide sequence of the upstream and downstream primer of described detection Enterobacter sakazakii primer pair is respectively by shown in SEQ ID NO.9, SEQ ID NO.10;
The nucleotide sequence of the upstream and downstream primer of described detection vibrio cholerae primer pair is respectively by shown in SEQ ID NO.11, SEQ ID NO.12;
The nucleotide sequence of the upstream and downstream primer of described detection yersinia entero-colitica primer pair is respectively by shown in SEQ ID NO.13, SEQ ID NO.14;
The nucleotide sequence of the upstream and downstream primer of described detection Campylobacter Coli primer pair is respectively by shown in SEQ ID NO.15, SEQ ID NO.16;
The nucleotide sequence of the upstream and downstream primer of described detection Aeromonas hydrophila primer pair is respectively by shown in SEQ ID NO.17, SEQ ID NO.18;
The nucleotide sequence of the upstream and downstream primer of described detection Vibrio parahemolyticus primer pair is respectively by shown in SEQ ID NO.19, SEQ ID NO.20;
The nucleotide sequence of the upstream and downstream primer of described detection bacillus cereus primer pair is respectively by shown in SEQ ID NO.21, SEQ ID NO.22;
The nucleotide sequence of the upstream and downstream primer of described detection colon bacillus primer pair is respectively by shown in SEQ ID NO.23, SEQ ID NO.24;
The nucleotide sequence of the upstream and downstream primer of described detection Listeria monocytogenes primer pair is respectively by shown in SEQ ID NO.25, SEQ ID NO.26;
The nucleotide sequence of the upstream and downstream primer of described detection campylobacter jejuni primer pair is respectively by shown in SEQ ID NO.27, SEQ ID NO.28.
2. the method that 14 kinds of food-borne pathogens are carried out to the multiplex PCR detection, it is characterized in that, the DNA of the described primer sets of claim 1 and testing sample is carried out to multi-PRC reaction, and reaction is carried out electrophoretic analysis to determine in sample whether contain above-mentioned 14 kinds of food-borne pathogens to the PCR reaction product after finishing.
3. method according to claim 2, is characterized in that, when carrying out multi-PRC reaction, also adds positive internal reference primer pair in reaction system.
4. method according to claim 3, is characterized in that, the nucleotide sequence of the upstream and downstream primer of described positive internal reference primer pair is respectively by shown in SEQ ID NO.29 and SEQ ID NO.30.
5. according to the described method of claim 2-4 any one, it is characterized in that, described multi-PRC reaction carries out according to the following steps:
(1) 95 ℃ of denaturation 4min;
(2) 95 ℃ of sex change 15-60s, 55-65 ℃ of annealing 15-60s, 72 ℃ are extended 30-120s, carry out altogether 30-40 circulation;
(3) 72 ℃ are extended 5-10min.
6. a quick detection kit, is characterized in that, described test kit comprises primer sets claimed in claim 1, positive internal reference primer pair, positive internal reference template, archaeal dna polymerase, reaction system damping fluid, Mg
2+, dNTP, positive control and deionized water.
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