CN102634580B - Primers and method for simultaneously detecting various pathogenic bacteria - Google Patents
Primers and method for simultaneously detecting various pathogenic bacteria Download PDFInfo
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Abstract
The invention discloses primers and method for simultaneously detecting various pathogenic bacteria. The nucleotide sequences of the primers are shown in the specification, wherein an invA primer pair is used for detecting salmonella infection and salmonella paratyphi, a hly primer pair is used for detecting Listeria monocytogenes, a toxR primer pair is used for detecting vibrio parahaemolyticus, an ipaH primer pair is used for detecting shigella flexneri, and a vcc primer pair is used for detecting non-O1 Vibrio cholerae. The invention also discloses a multiple PCR (Polymerase Chain Reaction) amplification system built by using all the primers pairs, the system can be used for quickly and accurately detecting whether the corresponding pathogenic bacteria exist in a sample to be detected or not, and the whole detection process costs less than 2 hours, so that the system is simple and convenient to use; as the specificity of each primer pair is very high, a false positive result is avoided; and the primers can be made into a kit and the like, thus being very suitable for the pathogenic bacteria detection of agricultural and sideline products.
Description
Technical field
The present invention relates to the agricultural byproducts safety detection technology, be specifically related to detect simultaneously primer and the detection method of various pathogens.
Background technology
Food safety is the link that should pay attention in public health problem, according to the World Health Organization's report whole world case of food origin disease occurs up to 100,000,000 examples every year, the whole world is annual approximately has 1,700,000 children to die from the dysentery that is caused by the microbial contamination of food source property, and grownup's cases of infection reach billions of.The research discovery, there are the following problems in present China's food safety field: (1) microbial contamination is the main factor that affects China's food safety.(2) supply with at input, agriculture home environment, the epidemic prevention system, still there is potential safety hazard in the links such as agricultural-food production, processing and sale.(3) food safety scientific and technological achievement and tachnical storage are not enough.(4) also there is obvious inadaptability in the aspects such as food safety standard system, test system, standard matter system, and food safety management system and legal and regulatory systems are left to be desired.(5) food poisoning and food origin disease have still formed obvious threat to the food safety of China, and great food safety accident occurs repeatedly.
For guaranteeing China's food safety and food trade, must strengthen energetically the dynamics of food test, set up, perfect the food safety rules in China, strengthen food security supervision, improve detection technique, make the corresponding rules of China, standard in line with international standards, particularly the food Fast Detection Technique is reached advanced world standards.
The World Health Organization is defined as food origin disease, enters human body by ingesting, and makes human body suffer from infectious or toxic disease.The mankind have 60% disease relevant with Animal diseases, and in the New Type of Diseases that occurs in recent years, this ratio was especially up to 75%, and, man and animal stage construction highly developed in aquaculture contact from now on, and this ratio probably also can raise.Common food-borne pathogens comprises salmonella, pathogenic colon bacillus, staphylococcus and toxin, proteus, Vibrio parahemolyticus, listeria bacteria, clostridium botulinum toxin, wax sample bud pole bacterium, Clostridium welchii, Campylobacter, the false pseudomonas bacillus of fermented flour coconut palm poison, colitis Yersinia, suis and Shigella etc.
Above-described pathogenic bacterium also do not have a cover can detect simultaneously its detection method whether at present.
Summary of the invention
The object of the invention is to the above-mentioned deficiency for prior art, provide a cover can detect simultaneously the detection primer of various pathogens.
Another object of the present invention is to provide a kind of detection method according to above-mentioned detection design of primers.
The present invention is achieved through the following technical solutions above-mentioned purpose:
In various food poisonings, bacterial food poisoning is the principal element of food poisoning.The present invention is directed to modal pathogenic bacterium in China's food poisoning, work out a kind of effective ways of poison by food diagnosis and food inspection.According to the related request in GB 4789.1 microbiological test of food hygiene general provisions and GB4789.17 meat and meat product check and in conjunction with the genesis situation of the present food-borne pathogens of China, choose following six kinds of bacteriums as detected object: Salmonella typhimurium, Salmonella paratyphi A, Listeria Monocytogenes, Vibrio parahemolyticus, shigella flexneri, non-O1 group cholera vibrio, at first filter out the PCR primer that detects above-mentioned each pathogenic bacterium.
Detect simultaneously the detection primer of various pathogens, nucleotide sequence is as follows:
InvAPrimer pair: SEQ ID NO:1 ~ 2(
InvA-up, down)
hlyPrimer pair: SEQ ID NO:3 ~ 4(
Hly-up, down);
ToxRPrimer pair: SEQ ID NO:5 ~ 6(
ToxR-up, down);
IpaHPrimer pair: SEQ ID NO:7 ~ 8(
IpaH-up, down);
vccPrimer pair: SEQ ID NO:9 ~ 10(
Vcc-up, down);
Above any combination of primers more than two pairs, can detect the corresponding pathogenic bacterium of this primer pair;
Wherein
InvAPrimer pair for detection of Salmonella typhimurium (
Salmonella typhimurium) and Salmonella paratyphi A (
Salmonella paratyphi),
hlyPrimer pair for detection of Listeria Monocytogenes (
Listeria monocytogenes),
ToxRPrimer pair for detection of Vibrio parahemolyticus (
Vibrio parahaemolyticus),
IpaHPrimer pair for detection of shigella flexneri (
Shigella flexneri),
vccPrimer pair is for detection of non-
O1Group cholera vibrio (
Vibrio Cholerae non-O1).
Above primer is the Salmonellas that filters out by Blast, MegAlign and DNAStar program
invThe gene cluster conserved sequence (GenBank:
FR775234.1), the hemolysin O gene of Listeria Monocytogenes
hlyConserved sequence (GenBank:
NC_012488.1), Vibrio parahemolyticus toxR gene conserved sequence (GenBank:
L11929.1), shigella flexneri aggressive plasmid antigen H gene ipaH7 conserved sequence (GenBank:
NC_004337.1), non-
O1Group cholera vibrio
vccThe gene conserved sequence (GenBank:
NC_012578.1) as template, with Primer Explorer V5 software design gained.
The selection principle of target gene of the present invention has two: select virulence gene, select to be positioned at the gene of bacterial genomes.Because the non-pathogenic type that various pathogenic bacterium belong to together with it is as broad as long on biochemical reaction, between genus, genomic homology is also very high, but high to pathogenic relevant virulence gene specificity.Just can regard as pathogenic bacterium and only detect virulence on food inspection, so the selection virulence gene is target gene.Bacterium belongs to prokaryotic organism, and its hereditary thing material has genome and plasmid two classes, and a lot of virulence genes are positioned on plasmid.But plasmid instability, may shift between bacterium, also easily loses in the cultivation of going down to posterity.The size of plasmid DNA and genomic dna differs greatly, and can not extract with same method.So the virulence gene of selecting to be positioned on bacterial genomes is target gene.
Owing to carrying out simultaneously the amplification of at least two goal gene in same PCR body is, the design of primers more complicated, and the specificity in primer amplification district is had relatively high expectations.So design of primers does not have the general primer simplicity of design, design good primer, need to carry out time-consuming screening on the basis of various bioinformatics software assessments.The selection specificity that has height due to designed primer, carrying out cause of disease while detecting, require primer for the target sequence site should be quite conservative, such method of setting up just has suitability.Also can design some degenerated primers in research to strengthen the popularity of its application, but the high primer of degeneracy can cause negative impact to amplified reaction.Have the aim sequence of the characteristics such as GC content is high, variability strong, primer dimer for some, have no idea to design well-adapted primer, the application of Multiple detection technology is restricted.
Because designed primer will carry out the multiplex PCR reaction at next step, the sequence that therefore each gene pairs will be answered when the design primer together compares.Except following the rule of design of primers, also to make the Tm value close between primer pair inside and primer pair as far as possible, guarantee all to obtain amplification preferably under identical conditions; Each, to can not be complementary between primer inside and primer pair, especially avoids the complementation of 3 ' end, to reduce the formation of primer dimer; Each primer and other amplified fragments and template have specificity preferably; The size of amplified production fragment is variant, guarantees to distinguish with agarose gel electrophoresis method for detecting.The primer sequence that designs is submitted to the NCBI website and carries out the BLAST analysis, in order to verify the theoretical specificity of primer.
Another technical scheme that the present invention will protect is a kind of detection method that detects simultaneously various pathogens; the method is used and toply to be detected and to use primer pair more than two pairs arbitrarily; carry out pcr amplification take the DNA that extracts in sample to be checked as template; following band appears in the amplified production electrophoresis result, shows in sample to be checked and has corresponding pathogenic bacterium:
214bp: Vibrio parahemolyticus;
394bp: shigella flexneri;
490bp: non-O1 group cholera vibrio;
568bp: Salmonella typhimurium and Salmonella paratyphi A;
797bp: Listeria Monocytogenes.
The present invention also protects a kind of double PCR reaction system that detects simultaneously two kinds of pathogenic bacterium, this reaction system is 25 μ L, 2 * PCRmix, 12.5 μ L wherein, each 0.4 μ L of the upstream and downstream primer of 10 μ mol/L, detected sample DNA profiling 1.0 μ L, ultrapure water is supplied 25 μ L; Described upstream and downstream primer is the upstream and downstream primer in any two kinds of detections use primer pair in SEQ ID NO:1 ~ 10, if select
InvAPrimer pair and/or
hlyPrimer pair, its upstream and downstream primer need respectively add 1 μ L; Described pathogenic bacterium be selected primer pair the pathogenic bacterium to detecting.
The amplification method of above-mentioned double PCR reaction system, amplification condition are 56 ℃ of annealing 30s, and 72 ℃ are extended 45s, totally 28 circulations.
When the pathogenic bacterium kind that detects increases, the cumulative volume of above-mentioned PCR reaction system and component also have corresponding change, as a kind of PCR system preference scheme that can detect pathogenic bacterium more than three kinds, this multi-PRC reaction system that detects simultaneously various pathogens is 50 μ L, 2 * PCRmix, 12.5 μ L wherein, each 0.4 μ L of the upstream and downstream primer of 10 μ mol/L, detected sample DNA profiling 1.0 μ L, ultrapure water is supplied 50 μ L; Described upstream and downstream primer is any detection more than three kinds with the upstream and downstream primer in primer pair in SEQ ID NO:1 ~ 10, if select
InvAPrimer pair and/or
hlyPrimer pair, its upstream and downstream primer need respectively add 1 μ L; Described pathogenic bacterium be selected primer pair the pathogenic bacterium to detecting.
The amplification method of above-mentioned multi-PRC reaction system, amplification condition are 56 ℃ of annealing 30s, and 72 ℃ are extended 45s, totally 28 circulations.
Increased primer and template in the multiplex PCR system, the factor of impact reaction increases, and has increased interference between mutually.Therefore increased the difficulty of design primer.In addition in system, the amount of each composition and loop parameter also need to make the appropriate adjustments in experiment, and to reduce the impact of non-specific amplification, the amount of each product of balance, obtain each section electrophoretic band clearly.In actual the detection, the amount of template is unascertainable, so the concentration of primer and other compositions will guarantee Enough supply, can not be excessive, and this has a direct impact detected result.Therefore in system the emphasis adjustment be the concentration of primer.
The detection that detects simultaneously various pathogens of the present invention is strong with primer specificity, can be used for preparing the especially detection reagent of fishery products pathogenic bacterium of agricultural byproducts.
Compared with prior art, the present invention has following beneficial effect:
(1) the present invention has filtered out the pathogen PCR amplimer of multiple high specificity, has set up a kind of multiple PCR method that detects simultaneously various pathogens, for various pathogens in quick, efficient detection agricultural byproducts provides a kind of simple and rapid method.
(2) 5 pairs of primers of the present invention
InvA-up, down, hly-up, down, toxR-up, down, ipaH-up, down, vcc-up, downOnly cause the specific amplification of corresponding target genes,, without any non-specific amplification, shown good specificity, can avoid in practice the appearance of false positive results.
(3) detection method amplified reaction of the present invention is quick, efficient, easy, and whole amplification can be completed less than 2 hours.
(4) 5 pairs of primers of the present invention detect medium sensitivity all 10 at six kinds of bacterium substance PCR
3Cfu/mL ~ 10
4Cfu/mL, shown highly sensitive.
(5) can detect simultaneously multiple pathogenic bacteria: by the selection of the specific gene fragment to six kinds of pathogenic bacterium, foundation and debugging by a multiplex PCR system, success has amplified six kinds of bacterium simultaneously, and the amplification of six kinds of bacterium random combines is success also, the strong phenomenon that suppresses each other do not occur.
Description of drawings
Fig. 1. Salmonella typhimurium DNA is the primer of template
InvA-up, downAnnealing temperature gradient experimental result electrophoresis result figure, annotate: 1 ~ 10 road from be respectively from left to right annealing temperature (℃): 50,50.5,51.2,52.1,53.5,54.7,55.8,56.9,58.1,59.1.
Fig. 2. Salmonella paratyphi A DNA is the primer of template
InvA-up, downAnnealing temperature gradient experimental result electrophoresis result figure, annotate: 1 ~ 10 road from be respectively from left to right annealing temperature (℃): 50,50.5,51.2,52.1,53.5,54.7,55.8,56.9,58.1,59.1.
Fig. 3. primer
Hly-up, downAnnealing temperature gradient experimental result electrophoresis result figure, annotate: 1 ~ 10 road from be respectively from left to right annealing temperature (℃): 50.5,51.2,52.1,53.5,54.7,55.8,56.9,58.1,59.1,59.7.
Fig. 4. primer
ToxR-up, downAnnealing temperature gradient experimental result electrophoresis result figure, annotate: 1 ~ 10 road from be respectively from left to right annealing temperature (℃): 50,50.5,51.2,52.1,53.5,54.7,55.8,56.9,58.1,59.1.
Fig. 5. primer
IpaH-up, downAnnealing temperature gradient experimental result electrophoresis result figure, annotate: 1 ~ 10 road from be respectively from left to right annealing temperature (℃): 50,50.5,51.2,52.1,53.5,54.7,55.8,56.9,58.1,59.1.
Fig. 6. primer
Vcc-up, downAnnealing temperature gradient experimental result electrophoresis result figure, annotate: 1 ~ 10 road from be respectively from left to right annealing temperature (℃): 50,50.5,51.2,52.1,53.5,54.7,55.8,56.9,58.1,59.1.
Fig. 7. primer
InvA-up, down, to the amplification electrophoresis result figure of several bacterium, annotate: 1 negative contrast, 2 ~ 7 roads are respectively the different strains genomic templates: 2. Salmonella typhimurium, 3. Salmonella paratyphi A, 4. Listeria Monocytogenes, 5. Vibrio parahemolyticus, 6. shigella flexneri, 7. non-
O1Group cholera vibrio.
Fig. 8. primer
InvA-up, down, to the amplification electrophoresis result figure of six kinds of bacterium, annotate: 1 negative contrast, 2 ~ 7 roads are respectively the different strains genomic templates: 2. Salmonella paratyphi A, 3. Salmonella typhimurium, 4. Listeria Monocytogenes, 5. Vibrio parahemolyticus, 6. shigella flexneri, 7. non-
O1Group cholera vibrio.
Fig. 9. primer
Hly-up, down, to the amplification electrophoresis result figure of six kinds of bacterium, annotate: 1 negative contrast, 2 ~ 7 roads are respectively the different strains genomic templates: 2. Listeria Monocytogenes, 3. Salmonella typhimurium, 4. Salmonella paratyphi A, 5. Vibrio parahemolyticus, 6. shigella flexneri, 7. non-
O1Group cholera vibrio.
Figure 10. primer
ToxR-up, down, to the amplification electrophorogram of six kinds of bacterium, annotate: 1 negative contrast, 2 ~ 7 roads are respectively the different strains genomic templates: 2. Vibrio parahemolyticus, 3. Salmonella typhimurium, 4. Salmonella paratyphi A, 5. Listeria Monocytogenes, 6. shigella flexneri, 7. non-
O1Group cholera vibrio.
Figure 11. primer
IpaH-up, down, to the amplification electrophorogram of six kinds of bacterium, annotate: 1 negative contrast, 2 ~ 7 roads are respectively the different strains genomic templates: 2. shigella flexneri, 3. Salmonella typhimurium, 4. Salmonella paratyphi A, 5. Listeria Monocytogenes, 6. Vibrio parahemolyticus, 7. non-
O1Group cholera vibrio.
Figure 12. primer
Vcc-up, down, to the amplification of six kinds of bacterium, annotate: 1 negative contrast, 2 ~ 7 roads are respectively the different strains genomic templates: 2. non-O1 group cholera vibrio, 3. Salmonella typhimurium, 4. Salmonella paratyphi A, 5. Listeria Monocytogenes, 6. Vibrio parahemolyticus, 7. shigella flexneri.
Figure 13. Salmonella typhimurium sensitivity detected result, 1 ~ 8 road is respectively 10
8, 10
7, 10
6, 10
5, 10
4, 10
3, 10
2, 10
1Cfu/mL.
Figure 14. Salmonella paratyphi A sensitivity detected result, 1 ~ 8 road is respectively 10
8, 10
7, 10
6, 10
5, 10
4, 10
3, 10
2, 10
1Cfu/mL.
Figure 15. Listeria Monocytogenes sensitivity detected result, 1 ~ 8 road is respectively 10
8, 10
7, 10
6, 10
5, 10
4, 10
3, 10
2, 10
1Cfu/mL.
Figure 16. Vibrio parahemolyticus sensitivity detected result, 1 ~ 8 road is respectively 10
8, 10
7, 10
6, 10
5, 10
4, 10
3, 10
2, 10
1Cfu/mL.
Figure 17. shigella flexneri sensitivity detected result, 1 ~ 8 road is respectively 10
8, 10
7, 10
6, 10
5, 10
4, 10
3, 10
2, 10
1Cfu/mL.
Figure 18. non-O1 group cholera vibrio sensitivity detected result, 1 ~ 8 road is respectively 10
8, 10
7, 10
6, 10
5, 10
4, 10
3, 10
2, 10
1Cfu/mL.
Figure 19. double PCR electrophoresis detection result, 1,2 amplification
InvAWith
hlyGene, 3,4 amplifications
InvAWith
ToxRGene, 5,6 amplifications
InvAWith
IpaHGene, 7,8 amplifications
InvAWith
vccGene.
Figure 20. double PCR electrophoresis detection result, 1,2 amplification
InvAWith
hlyGene, 3,4 amplifications
InvAWith
ToxRGene, 5,6 amplifications
InvAWith
IpaHGene, 7,8 amplifications
InvAWith
vccGene.
Figure 21. double PCR electrophoresis detection result, 1,2 amplification
hlyWith
ToxRGene, 3,4 amplifications
hlyWith
IpaHGene, 5,6 amplifications
hlyWith
vccGene.
Figure 22. double PCR electrophoresis detection result, 1,2 amplification
ToxRWith
IpaHGene, 3,4 amplifications
ToxRWith
vccGene, 5,6 amplifications
IpaHWith
vccGene.
Figure 23. double PCR electrophoresis detection result, 1 amplification
InvAWith
ToxRGene, 2 amplifications
InvAWith
IpaHGene, 3 amplifications
InvAWith
vccGene, 4 amplifications
hlyWith
ToxRGene, 5 amplifications
hlyWith
ToxRGene, 6 amplifications
hlyWith
ToxRGene.
Figure 24. double PCR electrophoresis detection result, 1 amplification
InvAWith
ToxRGene, 2 amplifications
InvAWith
IpaHGene, 3 amplifications
InvAWith
vccGene, 4 amplifications
hlyWith
ToxRGene, 5 amplifications
hlyWith
ToxRGene, 6 amplifications
hlyWith
ToxRGene.
Figure 25. triple PCR electrophoresis detection result, 1,2 amplification
InvA, ipaHWith
vccGene, 3,4 amplifications
InvA, vccWith
ToxRGene, 5,6 amplifications
InvA, toxRWith
IpaHGene, 7,8 amplifications
InvA, hlyWith
vccGene, 9,10 amplifications
InvA, ipaHWith
hlyGene, 11,12 amplifications
InvA, hlyWith
ToxRGene.
Figure 26. triple PCR electrophoresis detection result, 1,2 amplification
InvA, hlyWith
ToxRGene, 3,4 amplifications
InvA, ipaHWith
hlyGene, 5,6 amplifications
InvA, hlyWith
vccGene, 7,8 amplifications
InvA, toxRWith
IpaHGene, 9,10 amplifications
InvA, vccWith
ToxRGene, 11,12 amplifications
InvA, ipaHWith
vccGene.
Figure 27. triple PCR electrophoresis detection result, 1,2 amplification
Hly, ipaHWith
ToxRGene, 3,4 amplifications
Hly, toxRWith
vccGene, 5,6 amplifications
Hly, ipaHWith
vccGene, 7,8 amplifications
Vcc, toxRWith
IpaHGene.
Figure 28. Quadruple-PCR electrophoresis detection result, 1,2 amplification
InvA, hly, ipaHWith
ToxRGene, 3,4 amplifications
Hly, toxR, ipaHWith
vccGene.
Figure 29. Quadruple-PCR electrophoresis detection result, 1,2,3 amplifications
Hly, toxR, ipaHWith
vccGene.
Figure 30. five heavy PCR electrophoresis detection results, 1,2,3,4 amplifications
InvA, hly, toxR, ipaHWith
vccGene.
Figure 31. five heavy PCR electrophoresis detection results, 1,2,3, amplification
InvA, hly, toxR, ipaHWith
vccGene.
Figure 32. five heavy PCR electrophoresis detection results, 1,2,3,4,5,6 amplifications
InvA, hly, toxR, ipaHWith
vccGene, 1,2 amplification
InvA, hlyThe gene primer amount is
ToxR, ipaHWith
vcc2 times of genes, 3,4 amplifications
InvA, hlyThe gene primer amount is
ToxR, ipaHWith
vcc3 times of genes, 5,6 amplifications
InvA, hlyGene primer and template amount are
ToxR, ipaHWith
vcc2 times of genes.
Embodiment
If no special instructions, be this area normal experiment reagent and routine operation step in following examples.
Design and the screening of 1 five kinds of pathogenic microbes detect primers of embodiment
1.1 experiment material
Main tool enzyme, biochemical reagents and test kit:
The reagent such as LB meat soup, various biochemical tube, Li Shi enrichment liquid are available from the Guangzhou triumphant biological reagent of ring company limited.
Taq DNA polysaccharase (Fermentas), N,O-Diacetylmuramidase, bacterial genomes DNA extracts test kit (day root), plain agar sugar gel, DNA reclaims test kit (day root), and pGM-T19 connects and contains test kit (containing carrier, ligase enzyme) available from precious Tyke, Guangzhou bio tech ltd, plasmid DNA is extracted test kit (day root), EB substituted dyes (Beijing Puli's Lay) in a small amount; The consumptive materials such as Tip and Tip box, centrifuge tube, glass test tube, plate, triangular flask are available from Guangzhou rhythm luxuriant growth test apparatus company; Other reagent are the conventional reagent of analytical pure.
The LB solid medium: additional proportion is the agar powder of 15g L-1 on the LB Mycoplasma Broth Base, mix boil the fusing sterilizing.
Nutrient broth NB substratum (1L): extractum carnis 3g, peptone 10g, NaCl 5g, pH7.4.
Experimental strain source and numbering are in Table 1
Table 1 experimental strain and source
1.2 design of primers
Selecting of PCR amplification bacterium specific target gene: Salmonellas inv gene cluster-coding absorption and invasion and attack surface epithelial cell protein gene
InvA, the hemolysin O gene of Listeria Monocytogenes
hly, Vibrio parahemolyticus
ToxRGene, shigella flexneri aggressive plasmid antigen H gene
IpaH7, non-
O1Group cholera vibrio
vccGene.
, in NCBI retrieved web corresponding gene sequences,, with Primer premier 5.0 software design multi-primerses, see table 2 for details.Primer is synthetic by Beijing AudioCodes biotechnology company limited, and the primer after synthesizing is diluted to 10 mmol/mL solution with ultrapure water ,-20 ℃ of preservations.Primer sequence is as follows:
Table 2 primer and relevant information
1.3
InvA, hly, toxR, ipaH and vccThe substance PCR amplification of gene
The PCR reaction composition is as follows:
2×PCRmix 12.5μL
Upstream primer (10 μ mol/L) 0.4 μ L
Downstream primer (10 μ mol/L) 0.4 μ L
DNA template 1.0 μ L
Ultrapure water 10.7 μ L
Add successively said components in the PCR pipe, the total reaction system is 25 μ L, carries out amplified reaction on the PCR instrument.
Test between the thermograde of 10 ℃ of synthetic primer annealing medial temperature up and down, the reaction heat loop parameter is: 94 ℃ of denaturation 5 min, and 94 ℃ of sex change 30 s, 56 ℃ of annealing 30s, 72 ℃ are extended 45s, totally 28 circulations, last 72 ℃ are extended 10min.After amplification finishes, PCR product-20 ℃ preservation.The agarose gel electrophoresis of PCR product detects.
1.4 the suitableeest annealing temperature of primer
Respectively with the experimental strain Salmonella typhimurium, Salmonella paratyphi A, Listeria Monocytogenes, Vibrio parahemolyticus, shigella flexneri, non-
O1(it is 10 that colony counting method is measured bacteria containing amount to the fresh medium of group cholera vibrio
8Cfu/mL) 2mL, the extraction genomic dna is template, with in table 2, each carries out the PCR reaction to primer, it is 50,50.5,51.2,52.1 that annealing temperature is set gradient, 53.5,54.7,55.8,56.9,58.1,59.1 ℃.
Result shows: Fig. 1, and Fig. 2, Fig. 3, Fig. 4, Fig. 5 annealing region is 50 ~ 59.1 ℃, Fig. 6 annealing region is 50 ~ 58.1 ℃.The common annealing temperature of several goal gene is 56 ℃.
1.5 primer specificity detects
Respectively with the experimental strain Salmonella typhimurium, Salmonella paratyphi A, Listeria Monocytogenes, Vibrio parahemolyticus, shigella flexneri, non-
O1(it is 10 that colony counting method is measured bacteria containing amount to the fresh medium of group cholera vibrio
8Cfu/mL) 2mL, the extraction genomic dna is template, with in table 2, each carries out the PCR reaction to primer, PCR system and thermal circulation parameters are with 1.3.Amplification finishes rear electrophoresis, and result is with the gel imaging system analysis preservation of taking pictures.
Result shows: Fig. 7, and Fig. 8, Fig. 9, Figure 10, Figure 11, Figure 12 only cause the corresponding target genes specific amplification,, without any non-specific amplification, have shown good specificity.
5 pairs of primers that specificity is good have been determined in this experiment thus
InvA-up, down, hly-up, down, toxR-up, down, ipaH-up, down, vcc-up, downBe used for subsequent experimental.
1.6 sensitivity detects
Get respectively the experimental bacteria Salmonella typhimurium, Salmonella paratyphi A, Listeria Monocytogenes, Vibrio parahemolyticus, shigella flexneri, (it is 10 that colony counting method is measured bacteria containing amount to the fresh medium of non-O1 group cholera vibrio
8Cfu/mL) 2mL extracts genomic dna with test kit and does template, uses ddH
2Ten times of gradient dilutions of O, produce 10
8The template of cfu/mL concentration, carry out the PCR reaction with corresponding primer separately, and PCR system and thermal circulation parameters are with 1.3.Amplification finishes rear electrophoresis and detects positive findings, and the minimum concentration that detects bacterium liquid is the sensitivity of PCR reaction.
The process that in experimental implementation, all of not specified (NS) relate to viable bacteria inoculation, DNA operation is all undertaken by the aseptic technique rules in Bechtop, abandon after the equal autoclaving harmless treatment of all band bacteria liquids.The operation that relates to electrophoresis EB pollution is strictly undertaken by the laboratory room managing rules, and gurry is unified collection and treatment.
Result shows: by Figure 13 to Figure 18 as can be known, and primer
InvA-up, downThe PCR reaction that causes, can make Salmonella typhimurium, and the Salmonella paratyphi A detection sensitivity reaches respectively 10
3Cfu/mL and 10
4Cfu/mL; Listeria Monocytogenes and shigella flexneri detection sensitivity all reach 10
3Cfu/mL; Vibrio parahemolyticus and non-
O1The group cholera vibrio detection sensitivity all reaches 10
4Cfu/mL.
In this research, five kinds of bacterium substance PCR detection sensitivities are 10
3Cfu/mL ~ 10
4Cfu/mL, highly sensitive.
The foundation of five kinds of pathogenic bacteria five heavy PCR detection methods in embodiment 2 fishery products
In test, material used, reagent are equal to embodiment 1.
1 multiplex PCR amplification
First from double PCR on-test, combination of two, introduce the template of the 3rd group and primer, template and the primer of the 4th group again after the system stable testing.
InvA, hly, toxR, ipaH and vccThe double PCR amplification PCR reaction composition of gene is as follows:
2×PCRmix 12.5μL
Upstream primer (10 μ mol/L) 0.4 μ L, (2 ~ 4 μ L) altogether
Downstream primer (10 μ mol/L) 0.4 μ L, (2 ~ 4 μ L) altogether
(annotate: primer
InvA-up, downWith
hly-
Up, downRespectively add 1 μ L, primer
ToxR-
Up, down,
IpaH-
Up, downWith
vcc-
Up, downRespectively add 0.4 μ L)
DNA template 1.0 μ L
Template is the experimental bacteria Salmonella typhimurium, Salmonella paratyphi A, Listeria Monocytogenes, Vibrio parahemolyticus, shigella flexneri, 10 of non-O1 group cholera vibrio
8The genomic dna that cfu/mL concentration nutrient solution 3mL test kit method is extracted.Add successively said components in the PCR pipe, add dd H
2O to the total reaction system be 25 μ L, carry out amplified reaction on the PCR instrument.
The reaction heat loop parameter is: 94 ℃ of denaturation 5 min, and 94 ℃ of sex change 30 s, 56 ℃ of annealing 30s, 72 ℃ are extended 45s, totally 28 circulations, last 72 ℃ are extended 10min.After amplification finishes, PCR product-20 ℃ preservation.
Triple PCR is added one group of DNA template (1 μ L) and upstream and downstream primer (each 0.4 μ L, primer
InvA-up, downWith
hly-
Up, downNeed respectively add 1 μ L), all the other components are constant, add ddH
2O to the total reaction system be 50 μ L.
Quadruple-PCR is four groups of templates (each 1 μ L) and corresponding upstream and downstream primer (each 0.4 μ L, primer
InvA-up, downWith
hly-
Up, downNeed respectively add 1 μ L), all the other components are constant, add dd H
2O to the total reaction system be 50 μ L.The reaction heat loop parameter is constant.After amplification finishes, PCR product electrophoresis detection ,-20 ℃ of preservations.
Five heavy PCR are five groups of templates (each 1 μ L) and corresponding upstream and downstream primer (each 0.4 μ L, primer
InvA-up, downWith
hly-
Up, downNeed respectively add 1 μ L), all the other components are constant, add dd H
2O to the total reaction system be 50 μ L.The reaction heat loop parameter is constant.After amplification finishes, PCR product electrophoresis detection ,-20 ℃ of preservations.
2 results and analysis
2.1 double pcr amplification result
The experimental bacteria Salmonella typhimurium, Salmonella paratyphi A, Listeria Monocytogenes, Vibrio parahemolyticus, shigella flexneri, the genomic templates of non-O1 group cholera vibrio are incorporated into the performing PCR amplification in twos, and amplification system and loop parameter are the same, the results are shown in Figure 19, Figure 20, Figure 21, Figure 22, wherein Figure 19 is that Salmonella typhimurium DNA is the experimental result of template, and Figure 20 is that Salmonella paratyphi A DNA is the experimental result of template.
For goal gene that to inquire at same PC body be different fragments length in amplification procedure, each primer, the impact of template amount difference on amplified production concentration, add
InvAWith
hlyGene primer and template amount are respectively 2 times and 3 times of other gene dosages, the results are shown in Figure 23, Figure 24, and wherein Figure 23 is 2 times of primers and template amount result, Figure 24 is 3 times of primers and template amount result.
Result shows: different primers template combination of two in Figure 19, can individual features amplify goal gene fragment separately, and
InvAWith
IpaHOther purpose bands are bright relatively for the band that gene amplification goes out.
Different primers template combination of two in Figure 20, can individual features amplify goal gene fragment separately, 7,8 amplifications
InvAWith
vccOther purpose bands are bright relatively for the band that gene amplification goes out.
Different primers template combination of two in Figure 21, can individual features amplify goal gene fragment separately, and each band brightness is much the same.
Different primers template combination of two in Figure 22, can individual features amplify goal gene fragment separately, and each band brightness is much the same.
Different primers template combination of two in Figure 23, can individual features amplify pigeon goal gene fragment, and should scheme each purpose band than Figure 19, Figure 20, and Figure 21, Figure 22, corresponding band is all bright.
Different primers template combination of two in Figure 24, can individual features amplify pigeon goal gene fragment, and this Fig. 1 amplification
InvAWith
ToxRGene, 2 amplifications
InvAWith
IpaHGene, 3 amplifications
InvAWith
vccGene purpose band is than Figure 19, Figure 20, and Figure 21, Figure 22, the corresponding band of Figure 23 is all bright.
2.2 triple PCR amplification
The experimental bacteria Salmonella typhimurium, Salmonella paratyphi A, Listeria Monocytogenes, Vibrio parahemolyticus, shigella flexneri, in the genomic templates of non-O1 group cholera vibrio, three kinds are incorporated into the performing PCR amplification, and amplification system and loop parameter are the same, the results are shown in Figure 25, Figure 26, Figure 27, wherein Figure 25 is that Salmonella typhimurium DNA is the experimental result of template, Figure 26 is that Salmonella paratyphi A DNA is the experimental result of template.
Result shows: Figure 25, and Figure 26, in Figure 27, specificity purpose band separately all appears in the combination of three pairs of primer templates, and band is clear.
2.3 Quadruple-PCR amplification
The experimental bacteria Salmonella paratyphi A, Listeria Monocytogenes, Vibrio parahemolyticus, shigella flexneri, in the genomic templates of non-O1 group cholera vibrio, four kinds are incorporated into the performing PCR amplification, and amplification system and loop parameter are the same, the results are shown in Figure 28, Figure 29, wherein Figure 28 is that Salmonella paratyphi A DNA is the experimental result of template.
Result shows: Figure 28, in Figure 29, specificity purpose band separately appears in the combination office of the four pairs of primers and template, and band is clear, wherein amplification
IpaHThe purpose band of gene is the brightest in the band of four entries of same swimming lane.
2.4 five heavy pcr amplification results
The experimental bacteria Salmonella typhimurium, Salmonella paratyphi A, Listeria Monocytogenes, Vibrio parahemolyticus, shigella flexneri, in the genomic templates of non-O1 group cholera vibrio, four kinds are incorporated into the performing PCR amplification, amplification system and loop parameter are the same, the results are shown in Figure 30, Figure 31, wherein Figure 30 is that Salmonella typhimurium DNA is the experimental result of template, and Figure 31 is that Salmonella paratyphi A DNA is the experimental result of template.
For goal gene that to inquire at same PC body be different fragments length in amplification procedure, each primer, the impact of template amount difference on amplified production concentration, add
InvAWith
hlyGene primer and template amount are respectively 3 times of other gene dosages, the results are shown in Figure 32.
Result shows: in Figure 30, specificity purpose band separately appears in the combination office of the five pairs of primers and template, and band is clear, wherein amplification
IpaHThe purpose band of gene is the brightest in the band of four entries of same swimming lane.
In Figure 31, specificity purpose band separately appears in the combination office of the five pairs of primers and template, and band is clear, wherein amplification
IpaHThe purpose band of gene is the brightest in the band of four entries of same swimming lane.
Specificity purpose band separately appears in the combination office of five pairs of primers of Figure 32 and template, and band is clear, 3,4 amplifications
InvA, hlyThe gene primer amount is
ToxR, ipaH With
vccIn 3 times of genes each purpose band all relatively other two kinds bright.
The five kinds of heavy PCR detection method check of pathogenic bacteria five artificial challenge samples in embodiment 3 fishery products
In test, material used, reagent are equal to embodiment 1; Five re-detection reaction systems and thermal circulation parameters are with embodiment 2.
In order to verify the validity of five heavy PCR detection methods, this enforcement detects respectively 21 parts and uses Salmonella typhimurium, Salmonella paratyphi A, Listeria Monocytogenes, Vibrio parahemolyticus, shigella flexneri, non-O1 group cholera vibrio infects fresh sample.Infected sample shaking culture 24 hours under 37 ℃ of temperature condition.
Result shows: in table 3, the sample positive rate of strains separation method is 100%(21/21), and five heavy PCR detection method sample positive rates are also 100%(21/21), two kinds of method detected results meet fully.
Table 3 compares with the sample result that strains separation and two kinds of methods of five heavy PCR detections detect respectively five kinds of pathogenic bacterial infections
S.S.The expression Salmonellas
, M.P.L. expression monokaryon hyperplasia listeria bacteria,
V.P.The expression Vibrio parahemolyticus,
S.F.The expression shigella flexneri
V.C.Representing non-O1 group cholera vibrio, positive findings is expressed as+, negative findings is expressed as-.
SEQUENCE LISTING
＜110〉Agricultural University Of South China
＜120〉detection that detects simultaneously various pathogens is with primer and detection method
<130>
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
＜213〉artificial sequence
<400> 1
catcgacaga cgtaaggagg 20
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<211> 20
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＜213〉artificial sequence
<400> 2
catgaagagg gggagaaact 20
<210> 3
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<212> DNA
＜213〉artificial sequence
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ttatgatgac gaaatggctt a 21
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<211> 20
<212> DNA
＜213〉artificial sequence
<400> 4
gcaaatagat ggacgatgtg 20
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<212> DNA
＜213〉artificial sequence
<400> 5
caaggttttg aggtggat 18
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<212> DNA
＜213〉artificial sequence
<400> 6
cagaggcgtc attgttat 18
<210> 7
<211> 17
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＜213〉artificial sequence
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tgaggcggta agaagac 17
<210> 8
<211> 18
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＜213〉artificial sequence
<400> 8
aggcagagat ggaagagt 18
<210> 9
<211> 18
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＜213〉artificial sequence
<400> 9
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Claims (2)
1. detect simultaneously the primer of multiple fishery products pathogenic bacterium, it is characterized in that nucleotide sequence is as follows:
InvAPrimer pair: SEQ ID NO:1 ~ 2;
hlyPrimer pair: SEQ ID NO:3 ~ 4;
ToxRPrimer pair: SEQ ID NO:5 ~ 6;
IpaHPrimer pair: SEQ ID NO:7 ~ 8;
vccPrimer pair: SEQ ID NO:9 ~ 10;
Above any combination of primers more than two pairs, can detect the corresponding pathogenic bacterium of this primer pair;
Wherein
InvAPrimer pair is for detection of Salmonella typhimurium or Salmonella paratyphi A,
hlyPrimer pair is for detection of Listeria Monocytogenes,
ToxRPrimer pair is for detection of Vibrio parahemolyticus,
IpaHPrimer pair is for detection of shigella flexneri,
vccPrimer pair is for detection of non-
O1Group cholera vibrio.
2. the described application of primer in preparing agricultural byproducts pathogenic microbes detect reagent that detects simultaneously various pathogens of claim 1.
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| CN104975086A (en) * | 2015-06-15 | 2015-10-14 | 浙江省海洋开发研究院 | Fast detection kit of aquatic product bacterial diseases, and application thereof |
| CN105950756B (en) * | 2016-06-20 | 2019-09-20 | 山西农业大学 | The multiple PCR identification method of bafillus natto and lactobacillus acidophilus fusant |
| CN108424976A (en) * | 2018-02-08 | 2018-08-21 | 杭州富集生物科技有限公司 | Happen suddenly acute and severe infectious disease Emergent detection deposit kit and the method for inspection |
| CN113957162A (en) * | 2021-09-01 | 2022-01-21 | 上海海关动植物与食品检验检疫技术中心 | Primer probe combination, kit and method for detecting salmonella based on fluorescence RAA technology |
| CN114277170B (en) * | 2022-02-17 | 2024-02-13 | 重庆市计量质量检测研究院 | Primer and kit for detecting nine food-borne pathogenic bacteria through multiplex PCR (polymerase chain reaction) |
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