CN103981275A - Multi-PCR (Polymerase Chain Reaction) primers used for simultaneously detecting four types of pathogenic bacteria in ocean and design method thereof - Google Patents
Multi-PCR (Polymerase Chain Reaction) primers used for simultaneously detecting four types of pathogenic bacteria in ocean and design method thereof Download PDFInfo
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Abstract
The invention relates to multi-PCR (Polymerase Chain Reaction) primers used for simultaneously detecting four types of pathogenic bacteria in an ocean and a design method thereof. The design method comprises the following steps: designing a multi-PCR primer combination for simultaneously detecting enterobacter cloacae, listeria monocytogenes, aeromonas hydrophila and vibrio parahaemolyticus by utilizing primer design software; screening primers superior in competitive states and performing amplification validation on the primers superior in competitive states by utilizing a PCR method; and finally, removing the primers weak in competitive states from the amplification primers, thereby obtaining the multi-PCR primer combination. In addition, the invention further discloses a multi-PCR detecting method. Compared with the prior art, the method for establishing the multi-PCR primers for simultaneously detecting the enterobacter cloacae, the listeria monocytogenes, the aeromonas hydrophila and the vibrio parahaemolyticus, which is provided by the invention, has the advantages of being simple, convenient and rapid to operate, strong in specificity, high in sensitivity and the like, i.e., the four types of bacteria can be simultaneously detected by virtue of one PCR reaction.
Description
Technical field
The invention belongs to microorganism detection field, be specifically related to a kind of for detect multiple PCR primer and the method for design thereof of ocean enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus simultaneously.
Background technology
Enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus, belong to the pathogenic bacteria that can propagate by food, water.Their biological characteristics and pathogenicly have certain difference, but can cause food poisoning and digestive system.
Enterobacter cloacae belongs to conditioned pathogen, can not cause pathogenicly under general condition, but also can cause the symptoms such as septicemia, lower respiratory infection, wound infection, soft tissue infection, endocarditis, urinary tract infection.Enterobacter cloacae can cause that the mankind pay attention to, and is mainly that resistance situation is serious, and possesses multiple other resistance mechanisms because it can produce extended spectrumβ-lactamase, AmpC enzyme, and to clinical antibiotics, treatment brings new challenge.Enterobacter cloacae resistance mechanism mainly comprises and produces β-lactamase, qnr gene mediated resistance, integron, the initiatively change, outer membrane permeability change etc. of extra-pumping system effect, drug effect target position.These complicated resistance mechanisms exist, and for the antibiotic therapy of other bacterium mixed infections, cause invalid or curative effect is very micro-.Enterobacter cloacae is extensively present in occurring in nature, and the seafood poisoning being caused by it occasionally has generation, but the propagation in hospital is only second to intestinal bacteria and klebsiella.
Monocytogenes hyperplasia Listeria is a kind of pathogenic bacteria of important zoonosis.It is septicemia, meningitis and monocytosis etc. that people infects rear main manifestations.This bacterium is extensively present in occurring in nature, and the monocytogenes hyperplasia Listeria existing in food has danger to the mankind's safety, still can growth and breeding in the environment of 4 ℃, and be one of refrigerated food the main pathogenic fungi of threatening human health.In clinical disease and food pollution problem American-European, that Japan is caused by this bacterium, surpass and in bacterial food poisoning, account for the Salmonellas of the 1st.The many ground of China also has this bacterium diseases induced and be in hospital and dead report.Because this bacterium is a kind of intracellular parasitic bacteria, antibiotic therapy can only play a role within for some time, finds that at present its resistance presents loose shape and distributes, and some antibiotic resistances are constantly strengthened.
Aeromonas hydrophila is distributed widely in natural various water body, is the primary pathogenic bacterium of multiple hydrocoles, for people, is conditioned pathogen, is typical people-beast-fish ill pathogenic bacteria altogether.Aeromonas hydrophila can produce the extracellular toxin that toxicity is very strong, as hemolysin, histotoxin, necrotoxin, enterotoxin and proteolytic enzyme etc., mainly causes that enteron aisle sexuality dyes.Infected by Aeromonas hydrophila people's the rare record of case, once but outbreak, mortality ratio is very high, and curative ratio is lower, and other bacterium of Chang Huiyu (as gentle aerogenesis Zymomonas mobilis, vibrios etc.) polyinfection aggravates disease.In China, also there is the food poisoning repeatedly being caused by Aeromonas hydrophila.
Vibrio parahaemolyticus all has stronger virulence to humans and animals, and its morbid substance mainly contains thermotolerance hemolysin (TDH) toxin relevant with heat-resisting haemolysis (TRH), and these toxin have hemolytic activity, intestines toxicity and lethal effect.This bacterium is because of containing growing in the substratum of salt denseer (more than 3%-4%), and irreproducible in salt-free substratum, so claim again halophilic bacterium.Vibrio parahaemolyticus all has distribution all over the world, be conventionally present in region, shoreline and sea-food, and people Duo Yin is edible pollutes this bacterium again without the sea-food of well processed, as crab class, oyster, shrimp etc., causes food poisoning.The food poisoning that Vibrio parahaemolyticus causes, in the bacterial food poisoning of coastland, proportion is higher, is one of topmost food poisoning bacterium in coastal region.The bacterial food poisoning harm that it causes is only second to Salmonellas, intestinal bacteria, staphylococcus and Clostridium botulinum.China's Coastal Areas has every year in a large number by Vibrio parahemolyticus and causes food poisoning report.
Polymerase chain reaction (Polymerase Chain Reaction, be called for short PCR) be the method for external rapid amplifying DNA a kind of, be used for amplifying specific DNA fragmentation, in a few hours, can make goal gene fragment amplification to the Protocols in Molecular Biology of millions of copies.The specificity of round pcr depends on the Oligonucleolide primers with the complementation of target sequence two ends.PCR consists of sex change, annealing (renaturation), three primitive reaction steps of extension: the 1. sex change of template DNA: template DNA is through being heated to 93 ℃ of left and right after for some time, template DNA double-stranded DNA double-stranded or that form through pcr amplification is dissociated, make it to become strand, so that it is combined with primer, for lower whorl reaction is prepared; 2. the annealing (renaturation) of template DNA and primer: template DNA is after heat denatured becomes strand, and temperature is down to 55 ℃ of left and right, the complementary sequence pairing combination of primer and template DNA strand; 3. the extension of primer: the binding substances of DNA profiling and primer is at 72 ℃, under the effect of Taq archaeal dna polymerase, take dNTP as raw material, target sequence is template, press base complementrity pairing and semiconservative replication principle, synthetic new and the semiconservative replication chain complementation of template DNA chain.Recirculation sex change, annealing (renaturation), three processes of extension just can obtain more " semiconservative replication chain ", and this new chain can become again the template of circulation next time.Multiplex PCR is transformed on the basis of regular-PCR, in a PCR reaction system, adds multipair Auele Specific Primer simultaneously, for the increase round pcr of a plurality of object fragments of the different zones of a plurality of DNA profilings or same template.Can simultaneously increase in primary first-order equation a plurality of target sequences of a plurality of object fragments of multiplex PCR.When this technology can be used for multiple pathogenic microorganisms, detect or identify, in same PCR reaction tubes, adding the Auele Specific Primer of multiple pathogenic microorganisms simultaneously, carrying out pcr amplification.The problem of multiplex PCR maximum is the design of primer and the optimization of reaction conditions.Compare with general PCR, multiplex PCR needs to amplify the object fragment of a plurality of pathogenic bacterias in same PCR reaction tubes simultaneously, once completes the amplification of a plurality of templates.In the present invention, detect enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus in same reaction tubes, will greatly save time and reagent, reduction of expenditure, for more information more accurately that provides is provided.The design of primers of multiplex PCR requires consistent with the primer of general PCR generally, as primer self need to have stable secondary structure, between primer, can not form hair clip and primer dimer etc.This is because list can amplify band object fragment clearly to the single template of primer amplification, but the mixed rear amplification of various primers has the appearance of band Loss.Between each primer, also have competitive relation, surging primer may be covered weak tendency primer, causes object fragment loss.Even between primer, interact and produce serious primer dimer, cause object fragment loss.
Multiplex PCR is with its feature quick, efficient, specificity is good, highly sensitive, in the detections such as food pathogenic micro-organism, non-pathogenic microorganism and environmental microorganism, there is vital role, the needs that meet the part rapid detection such as port, health, quality inspection, be a kind of accurately, the molecular biology method of reliable, science.In view of this, develop as early as possible a kind of imperative for detect the multiplex PCR of enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus simultaneously.This is for the effect of strengthening the quick test quarantine of these pathogenic bacterium in fishery products, the aspects such as the investigation of aquiculture disease and control, nuisanceless fishery products detection and production and fishery products hygienic quality supervision and inspection all have prompting and warn.
Summary of the invention
A technical problem to be solved by this invention is that the present situation for prior art provides a kind of for detect multiple PCR primer and the method for design thereof of the four kinds of pathogenic bacterium in ocean simultaneously.
Another technical problem to be solved by this invention is that the present situation for prior art provides a kind of for detect the multi-PCR detection method of the four kinds of pathogenic bacterium in ocean simultaneously, and it has feature simple, sensitive, quick, high specificity.
The present invention solves the problems of the technologies described above adopted technical scheme to be: should, for detect the multiple PCR primer of the four kinds of pathogenic bacterium in ocean simultaneously, it is characterized in that: the multiple PCR primer of these four kinds of pathogenic bacterium comprises respectively the multiple PCR primer of enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus;
Wherein, the upstream and downstream primer of enterobacter cloacae is:
P1：5’-AAGCGHCCNGSNATGTAYATHGG-3’；
P2：5’-CCNGCNGARTCNCCYTCNAC-3’；
The upstream and downstream primer of monocytogenes hyperplasia Listeria is:
P3：5’-TTCCGCAGAGGACAGTGATG-3’；
P4：5’-CATATTCCACTTTAACCGTG-3’；
The upstream and downstream primer of Aeromonas hydrophila is:
P5：5’-AAAGCGGATTATGCAGAAGCACTG-3’；
P6：5’-GCTACTTTCTAGCATTTTCTCTGC-3’；
The upstream and downstream primer of Vibrio parahaemolyticus is:
P7：5’-TGAGTTGCTGTTGTTGGATGC-3’；
P8：5’-GTTGATGACACTGCCAGATGC-3’；
Further, for detect the multiple PCR primer of the four kinds of pathogenic bacterium in ocean simultaneously, it is characterized in that: the pcr amplified fragment size that described enterobacter cloacae primer pair is answered is 1250bp, the pcr amplified fragment size that described monocytogenes hyperplasia Listeria primer pair is answered is 768bp and 272bp, the pcr amplified fragment size that described Aeromonas hydrophila primer pair is answered is 483bp, and the pcr amplified fragment size that described Vibrio parahaemolyticus primer pair is answered is 454bp.
In the present invention, for detect the method for design of the multiple PCR primer of the four kinds of pathogenic bacterium in ocean simultaneously, it is characterized in that: comprise the following steps:
Step 1, utilize primer-design software design to detect the multiple PCR primer combination of enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus simultaneously, the primer base number that formed combination of primers comprises is 20-24, the melting temperature (Tm) Tm value of primer is 55 ℃-65 ℃, and the GC% of primer is 40%-60%;
Step 2, screens the primer in combination of primers, retains the primer that does not form primer dimer;
Step 3, the good and bad state of competition of primer that judgement retains, the GC% of above-mentioned retained primer and base number are compared, when the base number of primer all GC% identical and inner side primer be less than the GC% of outside primer, or when the base number of primer is incomplete same and the base number of inner side primer during than the few base of outside primer, be judged as race condition inferior position primer, carry out step 4; Otherwise be judged as race condition advantage primer, carry out step 5;
Step 4, screens race condition primer again, and concrete steps are: repeating step 2, judges according to step 3 again to retained primer;
Step 5, utilizes PCR method to the checking of increasing of race condition advantage primer;
Step 6, removes the race condition inferior position primer in amplimer, obtains multiple PCR primer combination.
In the present invention, for detect the multi-PCR detection method of the multiple PCR primer of the four kinds of pathogenic bacterium in ocean simultaneously, it is characterized in that comprising the steps:
1) initial gross separation of bacterium: extract genomic dna from substratum, the standby pcr template of doing;
2) multiplex PCR amplification comprises: a, multiplex PCR amplification reaction system: get above-mentioned DNA profiling solution 38-60ng/ μ L, with above-mentioned test kit, detect, reaction system is 20-30.0 μ L, each reaction product is respectively TaqDNA polysaccharase 0.02U/ μ L, PCR damping fluid 2.5 μ L, deoxyribonucleoside triphosphate mixture 10mmol/L, each 0.5-2 μ L of primer P1, P2, each 0.75-1.5 μ L of P3, P4, each 0.75-1.5 μ L of P5, P6, each 0.25-1.0 μ L of P7, P8, surplus is supplied with redistilled water; B, amplification reaction condition: 94 ℃ of denaturation 4min, 94 ℃ of sex change 30s, 56 ℃ of annealing 45s, 72 ℃ are extended 40s, circulate 30 times, and 10min is extended at 72 ℃ of whole ends; C, the amplified production of step b is carried out to electrophoresis detection and interpretation of result.
As preferably, each 2 μ L of primer P1, P2 in described step a, each 0.75 μ L of P3, P4, each 0.75 μ L of P5, P6, each 0.25 μ L of P7, P8.
Compared with prior art, the invention has the advantages that: the multiplex PCR that adopts the present invention to set up detects the method for enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus simultaneously, have easy and simple to handle, quick, high specificity, the advantages such as sensitivity height, only need a PCR reaction just can detect these four kinds of bacteriums simultaneously, detection by application the method to varying environment seawater sample and breeding seawater sample, compare multiplex PCR with ordinary method detection simultaneously and detect result, fit like a glove with conventional bacterium isolation identification result; Adopt multiplex PCR of the present invention to detect the method for enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus simultaneously, can prompting and warning sample whether there is hazardness, this is for prevention and control China's fishery products and Marine Environmental Security has important practical significance.Have no at present the report that utilizes enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus in this technology for detection ocean and fishery products thereof both at home and abroad, this technology is expected to for these four kinds of bacteriums of multidisciplinary detection provide a kind of rapid sensitive, easy reliable method, and the departments such as quarantine, health that are particularly suitable for implement quick test needs.
Accompanying drawing explanation
Fig. 1 is the electrophoresis detection result that multiplex PCR is differentiated enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus.
Embodiment
Below in conjunction with the drawings and embodiment the invention will be further described.
1) utilize simultaneously the increase multi-primers combination of enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus of primer express2.0 primer-design software design;
2) primer of PCR detection enterobacter cloacae in the combination of screening multi-primers;
3) primer of PCR detection monocytogenes hyperplasia Listeria in the combination of screening multi-primers;
4) primer of PCR detection Aeromonas hydrophila in the combination of screening multi-primers;
5) primer of PCR detection Vibrio parahaemolyticus in the combination of screening multi-primers;
6) screening multiplex PCR detects the combination of primers of enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus simultaneously.
Described step 1), on the corresponding gene order of primer reference laboratories ocean strain isolated wherein relating to and GenBank, deliver that (E.bact.gyrB is GenBank AF302677, AY370837, L.mono.ppbp is GenBank FR733647, HE999705, A.hydr.hlyA is GenBank L36462, GU229025, V.para.tlh is GenBank GU971655, AB012596) four kinds of gene orders that bacterium is corresponding, in the time of application primer express2.0 design, detect the Auele Specific Primer combination of four kinds of bacteriums.
In this patent text, the english abbreviation that E.bact. is enterobacter cloacae, L.mono. is the english abbreviation of monocytogenes hyperplasia Listeria, the english abbreviation that A.hydr. is Aeromonas hydrophila, the english abbreviation that V.para. is Vibrio parahaemolyticus, E.bact. (gyrB) P1 and E.bact. (gyrB) P2 is according to the upstream primer P1 of enterobacter cloacae gyrB gene design and downstream primer P2, L.mono. (ppbp) P3 and L.mono. (ppbp) P4 is according to upstream primer P3 and the downstream primer P4 of monocytogenes hyperplasia Listeria ppbp gene design, A.hydr. (hlyA) P5 and A.hydr. (hlyA) P6 is according to the upstream primer P5 of Aeromonas hydrophila hlyA gene design and downstream primer P6, V.para. (tlh) P7 and V.para. (tlh) P8 is according to upstream primer P7 and the downstream primer P8 of tlh gene of vibrio parahaemolyticus design.
Described step 2), 3), 4), 5) and 6), through screening, show that the pcr amplified fragment size that enterobacter cloacae primer pair is answered is 1250bp, the pcr amplified fragment size that monocytogenes hyperplasia Listeria primer pair is answered is 768bp and 272bp, the pcr amplified fragment size that Aeromonas hydrophila primer pair is answered is 483bp, and the pcr amplified fragment size that Vibrio parahaemolyticus primer pair is answered is 454bp.Concrete primer sequence is as follows:
The upstream and downstream primer of enterobacter cloacae is:
E.bact.(gyrB)P1：5’-AAGCGHCCNGSNATGTAYATHGG-3’；
E.bact.(gyrB)P2：5’-CCNGCNGARTCNCCYTCNAC-3’；
The upstream and downstream primer of monocytogenes hyperplasia Listeria is:
L.mono.(ppbp)P3：5’-TTCCGCAGAGGACAGTGATG-3’；
L.mono.(ppbp)P4：5’-CATATTCCACTTTAACCGTG-3’；
The upstream and downstream primer of Aeromonas hydrophila is:
A.hydr.(hlyA)P5：5’-AAAGCGGATTATGCAGAAGCACTG-3’；
A.hydr.(hlyA)P6：5’-GCTACTTTCTAGCATTTTCTCTGC-3’；
The upstream and downstream primer of Vibrio parahaemolyticus is:
V.para.(tlh)P7：5’-TGAGTTGCTGTTGTTGGATGC-3’；
V.para.(tlh)P8：5’-GTTGATGACACTGCCAGATGC-3’；
Described step 6), multiplex PCR detects the mensuration of enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus method optimum reaction condition simultaneously, utilize respectively 4 pairs of primers to carry out the mensuration of 4 kinds of bacterium template multiplex PCR optimum annealing temperatures, primer concentration, template concentrations etc., then according to experimental result, carry out the mensuration of multiplex PCR optimum cycle number of times.
Described step 6), the reaction system that multiplex PCR detects enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus method is simultaneously 25 μ L, specific as follows: 10 * PCR Buffer is (containing 25mmol/L MgCl
2) 2.5 μ L, 10mmol/L deoxyribonucleoside triphosphate mixture (dNTP) 3 μ L, each 0.5 μ L of 10mmol/L E.bact. (gyrB) upstream and downstream primer, each 1.5 μ L of 10mmol/L L.mono. (ppbp) and A.hydr. (hlyA) upstream and downstream primer, each 1.0 μ L of 10mmol/L V.para. (tlh) upstream and downstream primer, 5U/ μ L Taq enzyme 0.25 μ L, E.bact. DNA profiling 3 μ L, L.mono. DNA profiling 4 μ L, A.hydr. DNA profiling 5 μ L, V.para. DNA profiling 2 μ L, ddH
2o complements to 25 μ L.Amplification condition is 94 ℃ of 5min of denaturation; 94 ℃ of 30s of sex change, the 56 ℃ of 30s that anneal, extend 72 ℃ of 60s, circulate 30 times; Extend eventually 72 ℃ of 10min; 4 ℃ of preservations.After reaction finishes, extract reaction solution each 5 μ L and carry out 1.5% agarose gel electrophoresis, deposition condition: Tris boric acid (TBE), electric current 50mA, voltage 150v, after electrophoresis time 30min, with Bio-Rad gel imaging system, take imaging, in Fig. 1, shown the present embodiment sample is carried out respectively to multiple and detected result substance PCR, wherein M swimming lane is DL2000DNA Marker; No. 1 swimming lane is Quadruple-PCR amplification, at molecular weight, be about 1250bp, 768bp and 272bp, 483bp and 454bp place and present respectively bright object amplified band, illustrate in the sample of the present embodiment and have enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus; The detected result that No. 2 swimming lanes are enterobacter cloacae, presents bright, the single amplified band that molecular weight is about 1250bp, contains enterobacter cloacae in interpret sample; No. 3 swimming lanes are the detected result of monocytogenes hyperplasia Listeria, present the amplified band bright, feature that molecular weight is about 768bp and 272bp, contain monocytogenes hyperplasia Listeria in interpret sample; The detected result that No. 4 swimming lanes are Aeromonas hydrophila, presents bright, the single amplified band that molecular weight is about 483bp, contains Aeromonas hydrophila in interpret sample; The detected result that No. 5 swimming lanes are Vibrio parahaemolyticus, presents bright, the single amplified band that molecular weight is about 454bp, contains Vibrio parahaemolyticus in interpret sample.
Embodiment 1
For detect the multiple PCR primer method of design of enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus simultaneously, the method comprises the following steps:
Step 1, utilize primer-design software Oligo6.0 design to detect the multiple PCR primer combination of enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus simultaneously, the primer base number that formed combination of primers comprises is 24, the melting temperature (Tm) Tm value of primer is 65 ℃, and the GC% of primer is 40%;
Step 2, screens the primer in combination of primers, retains the primer that does not form primer dimer;
Step 3, the good and bad state of competition of primer that judgement retains, the GC% of above-mentioned retained primer and base number are compared, when the base number of primer all GC% identical and inner side primer be less than the GC% of outside primer, or when the base number of primer is incomplete same and the base number of inner side primer during than the few base of outside primer, be judged as race condition inferior position primer, carry out step 4; Otherwise be judged as race condition advantage primer, carry out step 5;
Step 4, screens race condition primer again, and concrete steps are: repeating step 2, judges according to step 3 again to retained primer;
Step 5, utilizes PCR method to the checking of increasing of race condition advantage primer;
Step 6, removes the race condition inferior position primer in amplimer, obtains multiple PCR primer combination.
Concrete primer sequence is as follows:
E.bact.(gyrB)P1：5’-AAGCGHCCNGSNATGTAYATHGG-3’
E.bact.(gyrB)P2：5’-CCNGCNGARTCNCCYTCNAC-3’
L.mono.(ppbp)P3：5’-TTCCGCAGAGGACAGTGATG-3’
L.mono.(ppbp)P4：5’-CATATTCCACTTTAACCGTG-3’
A.hydr.(hlyA)P5：5’-AAAGCGGATTATGCAGAAGCACTG-3’
A.hydr.(hlyA)P6：5’-GCTACTTTCTAGCATTTTCTCTGC-3’
V.para.(tlh)P7：5’-TGAGTTGCTGTTGTTGGATGC-3’
V.para.(tlh)P8：5’-GTTGATGACACTGCCAGATGC-3’。
Embodiment 2
For detect the multiple PCR primer method of design of enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus simultaneously, the method comprises the following steps:
Step 1, utilize primer-design software Primer Premier5.0 design to detect the multiple PCR primer combination of enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus simultaneously, the primer base number that formed combination of primers comprises is 20, the melting temperature (Tm) Tm value of primer is 55 ℃, and the GC% of primer is 60%;
Step 2, screens the primer in combination of primers, retains the primer that does not form primer dimer;
Step 3, the good and bad state of competition of primer that judgement retains, the GC% of above-mentioned retained primer and base number are compared, when the base number of primer all GC% identical and inner side primer be less than the GC% of outside primer, or when the base number of primer is incomplete same and the base number of inner side primer during than the few base of outside primer, be judged as race condition inferior position primer, carry out step 4; Otherwise be judged as race condition advantage primer, carry out step 5;
Step 4, screens race condition primer again, and concrete steps are: repeating step 2, judges according to step 3 again to retained primer;
Step 5, utilizes PCR method to the checking of increasing of race condition advantage primer;
Step 6, removes the race condition inferior position primer in amplimer, obtains multiple PCR primer combination.
Concrete primer sequence is as follows:
E.bact.(gyrB)P1：5’-AAGCGHCCNGSNATGTAYATHGG-3’
E.bact.(gyrB)P2：5’-CCNGCNGARTCNCCYTCNAC-3’
L.mono.(ppbp)P3：5’-TTCCGCAGAGGACAGTGATG-3’
L.mono.(ppbp)P4：5’-CATATTCCACTTTAACCGTG-3’
A.hydr.(hlyA)P5：5’-AAAGCGGATTATGCAGAAGCACTG-3’
A.hydr.(hlyA)P6：5’-GCTACTTTCTAGCATTTTCTCTGC-3’
V.para.(tlh)P7：5’-TGAGTTGCTGTTGTTGGATGC-3’
V.para.(tlh)P8：5’-GTTGATGACACTGCCAGATGC-3’。
Embodiment 3
1, the synthetic enterobacter cloacae of design, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus primer
(E.bact.gyrB is GenBank AF302677 to the gene order corresponding with reference to four kinds of bacteriums of delivering on the corresponding gene order of ocean strain isolated and GenBank, AY370837, L.mono.ppbp is GenBank FR733647, HE999705, A.hydr.hlyA is GenBank L36462, GU229025, V.para.tlh is GenBank GU971655, AB012596) four kinds of gene orders that bacterium is corresponding, application primer express2.0 designs a pair of Auele Specific Primer, the pcr amplified fragment size that enterobacter cloacae primer pair is answered is 1250bp, the pcr amplified fragment size that monocytogenes hyperplasia Listeria primer pair is answered is 768bp and 272bp, the pcr amplified fragment size that Aeromonas hydrophila primer pair is answered is 483bp, the pcr amplified fragment size that Vibrio parahaemolyticus primer pair is answered is 454bp, primer is synthetic by giving birth to work biotechnology (Shanghai) limited-liability company, specifically referring to table 1:
2, grope bacterium template extraction method
Adopt traditional method (phenol-chloroform extraction method), pyrolysis method and 3 kinds of methods of isolation kit method respectively DNA of bacteria to be extracted, and the DNA profiling of extraction is carried out to pcr amplification, result is contrasted, select optimum extracting method.Isolation kit method, pyrolysis method and traditional extraction method extraction effect are basic identical, and effect is better.But operated in accordance with conventional methods is comparatively loaded down with trivial details, isolation kit method is because relative cost is higher, and therefore final selection pyrolysis method is optimum extracting method.
Wherein, pyrolysis method concrete scheme is as follows: enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus bacterial strain are inoculated into respectively with in Luria Broth (LB) substratum and 3.5% basic peptone water to incubated overnight 16 hours; Pour bacterium liquid after cultivation in 1.5mLEp pipe about 1.2mL, the centrifugal about 1min of 12000rpm, removes supernatant, the more centrifugal about 1min of 13000rpm, sucks raffinate; By the resuspended precipitation of aseptic PBS120 μ L, put into boiling water and suspend after heating 10min, immediately ice bath 5min; Put into again boiling water suspension heating 5min, then ice bath 5min; By the Ep pipe after ice bath, the centrifugal 1min of 10000rpm, gets supernatant liquor and does template.
3, the foundation of multi-PCR detection method
The reaction system of pcr amplification is 25 μ L, specific as follows: 10 * PCR Buffer is (containing 25mmol/L MgCl
2) 2.5 μ L, 10mmol/L deoxyribonucleoside triphosphate mixture (dNTP) 3 μ L, each 2 μ L of 10mmol/L E.bact. (gyrB) upstream and downstream primer P1, P2, each 0.75 μ L of 10mmol/L L.mono. (ppbp) upstream and downstream primer P3, P4 and A.hydr. (hlyA) upstream and downstream primer P5, P6, each 0.25 μ L of 10mmol/L V.para. (tlh) upstream and downstream primer P7, P8,5U/ μ L Taq enzyme 0.2 μ L, E.bact. DNA profiling 4 μ L, L.mono. DNA profiling 5 μ L, A.hydr. DNA profiling 6 μ L, V.para. DNA profiling 2 μ L, ddH
2o complements to 25 μ L.Amplification condition is 94 ℃ of 5min of denaturation; 94 ℃ of 30s of sex change, the 56 ℃ of 30s that anneal, extend 72 ℃ of 60s, 35 circulations; Extend eventually 72 ℃ of 10min; 4 ℃ of preservations.Get pcr amplification product 5 μ L and carry out sepharose (1.5%) electrophoresis detection amplification;
By above PCR product electrophoresis 30min under 0.5 * Tris boric acid (TBE), 2.0% sepharose, 150V voltage conditions, then in gel imaging system, observe.
4, multiplex PCR optimum reaction condition gropes
With 4 pairs of primers, carry out respectively the mensuration of 4 kinds of bacterium template multiplex PCR optimum annealing temperatures, primer concentration, template concentrations etc., then according to experimental result, carry out the mensuration of multiplex PCR optimum cycle number of times.Result records multiplex PCR, annealing temperature be 54 ℃-58 ℃ all can, wherein with 56 ℃ of best results; Dosage 2 μ L, the 2.5 μ L of 10mmol/L dNTP, 3 μ L, 3.5 μ L all can, finally selecting 3 μ L is optimum quantum of utilization, because usage quantity is less and band is clear; Added 5U/ μ L Taq enzyme dosage 0.25 μ L, 0.20 μ L, 0.15 μ L, 0.10 μ L, 0.05 μ L in 25 μ L reaction systems, wherein with 0.20 μ L and 0.25 μ L best results, because of 0.20 μ L consumption less as optimum addition; 28～35 of multiplex PCR cycle indexes all can, but 35 cycle index successfuls.Multi-PRC reaction condition and range: 10mmol/L dNTP2-3.5 μ L, 5U/ μ L Taq enzyme 0.10-0.25 μ L, 28～35 cycle indexes.Multiplex PCR optimum reaction condition: 10mmol/L dNTP3 μ L, 5U/ μ LTaq enzyme 0.20 μ L, 35 cycle indexes.
5, multiplex PCR sensitivity detects
Getting 28 ℃ of enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus bacterium liquid of cultivating 18h selects pyrolysis method to extract each thallus DNA template, after 10 times of gradient dilutions, the DNA profiling of the different bacterium of identical extent of dilution mixes in proportion, adopts the multiplex PCR system of having optimized to carry out the detection of sensitivity.In multiplex PCR energy while detection reaction system, DNA minimum concentration is enterobacter cloacae 3.42 * 102pg/mL, monocytogenes hyperplasia Listeria 4.55 * 102pg/mL, Aeromonas hydrophila 4.20 * 102pg/mL, Vibrio parahaemolyticus 2.23 * 102pg/mL.
6, multiplex PCR specific assay
Respectively the DNA of bacteria such as Vibrio parahaemolyticus, vibrio alginolyticus, enterococcus faecalis, Wdwardsiella tarda, meat Bacillaceae, Bacillus thuringiensis, Salmonellas, intestinal bacteria are extracted, utilize the multiplex PCR condition of optimizing respectively the random dna combination of single DNA of bacteria and enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus and other bacteriums to be increased, to detect its specificity.In 10 bacterial strains, only have as a result enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus can amplify corresponding fragment, size is respectively enterobacter cloacae 1250bp, monocytogenes hyperplasia Listeria 768bp and 272bp, Aeromonas hydrophila 483bp, Vibrio parahaemolyticus 454bp, all conform to expection, and other does not all amplify corresponding fragment.Illustrate that this research multiple PCR method specificity is good.
7, multiplex PCR Stability Determination
Packing after the PCR reaction system beyond removing template being mixed according to optimum multiplex PCR condition, put-20 ℃ frozen, according to best PCR reaction conditions, regularly take out detection.Between detection period, the PCR mixed solution being pre-mixed is placed on and under-20 ℃ of conditions, preserves the PCR blended liquid phase that still can amplify and now join for 1,2,3,4,5 month like band clearly, this test is also underway, and the final time is also finally not definite.But by the detection of half a year nearly, can find out that the stability of this reagent is better, at least can keep more than 5 months;
8, multiplex PCR seawater sample detects
Take 12 parts of environment seawater samples and 9 parts of breeding seawater samples, with multiplex PCR, detect, detect and compare with ordinary method simultaneously.PCR detected result and conventional bacterium isolation identification result fit like a glove.
Claims (5)
1. for detect a multiple PCR primer for the four kinds of pathogenic bacterium in ocean simultaneously, it is characterized in that: the multiple PCR primer of these four kinds of pathogenic bacterium comprises respectively the multiple PCR primer of enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus;
Wherein, the upstream and downstream primer of enterobacter cloacae is:
P1：5’-AAGCGHCCNGSNATGTAYATHGG-3’；
P2：5’-CCNGCNGARTCNCCYTCNAC-3’；
The upstream and downstream primer of monocytogenes hyperplasia Listeria is:
P3：5’-TTCCGCAGAGGACAGTGATG-3’；
P4：5’-CATATTCCACTTTAACCGTG-3’；
The upstream and downstream primer of Aeromonas hydrophila is:
P5：5’-AAAGCGGATTATGCAGAAGCACTG-3’；
P6：5’-GCTACTTTCTAGCATTTTCTCTGC-3’；
The upstream and downstream primer of Vibrio parahaemolyticus is:
P7：5’-TGAGTTGCTGTTGTTGGATGC-3’；
P8：5’-GTTGATGACACTGCCAGATGC-3’。
2. according to claim 1 for detect the multiple PCR primer of the four kinds of pathogenic bacterium in ocean simultaneously, it is characterized in that: the pcr amplified fragment size that described enterobacter cloacae primer pair is answered is 1250bp, the pcr amplified fragment size that described monocytogenes hyperplasia Listeria primer pair is answered is 768bp and 272bp, the pcr amplified fragment size that described Aeromonas hydrophila primer pair is answered is 483bp, and the pcr amplified fragment size that described Vibrio parahaemolyticus primer pair is answered is 454bp.
3. according to claim 1 for detect a method of design for the multiple PCR primer of the four kinds of pathogenic bacterium in ocean simultaneously, it is characterized in that: comprise the following steps:
Step 1, utilize primer-design software design to detect the multiple PCR primer combination of enterobacter cloacae, monocytogenes hyperplasia Listeria, Aeromonas hydrophila and Vibrio parahaemolyticus simultaneously, the primer base number that formed combination of primers comprises is 20-24, the melting temperature (Tm) Tm value of primer is 55 ℃-65 ℃, and the GC% of primer is 40%-60%;
Step 2, screens the primer in combination of primers, retains the primer that does not form primer dimer;
Step 3, the good and bad state of competition of primer that judgement retains, the GC% of above-mentioned retained primer and base number are compared, when the base number of primer all GC% identical and inner side primer be less than the GC% of outside primer, or when the base number of primer is incomplete same and the base number of inner side primer during than the few base of outside primer, be judged as race condition inferior position primer, carry out step 4; Otherwise be judged as race condition advantage primer, carry out step 5;
Step 4, screens race condition primer again, and concrete steps are: repeating step 2, judges according to step 3 again to retained primer;
Step 5, utilizes PCR method to the checking of increasing of race condition advantage primer;
Step 6, removes the race condition inferior position primer in amplimer, obtains multiple PCR primer combination.
4. application is as claimed in claim 1 for detect a multi-PCR detection method for the multiple PCR primer of the four kinds of pathogenic bacterium in ocean simultaneously, it is characterized in that comprising the steps:
1) initial gross separation of bacterium: extract genomic dna from substratum, the standby pcr template of doing;
2) multiplex PCR amplification comprises: a, multiplex PCR amplification reaction system: get above-mentioned DNA profiling 2～8 μ L, reaction system is 20-30.0 μ L, and each reaction product is respectively TaqDNA polysaccharase 5U/ μ L, 0.1-0.25 μ L, PCR damping fluid 2.5 μ L, deoxyribonucleoside triphosphate mixture 10mmol/L, 2-3.5 μ L, each 0.5-2 μ L of primer P1, P2, each 0.75-1.5 μ L of P3, P4, each 0.75-1.5 μ L of P5, P6, each 0.25-1.0 μ L of P7, P8, surplus is supplied with redistilled water; B, amplification reaction condition: 94 ℃ of denaturation 5min, 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 40s, circulate 30 times, and 10min is extended at 72 ℃ of whole ends; C, the amplified production of step b is carried out to electrophoresis detection and interpretation of result.
5. according to claim 4 for detect the multi-PCR detection method of the multiple PCR primer of the four kinds of pathogenic bacterium in ocean simultaneously, it is characterized in that: each 2 μ L of primer P1, P2 in described step a, each 0.75 μ L of P3, P4, each 0.75 μ L of P5, P6, each 0.25 μ L of P7, P8.
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