CN108384868A

CN108384868A - Multiple PCR primer group and detection method and kit for four kinds of morbid vibrios of detection simultaneously

Info

Publication number: CN108384868A
Application number: CN201810483056.5A
Authority: CN
Inventors: 陈鲤群; 黄蓝青; 林峻; 蔡伟文; 赵依依
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2018-05-18
Filing date: 2018-05-18
Publication date: 2018-08-10

Abstract

The invention discloses a kind of multiple PCR primer group for detecting four kinds of morbid vibrios simultaneously and detection method and kit, detection primer group include detect vibrio parahemolyticus, Vibrio vulnificus, vibrio harveyi and vibrio mimicus primer pair.The present invention has also set up a kind of multi-PCR detection method and kit being used for while detecting four kinds of morbid vibrios, the primer sets obtained using design, genomic DNA to the bacterium extracted in sample to be tested, multi-PRC reaction is carried out in same reaction system, judge in sample whether to contain above-mentioned morbid vibrio by the electrophoretic analysis to reaction product, have the advantages that quick and precisely, at low cost, specific and high sensitivity.

Description

Multiple PCR primer group and detection method for detecting four kinds of morbid vibrios simultaneously and Kit

Technical field

The invention belongs to microorganism detection field, be related to detecting simultaneously the multiple PCR detection primer group of four kinds of morbid vibrios, Detection method and kit.

Background technology

Vibrios (Vibrio) is under the jurisdiction of vibrionaceae (Vibrionaceae) vibrio, and vibrionaceae further includes Aeromonas (Aeromonas), three categories such as Plesiomonas (Plesiomonas) and Photobacterium (Photorhabdus), they Common feature is that a group form is in straight or micro-bend leather with extreme flagellum, the power positive, oxidase positive, glucose fermentation Blue negative bacillus, in addition to luminous bacillus, excess-three belongs to the mankind with pathogenic.

Vibrios is one of predominant bacteria flora common in marine environment, in recent years, as sea fishery cultivates scale Grow the negative Effects of Factors brought with high density intensive culture, fish and shellfish vibrios disease happens occasionally, and seriously hinders ocean Expanding economy.Kinds of pathogenic vibrio becomes Marine cultured fish and shrimp crab all over the world because of the features such as its Epidemic Scope is wide, incidence is high The primary pathogen of shellfish bacteriosis, one of the hot spot as sea fishery disease research field receive scholar pass at home and abroad Note.There is early 20th century researcher to being named after having done Physiology and biochemistry identification from the kinds of pathogenic vibrio being separated in European eel For Vibrio anguillarum (V.anguillarum), which is considered as the arch-criminal for causing the red pest of Baltic Sea sea eel, becomes relatively early It was found that one of marine bacteria pathogen, so far, the Sea central platform being reported has as many as tens of kinds.

The ocean fish for suffering from vibriosis mostly can be in body surface inflammation, erythema hyperemia, and it is rotten that some even can cause body surface ulcer, rotten fin The symptoms such as tail, even septicemia；The prawn of infection vibrios will appear red leg disease, white black spot, muscle white oil disease, rotten Bao disease, lose The symptoms such as mass formed by blood stasis；Its abdominal foot flesh will appear warts after abalone infection vibrios, and internal organ are exposed, muscle ulceration, and upper and lower myomalacia is rotted It is lethal；The mankind infect vibrios since wound contacts seawater or eats seafood raw, it may appear that diarrhea, chordapsus, limbs infection are drawn The septicemia risen.

Vibrio parahemolyticus (Vibrio Parahemolyticus, VP) is widely present in seawater and marine product, is me State coastal area common meal poisoning pathogen.Vibrio parahaemolytisus poisoning is caused by feeding the food containing the bacterium, Essentially from marine product, such as inkfish, ocean fish, sea shrimp, sea crab, jellyfish, and the higher cure foods containing salinity, such as salted vegetables salt down Meat etc..Clinically using Acute onset, abdominal pain, vomiting, diarrhea and watery stool as cardinal symptom.Arizona, USA university Donald V. Lightner teach the viewpoint delivered in global aquaculture alliance, claim to find that vibrio parahaemolytious is to cause pair The pathogen of shrimp Acute Hepatic pancreatic necrosis, which orally propagates and place is in prawn gastrointestinal tract, generates toxin and tissue is caused to damage Bad and prawn liver function, digestive system obstacle.

Vibrio vulnificus (vibrio vulnificus) is a kind of bacterium inhabited in Yu Haiyang, including two bions, life Object type 1 is human condition pathogenic bacteria, and shellfish is related with contacting and eating raw, and bion 2 is sea eel pathogenic bacteria, easily causes the gill, stomach and intestine The corresponding lesion of the appearance such as road, liver, kidney.The mankind once infect, and morbidity is anxious, and quickly, 75% patient is entering progression of the disease Because multi-organ function is not full and dead in institute 48 hours, it is referred to as " the noiseless killer in ocean ", metainfective symptom includes vomitting Spit, have a fever, diarrhea, low blood pressure, swelling and pain etc..There are mainly two types of routes of infection, and one is feed life or not processed Ripe shellfish Class A marine product (especially oyster), harm does not lie in it and causes gastroenteritis, and is its caused celluar tissue Scorching and septicemia, the death rate are up to 50% or more；Another route of infection is that damaged extremity seawater or marine product are stabbed Skin and infect.Bacterium is by damaged skin very fast propagation, and it is serious bad to cause serious myositis and myofascitis to cause Septicemia then occurs for subcutaneous ulcer.

Vibrio harveyi (Vibrio harveyi) is also known as Vibrio harveyi, and also some is denoted by Hawaii vibrios, in the past Once kirschner bacterium in Kazakhstan shellfish was named as by Johnson etc., it is formally attributed to vibrio and ordered by Baumann in 1981 etc. Entitled vibrio harveyi.The bacterium is a kind of vibrios with the characteristics of luminescence, is normal member and the seawater of seawater microbiota A kind of middle common pathogenic bacteria, mainly cause the infection morbidity of shrimp and fish.

Vibrio mimicus (Vibrio mimicus) is a kind of common pathogenic vibrio, close with comma bacillus affiliation, Be initially considered that be atypia biochemical characteristic comma bacillus, become the important vibriosis cause of disease of the mankind, human infection is main Because eating the raw fish or shellfish that are polluted by the bacterium, symptom is similar with Vibrio cholerae infection, be mainly shown as diarrhea, nausea, Vomiting and tormina and the heat that occurs together etc., report vibrio mimicus infection crayfish causes to cultivate a large amount of dead of crayfish for the first time since 1991 Since dying, which has been considered as the pathogen of a variety of shell-fish and seawater fish.

According to statistics, the annual aquaculture in China economic loss caused by bacterium class disease surpasses 10,000,000,000, and the U.S. has nearly 8 every year Ten thousand people infect vibrios case, and food poisoning case growth rate has more than greatly Escherichia coli and Salmonella caused by kinds of pathogenic vibrio The gesture of bacterium is reinforced extremely urgent to the detection and identification of Sea central platform.

Currently, the detection of vibrios is mainly the following method with identification：

1, traditional Physiology and biochemistry identification

So far, domestic country still using traditional microbiology Physiology and biochemistry identification method as kinds of pathogenic vibrio and Professional standard detection method.Traditional phenotypic evaluation technology needs the form for pathogen, physiology, Biochemical Characteristics to carry out a system Row appraisal includes the separation of selection tablet, biochemical test, kanagawa phenomenon etc., and the period is longer, heavy workload, cumbersome, It takes time and effort, is unfavorable for the quick Testing and appraisal of pathogenic bacteria.The full-automatic fast microbiological identification of VITEK series to come out in recent years Intelligent analysis system and API20E biochemical identifications reagent strip because of the advantages that its is fast and convenient accurate by more and more favors and Approve, defect is that testing cost is higher, it is possible that the qualification result mistake caused by part bacterium short in size.

2, immunological technique

Often it is using special with the immunological technique of one of checkout and diagnosis method as ocean fishes and shrimps shellfish bacteriosis Property antigen-antibody reaction capture pathogenic bacteria, be related to elisa technique, immunofluorescence technique and immunoblotting (Western Blot), accuracy is high, and detection speed is fast, the disadvantage is that cost is slightly higher, requires experimental skill high.

3, nucleic acid detection technique

With the rise of Development of Molecular Biology and technique for gene engineering, the detection technique based on nucleic acid is successfully used It is spy in the quick detection of vibrios, including PCR, nucleic acid probe and 16S rRNA technologies, high sensitivity, high specificity, difficult point Can the design of specific primer close to affiliation, and the inadequate species of differentiation degree distinguish.Multiplex polymerase chain formula is anti- It is to utilize well-designed multipair primer same to answer (multiplex polymerase chain reaction, M-PCR) Multiple target gene are expanded simultaneously in a PCR system, to make up the deficiency that single primer amplification is easy to miss inspection, reduce experiment material damage Consumption shortens detection time, and from Chamberlian J S since 1988 are put forward for the first time this concept, M-PCR technologies are gradually It is used for the diagnosis of bacterial disease and the identification of pathogen etc., application prospect is quite wide.In actual application, target sequence The control of selection, design of primers and experiment condition is all the difficult point of this technology.In addition, the pollution of micro exogenous DNA is led Cause false positive results and enriched medium selection it is improper caused by false negative result be also the problem of it is easy tod produce.

There are numerous advantages in view of multiple PCR technique, is gradually applied to the detection of pathogenic microorganism.Double sum is triple PCR detections have relevant report repeatly, but since multiplex PCR influence factor is complicated, different primers, template, primer concentration, template Concentration, Mg²⁺Concentration, dNTP concentration and its ratio etc. will produce complicated comprehensive effect, therefore the objective microbe detected simultaneously Type is more, and the difficulty that PCR system is established is bigger.It is inquired through domestic and foreign literature, there is not yet multiplex PCR is applied to parahemolyticas The relevant report that vibrios, Vibrio vulnificus, vibrio harveyi and vibrio mimicus quickly detect.

Invention content

In order to solve the problems in the existing technology, it is used for the object of the present invention is to provide one kind while four kinds of detection causes Multiple PCR primer group, detection method and the kit of sick vibrios are, it can be achieved that vibrio parahemolyticus, Vibrio vulnificus, vibrio harveyi With the quick detection of vibrio mimicus.

In order to realize the object of the invention, present invention firstly provides the technical solutions and one for designing and screening specific primer The primer sets of kind multiplex PCR detection morbid vibrio：

1, positive internal reference (IAC) amplimer and template are built

Using the 16S rDNA sequences of vibrios as stencil design pair of primers, the segment of 663bp sizes therein is expanded, as The IAC of detection.IAC upstream primer sequences are that the sequence of 5 '-CGGTGA AAT GCG TAG AGA T-3 ', IAC downstream primers is 5’-TTA CTA GCG ATT CCG AGT TC-3’。

2, the specific target gene for screening different vibrios is used for design of primers

In vibrios identification method based on by PCR, the selection of target gene is primary difficult point and main points, this kind of target base Because including mainly virulence gene and house-keeping gene, it should ensure that the target gene is relatively conservative within belonging to, make it have versatility, again It is required that it is relatively low to realize specificity in the homology of inter-species.It is analyzed by document analysis and detection demand, obtains each The gene or DNA fragmentation of morbid vibrio candidate amplification：The flaE genes of vibrio parahemolyticus；The vvc genes of Vibrio vulnificus；It breathes out The vhhp2 genes of family name vibrios；The sodB genes of vibrio mimicus.

3, the design of primer alternative scheme

The difficult point of multiplex PCR first consists in the design of primer, ensures that multipair primer melting temperature is similar as possible, avoids complementation The target fragment size of sequence, amplification cannot be too close to, and otherwise amplified production is inseparable when electrophoresis.

In design primer, it should be noted that following item：

(1) primer should design in the conservative region of sequence and have specificity.

(2) primer length is generally 15-30bp, because long can cause it to extend temperature more than 74 DEG C, is unsuitable for Taq DNA polymerases are reacted.

(3) primer should not form secondary structure, and the numerical value of primer dimer and hairpin structure is excessively high (more than 4.5kcal/ Mol it) easily leads to and generates primer dimer band, and reduce primer effective concentration and make PCR reactions that cannot be normally carried out.

(4) the Tm values of primer cannot it is too low can not be too high, the Tm values of template position sequence corresponding to primer are at 45-60 DEG C Left and right can make recovery condition best；The Tm values of two primers should be as equal as possible or close, and preferably difference is no more than 5 degree.Tm values A variety of computational methods are obtained, are such as calculated by formula Tm=4 (G+C)+2 (A+T).

(5) end of primer 3 ' can not be modified, and the last bit base that primer 3 ' is held has the DNA combined coefficienies of Taq polymerase larger Influence.3 ' ends should select A, T, select G, C, this primer that can be effectively prevented from false initiation, there is higher efficiency of initiation less.Draw 3 or more continuous bases cannot occur before in object sequence itself, therefore should avoid holding in primer 3 ' and be drawn using mistake Probability is sent out to increase.

The present invention is according to the virulence gene and house-keeping gene sequence of the various vibrios announced on GenBank, in conjunction with correlation Document report is shown, in main window using Oligo7 and Primer Primier5.0 software Design primers according to search result The middle secondary structure situation for checking the primer pair, analyzes, screens successively, one by one to all amplification target candidate genes or segment It is designed 1-2 set alternative schemes.

After upper and lower primer is chosen entirely, need to evaluate primer." Analyse " menu can be used to analyze primer：Than If any primer free dimer, hairpin structure etc..First check for primer dimer, the possibility that especially 3 ' end dimers are formed Property.It should be noted that primer dimer is likely to be upstream or downstream primer oneself generates, it is also possible to upstream and downstream primer Between formed (cross dinner).Capable of being worth for dimer formation is higher, more undesirable.General detection (non-clone) property PCR, requires primer location, primer size relatively low, thus chooses as far as possible and does not form dimer or other and can be worth lower draw Object.Section 2 inspection is hairpin structure (hairpin), identical as dimer, and the lower capable of being worth for hairpin structure the better.It is general next It says, this two structures can be worth no more than 4.5 preferably.Certainly, in the PCR primer of design clone's purpose, primer both ends are general Restriction enzyme site is all added, certainly exists hairpin structure, and can be worth will not be too low.This PCR needs to move back by flexible modulation Fiery temperature should not require the detection of the hairpin structure of primer too high with reaching best effects.Section 3 inspection is G/C content, with 45%-55% is advisable.There is the G/C content of some templates itself relatively low or higher, causes the G/C content of primer that cannot be controlled. Within the above range, the G/C content of upstream and downstream primer and Tm values at this moment should be made to keep close as possible, to be conducive to annealing temperature Selection.

4, Blast analyses are carried out to the primer candidate scheme for expanding target gene

Blast analyses are carried out to all primer candidate schemes in Genbank, the higher primer scheme of specificity is obtained and makees For experimental verification scheme, each morbid vibrio may select multiple amplification targets, and each target that expands has 1-2 in fact Test proof scheme.

5, substance PCR verifications are carried out to each available experimental verification scheme, determines that the availability of primer, including specificity are commented Estimate and sensitivity assessment.

6, the primer of all amplification targets is mixed and carries out multiplex PCR, verify availability when primer coamplification, therefrom The combination of annoyance level minimum between selection primer needs to draw according to step 2-4 design iterations are new to inoperable primer Object is replaced.Finally obtain a set of special primer sequence (referring to table 1) provided by the invention.

Table 1. is used for while detecting the multiple PCR primer group of four kinds of morbid vibrios

Based on the above-mentioned technical proposal, the present invention provides a kind of multiplex PCRs for being used for while detecting four kinds of morbid vibrios to draw Object group, it includes detection vibrio parahemolyticus, the primer pairs of Vibrio vulnificus, vibrio harveyi and vibrio mimicus；

The nucleotide sequence of the upstream and downstream primer of the primer pair of the detection vibrio parahemolyticus is respectively by SEQ ID Shown in NO.1, SEQ ID NO.2；

The nucleotide sequence of the upstream and downstream primer of the primer pair of the detection Vibrio vulnificus is respectively by SEQ ID NO.3, SEQ Shown in ID NO.4；

The nucleotide sequence of the upstream and downstream primer of the primer pair of the detection vibrio harveyi is respectively by SEQ ID NO.5, SEQ Shown in ID NO.6；

The nucleotide sequence of the upstream and downstream primer of the primer pair of the detection vibrio mimicus is respectively by SEQ ID NO.7, SEQ Shown in ID NO.8.

Further, the multiple PCR primer group for being used for while detecting four kinds of morbid vibrios also includes positive internal reference Primer pair, the nucleotide sequence of the upstream and downstream primer of the positive internal reference primer pair is respectively by SEQ ID NO.9, SEQ ID Shown in NO.10.

The present invention also provides a kind of using primer sets to vibrio parahemolyticus, Vibrio vulnificus, vibrio harveyi and mimicry arc The method that bacterium carries out multiplex PCR detection, which is characterized in that the detection by the detection primer of vibrio parahemolyticus to, Vibrio vulnificus It is anti-that the DNA of the detection primer pair of primer pair, the detection primer pair of vibrio harveyi and vibrio mimicus and sample to be tested carries out multiplex PCR It answers, electrophoretic analysis is carried out to PCR reaction products after reaction whether to determine in sample containing vibrio parahemolyticus, wound arc Bacterium, vibrio harveyi or vibrio mimicus；The upstream and downstream primer of the detection primer centering of the wherein described vibrio parahemolyticus is respectively provided with The nucleotide sequence as shown in SEQ ID NO.1, SEQ ID NO.2, the upstream and downstream of the detection primer centering of the Vibrio vulnificus Primer is respectively provided with the nucleotide sequence as shown in SEQ ID NO.3, SEQ ID NO.4, the detection primer of the vibrio harveyi The upstream and downstream primer of centering is respectively provided with the nucleotide sequence as shown in SEQ ID NO.5, SEQ ID NO.6, the mimicry arc The upstream and downstream primer of the detection primer centering of bacterium is respectively provided with the nucleotides sequence as shown in SEQ ID NO.7, SEQ ID NO.8 Row.

Further, when carrying out multi-PRC reaction, positive internal reference primer pair and corresponding is additionally added in reaction system Template.

Further, the nucleotide sequence of the upstream and downstream primer of the positive internal reference primer pair is respectively by SEQ ID Shown in NO.9, SEQ ID NO.10.

The comprehensive Rapid Screening vibrio parahemolyticus of the method energy, Vibrio vulnificus, vibrio harveyi and vibrio mimicus, technology Scheme is specific as follows：

1, primer synthesizes

According to the primer sequence in table 1, the oligonucleotide sequences of all primers are synthesized in gene chemical synthesis company.

2, DNA profiling extracts

Select vibrio parahemolyticus, Vibrio vulnificus, vibrio harveyi and vibrio mimicus micro- as the target for establishing detection method Biology carries out Zengjing Granule, using bacterial genomes according to national standard or industry standard methods to above-mentioned four kinds of microorganisms respectively Extracts kit extracts DNA profiling.

3, multiplex PCR detection architecture is built

In determining multi-PRC reaction system after primer sequence, add according to the M-PCR reaction systems (table 2) of following 20 μ l Enter reagent, mixing respectively refers to the PCR reaction conditions (table 3) through repeatedly groping optimization and carries out PCR reactions.

2. multi-PRC reaction system of table

3. multi-PRC reaction condition of table

4, the sequence analysis of multiplex PCR amplification product and result judgement

By 4ul multiple PCR products into row agarose gel electrophoresis analyze, multiplex PCR amplification product through electrophoresis gel at After observation result under instrument, the PCR product of positive amplification is sent into genetic test company and is sequenced, sequence assembly result is used After ChromasPro corrections, sequence comparison is carried out by the Blast of NCBI, occurs comparing score value, that is, the Max having in Blast The comparison result of the highest bacterial species of Ident values, and the sequencing result of 16SrDNA PCR is combined to be speculated as accordingly belonging to kind.

5, the verification of detection architecture

The multi-PCR detection method of four kinds of morbid vibrios of detection while being had built up as template verification using reference culture Specificity and validity.

Specificity verification：Comma bacillus, vibrio alginolyticus, vibrio fluvialis, Vibrio anguillarum is selected to be used as specific assessment bacterial strain, Nucleic acid extraction is carried out, the reaction condition established and optimized using early period is detected using detection reagent of the present invention.The result shows that IAC is the positive.In addition to IAC, specific band expands nothing but for negative control and specificity verification bacterial strain, repeats testing result It is all consistent, show that the detection method has preferable specificity, non-targeted bacterium can effectively be distinguished.

Sensibility is verified：It is 10 to initial concentration⁹Four kinds of object bacteria DNA of CFU/mL are diluted to 10 respectively⁸CFU/mL, 10⁷CFU/mL, 10⁶CFU/mL, 10⁵CFU/mL, 10⁴CFU/mL.The reaction condition established and optimized using early period is detected, The detection limit of each object bacteria can reach 10⁵CFU/mL, repetition testing result is all consistent, shows that the detection method has height The sensibility of degree and good repeatability.

The present invention also provides a kind of kits being used for while detecting four kinds of morbid vibrios, which is characterized in that the examination Agent box includes aforementioned primer sets, positive internal reference primer pair, positive internal reference template, Taq archaeal dna polymerases, reaction system buffering Liquid, dNTP and distilled water.

The beneficial effects of the present invention are：

1, Rapid identification Vibrio

The multiple PCR method that the present invention establishes can be identified vibrio parahemolyticus, Vibrio vulnificus, vibrio harveyi and be intended with a step Four kinds of morbid vibrios of state vibrios, and introduce the false negative of amplification interior label instruction PCR reactions, the side established compared with the existing technology Both the flux that detection had been increased for method, also ensures the accuracy of result, while having and saving human cost and time cost Advantage.

2, specificity and high sensitivity

The multiple PCR detection primer group of the present invention has high degree of specificity, and all primers all pass through Blast and compare analysis, Conservative with height and specificity；Pass through specificity verification simultaneously, can be good at distinguishing it is close with detection target species symbolic animal of the birth year, The identical bacterium of living environment, it was demonstrated that detection method has the specificity of height, can accurately distinguish non-targeted bacterium. The screening while detection method that the present invention is established can realize four kinds of object bacterias, the in the reaction system inspection of each object bacteria It surveys sensitivity and can reach 10⁵CFU/mL。

Description of the drawings

Fig. 1 is that four kinds of morbid vibrio multiplex PCRs detect agarose gel electrophoresis result in the embodiment of the present invention 3.

Wherein, 1：Negative control；2：Vibrio mimicus；3：Vibrio harveyi；4：Vibrio vulnificus；5：Vibrio parahemolyticus；6：Its His vibrios；M：250bp DNA ladder Marker.

Specific implementation mode

The embodiment of the present invention is described below in detail, the examples of the embodiments are intended to be used to explain the present invention, and cannot It is interpreted as limitation of the present invention.In the examples where no specific technique or condition is specified, according to described by document in the art Technology or condition or carried out according to product description.Reagents or instruments used without specified manufacturer is that can lead to Cross the conventional products of acquisition purchased in market.

The design of 1 primer of embodiment and screening (using vibrio parahemolyticus as representative)

According to the virulence gene for the vibrio parahemolyticus announced on GenBank and house-keeping gene sequence, in conjunction with related text Report is offered, the DNA fragmentation of the candidate amplification of vibrio parahemolyticus, i.e. the flaE genes of vibrio parahemolyticus is obtained, utilizes Oligo7 and Primer Primier5.0 software Design primers are screened in the high primer that scores, by appropriate manual Adjustment and modification, then carry out Blast analyses, if any of which primer by the design of primers scheme of acquisition on the websites NCBI Pair with non-targeted bacterium there are cross reaction, the position for readjusting primer and sequence length are needed, until obtaining high specific FlaE gene primer sequences.The final two kinds of primer alternatives obtained such as table 4.

When primer is screened in design, it should be noted that some once：

(5) end of primer 3 ' can not be modified, and the last bit base that primer 3 ' is held has the DNA combined coefficienies of Taq polymerase larger Influence.3 ' ends should select A, T, select G, C, this primer that can be effectively prevented from false initiation less as possible, have higher initiation to imitate Rate.3 or more continuous bases cannot occur before in primer sequence itself, therefore should avoid holding using wrong in primer 3 ' Hair probability is misquoted to increase.

The primer alternative of 4. vibrio parahemolyticus of table

Then substance PCR verifications are carried out to above-mentioned alternative, determines the availability of primer, including specificity assessment and quick Perception assessment：

The bacterium group of specificity assessment：Including the detection in three kinds of Vibrio vulnificus, vibrio harveyi and vibrio mimicus kits Object bacteria further includes, living environment identical bacterium close with detection target species symbolic animal of the birth year, such as comma bacillus, vibrio alginolyticus, river arc Bacterium, Vibrio anguillarum.

It is spare using bacterial genomes extracts kit acquisition DNA profiling to above-mentioned bacterium.The template of extraction is respectively taken into 1 μ l Mixing, the template as specificity verification.

The specificity analysis experiment of vibrio parahemolyticus：The primer pair completed using above-mentioned design is matched according to following project Set PCR reaction systems：10 × PCR buffer (100mM Tris-HCl buffer solutions (pH8.3), 15mM Mg2+, 500mM KCl) 0.5 μ l, dNTP (2.5mM) 1.6 μ l, each pair of 2 μ l of primer (10 μM) of 2 μ l, Taq archaeal dna polymerase (5U/ μ l), specificity 1 μ l of validating DNA template, distilled water are mended to 20 μ l.PCR amplification is carried out according to following reaction condition：94 DEG C of pre-degeneration 5min；94 DEG C denaturation 40s, 57 DEG C annealing 1min, 72 DEG C extension 1.5min, recycle 35 times；72 DEG C of extension 10min.

4 μ l of pcr amplification product are analyzed using 2% agarose gel electrophoresis, the results show that (being added secondary molten in positive control The primer and template of courageous and upright vibrios) set up under the premise of, two vibrio parahemolyticus primer flaE-1 and flaE-2 of design with The habitats such as Vibrio vulnificus, vibrio harveyi, vibrio mimicus, comma bacillus, vibrio alginolyticus, vibrio fluvialis, Vibrio anguillarum are similar, high frequency simultaneously The equal no cross reaction of bacterium deposited.It can thus be assumed that two primers flaE-1 and flaE-2 all have preferable specificity.

Sensitivity assessment：The template of 1 plant of vibrio parahemolyticus is selected, initial concentration is about 10⁹CFU/mL, gradient are dilute It releases to 10⁴CFU/mL, the template as flaE gene magnification primer sensitivity assessments.System configurations are as follows：10×PCR 2 μ l, Taq archaeal dna polymerases (5 of buffer (100mM Tris-HCl buffer solutions (pH8.3), 15mM Mg2+, 500mM KCl) U/ μ l) 0.5 μ l, dNTP (2.5mM) 1.6 μ l, each pair of 2 μ l of primer (10 μM), 1 μ l of vibrio parahemolyticus template, distilled water mend to 20μl.PCR reaction condition homospecificities are tested.

4 μ l of pcr amplification product are analyzed using 2% agarose gel electrophoresis, the results show that two primer pairs can reach 10⁵The band brightness ratio flaE-2 of CFU/mL, wherein primer pair flaE-1 are bright very much.

The assessment result of overall sensitivity and specificity, selects flaE-1 as the primer of flaE gene magnifications.

The design screening technique of the primer of other vibrios is identical as vibrio parahemolyticus.Primer is by Invitrogen companies Synthesis.

Embodiment 2 is used for while detecting the establishment of the multiple PCR detection kit of four kinds of morbid vibrios

Kit is by PCR reaction systems buffer solution (100mM Tris-HCl buffer solutions (pH8.3), 15mM Mg²⁺、500mM KCl), Taq archaeal dna polymerases (5U/ μ l), dNTP, primer mixed liquor (four kinds of morbid vibrio primers and IAC primer mixed liquors), (hybrid template of four kinds of morbid vibrios, each 10 for positive control template⁶CFU/ml), distilled water is constituted, the reactant of kit System is 20ul, and concrete configuration is as follows：

The configuration of 5. multiple PCR detection kit of table

Embodiment 3 detects kit multiplex PCR in the gel electrophoresis of plain agar sugar and tests

Detect sample：Select vibrio parahemolyticus, Vibrio vulnificus, vibrio harveyi and vibrio mimicus pathogen genome Templates of the DNA as experiment, the concentration of each template is about 10⁵CFU/ml。

Kit is set up：Using the kit in embodiment 2.

Kit operates：2mL bacterial suspensions extract template DNA according to bacterial genomes extracts kit specification.It will PCR pipe is put into Bio-Rad C1000 type PCR instruments, and after opening heat lid, PCR reactions are carried out according to following procedure：94 DEG C of pre-degenerations 5min；94 DEG C of denaturation 40s, 57 DEG C of annealing 1min, 72 DEG C extend 1.5min, recycle 35 times；72 DEG C of extension 10min.

It is as shown in Figure 1 that multiplex PCR detects test result.

As shown in Figure 1：The correspondence purpose band of multiplex PCR system reaction product is clear, and without miscellaneous band generate, show by The reaction condition and reacted constituent ratio repeatedly established after optimization are suitable, do not generate interference, five pairs of primer combination ginsengs between each other With multi-PRC reaction system have well specificity.According to the target fragment size after electrophoresis, (there is 121bp in the 2nd swimming lane There are 467bp amplified bands in amplified band, the 3rd swimming lane, and 310bp amplified bands occurs in the 4th swimming lane, and 897bp occurs in the 5th swimming lane Amplified band), in conjunction with the sequencing result of amplified production, logs in gene database and be compared, it is known that the 2nd swimming lane is mimicry arc Bacterium, the 3rd swimming lane is vibrio harveyi, and the 4th swimming lane is Vibrio vulnificus, and the 5th swimming lane is vibrio parahemolyticus.

The specific test of 4 kit of embodiment

Selection comma bacillus, vibrio alginolyticus, vibrio fluvialis, Vibrio anguillarum etc. are close with detection object bacteria kind, there are environment Similar bacterium only has an IAC bands amplification, no miscellaneous band generates, and is detected to object bacteria after testing as bacterium to be checked When, in addition to specific band, no miscellaneous band generates.Show that kit of the present invention can effectively distinguish non-targeted bacterium, has preferable Specificity.

The sensitivity tests of 5 kit of embodiment

Assessment detection sample：By the concentration of vibrio parahemolyticus, Vibrio vulnificus, vibrio harveyi and vibrio mimicus adjust to 10⁹The CFU/mL orders of magnitude extract four kinds of detection object bacteria DNA using bacterial genomes extracts kit.Each template gradient is dilute It is interpreted into 10⁸CFU/mL, 10⁷CFU/mL, 10⁶CFU/mL, 10⁵CFU/mL, 10⁴The detection sample of CFU/mL.

Detect the template of four object bacteria difference dilutions respectively using kit of the present invention.Sentenced according to 3 result of embodiment Disconnected method, the results are shown in Table 6 for the sensitivity tests of kit four kinds of object bacterias of detection：

6. kit of table detects four kinds of object bacteria sensitivity tests results

As seen from Table 6, the sensibility that kit detects four kinds of morbid vibrios is 10⁵CFU/mL。

Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example Property, it is not considered as limiting the invention, those skilled in the art are not departing from the principle of the present invention and objective In the case of can make changes, modifications, alterations, and variations to the above described embodiments within the scope of the invention.

110>University of Fuzhou

<120>Multiple PCR primer group and detection method and kit for four kinds of morbid vibrios of detection simultaneously

<130> SC-ZL20188412

<160> 10

<170> PatentIn version 3.3

<210> 1

<211> 22

<212> DNA

<213> Vp-F

<400> 1

gcagctgatc aaaacgttga gt 22

<210> 2

<211> 21

<212> DNA

<213> Vp-R

<400> 2

attatcgatc gtgccactca c 21

<210> 3

<211> 19

<212> DNA

<213> Vv-F

<400> 3

gtcttaaagc ggttgctgc 19

<210> 4

<211> 21

<212> DNA

<213> Vv-R

<400> 4

cgcttcaagt gctggtagaa g 21

<210> 5

<211> 22

<212> DNA

<213> Vh-F

<400> 5

gcaccatggg ttaaaagccg ct 22

<210> 6

<211> 25

<212> DNA

<213> Vh-R

<400> 6

agagaactga cgcacaacgt ggttc 25

<210> 7

<211> 20

<212> DNA

<213> Vm-F

<400> 7

cattcggttc tttcgctgat 20

<210> 8

<211> 24

<212> DNA

<213> Vm-R

<400> 8

gaagtgttag tgattgctag agat 24

<210> 9

<211> 19

<212> DNA

<213> V16s-F

<400> 9

cggtgaaatg cgtagagat 19

<210> 10

<211> 20

<212> DNA

<213> V16s-R

<400> 10

ttactagcga ttccgagttc 20

Claims

1. a kind of multiple PCR primer group being used for while detecting four kinds of morbid vibrios, which is characterized in that it includes the secondary haemolysis of detection Property vibrios, Vibrio vulnificus, vibrio harveyi and vibrio mimicus primer pair；

The nucleotide sequence of the upstream and downstream primer of the primer pair of the detection vibrio parahemolyticus is respectively by SEQ ID NO.1, SEQ Shown in ID NO.2；

The nucleotide sequence of the upstream and downstream primer of the primer pair of the detection Vibrio vulnificus is respectively by SEQ ID NO.3, SEQ ID Shown in NO.4；

The nucleotide sequence of the upstream and downstream primer of the primer pair of the detection vibrio harveyi is respectively by SEQ ID NO.5, SEQ ID Shown in NO.6；

The nucleotide sequence of the upstream and downstream primer of the primer pair of the detection vibrio mimicus is respectively by SEQ ID NO.7, SEQ ID Shown in NO.8.

2. the multiple PCR primer group according to claim 1 being used for while detecting four kinds of morbid vibrios, which is characterized in that Also include positive internal reference primer pair, the nucleotide sequence of the upstream and downstream primer of the positive internal reference primer pair is respectively by SEQ Shown in ID NO.9, SEQ ID NO.10.

3. a kind of carrying out multiplex PCR detection using primer sets to vibrio parahemolyticus, Vibrio vulnificus, vibrio harveyi and vibrio mimicus Method, which is characterized in that by the detection primer of vibrio parahemolyticus to the detection primer of, Vibrio vulnificus to, vibrio harveyi The DNA of detection primer pair and the detection primer pair of vibrio mimicus and sample to be tested carries out multi-PRC reaction, right after reaction PCR reaction products carry out electrophoretic analysis whether to determine in sample containing vibrio parahemolyticus, Vibrio vulnificus, vibrio harveyi or quasi- State vibrios；The upstream and downstream primer of the detection primer centering of the wherein described vibrio parahemolyticus be respectively provided with as SEQ ID NO.1, Nucleotide sequence shown in SEQ ID NO.2, the upstream and downstream primer of the detection primer centering of the Vibrio vulnificus be respectively provided with as The upstream and downstream of nucleotide sequence shown in SEQ ID NO.3, SEQ ID NO.4, the detection primer centering of the vibrio harveyi is drawn Object is respectively provided with the nucleotide sequence as shown in SEQ ID NO.5, SEQ ID NO.6, the detection primer pair of the vibrio mimicus In upstream and downstream primer be respectively provided with the nucleotide sequence as shown in SEQ ID NO.7, SEQ ID NO.8.

4. according to the method described in claim 3, it is characterized in that, when carrying out multi-PRC reaction, it is additionally added in reaction system Positive internal reference primer pair.

5. according to the method described in claim 4, it is characterized in that, the core of the upstream and downstream primer of the positive internal reference primer pair Nucleotide sequence is respectively shown in SEQ ID NO.9, SEQ ID NO.10.

6. according to claim 3-5 any one of them methods, which is characterized in that the multi-PRC reaction according to the following steps into Row：

(1) 94 DEG C of pre-degeneration 5min；

(2) 94 DEG C of denaturation 40s, 57 DEG C of annealing 1min, 72 DEG C extend 1.5min, recycle 35 times；

(3) 72 DEG C of extension 10min.

7. a kind of kit being used for while detecting four kinds of morbid vibrios, which is characterized in that the kit includes claim 1 The primer sets, positive internal reference primer pair, positive internal reference template, Taq DNA polymerase, reaction system buffer solution, dNTP And distilled water.