CN104017877A - Vibrio parahaemolyticus molecule detection method and primer pair used by same - Google Patents
Vibrio parahaemolyticus molecule detection method and primer pair used by same Download PDFInfo
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Abstract
The invention discloses a Vibrio parahaemolyticus molecule detection method and a primer pair used by the same. The specific primer pair for Vibrio parahaemolyticus molecule detection comprise a forward primer toxRS-F disclosed as SEQ ID NO.1 and a reverse primer toxRS-R disclosed as SEQ ID NO.2.According to the Vibrio parahaemolyticus molecule detection method, the specific primers are utilized to perform PCR (polymerase chain reaction) amplification on the DNA (deoxyribonucleic acid) of the sample to be detected, and the implementation of the amplification to the 679bp segment indicates that the sample to be detected contains the Vibrio parahaemolyticus. The method is simple to operate, has the advantages of short detection period, favorable specificity, low detection limit and accurate and stable detection result, is suitable for the demands for food microbe inspection development, and has higher application and popularization values.
Description
Technical field
The invention belongs to field of biological detection, relate to the primer pair of a kind of Vibrio parahemolyticus molecular detecting method and use.
Background technology
Vibrio parahemolyticus (vibrio parahaemolyticus, Vp) be a kind of Gram-negative halophilism bacterium, belong to vibrionaceae (Vibrio) Vibrio, be mainly present in the sea-foods such as inshore seawater, marine bottom sediment and fish, shrimps, shellfish, oyster.People Duo Yin is edible to be caused poisoning by this fungi pollution and the sea-food that do not boil.China has Vibrio parahemolyticus to cause the report of food poisoning every year, and world's sickness rate is in rising trend in recent years.At present, the traditional detection method for Vibrio parahaemolyticus will pass through the processes such as front increasing bacterium, selective enrichment, selectivity cultivation, biochemical identification, Kahagawa phenomenon and serological reaction mostly.These experimental implementation are loaded down with trivial details, need 5-6 days consuming time, to sea-food production, inspection and quarantining for import/export, Food Hygiene Surveillance etc., bring difficulty, affect Economic development.At present, scholar is to the method for the Molecular Detection of Vibrio parahaemolyticus mostly based on genes such as tlh, tdh, gyrb, pr72H, but large multi-method by testing data afterwards negated, and shortage can be by the molecular detecting method of public acceptance now.Therefore it is extremely urgent to set up a kind of easy, special and rigorous fast molecular detection technology.
Summary of the invention
The object of the invention is the above-mentioned deficiency for prior art, a pair of Auele Specific Primer pair for Vibrio parahemolyticus Molecular Detection is provided.
Another object of the present invention is to provide the application of this primer pair.
Another object of the present invention is to provide a kind of Vibrio parahemolyticus molecular detecting method.
Object of the present invention can be achieved through the following technical solutions:
The toxRS gene of the foundation of design of primers of the present invention and molecular detecting method based on Vibrio parahemolyticus.ToxRS, toxR and toxS, be the toxin operon gene of vibrios, and toxS gene order is positioned at toxR downstream, and both consecutive genes are collectively referred to as toxRS.Variable region design primer based on this gene, sets up the molecular detecting method of Vibrio parahaemolyticus.
The a pair of Auele Specific Primer pair for Vibrio parahemolyticus Molecular Detection, upstream primer toxRS-F is SEQ ID NO.1, downstream primer toxRS-R is SEQ ID NO.2.
The application of primer pair of the present invention in the detection reagent of preparation detection Vibrio parahemolyticus.
For a test kit for Vibrio parahemolyticus Molecular Detection, comprise Auele Specific Primer pair of the present invention.
Test kit of the present invention, also comprises Taq PCR Master Mix.
The application of primer pair of the present invention in detecting Vibrio parahemolyticus.
A Vibrio parahemolyticus molecular detecting method, the DNA of the Auele Specific Primer pcr amplification sample to be checked described in using, obtains the fragment of 679bp if can increase, and illustrates in sample to be checked and contains Vibrio parahemolyticus.
The reaction system of described pcr amplification is 25 μ l:2 * Taq PCR Master Mix12.5 μ l, dd H
2o9.5 μ l, upstream, each 1 μ l of downstream primer, DNA profiling 1 μ l; Response procedures is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 59 ℃ of annealing 30s, 72 ℃ are extended 45s, totally 30 circulations, last 72 ℃ are extended 10min.
Beneficial effect:
The foundation of Vibrio parahemolyticus PCR detection method, can not there is false positive in requirement---be strict species specificity, can not occur undetected---repeatability is good, thus to select species specific target gene, and get rid of special serotype, hypotype and virulence gene sequence.The present invention, on the basis with reference to lot of documents and analyzing gene sequence, gets rid of virulence gene, hypotype and serotype specific gene, the species specificity sequence that locking Vibrio parahaemolyticus may exist.It is of equal importance that the specificity of primer and target gene specific plays a part, and for detection specificity and the sensitivity of Vibrio parahemolyticus, has great impact.The present invention is through repeatedly comparing and screening, determined and usingd the 579-1258 base of toxRS gene (GenBank numbers L11929.1) as target sequence, and designed Auele Specific Primer to toxRS-F/toxRS-R, from theoretical aspect, guarantee that present method has very high specificity.
2 * Taq PCR Master Mix substitutes 10 * buffer, MgCl2, dNTP, the Taq enzyme in normal PCR system for this test, time saving and energy saving and reduce operate miss.In PCR program, higher annealing temperature can improve the specificity of primer to a certain extent, but do not represent more high better.Therefore Ying Neng follows on the good basis of specificity, repeatability, susceptibility, selects the highest annealing temperature.Experiment results proved, 59 ℃ is the suitableeest annealing temperature.Final definite PCR program parameter is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 59 ℃ of annealing 30s, 72 ℃ are extended 45s, totally 30 circulations, last 72 ℃ are extended 10min.
Detection through 51 strain Vibrio parahemolyticus, the close bacterium of 10 strain relationship and 9 strain food-borne pathogenic bacteriums shows, primer pair pcr amplification of the present invention has good Vibrio parahaemolyticus specificity and repeatability, does not find that the food-borne pathogens such as the close bacterium of the relationships such as vibrio cholerae, vibrio fluvialis and intestinal bacteria, streptococcus aureus, salmonella amplify any band.Through test, the limit of detection of present method can reach 2pg/ μ l, has the extremely positive gene group of trace can detect positive findings.The bacterium of pure culture, qualification time only needs 3-4h, is significantly shorter than routine inspection method.Show thus, the inventive method is easy and simple to handle, and sense cycle is short, and specificity is good, and detection limit is low, and detected result is accurate, stable, adapts to the needs of food microorganisms check development, has higher use promotional value.
Accompanying drawing explanation
Fig. 1 GB4789.7-2013 < < food safety national standard food microbiological analysis Vibrio parahemolyticus check > > Vibrio parahemolyticus isolation identification basic procedure.
Fig. 2 thermograde PCR result
M:Marker，1：58℃，2：59℃，3：60℃，4：61℃，5：62℃，6：63℃，7：64℃。
The close bacterium pcr amplification of Fig. 3 relationship result
M:Marker, 1: Vibrio parahemolyticus (ATCC33847), 2: vibrio alginolyticus, 3: vibrio cholerae, 4: Vibrio vulnificus, 5: Vibrio mimicus, 6: Vibrio harveyi, 7: vibrio fluvialis, 8: Fu Shi vibrios, 9: Aeromonas hydrophila, 10: Pseudomonas fluorescens, 11: Pseudomonas aeruginosa, 12: negative control (without template)
Fig. 4 food-borne pathogens simultaneous test
M:Marker, 1: Vibrio parahemolyticus (ATCC33847), 2: produce monokaryon Listeria monocytogenes, 3: intestinal bacteria, 4: Salmonellas, 5: enterococcus faecalis, 6: Shigella flexneri, 7: streptococcus aureus, 8: Proteus mirabilis, 9: clostridium perfringens,
10: pasteurella multocida, 11: negative control (without template)
The pcr amplification of Fig. 5 Vibrio parahaemolyticus strain isolated
M:Marker, 1: Vibrio parahemolyticus (ATCC33847), 2:Vp1,3:Vp2,4:Vp3,5:Vp4,6:Vp5,7:Vp6,8:Vp7,9:Vp8,10:Vp9,11Vp10,12:Vp117,13:Vp12,14:Vp13,15:Vp14,16:Vp15,17:Vp16,18:Vp17,19:Vp18,20:Vp19,21:Vp20,22:Vp21,23:Vp22,24:Vp23,25:Vp24,26:Vp27; 27:Vp26,28:Vp27,29:Vp28,30:Vp29,31:Vp30,32:Vp31,33:Vp32,34:Vp33,35:Vp34,36:Vp35,37:Vp36,38:Vp37,39:Vp38,40:Vp39,41:Vp40,42:Vp41,43:Vp42,44:Vp43,45:Vp44,46:Vp45,47:Vp46,48:Vp47,49:Vp48,50:Vp49,51:Vp50,52:Vp51,53: negative control (without template)
The pcr amplification of Fig. 6 different templates amount
M:Marker, 1:20ng, 2:2ng, 3:200pg, 4:20pg, 5:2pg, 6: negative control (without template)
Embodiment
1 test materials
1.1 bacterial strain
Bacterial strain is used in this test: Vibrio parahemolyticus Reference Strains ATCC33847, Vibrio parahemolyticus sample separation strain 51 strains (details are in Table 1), vibrio alginolyticus (ATCC17749), vibrio fluvialis (ATCC33809), Fu Shi vibrios (ATCC33841), the false unit cell (ATCC17571) of fluorescence, Shigella flexneri is purchased from Chinese microorganism strain preservation center, Vibrio vulnificus (ATCC27562), Vibrio mimicus is purchased from Chinese industrial microbial strains preservation center, non-O1 vibrio cholerae, Pseudomonas aeruginosa (ATCC27853) is purchased from Guangdong Province food microorganisms safety engineering research and development centre, produce monokaryon Listeria monocytogenes, streptococcus aureus (ATCC29213), intestinal bacteria, enterococcus faecalis (ATCC29212), Proteus mirabilis, Salmonella enteritidis, Vibrio harveyi, clostridium perfringens, Aeromonas hydrophila, killing property Pasteur bar more.
1.2 main agents and instrument
DNA of bacteria is extracted test kit purchased from Tian Gen bio tech ltd, and 2 * Taq PCR Master Mix is purchased from Nanjing Nuo Weizan Bioisystech Co., Ltd, dd H
2o, DNA Marker are purchased from Dalian precious biotechnology company limited, primer is synthetic by the synthetic portion of Beijing ancient cooking vessel state primer, agar Icing Sugar, TAE damping fluid, thiosulfuric acid saline citrate cholate sucrose agar (TCBS) substratum, the good vibrios substratum of Kerma (unit of kinetic energy) are purchased from the good company of Shanghai Kerma (unit of kinetic energy), Mei Liai full-automatic biochemical test board is purchased from Mei Liai Chinese companies, and GoldView nucleic acid staining agent etc. are purchased from the prosperous Bioisystech Co., Ltd of Beijing ancient cooking vessel state.Instrument mainly contains S1000TM ThermalCycler gene-amplificative instrament, gel imaging system (biorad), the full-automatic biological assay system of Mei Liai etc.
2 methods
The separation of 2.1 Vibrio parahemolyticus
This test is carried out isolation identification with reference to standard GB/T/T4789.7-2008 < < microbiological test of food hygiene Vibrio parahaemolyticus check > > and GB4789.7-2013 < < food safety national standard food microbiological analysis Vibrio parahemolyticus check > > (as Fig. 1) to the Vibrio parahaemolyticus in sample.This detection method need to pass through front increasing bacterium, increases bacterium, select the steps such as plate isolation, biochemical test, kanagawa phenomenon and serological typing.The Vibrio parahaemolyticus sample separation strain relating in following examples is in Table 1.
The strain of table 1 Vibrio parahaemolyticus sample separation
2.2 bacterial genomes are extracted
Get inoculum 5ml, centrifugal 1 minute of 10000rpm, abandons supernatant; For Gram-negative bacteria, to adding the thalline that suspends after 200 μ l damping fluid GA in bacterial sediment, for gram-positive microorganism, add the N,O-Diacetylmuramidase (damping fluid: 20mM Tris, pH8.0 that 180 μ l concentration are 20mg/ml; 2mM Na
2-EDTA; 1.2%Trition), process more than 30 minutes for 37 ℃; Add 4 μ l RNaseA (100mg/ml) solution, vibrate 15 seconds, room temperature is placed 5 minutes; Add 20 μ l Proteinase Ks, mix; Add 220 μ l damping fluid GB, vibrate 15 minutes, place 10 minutes for 70 ℃; Add 220 μ l dehydrated alcohols, fully vibrate 15 seconds; Liquid in pipe and flocks are added to (adsorption column is put into collection tube) in adsorption column, and centrifugal 30 seconds of 12000rpm, abandons waste liquid; To adsorption column, add 500 μ l damping fluid GD, centrifugal 30 seconds of 12000rpm, abandons waste liquid; In adsorption column, add 600 μ l rinsing liquid PW, centrifugal 30 seconds of 12000rpn, abandons waste liquid; Repeat previous action; Adsorption column is put back in collection tube, and centrifugal 2 minutes of 12000rpm, abandons waste liquid, and adsorption column room temperature is placed several minutes; Adsorption column is proceeded in clean centrifuge tube, to the middle part of adsorption film, drip 100 μ l elution buffer TE, room temperature is placed 2-5 minute, and centrifugal 2 minutes of 12000rpm, collects solution in centrifuge tube ,-20 ℃ of preservations.
2.3 design of primers
According to the research of the conservative gene of Vibrio parahaemolyticus and virulence gene, first selected several species specificity genes that may exist, from GenBank downloads corresponding gene sequences, log in NCBI BLAST website, by gene order successively BLAST, intercepting and vibrio parahaemolyticus gene 100% homology, and with other kind of bacterium homology gene order that is 0, thereby set up the Vibrio parahaemolyticus species specificity gene pool that may exist.In the gene pool finding, choose the gene fragment that length is 500bp-1500bp from aforesaid method, import in Primer Premier5.0 software and design corresponding primer, every fragment gene should design 3-5 pairs of primers.Primer is imported to olige7 its specificity is evaluated, choose the higher primer of scoring and test.
Pass through aforesaid method, finally choose the variable region gene of the toxRS complete sequence (GenBank numbers L11929.1) of Vibrio parahaemolyticus, based on this sequence, use Primer5.0 design primer, and by Oligo7, primer is evaluated, finally choose upstream primer toxRS-F:5 '-CGTAGAGCCGTCTTTAGC-3 ' (SEQ ID NO.1), downstream primer toxRS-R:5 '-AATCGCCATTCGGTAGGT-3 ' (SEQ ID NO.2) tests.Object band is 679bp, and position is the 579-1258 base of toxRS complete sequence (GenBank numbers L11929.1).
Embodiment 1
The genome of the positive Reference Strains of the Vibrio parahemolyticus of take (ATCC33847) carries out pcr amplification as template.Reaction system is 25 μ l:2 * Taq PCR Master Mix12.5 μ l, dd H
2o9.5 μ l, upstream, each 1 μ l of downstream primer, DNA profiling 1 μ l.Parameter: 94 ℃ of denaturation 5min, 94 ℃ of sex change 30s, 58-64 ℃ of annealing 30s, 72 ℃ are extended 45s, totally 30 circulations, last 72 ℃ are extended 10min.Pcr amplification product is carried out to agarose gel electrophoresis, get 7 μ l amplified productions and be added in the loading hole of 0.1% sepharose, electric current 150mA, electrophoresis 20min, observations under gel imaging system.The corresponding annealing temperature of PCR product that band is brighter, is the suitableeest possible annealing temperature.By the suitableeest possible annealing temperature from high to low, carry out successively specific test, replica test, susceptibility test, finally determine optimum temperuture.
Take respectively 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃ the genome of the positive Reference Strains of Vibrio parahemolyticus (ATCC33847) is increased as template as annealing temperature, 58 ℃ of-62 ℃ of amplified productions as annealing temperature are take in discovery, and what agarose gel electrophoresis all demonstrated near 679bp is single compared with bright wisp band (Fig. 2).
Embodiment 2 specific tests
The genome of take respectively the bacterium close with Vibrio parahaemolyticus relationship such as vibrio alginolyticus, non-O1 type vibrio cholerae and producing the food-borne pathogens such as monokaryon Listeria monocytogenes, intestinal bacteria carries out pcr amplification as template, the positive and negative control group are set simultaneously, positive controls template is Vibrio parahemolyticus ATCC33847, and negative control group is without template.Reaction system and program are the same.
The close bacterial strain simultaneous test of 2.1 relationship
The genome of vibrio alginolyticus, non-O1 type vibrio cholerae, Vibrio vulnificus, Vibrio mimicus, Vibrio harveyi, vibrio fluvialis, Fu Shi vibrios, Aeromonas hydrophila, Pseudomonas fluorescens, Pseudomonas aeruginosa of take is respectively template, carry out pcr amplification, product is through agarose gel electrophoresis, result shows (Fig. 3), only there is the positive Reference Strains of Vibrio parahemolyticus near 679bp, to demonstrate single compared with bright wisp band, other 10 kinds of close bacteriums of relationship all do not amplify any band, and demonstrating primer pair Vibrio parahemolyticus has good species specificity.
2.2 food-borne pathogens simultaneous tests
Respectively to produce monokaryon Listeria monocytogenes, intestinal bacteria, Salmonella enteritidis, enterococcus faecalis, Shigella flexneri, streptococcus aureus, Proteus mirabilis, clostridium perfringens, the genome of pasteurella multocida is template, carry out pcr amplification, product is through agarose gel electrophoresis, result shows (Fig. 4), only there is the positive Reference Strains of Vibrio parahemolyticus near 679bp, to demonstrate band, other 9 kinds of food-borne pathogens all do not amplify any band, show thus, during food borne bacteria PCR differentiates and identifies, primer pair Vibrio parahemolyticus has good specificity.
Embodiment 3 replica tests
The Vibrio parahemolyticus of 51 strain environment separation is carried out to replica test, to determine that PCR system and program can be reliably effective to the detection of all positive strains.The genome of the positive isolated strains of template arranges the positive and negative control simultaneously, and positive controls template is Vibrio parahemolyticus ATCC33847, and negative control group is without template.Reaction system and program are the same.
51 strain Vibrio parahemolyticus are carried out to pcr amplification, agarose gel electrophoresis shows (Fig. 5), when annealing temperature is 58 ℃ and 59 ℃, it is single compared with bright wisp band that 51 strain Vibrio parahemolyticus PCR products all demonstrate near 679bp, therefore annealing temperature in program is defined as higher 59 ℃.Meanwhile, 51 strain Vibrio parahemolyticus all amplify positive band, demonstrate the general applicability of primer pair Vibrio parahaemolyticus, show that primer pair is good to Vibrio parahemolyticus repeatability.
Embodiment 4 susceptibility tests
Genome by certain positive Reference Strains of concentration gradient dilution Vibrio parahemolyticus, carries out pcr amplification, to determine the susceptibility of primer, determines the minimum template amount that can amplify positive band.Reaction system and program are the same.
By concentration, be 686.8ng/ μ l, the Vibrio parahemolyticus ATCC33847 genome of OD (260)/OD (280)=1.92 dilutes respectively for 10.0ng/ μ l, 20.0ng/ μ l, 30.0ng/ μ l, 40.0ng/ μ l, 50.0ng/ μ l, 60.0ng/ μ l, 70.0ng/ μ l, 80.0ng/ μ l, 90.0ng/ μ l, each concentration is according to 4 extent of dilution of 10 doubling dilution, and the dilution gradient of 9 groups of concentration is: 104pg/ μ l, 103ng/ μ l, 102pg/ μ l, 10pg/ μ l, 1pg/ μ l.The genome of 9 groups of different concns of take carries out pcr amplification as template.Product is through agarose gel electrophoresis, and result shows (Fig. 6), and the minimum extent of dilution of 20.0ng/ μ l dilution group demonstrates good susceptibility.When positive template amount is 2pg, can record in test positive findings, show thus, primer pair Vibrio parahemolyticus has good susceptibility.
Embodiment 5
By the corresponding PCR product order-checking of the positive band of above-described embodiment, utilize blast program that the sequence in sequencing result and database is compared.Result shows, the sequence recording and former sequence 100% homology, and without sudden change.The sequence that shows PCR positive products all comes from Vibrio parahaemolyticus toxRS, and the bacterial strain of PCR positive findings is Vibrio parahemolyticus.
Claims (7)
1. a pair of Auele Specific Primer pair for Vibrio parahemolyticus Molecular Detection, is characterized in that upstream primer toxRS-F is SEQ ID NO.1, and downstream primer toxRS-R is SEQ ID NO.2.
2. the application of primer pair claimed in claim 1 in the detection reagent of preparation detection Vibrio parahemolyticus.
3. for a test kit for Vibrio parahemolyticus Molecular Detection, it is characterized in that comprising Auele Specific Primer pair claimed in claim 1.
4. test kit according to claim 3, is characterized in that also comprising Taq PCR Master Mix.
5. the application of primer pair claimed in claim 1 in detecting Vibrio parahemolyticus.
6. a Vibrio parahemolyticus molecular detecting method, is characterized in that right to use requires the DNA of the Auele Specific Primer pcr amplification sample to be checked described in 1, if can increase, obtains the fragment of 679bp, illustrates in sample to be checked and contains Vibrio parahemolyticus.
7. molecular detecting method according to claim 6, the reaction system that it is characterized in that described pcr amplification is 25 μ l:2 * Taq PCR Master Mix12.5 μ l, dd H
2o9.5 μ l, upstream, each 1 μ l of downstream primer, DNA profiling 1 μ l; Response procedures is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 59 ℃ of annealing 30s, 72 ℃ are extended 45s, totally 30 circulations, last 72 ℃ are extended 10min.
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| WO2022095922A1 (en) * | 2020-11-05 | 2022-05-12 | Becton, Dickinson And Company | Rapid identification and typing of vibrio parahaemolyticus |
| CN112646907A (en) * | 2020-12-30 | 2021-04-13 | 广东省微生物研究所（广东省微生物分析检测中心） | Vibrio parahaemolyticus standard strain containing specific molecular target and detection and application thereof |
| CN112646907B (en) * | 2020-12-30 | 2022-05-20 | 广东省微生物研究所（广东省微生物分析检测中心） | Vibrio parahaemolyticus standard strain containing specific molecular target and detection and application thereof |
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