CN105821133A - Kit for simultaneously detecting Vibrio parahaemolyticus, Escherichia coli O157:H7, Salmonella and Shigella - Google Patents
Kit for simultaneously detecting Vibrio parahaemolyticus, Escherichia coli O157:H7, Salmonella and Shigella Download PDFInfo
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Abstract
The invention relates to a multiplex amplification internal standard PCR (polymerase chain reaction) kit capable of simultaneously detecting Vibrio parahaemolyticus, Escherichia coli O157:H7, Salmonella and Shigella. The kit comprises a PCR reaction premix solution and a reference substance. The detection method comprises the following steps: acquiring a sample, carrying out amplification culture, extracting DNA (deoxyribonucleic acid), preparing a PCR reaction tube, putting the PCR reaction tube on a PCR instrument, carrying out PCR amplification, and carrying out detection result judgment on the PCR amplification product. The kit can simultaneously and quickly detect the four bacteria by one reaction, and can avoid the misjudgment caused by the false negative result due to the inhibiting factor for the DNA polymerase in the sample treatment process. Compared with the existing detection methods, the method provided by the invention is convenient to use, has the advantages of high reliability, short detection period, high sensitivity, high specificity, low cost and simple operation steps, is suitable for high-flux operation and standard operation, and is beneficial to enhancing the food safety detection level and preventing and controlling the foodborne diseases.
Description
Technical field
The present invention relates to biotechnology, particularly relate to one and can detect the test kit of the multiple system of vibrio parahaemolytious, Escherichia coli O 157: H7, Salmonella and shigella simultaneously, can carry out the sample containing above 4 kinds of antibacterials differentiating detection simultaneously.
Background technology
Food origin disease (foodbornedisease) covering scope widely, is all serious public health problem in worldwide developed country or developing country.Monitoring Data shows, 2011, just has 1 people that food origin disease once occurred in every 6.5 people of China.The global incidence of food origin disease is difficult to estimate, but it was reported, within only 2005, just has 1,800,000 people to die from diarrhoeal diseases.The major part of these cases is attributable to food and drinking water pollution, and food-borne pathogens is main root.
On JIUYUE 25th, 2012, Ministry of Public Health discloses " pathogenic bacterium limitation in food " exposure draft, it is proposed that the limitation requirement in many based foods of the Main Pathogenic Bacteria such as Salmonella, escherichia coli, announces that this standard will issue execution in latter 6 months formal simultaneously.Vibrio parahaemolytious, Escherichia coli O 157: H7, Salmonella and shigella are several modal food-borne pathogens, they can directly or indirectly contaminated food products and water source, people's peroral infection may result in the popular of the generation of infectious intestinal disease and alimentary toxicosis and poultry infectious disease.At present, the common method to this several frequently seen food-borne pathogens mainly has the detection of traditional biological method, colloidal gold strip, Enzyme-linked Immunosorbent Assay (ELISA) etc..Traditional biological method is the most long, detecting step is more complicated, colloidal gold strip detection price comparison is expensive, detection sensitivity is on the low side, enzyme linked immunosorbent assay (ELISA) is expensive, operation needs certain technology, it is thus desirable to set up a kind of simple to operate, quick, sensitive, special detection method, particularly to the high flux of a large amount of samples, quickly, detect simultaneously.Multiple PCR method be a kind of simple to operate, sensitivity is high, the molecular biology for detection of high specificity, in addition to the advantage with regular-PCR, the different genes of interest fragments of two parts or many parts DNA samples can also be expanded in a reaction system simultaneously, be particularly suitable for the quick diagnosis of the multiple system of food-borne pathogens.The present invention adds again pair for amplification interior label primer on the basis of multiple PCR method, can effectively getting rid of because sample handling processes existing the former results thus resulted in such as the material inhibited to DNA polymerase activity or operational error present false negative, the accuracy of testing result can be improved.
Summary of the invention
It is an object of the invention to provide the test kit that one can detect the multiple system containing amplification interior label of vibrio parahaemolytious, Escherichia coli O 157: H7, Salmonella and shigella simultaneously, its advantage be simple to operate, sensitivity is high, high specificity, it is suitable for high-throughout quick detection, it is possible to avoid the erroneous judgement caused because the inhibitive factor that there is archaeal dna polymerase in sample handling processes obtains false negative result.
The technical solution of the present invention is: one detects vibrio parahaemolytious, Escherichia coli O 157: H7, Salmonella and the test kit of 4 kinds of common food-borne pathogens of shigella simultaneously, and it is characterized in that
This test kit comprises: sterile deionized water, PCR reaction premixed liquid, reference substance and DNA dyestuff；
Described PCR reaction premixed liquid contains PCR reaction buffer, dideoxyribonucleotide triphosphate, Taq DNA polymerase, detection primer；
In described PCR reaction buffer, the concentration containing Mg2+ is 0.8mM, and the concentration of dideoxyribonucleotide triphosphate is 0.12mM, and the concentration of Taq DNA polymerase is 0.03U/ μ l；
Described detection primer is that the mixture of following 10 primers, its sequence and product sheet segment length are respectively as follows:
Amplification interior label 27F sequence is: AGAGTTTGATCCTGGCTCAG, 1492R sequence is: TACGGYTACCTTTGTTACGACTT, amplified production fragment length about 1500bp (see sequence table 11,16,17 and 18)；
Vibrio parahaemolytious aadSF sequence is: GGAATTCGCAGTGATCGCCGCTTGAG, aadSR sequence is: CGCGTCGACAGCATCGCGGTTATAGG, and amplified production fragment length is 1164bp (see sequence table 12)；
Escherichia coli O 157: H7rfbEF sequence is: CTACTGTAAGTAATGGAACGGTTGC, rfbER sequence is: TGCGGTCCTAGTTAGAATTGAGACC, amplified production fragment length is 713bp (see sequence table 13)；
Salmonella invAF sequence is: GGAACGAACTAATTCAGCGATA, invAR sequence is: AGATGTCATTAACCTTGTGGAG, and amplified production fragment length is 435bp (see sequence table 14)；
Shigella ipaHF sequence is: GAATTTACGGACTGGTTCTCCCTCTGG, ipaHR sequence is: GTGATTATGAATGGTGCAGTCGTGAGC, and amplified production fragment length is 298bp (see sequence table 15)；
Described reference substance is divided into negative controls and positive reference substance, negative controls template to be sterile deionized water, and positive reference substance template is the plasmon mixture obtained after each pair of corresponding amplified production of primer is connected to carrier T.
One detects vibrio parahaemolytious, Escherichia coli O 157: H7, Salmonella, the test kit of 4 kinds of common food-borne pathogens of shigella simultaneously, and method is:
(1) sample collecting: aseptic collection suspicious milk and milk products sample；
(2) Zengjing Granule: the suspicious milk and milk products sample mix uniformly rear sterile sampling that will gather, liquid milk and milk products is the most aseptic to measure, milk in solid form goods put into sterile mortar, tissue grinder or homogenizing bag homogenizing 2～5min, add the sterile vegetative broth bouillon being preheated to 45 DEG C, 37 ± 1 DEG C of shaken cultivation 6～8h, obtain enrichment liquid；
null(3) DNA extraction: take the enrichment liquid after cultivation，Vibration mixing，Take 1ml and 1000～1500r/mim be centrifuged 1～2min in centrifuge tube，Remove relatively large fragment，Take supernatant 8000～12000r/mim and be centrifuged 1～2min，Incline supernatant，It is inverted in positions different in absorbent paper，Blot residual liquid，Resuspended with sterile deionized water again、Washing precipitation，8000～12000r/mim are centrifuged 1～2min，Abandon supernatant，By the resuspended precipitation of a small amount of sterile deionized water，Boiling water bath 5～10min，Ice bath 5～10min，1000～5000r/mim are centrifuged 5～10min，Supernatant is sample template DNA，Adjust concentration standby to 10～30ng/ μ l，Or-20 DEG C of preservations；
(4) prepared by PCR reaction tube: all operations is both needed to carry out on ice；First PCR is reacted premixed liquid and be placed in thawed on ice, draw 50 μ l and be placed in PCR reaction tube, add 1 μ l sample template DNA, flick the PCR reaction tube prepared with finger or mix with micropipettor piping and druming, instantaneous high speed centrifugation 2s；PCR reaction tube is prepared respectively according to negative controls, actually detected sample, the order of positive reference substance；
(5) augmentation detection: be placed in PCR instrument by the PCR reaction tube of the negative controls of preparation, sample template DNA and positive reference substance, carry out PCR amplification, response procedures is: 95 DEG C of denaturations 5min；95 DEG C of degeneration 40s, 56.7 DEG C of annealing 40s, 72 DEG C extend 70s, carry out 35 circulations；72 DEG C extend 10min, 4 DEG C of preservations.Amplification obtains pcr amplification product and carries out sepharose electrophoresis, it is determined that testing result；
(6) testing result judges: being dissolved in 0.5 × TBE electrophoretic buffer of 100ml by 1g agarose, be subsequently adding 5 μ lDNA dyestuffs, mixing is boiled, and topples over gel slab；After gel cooled and solidified, draw in PCR primer 5 μ l addition gel pore with micropipettor and carry out electrophoresis；Electrophoresis takes out gel and is placed under uviol lamp and observes, take pictures after terminating, it is determined that result: the gel pore of (a) positive control have 5 electrophoretic bands and the gel pore of negative control without any band, then PCR reaction detection success；If b () negative control has band, illustrating there is cross-contamination in PCR reaction tube preparation process, result is unreliable, it is proposed that re-start PCR detection；C () positive control has 5 bands, negative control does not has band, and the gel pore of test sample only occurs that the electrophoretic band of 1 treaty 1500bp is consistent with the stripe size of the gel pore relevant position of positive control, then result is judged as feminine gender；D there are 2 or more than 2 electrophoretic bands in the gel pore of () test sample, and consistent with the stripe size of relevant position in the gel pore of positive control, and negative control is without band, then be judged to that the purpose antibacterial corresponding with respective strap size is positive；Vibrio parahaemolytious, Escherichia coli O 157: H7, Salmonella, specific band size that 4 kinds of pre biooxidation of shigella are corresponding are respectively 1164bp, 713bp, 435bp, 298bp；E the gel pore of () positive control has 5 electrophoretic bands, the gel pore of negative control is without any band, and the gel pore of test sample does not has electrophoretic band in 1500bp position, illustrate sample handling processes exists enzyme inhibitor, it is proposed that change additive method and carry out sample treatment；F () positive control does not has band, illustrate that test kit lost efficacy, and this testing result is invalid.
Beneficial effects of the present invention: detection primer is the specific gene according to purpose antibacterial or specific fragment and the characteristic primer that designs, may be used for the aspect such as the quick detection of purpose antibacterial, Epidemiological study, curative effect of medication monitoring in food after being assembled into test kit.Compared with existing detection method, the method is easy to use, good reliability, detection cycle are short, highly sensitive, high specificity, with low cost, operating procedure is simple, it is applicable to high flux operation, normalizing operation, and the erroneous judgement caused because the inhibitive factor that there is archaeal dna polymerase in sample handling processes obtains false negative result can be differentiated, to improving, food safety detection level is significant.
Accompanying drawing explanation
Fig. 1: detect successful pcr amplification product electrophoresis result.Negative control is without band, and positive control has 5 electrophoretic bands at about 1500bp, 1164bp, 713bp, 435bp and 298bp respectively, illustrates to detect successfully.Sample 1 only has 1 amplification interior label band at about 1500bp, consistent with positive control relevant position stripe size, in other positions without band, illustrates that vibrio parahaemolytious, Escherichia coli O 157: H7 in sample 1, Salmonella, 4 kinds of antibacterials of shigella are feminine gender；2nd swimming lane has 2 bands and positive control position consistency at 1500bp and 1164bp, illustrates that sample 1 vibrio parahaemolytious is positive；3rd swimming lane has 2 bands and positive control position consistency at 1500bp and 713bp, sample 3 Escherichia coli O 157 is described: H7 is positive；4th swimming lane has 2 bands and positive control position consistency at 1500bp and 435bp, illustrates that sample 4 Salmonella is positive；5th has 2 bands and positive control position consistency at 1500bp and 298bp, illustrates that sample 5 shigella is positive.
Fig. 2: occur cross-contamination to cause detecting failed pcr amplification product electrophoresis result during detection.Negative control has electrophoretic band to occur, illustrates that by cross-contamination in sample handling processes, testing result is insincere, needs to re-start detection.
Fig. 3: there is the suppression material of DNA polymerase activity or operational error in sample handling processes and cause detecting failed pcr amplification product electrophoresis result.Cloudy comparison is without band, positive control has 5 electrophoretic bands, and sample 1 at about 1500bp without amplification interior label band, illustrate sample handling processes exists material or the operational error of suppression DNA polymerase activity, testing result is insincere, detects after needing to re-start process.
Fig. 4: the part actual sample testing result of corresponding embodiment 1.
In Fig. 1 to Fig. 4, M:250bpDNA molecular weight standard；Sun: positive control；Cloudy: negative control；The part actual sample (numbering in figure is to arrange in order, and the same numeral numbering in different figures may be not with a sample) of 1-13: corresponding embodiment 1.
Detailed description of the invention
Embodiment 1
(1) sample collecting: according to meeting standard and the method for sampling of regulation, from various food samples such as aseptic collection vegetable, aquatic products, meat and meat products, milk and milk products, eggs and egg products such as the different market of farm produces, supermarkets；
(2) Zengjing Granule of sample: by the sample sterile sampling 25g of aseptic collection, put into sterile mortar or tissue grinder's homogenate, or it is placed in homogenizing 2min in aseptic homogenizing bag, add the pancreas peptone soybean broth containing 1% sodium chloride aseptic for 225ml to cultivate based on 37 DEG C of CMC model 8-12h, obtain enrichment liquid；
(3) DNA extraction: take the enrichment liquid after cultivation, after vibration mixing, take 1ml 1000r/mim in centrifuge tube and be centrifuged 1min, remove relatively large fragment, take supernatant 10000r/mim and be centrifuged 2min, incline supernatant, it is inverted in positions different in absorbent paper, blot residual liquid, resuspended with sterile deionized water again, washing precipitation, 10000r/mim is centrifuged 2min, abandon supernatant, by the resuspended precipitation of a small amount of sterile deionized water, the test kit using commercialization again extracts DNA or takes multigelation method to extract DNA, the most direct boiling water bath, ice bath, centrifugal, take supernatant, obtain sample template DNA, adjust to concentration with sterile distilled water and be about 10ng/ μ l, directly carry out PCR amplification or standby in-20 DEG C of freezen protective；
(4) prepared by PCR reaction tube: all operations is both needed to carry out on ice；First PCR is reacted premixed liquid and be placed in thawed on ice, draw 50 μ l and be placed in aseptic PCR reaction tube, add 1 μ l sample template DNA, flick the PCR reaction tube prepared with finger or mix with micropipettor piping and druming, instantaneous high speed centrifugation 2s；PCR reaction tube is prepared respectively according to negative controls, actually detected sample, the order of positive reference substance；
(5) augmentation detection: the PCR reaction tube of the negative controls of preparation, sample template DNA and positive reference substance is placed in PCR instrument, carries out PCR amplification, obtain pcr amplification product；Response procedures is: 95 DEG C of denaturations 5min；95 DEG C of degeneration 40s, 56.7 DEG C of annealing 40s, 72 DEG C extend 70s, carry out 35 circulations；72 DEG C extend 10min, 4 DEG C of preservations；
(6) testing result judges: being dissolved in 0.5 × TBE electrophoretic buffer of 100ml by 1g agarose, be subsequently adding 5 μ lDNA dyestuffs, mixing is boiled, and topples over gel slab；After gel cooled and solidified, draw in PCR primer 5 μ l addition gel pore with micropipettor and carry out electrophoresis；Electrophoresis takes out gel and is placed under uviol lamp and observes, take pictures after terminating, it is determined that result: the gel pore of (a) positive control have 5 electrophoretic bands and the gel pore of negative control without any band, then PCR reaction detection success；If b () negative control has band, illustrating there is cross-contamination in PCR reaction tube preparation process, result is unreliable, it is proposed that re-start PCR detection；C () positive control has 5 bands, negative control does not has band, and the gel pore of test sample only occurs that the electrophoretic band of 1 treaty 1500bp is consistent with the stripe size of the gel pore relevant position of positive control, then result is judged as feminine gender；D there are 2 or more than 2 electrophoretic bands in the gel pore of () test sample, and consistent with the stripe size of relevant position in the gel pore of positive control, and negative control is without band, then be judged to that the purpose antibacterial corresponding with respective strap size is positive；Vibrio parahaemolytious, Escherichia coli O 157: H7, Salmonella, specific band size that 4 kinds of pre biooxidation of shigella are corresponding are respectively 1164bp, 713bp, 435bp, 298bp；E the gel pore of () positive control has 5 electrophoretic bands, the gel pore of negative control is without any band, and the gel pore of test sample does not has electrophoretic band in 1500bp position, illustrate sample handling processes exists enzyme inhibitor, it is proposed that change additive method and carry out sample treatment；F () positive control does not has band, illustrate that test kit lost efficacy, and this testing result is invalid.
Sequence table
<110>Bohai University
<120>one often detects vibrio parahaemolyticus, Escherichia coli O 157: H7, Salmonella, shigella 4 kinds simultaneously
See the test kit of food-borne pathogens
<160>18
<170>PatentInversion3.5
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ccggtggcgaaggcggccccctggacgaagactgacgctcaggtgcgaaagcgtggggag780
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<213>Escherichia coli O 157: H7
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acacaggagccacccccattttcgttgattcagataatgaaacttggcaaatgtctgtta180
gtgacatagaacaaaaaatcactaataaaactaaagctattatgtgtgtccatttatacg240
gacatccatgtgatatggaacaaattgtagaactggccaaaagtagaaatttgtttgtaa300
ttgaagattgcgctgaagcctttggttctaaatataaaggtaaatatgtgggaacatttg360
gagatatttctacttttagcttttttggaaataaaactattactacaggtgaaggtggaa420
tggttgtcacgaatgacaaaacactttatgaccgttgtttacattttaaaggccaaggat480
tagctgtacataggcaatattggcatgacgttataggctacaattataggatgacaaata540
tctgcgctgctataggattagcccagttagaacaagctgatgattttatatcacgaaaac600
gtgaaattgctgatatttataaaaaaaatatcaacagtcttgtacaagtccacaaggaaa660
gtaaagatgtttttcacacttattggatggtctcaattctaactaggaccgca713
<210>14
<211>435
<212>DNA
<213>Salmonella
<400>14
ggaacgaactaattcagcgatatccaaatgttgcatagatcttttccttaattaagccct60
tatattgtttttataacattcactgacttgctatctgctatctcaccgaaagataaaacc120
tccagatccggaaaacgaccttcaatcattttcttaataaatcgacggacatcgacagac180
gtaaggaggacaagatctttatgtgcaatcaataaatcatccaacttaagtgtaatgaga240
tccatcaaattagcggaggcttccgggtcaaggctgaggaaggtactgccagaggtctga300
cggatccctttgcgaataacatcctcaacttcagcagataccattactgctcgtaattcg360
ccgccattggcgaatttatgacaaatataacgcgccattgctccacgaatatgctccaca420
aggttaatgacatct435
<210>15
<211>298
<212>DNA
<213>shigella
<400>15
gaatttacggactggttctccctctggggaccatggcatgctgtactgaagcgtacggaa60
gctgaccgctgggcgcaggcagaagagcagaagtatgagatgctggagaatgagtactct120
cagagggtggctgaccggctgaaagcatcaggtctgagcggtgatgcggatgcgcagagg180
gaagccggtgcacaggtgatgcgtgagactgaacagcagatttaccgtcagctgactgac240
gaggtactggccctgcgattgtctgaaaacggctcacgactgcaccattcataatcac298
<210>16
<211>1471
<212>DNA
<213>vibrio parahaemolytious
<400>16
attgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagt60
cgagcggaaacgagttatctgaaccttcggggaacgataacggcgtcgagcggcggacgg120
gtgagtaatgcctaggaaattgccctgatgtgggggataaccattggaaacgatggctaa180
taccgcatgatgcctacgggccaaagagggggaccttcgggcctctcgcgtcaggatatg240
cctaggtgggattagctagttggtgaggtaagggctcaccaaggcgacgatccctagctg300
gtctgagaggatgatcagccacactggaactgagacacggtccagactcctacgggaggc360
agcagtggggaatattgcacaatgggcgcaagcctgatgcagccatgccgcgtgtgtgaa420
gaaggccttcgggttgtaaagcactttcagtcgtgaggaaggtagtgtagttaatagctg480
cattatttgacgttagcgacagaagaagcaccggctaactccgtgccagcagccgcggta540
atacggagggtgcgagcgttaatcggaattactgggcgtaaagcgcatgcaggtggtttg600
ttaagtcagatgtgaaagcccggggctcaacctcggaattgcatttgaaactggcagact660
agagtactgtagaggggggtagaatttcaggtgtagcggtgaaatgcgtagagatctgaa720
ggaataccggtggcgaaggcggccccctggacagatactgacactcagatgcgaaagcgt780
ggggagcaaacaggattagataccctggtagtccacgccgtaaacgatgtctacttggag840
gttgtggccttgagccgtggctttcggagctaacgcgttaagtagaccgcctggggagta900
cggtcgcaagattaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgt960
ggtttaattcgatgcaacgcgaagaaccttacctactcttgacatccagagaactttcca1020
gagatggattggtgccttcgggaactctgagacaggtgctgcatggctgtcgtcagctcg1080
tgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatccttgtttgccagc1140
gagtaatgtcgggaactccagggagactgccggtgataaaccggaggaaggtggggacga1200
cgtcaagtcatcatggcccttacgagtagggctacacacgtgctacaatggcgcatacag1260
agggcggccaacttgcgaaagtgagcgaatcccaaaaagtgcgtcgtagtccggattgga1320
gtctgcaactcgactccatgaagtcggaatcgctagtaatcgtggatcagaatgccacgg1380
tgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggctgcaaaa1440
gaagtaggtagtttaaccttcggggggacgc1471
<210>17
<211>1546
<212>DNA
<213>Salmonella
<400>17
aattgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaag60
tcgaacggtaacaggaagcagcttgctgctttgctgacgagtggcggacgggtgagtaat120
gtctgggaaactgcctgatggagggggataactactggaaacggtggctaataccgcata180
acgtcgcaagaccaaagagggggaccttcgggcctcttgccatcagatgtgcccagatgg240
gattagcttgttggtgaggtaacggctcaccaaggcgacgatccctagctggtctgagag300
gatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggg360
gaatattgcacaatgggcgcaagcctgatgcagccatgccgcgtgtatgaagaaggcctt420
cgggttgtaaagtactttcagcggggaggaaggtgttgtggttaataaccgcagcaattg480
acgttacccgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagg540
gtgcaagcgttaatcggaattactgggcgtaaagcgcacgcaggcggtctgtcaagtcgg600
atgtgaaatccccgggctcaacctgggaactgcattcgaaactggcaggcttgagtcttg660
tagaggggggtagaattccaggtgtagcggtgaaatgcgtagagatctggaggaataccg720
gtggcgaaggcggccccctggacaaagactgacgctcaggtgcgaaagcgtggggagcaa780
acaggattagataccctggtagtccacgccgtaaacgatgtctacttggaggttgtgccc840
ttgaggcgtggcttccggagctaacgcgttaagtagaccgcctggggagtacggccgcaa900
ggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaatt960
cgatgcaacgcgaagaaccttacctggtcttgacatccacggaagttttcagagatgaga1020
atgtgccttcgggaaccgtgagacaggtgctgcatggctgtcgtcagctcgtgttgtgaa1080
atgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcggtccggcc1140
gggaactcaaaggagactgccagtgataaactggaggaaggtggggatgacgtcaagtca1200
tcatggcccttacgaccagggctacacacgtgctacaatggcgcatacaaagagaagcga1260
cctcgcgagagcaagcggacctcataaagtgcgtcgtagtccggattggagtctgcaact1320
cgactccatgaagtcggaatcgctagtaatcgtggatcagaatgccacggtgaatacgtt1380
cccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagtaggta1440
gcttaaccttcgggagggcgcttaccactttgtgattcatgactggggtgaagtcgtaac1500
aaggtaaccgtaggggaacctgcggttggatcacctccttacctta1546
<210>18
<211>1556
<212>DNA
<213>shigella
<400>18
ttaaattgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgc60
aagtcgaacggtaacaggaaacagcttgctgtttcgctgacgagtggcggacgggtgagt120
aatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaataccgc180
ataatgtcgcaagactaaagagggggaccttcgggcctcttgccatcggatgtgcccaga240
tgggattagctagtaggtggggtaacggctcacctaggcgacgatccctagctggtctga300
gaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagt360
ggggaatattgcacaatgggcgcaagcctgatgcagccatgccgcgtgtatgaagaaggc420
cttcgggttgtaaagtactttcagcggggaggaagggagtaaagttaatacctttactca480
ttgacgttacccgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacgg540
agggtgcaagcgttaatcggaattactgggcgtaaagcgcacgcaggcggtttgttaagt600
cagatgtgaaatccccgggctcaacctgggaactgcatctgatactggcaagcttgagtc660
tcgtagaggggggtagaattccaggtgtagcggtgaaatgcgtagagatctggaggaata720
ccggtggcgaaggcggccccctggacgaagactgacgctcaggtgcgaaagcgtggggag780
caaacaggattagataccctggtagtccacgccgtaaacgatgtcgacttggaggttgtg840
cccttgaggcgtggcttccggagctaacgcgttaagtcgaccgcctggggagtacggccg900
caaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggttta960
attcgatgcaacgcgaagaaccttacctggtcttgacatccacagaactttccagagatg1020
gattggtgccttcgggaactgtgagacaggtgctgcatggctgtcgtcagctcgtgttgt1080
gaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcggtccg1140
gccgggaactcaaaggagactgccagtgataaactggaggaaggtggggatgacgtcaag1200
tcatcatggcccttacgaccagggctacacacgtgctacaatggcgcatacaaagagaag1260
cgacctcgcgagagcaagcggacctcataaagtgcgtcgtagtccggattggagtctgca1320
actcgactccatgaagtcggaatcgctagtaatcgtggatcagaatgccacggtgaatac1380
gttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagtag1440
gtagcttaaccttcgggagggcgcttaccactttgtgattcatgactggggtgaagtcgt1500
aacaaggtaaccgtaggggaacctgcggttggatcacctccttaccttaaagaagc1556
Claims (8)
1. detect vibrio parahaemolyticus, Escherichia coli O 157: H7, Salmonella, a test kit for 4 kinds of common food-borne pathogens of shigella simultaneously, it is characterized in that:
This test kit comprises: sterile deionized water, PCR reaction premixed liquid, reference substance and DNA dyestuff；
Described PCR reaction premixed liquid contains PCR reaction buffer, dideoxyribonucleotide triphosphate, Taq DNA polymerase, detection primer.
One the most according to claim 1 detects vibrio parahaemolyticus, Escherichia coli O 157: H7, Salmonella, the test kit of 4 kinds of common food-borne pathogens of shigella simultaneously, it is characterized in that: containing Mg in described PCR reaction buffer2+Concentration be 0.8mM, the concentration of described dideoxyribonucleotide triphosphate is 0.12mM, and the concentration of described Taq DNA polymerase is 1.5U/ reaction tube.
One the most according to claim 1 detects vibrio parahaemolyticus, Escherichia coli O 157: H7, Salmonella, the test kit of 4 kinds of common food-borne pathogens of shigella simultaneously, it is characterized in that: described detection primer is the mixture of following 10 primers, and its sequence is respectively as follows:
Amplification interior label primer, 27F sequence: AGAGTTTGATCCTGGCTCAG, 1492R sequence: TACGGYTACCTTTGTTACGACTT；
Vibrio parahaemolytious characteristic primer, aadSF sequence: GGAATTCGCAGTGATCGCCGCTTGAG, aadSR sequence: CGCGTCGACAGCATCGCGGTTATAGG；
Escherichia coli characteristic primer, O157:H7rfbEF sequence: CTACTGTAAGTAATGGAACGGTTGC, rfbER sequence: TGCGGTCCTAGTTAGAATTGAGACC；
Salmonella characteristic primer, invAF sequence: GGAACGAACTAATTCAGCGATA, invAR sequence: AGATGTCATTAACCTTGTGGAG；
Shigella characteristic primer, ipaHF sequence: GAATTTACGGACTGGTTCTCCCTCTGG, ipaHR sequence: GTGATTATGAATGGTGCAGTCGTGAGC.
One the most according to claim 3 detects vibrio parahaemolyticus, Escherichia coli O 157: H7, Salmonella, the test kit of 4 kinds of common food-borne pathogens of shigella simultaneously, it is characterized in that: the concentration of described detection primer is respectively as follows:
Amplification interior label 27F and 1492R concentration are 0.1 μM, vibrio parahaemolytious characteristic primer aadSF and aadSR concentration are 0.16 μM, Escherichia coli O 157: H7 characteristic primer rfbEF and rfbER concentration are 0.1 μM, Salmonella characteristic primer invAF and invAR concentration are 0.1 μM, and shigella characteristic primer ipaHF and ipaHR concentration are 0.04 μM.
One the most according to claim 1 detects vibrio parahaemolyticus, Escherichia coli O 157: H7, Salmonella, the test kit of 4 kinds of common food-borne pathogens of shigella simultaneously; it is characterized in that: described reference substance is divided into negative controls and positive reference substance; negative controls template is sterile deionized water, and positive reference substance template is the plasmon mixture obtained after each pair of corresponding amplified production of primer is connected to carrier T.
One the most according to claim 1 detects vibrio parahaemolyticus, Escherichia coli O 157: H7, Salmonella, the test kit of 4 kinds of common food-borne pathogens of shigella simultaneously, it is characterized in that:
Use this test kit to detect vibrio parahaemolyticus, Escherichia coli O 157: H7, Salmonella, the method for 4 kinds of common food-borne pathogens of shigella are simultaneously:
(1) sample collecting: aseptic collection suspicious milk and milk products sample；
(2) Zengjing Granule: the suspicious milk and milk products sample mix uniformly rear sterile sampling that will gather, liquid milk and milk products is the most aseptic to measure, milk in solid form goods put into sterile mortar, tissue grinder or homogenizing bag homogenizing 2～5min, add the sterile vegetative broth bouillon being preheated to 45 DEG C, 37 ± 1 DEG C of shaken cultivation 6～8h, obtain enrichment liquid；
null(3) DNA extraction: take the enrichment liquid after cultivation，Vibration mixing，Take 1ml and 1000～1500r/mim be centrifuged 1～2min in centrifuge tube，Remove relatively large fragment，Take supernatant 8000～12000r/mim and be centrifuged 1～2min，Incline supernatant，It is inverted in positions different in absorbent paper，Blot residual liquid，Resuspended with sterile deionized water again、Washing precipitation，8000～12000r/mim are centrifuged 1～2min，Abandon supernatant，By the resuspended precipitation of a small amount of sterile deionized water，Boiling water bath 5～10min，Ice bath 5～10min，1000～5000r/mim are centrifuged 5～10min，Supernatant is sample template DNA，Adjust concentration standby to about 10～30ng/ μ l，Or-20 DEG C of preservations；
(4) prepared by PCR reaction tube: all operations is both needed to carry out on ice；First PCR is reacted premixed liquid and be placed in thawed on ice, draw 50 μ l and be placed in aseptic PCR reaction tube, add 1 μ l sample template DNA, flick the PCR reaction tube prepared with finger or mix with micropipettor piping and druming, instantaneous high speed centrifugation 2s；PCR reaction tube is prepared respectively according to negative controls, actually detected sample, the order of positive reference substance；
(5) augmentation detection: the PCR reaction tube of the negative controls of preparation, actually detected sample and positive reference substance is placed in PCR instrument, carries out PCR amplification, obtain pcr amplification product；
(6) testing result judges: being dissolved in 0.5 × TBE electrophoretic buffer of 100ml by 1g agarose, be subsequently adding 5 μ lDNA dyestuffs, mixing is boiled, and topples over gel slab；After gel cooled and solidified, draw in PCR primer 5 μ l addition gel pore with micropipettor and carry out electrophoresis；Electrophoresis takes out gel and is placed under uviol lamp and observes, take pictures after terminating, it is determined that result: the gel pore of (a) positive control have 5 electrophoretic bands and the gel pore of negative control without any band, then PCR reaction detection success；If b () negative control has band, illustrating there is cross-contamination in PCR reaction tube preparation process, result is unreliable, it is proposed that re-start PCR detection；C () positive control has 5 bands, negative control does not has band, and the gel pore of test sample only occurs that the electrophoretic band of 1 treaty 1500bp is consistent with the stripe size of the gel pore relevant position of positive control, then result is judged as feminine gender；D there are 2 or more than 2 electrophoretic bands in the gel pore of () test sample, and consistent with the stripe size of relevant position in the gel pore of positive control, and negative control is without band, then be judged to that the purpose antibacterial corresponding with respective strap size is positive；Vibrio parahaemolyticus, escherichia coli, Salmonella, specific band size that 4 kinds of pre biooxidation of shigella are corresponding are respectively 1164bp, 713bp, 435bp, 298bp；E the gel pore of () positive control has 5 electrophoretic bands, the gel pore of negative control is without any band, and the gel pore of test sample does not has electrophoretic band in 1500bp position, illustrate sample handling processes exists enzyme inhibitor, it is proposed that change additive method and carry out sample treatment；F () positive control does not has band, illustrate that test kit lost efficacy, and this testing result is invalid.
One the most according to claim 6 detects vibrio parahaemolyticus, Escherichia coli O 157: H7, Salmonella, the test kit of 4 kinds of common food-borne pathogens of shigella simultaneously, it is characterized in that: the enriched medium pancreas peptone soybean broth culture medium of sample contains 1% sodium chloride, to guarantee to make vibrio parahaemolytious normal growth, the most do not suppress the growth of other antibacterials simultaneously.
One the most according to claim 6 detects vibrio parahaemolyticus, Escherichia coli O 157: H7, Salmonella, the test kit of 4 kinds of common food-borne pathogens of shigella simultaneously, it is characterized in that: response procedures during augmentation detection is: 95 DEG C of denaturations 5min；95 DEG C of degeneration 40s, 56.7 DEG C of annealing 40s, 72 DEG C extend 70s, carry out 35 circulations；72 DEG C extend 10min, 4 DEG C of preservations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610279017.4A CN105821133A (en) | 2016-05-03 | 2016-05-03 | Kit for simultaneously detecting Vibrio parahaemolyticus, Escherichia coli O157:H7, Salmonella and Shigella |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610279017.4A CN105821133A (en) | 2016-05-03 | 2016-05-03 | Kit for simultaneously detecting Vibrio parahaemolyticus, Escherichia coli O157:H7, Salmonella and Shigella |
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| CN105821133A true CN105821133A (en) | 2016-08-03 |
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| CN201610279017.4A Pending CN105821133A (en) | 2016-05-03 | 2016-05-03 | Kit for simultaneously detecting Vibrio parahaemolyticus, Escherichia coli O157:H7, Salmonella and Shigella |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967816A (en) * | 2017-04-25 | 2017-07-21 | 暨南大学 | Detect the RPA specific primers of vibrio parahaemolytious and kit and application in food |
| CN107488720A (en) * | 2017-09-15 | 2017-12-19 | 重庆市动物疫病预防控制中心 | For detecting the primer sets and its detection method of Escherichia coli O 157 in feed |
| CN108728558A (en) * | 2018-05-31 | 2018-11-02 | 广西壮族自治区兽医研究所 | A kind of PCR amplification primer of quick detection Mannheimia haemolytica and its application |
| CN116286317A (en) * | 2023-04-14 | 2023-06-23 | 安徽国泰众信检测技术有限公司 | High-sensitivity detection kit for food-borne pathogenic bacteria and application method of high-sensitivity detection kit |
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| CN103409512A (en) * | 2013-07-10 | 2013-11-27 | 渤海大学 | Kit and method for detecting vibrio parahaemolyticus in aquatic product |
| CN103484546A (en) * | 2013-09-17 | 2014-01-01 | 北京卓诚惠生生物科技有限公司 | Fourteen-food-borne pathogenic bacterium multiplex PCR detection primer set and kit |
| CN104593495A (en) * | 2015-01-09 | 2015-05-06 | 渤海大学 | PCR method and kit for detecting internal amplification control of salmonella in foods |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103409512A (en) * | 2013-07-10 | 2013-11-27 | 渤海大学 | Kit and method for detecting vibrio parahaemolyticus in aquatic product |
| CN103484546A (en) * | 2013-09-17 | 2014-01-01 | 北京卓诚惠生生物科技有限公司 | Fourteen-food-borne pathogenic bacterium multiplex PCR detection primer set and kit |
| CN104593495A (en) * | 2015-01-09 | 2015-05-06 | 渤海大学 | PCR method and kit for detecting internal amplification control of salmonella in foods |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967816A (en) * | 2017-04-25 | 2017-07-21 | 暨南大学 | Detect the RPA specific primers of vibrio parahaemolytious and kit and application in food |
| CN106967816B (en) * | 2017-04-25 | 2020-01-17 | 暨南大学 | RPA specific primer for detecting vibrio parahaemolyticus in food, kit and application |
| CN107488720A (en) * | 2017-09-15 | 2017-12-19 | 重庆市动物疫病预防控制中心 | For detecting the primer sets and its detection method of Escherichia coli O 157 in feed |
| CN108728558A (en) * | 2018-05-31 | 2018-11-02 | 广西壮族自治区兽医研究所 | A kind of PCR amplification primer of quick detection Mannheimia haemolytica and its application |
| CN116286317A (en) * | 2023-04-14 | 2023-06-23 | 安徽国泰众信检测技术有限公司 | High-sensitivity detection kit for food-borne pathogenic bacteria and application method of high-sensitivity detection kit |
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