CN104513857A - Loop-mediated isothermal amplification detection primer group, detection method and kit of vibrio parahaemolyticus - Google Patents
Loop-mediated isothermal amplification detection primer group, detection method and kit of vibrio parahaemolyticus Download PDFInfo
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Abstract
The invention discloses a loop-mediated isothermal amplification detection primer group, a detection method and a kit of vibrio parahaemolyticus. The detection primer group is as follows: an upstream outer primer F3: 5'-GCAAAGAAACGCTTGGCG-3', a downstream outer primer B3: 5'-TGCATAGCAATGTTGTCGCT-3', an upstream inner primer FIP: 5'-TCTCTCGGGTGGTGGATGGGTTTCGTTACACTCCGTTCGC-3'; a downstream inner primer BIP: 5'-ATGGTTTGCTACTCTCGCACCCTCGGCTGACAAATGGCTCTA-3'. The detection method disclosed by the invention has the advantages of high sensitivity, high specificity, good accuracy rate and short detection time, only 12 hours are taken from sample treatment to result report, no PCR instrument or electrophoresis instrument is needed, the operation process is simple, and compared with other PCR techniques, the loop-mediated isothermal amplification detection primer group is relatively high in specificity and is applicable to detection of primary testing organizations and food companies.
Description
Technical field:
The invention belongs to biological technical field, the ring mediated isothermal amplification being specifically related to a kind of Vibrio parahemolyticus detects primer sets, detection method and test kit.
Background technology:
It is a kind of important food-borne pathogens that Vibrio parahemolyticus (Vibrio parahaemolyticus) is worldwide recognized, and is also the No.1 pathogenic bacterium of China's microbes food poisoning in recent years.Vibrio parahaemolytisus poisoning can cause acute gastroenteritis, and a few cases can also cause septicemia, threat to life.Vibrio parahemolyticus pollution rate higher in fishery products result in and happened occasionally all over the world by this microbial food poisoning case.In only summer in 2006, the relevant diarrhoea case of 900 many cases VP has just been reported in Chilean somewhere; On 25 days ~ September 25 August in 2007, Xiamen City of China recurs two VP food poisonings, poisoning more than 2000 people.
At present, the U.S. and European Union to import fishery products all Compulsory Feature carry out the detection of Vibrio parahemolyticus, assay is that feminine gender can clearance, otherwise the fishery products of outlet " all automatically will be detained " and destroyed on the spot.This is provided with technology barriers to the outlet of China's fishery products.The states such as the international microbial standard council and Japan, Britain, Canada and China Hongkong all propose testing fishery products being carried out to Vibrio parahemolyticus.China also requires the examination criteria (GB 4789.7-2013, SNT 2142-2008, SN0173-1992, GB18406.4-2001) of the detection of Vibrio parahemolyticus as aquatic product quality.At present, China is to the detection of Vibrio parahemolyticus mainly according to GB 4789.7-2013, and this needs 5 ~ 7d time, is difficult to adapt to requirement fast and accurately.Along with the development of modern science and technology, particularly immunology, biological chemistry, molecular biological development, people have created Vibrio parahemolyticus detection method that is much quick, easy, special, responsive and that be suitable for.
Although the existing patent report (number of patent application: 2007100304416) setting up LAMP method for Vibrio parahemolyticus tlh gene and some other sequences, but due to special not (the Croci et al. of the genes such as tlh, 2007), with this LAMP method set up, there is certain limitation in actual applications.
Summary of the invention:
The first object of the present invention is to provide the detection primer sets that a kind of quick, special good, sensitive high application ring mediated isothermal amplification detects Vibrio parahemolyticus (Vibrio parahaemolyticus), utilizes this detection primer sets can specific detection Vibrio parahemolyticus.
The present invention is (Yu et al. on the basis selecting new specific target gene irgB, 2010), by screening and optimizing primer, establish a kind of LAMP amplification method, by groping sample pre-treatments condition (increasing bacterium), sample preparation combine with technique LAMP technology is organically combined, sets up the method for quick being used for Vibrio parahemolyticus in fishery products.The application of the method is for fishery products rapid detection and ensure that fish quality is significant, thus achieves object of the present invention.
Application ring mediated isothermal amplification of the present invention detects the detection primer sets of Vibrio parahemolyticus (Vibrio parahaemolyticus), it is characterized in that, is made up of following primer:
Upstream outer primer is F3:5 '-GCAAAGAAACGCTTGGCG-3 ' (as shown in SEQ ID NO.1);
Downstream outer primer B3:5 '-TGCATAGCAATGTTGTCGCT-3 ' (as shown in SEQ ID NO.2);
Upstream inner primer FIP:5 '-TCTCTCGGGTGGTGGATGGGTTTCGTTACACTCCGTTCGC-3 ' (as shown in SEQ ID NO.3);
Downstream inner primer BIP:5 '-ATGGTTTGCTACTCTCGCACCCTCGGCTGACAAATGGCTCTA-3 ' (as shown in SEQ ID NO.4).
Second object of the present invention is to provide the detection method of a kind of Vibrio parahemolyticus of Diagnosis and Treat object of non-diseases, it is characterized in that, Zengjing Granule is carried out to sample and obtains enrichment liquid, extract the DNA of bacteria in enrichment liquid, then by above-mentioned detection primer sets, selective amplification is carried out to DNA of bacteria by the method for ring mediated isothermal amplification, be confirmed whether to have amplified production.
Described sample can be the various samples that may contain Vibrio parahemolyticus, as fishery products.
Described carries out Zengjing Granule preferably by sample cut-in quality mark 3%Nacl basic peptone water to sample, cultivates 10h for 36 DEG C ± 1 DEG C, obtains enrichment liquid.
Described by above-mentioned detection primer sets, selective amplification carried out to DNA of bacteria by the method for ring mediated isothermal amplification and be specially: ring mediated isothermal amplification system is system cumulative volume 25 μ L, comprises 2.5 μ L 10 × Thermopol reaction buffers, 0.4 μ L10mmol/LdNTPs, 0.5 μ L, 10 μm of ol/L upstream outer primer F3,0.5 μ L, 10 μm of ol/L downstream outer primer B3,1 μ L40 μm ol/L upstream inner primer FIP, 1 μ L, 40 μm of ol/L downstream inner primer BIP, 0.6 μ L100mmol/L MgSO
4, 12.5 μ L2mol/L trimethyl-glycines, 2 μ L sterilizing distilled water ddH
2o, 1 μ L Bst archaeal dna polymerase (8U/ μ L) and 3 μ L DNA of bacteria templates, reaction conditions hatches 45 ~ 60min for being 60 ~ 65 DEG C in temperature.
Described is confirmed whether that having amplified production can utilize the detection of electrophoresis detection, fluorescence developing or Turbidity measurement loop-mediated isothermal amplification product whether to have amplified production, preferably detect with fluorescence developing, specifically at 80 DEG C of termination reaction 1 ~ 2min, SYBR Green I developer is added in amplified reaction pipe, observations after 1 ~ 5min, if color is orange, then sample Vibrio parahemolyticus is negative, without Vibrio parahemolyticus, if color is green, then sample Vibrio parahemolyticus is positive, containing Vibrio parahemolyticus.
Described 10 × Thermopol reaction buffer is material of the prior art, can buy from Reagent Company, it contains trishydroxymethylaminomethane-hydrochloric acid (Tris-HCl), 100mmol/L Repone K (KCl), the 100mmol/L ammonium sulfate (NH of 200mmol/L pH8.8
4)
2sO
4, 20mmol/L magnesium sulfate (MgSO
4) and 1% triton x-100 (TtitonX-100), surplus is water.
3rd object of the present invention is to provide the detection kit of a kind of Vibrio parahemolyticus, and comprise ring mediated isothermal amplification reagent and detect primer sets, it is characterized in that, described detection primer sets is made up of following primer:
Upstream outer primer is F3:5 '-GCAAAGAAACGCTTGGCG-3 ';
Downstream outer primer B3:5 '-TGCATAGCAATGTTGTCGCT-3 ';
Upstream inner primer FIP:5 '-TCTCTCGGGTGGTGGATGGGTTTCGTTACACTCCGTTCGC-3 ';
Downstream inner primer BIP:5 '-ATGGTTTGCTACTCTCGCACCCTCGGCTGACAAATGGCTCTA-3 '.
The specific target gene irgB that the present invention is directed to Vibrio parahemolyticus designs Auele Specific Primer, has stronger specificity, is suitable for the detection of Vibrio parahemolyticus.The present invention selects new specific gene irgB to be target gene, design and screened a set of specific detection primer group and the detection kit containing this detection primer sets and utilize this detection kit by the detection method of ring mediated isothermal amplification, and then determining the detection method that whether there is Vibrio parahemolyticus in detected sample.Detection kit of the present invention and detection method have highly sensitive, that high specificity, accuracy rate are good, detection time is short advantage, only 12h need be spent to reporting the result from sample preparation, do not need PCR instrument and electrophoresis apparatus, operating process is simple, than other round pcrs, there is higher specificity, be particularly suitable for whether containing Vibrio parahemolyticus in feeler mechanism of basic unit and food enterprise detection fishery products, significant with guarantee fish quality for fishery products rapid detection.
Accompanying drawing illustrates:
Fig. 1 is the electrophoresis result of the LAMP amplified production in pure bacterium sensitivity experiment: 1:1.02 X 10
5; 2:1.02 X 10
4; 3:1.02 X 10
3; 4:1.02 X 10
2; 5:1.02 X 10
1; 6:1.02 X 10
0; 7:1.02 X 10
-1; 8: negative control; M:DL2000;
Fig. 2 is the display liquid result of the LAMP amplified production in pure bacterium sensitivity experiment: 1:1.02 X 10
5; 2:1.02 X 10
4; 3:1.02 X 10
3; 4:1.02 X 10
2; 5:1.02 X 10
1; 6:1.02 X 10
0; 7:1.02 X 10
-1; 8: negative control;
Fig. 3 is the pure bacterium sensitivity experiment of PCR: 1:1.02 X 10
5; 2:1.02 X 10
4; 3:1.02 X 10
3; 4:1.02 X 10
2; 5:1.02 X 10
1; 6:1.02 X 10
0; 7:1.02 X 10
-1; 8: negative control; M:DL2000.
Embodiment:
Following examples further illustrate of the present invention, instead of limitation of the present invention.
Embodiment 1: in shrimps, Vibrio parahemolyticus detects
1, sample increases bacterium
By the shrimp homogeneous that market is bought, take homogeneous samples 25g, put into 225mL massfraction 3%NaCl basic peptone water, cultivate 10h, obtain bacterial cultures for 36 DEG C ± 1 DEG C.
2, the extraction of DNA of bacteria
Get 1mL bacterial cultures in the centrifugal 5min of 12000r/min, abandon supernatant, collect thalline, add 50uLTE and fully to suspend mixing, in 100 DEG C of water-bath 10min, the centrifugal 5min of ice bath 3min, 12000r/min, gets supernatant for subsequent use, namely obtains DNA of bacteria.
3, LAMP amplification
Ring mediated isothermal amplification system is system cumulative volume 25 μ L, comprises 2.5 μ L 10 × Thermopol reaction buffers, 0.4 μ L10mmol/L dNTPs, 0.5 μ L, 10 μm of ol/L upstream outer primer F3,0.5 μ L, 10 μm of ol/L downstream outer primer B3,1 μ L40 μm ol/L upstream inner primer FIP, 1 μ L, 40 μm of ol/L downstream inner primer BIP, 0.6 μ L100mmol/L MgSO
4, 12.5 μ L2mol/L trimethyl-glycines, 2 μ L sterilizing distilled water ddH
2o, 1 μ L Bst archaeal dna polymerase (8U/ μ L) and 3 μ L DNA of bacteria templates, reaction conditions hatches 60min for being 60 DEG C in temperature.
LAMP detection primer is:
Upstream outer primer is F3:5 '-GCAAAGAAACGCTTGGCG-3 ';
Downstream outer primer B3:5 '-TGCATAGCAATGTTGTCGCT-3 ';
Upstream inner primer FIP:5 '-TCTCTCGGGTGGTGGATGGGTTTCGTTACACTCCGTTCGC-3 ';
Downstream inner primer BIP:5 '-ATGGTTTGCTACTCTCGCACCCTCGGCTGACAAATGGCTCTA-3 '.
80 DEG C of termination reaction 1-2min, take out reaction tubes.
4, developer is observed
SYBR Green I developer of 1 μ L 10% is added in reaction tubes, directly detect by an unaided eye colour-change, reaction tubes color becomes green, and [negative control (genomic dna of Vibrio vulnificus ATCC27562) is orange, positive control (genomic dna of Vibrio parahemolyticus ATCC33847) is green], interpret sample has Vibrio parahemolyticus to pollute.This and conventional biochemical authentication method detected result are coincide, and prove that this method data are reliable.
Embodiment 2: in yellow croaker, Vibrio parahemolyticus detects
By the yellow croaker homogeneous that market is bought, take homogeneous samples and carry out LAMP detection according to the method for embodiment 1, just the temperature of reaction of LAMP is 65 DEG C and hatches 45min, and other are identical with embodiment 1.
Result is: reaction tubes color becomes that orange (negative control (genomic dna of Vibrio vulnificus ATCC27562) is orange, positive control (genomic dna of positive strain Vibrio parahemolyticus ATCC33847) is green), interpret sample does not have Vibrio parahemolyticus to pollute.This and conventional biochemical method detected result are coincide, and prove that this method data are reliable.
The specificity of embodiment 3:LAMP method
Collect Vibrio parahemolyticus and non-Vibrio parahemolyticus 110 strain, by bacterial strain after Tryptones meat soup TSB (Vibrio parahemolyticus is at 3%Nacl TSB) 37 DEG C cultivates 24h, get 1mL bacterial cultures respectively in the centrifugal 5min of 12000r/min, abandon supernatant, collect thalline, add 50 μ L TE fully to suspend mixing, in 100 DEG C of water-bath 10min, the centrifugal 5min of ice bath 3min, 12000r/min, get supernatant for subsequent use, obtain the DNA of bacteria of 110 strains thus respectively.
LAMP increases: ring mediated isothermal amplification (LAMP) reaction system is 25 μ L, comprising: 2.5 μ L 10 × Thermopol reaction buffers, 0.4 μ L 10mmol/LdNTPs, 0.5 μ L, 10 μm of ol/L upstream outer primer F3,0.5 μ L, 10 μm of ol/L downstream outer primer B3,1 μ L, 40 μm of ol/L upstream inner primer FIP, 1 μ L, 40 μm of ol/L downstream inner primer BIP, 0.6 μ L100mmol/L MgSO
4, 12.5 μ L 2mol/L trimethyl-glycines, 2 μ L sterilizing distilled water ddH
2o, 1 μ L Bst archaeal dna polymerase (8U/ μ L) and 3 μ L DNA of bacteria templates, reaction conditions hatches 45min for being 65 DEG C in temperature.80 DEG C of termination reaction 1-2min, take out reaction tubes.
Upstream outer primer is F3:5 '-GCAAAGAAACGCTTGGCG-3 ';
Downstream outer primer B3:5 '-TGCATAGCAATGTTGTCGCT-3 ';
Upstream inner primer FIP:5 '-TCTCTCGGGTGGTGGATGGGTTTCGTTACACTCCGTTCGC-3 ';
Downstream inner primer BIP:5 '-ATGGTTTGCTACTCTCGCACCCTCGGCTGACAAATGGCTCTA-3 '.
Carry out LAMP amplification according to the method described above with above-mentioned 110 strain DNA of bacteria respectively, 80 DEG C of termination reaction 1-2min, take out reaction tubes.
Developer is observed: fluorescence dye SYBR GREEN I developer adding 1 μ L10% in reaction tubes, and directly detect by an unaided eye colour-change, if color is orange, negative, as color becomes green, positive.Result is as table 1.As can be seen from Table 1, utilize ring mediated isothermal amplification of the present invention to detect the detection primer sets of Vibrio parahemolyticus, detect according to detection method of the present invention, it has good specificity, to 2 strain Vibrio parahemolyticus type strains and the 80 strain strain isolateds positive; Negative to the vibrio cholerae of Vibrio, Vibrio vulnificus and vibrio alginolyticus; Negative to other 25 strain bacterium of non-Vibrio.
Table 1: specific test bacterial strain uses therefor and experimental result
ATCC, American Type Culture collection warehousing; CMCC, China General Microbiological culture presevation administrative center; NCTC: National Collection of Type Cultures (Britain); GIM: Guangdong Microbes Inst; CAS, the Chinese Academy of Sciences;
bthe results were shown as ' Number of positivestrains/number of strains tested.
The sensitivity of embodiment 4:LAMP method
(1) pure bacterium sensitivity
With Vibrio parahemolyticus type strain ATCC33847 for reference strain, by its in 3%Nacl TSB enrichment liquid 37 DEG C cultivate after 24h, get 1mL bacterium liquid, 10 times of gradient dilutions are carried out with sterile saline, choose 3 suitable extent of dilution and carry out plate count, each extent of dilution 2 flat boards, test repetition 3 times.The concentration of plate count original bacteria liquid is 1.02 X 10
8cFU/ml.To each dilution bacterium liquid, get 1mL bacterium liquid respectively in the centrifugal 5min of 12000r/min, abandon supernatant, collect thalline, add 50 μ L TE and fully to suspend mixing, in 100 DEG C of water-bath 10min, ice bath 3min, the centrifugal 5min of 12000r/min, gets supernatant for subsequent use, obtains each dilution DNA of bacteria thus respectively.
LAMP increases: ring mediated isothermal amplification (LAMP) reaction system is 25 μ L, comprising: 2.5 μ L 10 × Thermopol reaction buffers, 0.4 μ L 10mmol/LdNTPs, 0.5 μ L, 10 μm of ol/L upstream outer primer F3,0.5 μ L, 10 μm of ol/L downstream outer primer B3,1 μ L, 40 μm of ol/L upstream inner primer FIP, 1 μ L, 40 μm of ol/L downstream inner primer BIP, 0.6 μ L100mmol/L MgSO
4, 12.5 μ L 2mol/L trimethyl-glycines, 2 μ L sterilizing distilled water ddH
2o, 1 μ L Bst archaeal dna polymerase (8U/ μ L) and 3 μ L DNA of bacteria templates, reaction conditions hatches 45min for being 65 DEG C in temperature.80 DEG C of termination reaction 1-2min, take out reaction tubes.
Upstream outer primer is F3:5 '-GCAAAGAAACGCTTGGCG-3 ';
Downstream outer primer B3:5 '-TGCATAGCAATGTTGTCGCT-3 ';
Upstream inner primer FIP:5 '-TCTCTCGGGTGGTGGATGGGTTTCGTTACACTCCGTTCGC-3 ';
Downstream inner primer BIP:5 '-ATGGTTTGCTACTCTCGCACCCTCGGCTGACAAATGGCTCTA-3 '.
Carry out LAMP amplification according to the method described above with above-mentioned each dilution DNA of bacteria respectively, 80 DEG C of termination reaction 1-2min, take out reaction tubes.
To verify the sensitivity of loop-mediated isothermal amplification detection method of the present invention and conventional PCR method.
As shown in Figure 1, Figure 2 and Figure 3, Fig. 1-3 shows result, and the sensitivity of loop-mediated isothermal amplification detection method of the present invention is 102CFU/mL (Fig. 1-Fig. 2), and developer detected result is: 1:1.02 X 10
5cFU/ml (green); 2:1.02 X 10
4cFU/ml (green); 3:1.02 X 10
3cFU/ml (green); 4:1.02 X 10
2cFU/ml (green); 5:1.02 X 10
1cFU/ml (orange); 6:1.02 X 10
0cFU/ml (orange); 7:1.02 X 10
-1(orange), 8: negative control (orange), illustrates thus, the sensitivity of loop-mediated isothermal amplification detection method of the present invention (irgB-LAMP) is 102CFU/mL.
Negative control is: the genomic dna of Vibrio vulnificus ATCC27562.
And the sensitivity of PCR is 1020CFU/mL (Fig. 3-agarose electrophoresis detects), highly sensitive 10 times of LAMP remolding sensitivity PCR.
Claims (7)
1. apply the detection primer sets that ring mediated isothermal amplification detects Vibrio parahemolyticus (Vibrio parahaemolyticus), it is characterized in that, be made up of following primer:
Upstream outer primer is F3:5 '-GCAAAGAAACGCTTGGCG-3 ';
Downstream outer primer B3:5 '-TGCATAGCAATGTTGTCGCT-3 ';
Upstream inner primer FIP:5 '-TCTCTCGGGTGGTGGATGGGTTTCGTTACACTCCGTTCGC-3 ';
Downstream inner primer BIP:5 '-ATGGTTTGCTACTCTCGCACCCTCGGCTGACAAATGGCTCTA-3 '.
2. the detection method of the Vibrio parahemolyticus of the Diagnosis and Treat object of a non-diseases, it is characterized in that, Zengjing Granule is carried out to sample and obtains enrichment liquid, extract the DNA of bacteria in enrichment liquid, then by detection primer sets according to claim 1, selective amplification is carried out to DNA of bacteria by the method for ring mediated isothermal amplification, be confirmed whether to have amplified production.
3. detection method according to claim 2, is characterized in that, described sample is fishery products.
4. detection method according to claim 2, is characterized in that, described to carry out Zengjing Granule to sample be by sample cut-in quality percentage ratio 3%Nacl basic peptone water, cultivates 10h for 36 DEG C ± 1 DEG C, obtain enrichment liquid.
5. detection method according to claim 2, it is characterized in that, described by detection primer sets according to claim 1, selective amplification carried out to DNA of bacteria by the method for ring mediated isothermal amplification and be specially: ring mediated isothermal amplification system is system cumulative volume 25 μ L, comprise 2.5 μ L 10 × Thermopol reaction buffers, 0.4 μ L 10mmol/LdNTPs, 0.5 μ L10 μm of ol/L upstream outer primer F3, 0.5 μ L 10 μm ol/L downstream outer primer B3, 1 μ L 40 μm ol/L upstream inner primer FIP, 1 μ L 40 μm ol/L downstream inner primer BIP, 0.6 μ L100mmol/L MgSO
4, 12.5 μ L 2mol/L trimethyl-glycines, 2 μ L sterilizing distilled water ddH
2o, 1 μ L 8U/ μ L Bst archaeal dna polymerase and 3 μ L DNA of bacteria templates, reaction conditions hatches 45 ~ 60min for being 60 ~ 65 DEG C in temperature.
6. detection method according to claim 2, it is characterized in that, described is confirmed whether that having amplified production selects fluorescence developing to detect, specifically at 80 DEG C of termination reaction 1 ~ 2min, SYBR Green I developer is added in amplified reaction pipe, observations after 1 ~ 5min, if color is orange, then sample Vibrio parahemolyticus is negative, without Vibrio parahemolyticus, if color is green, then sample Vibrio parahemolyticus is positive, containing Vibrio parahemolyticus.
7. a detection kit for Vibrio parahemolyticus, comprise ring mediated isothermal amplification reagent and detect primer sets, it is characterized in that, described detection primer sets is made up of following primer:
Upstream outer primer is F3:5 '-GCAAAGAAACGCTTGGCG-3 ';
Downstream outer primer B3:5 '-TGCATAGCAATGTTGTCGCT-3 ';
Upstream inner primer FIP:5 '-TCTCTCGGGTGGTGGATGGGTTTCGTTACACTCCGTTCGC-3 ';
Downstream inner primer BIP:5 '-ATGGTTTGCTACTCTCGCACCCTCGGCTGACAAATGGCTCTA-3 '.
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| CN105803057A (en) * | 2015-11-26 | 2016-07-27 | 青岛大学 | Novel isothermal loop-mediated vibrio parahaemolyticus nucleic acid marker detection reagent |
| CN106222274A (en) * | 2016-08-05 | 2016-12-14 | 集美大学 | A kind of method for quick of Martin Hollis Ge Limengte Salmonella |
| CN109680079A (en) * | 2018-06-08 | 2019-04-26 | 深圳市计量质量检测研究院（国家高新技术计量站、国家数字电子产品质量监督检验中心） | Detect RPA primer, probe, kit and the method for vibrio parahemolyticus |
| CN111057780A (en) * | 2015-09-02 | 2020-04-24 | 上海旺旺食品集团有限公司 | Rapid isothermal nucleic acid detection method and kit for 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 |
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| CN106222274A (en) * | 2016-08-05 | 2016-12-14 | 集美大学 | A kind of method for quick of Martin Hollis Ge Limengte Salmonella |
| CN106222274B (en) * | 2016-08-05 | 2020-01-31 | 集美大学 | quick detection method for hollisgilettia |
| CN109680079A (en) * | 2018-06-08 | 2019-04-26 | 深圳市计量质量检测研究院（国家高新技术计量站、国家数字电子产品质量监督检验中心） | Detect RPA primer, probe, kit and the method for vibrio parahemolyticus |
| 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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