CN101082063A - One-tube half-nest type PCR detection reagent case for injury vibrio and detection method - Google Patents

One-tube half-nest type PCR detection reagent case for injury vibrio and detection method Download PDF

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CN101082063A
CN101082063A CNA2007101007582A CN200710100758A CN101082063A CN 101082063 A CN101082063 A CN 101082063A CN A2007101007582 A CNA2007101007582 A CN A2007101007582A CN 200710100758 A CN200710100758 A CN 200710100758A CN 101082063 A CN101082063 A CN 101082063A
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electrophoresis
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CN100572561C (en
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谢珍玉
周永灿
冯永勤
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Hainan University
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Abstract

The present invention is one-pipe and semi-nest type PCR detection kit and process for fast detecting Vibrio vulnificus. The kit includes: one DNA extracting reagent containing buffering reagent and cell cracking reagent; one PCR reagent including one reaction pre-mixing reagent, one first reaction primer reagent with specific primers Vvg1 of 5'-CATCGT GGTGGTCATACTCATAGC-3' and Vvg2 of 5'-GGTGCTCGGCTAAGAAGTCGTT-3', and one second reaction primer reagent with specific primer Vvg3 of 5'-AAGACGGGATTGCGGTTGAAGT-3'; and one electrophoresis and color-producing reagent containing for reagent components. The detection process includes the steps of: treating sample, extracting DNA, PCR amplifying, and electrophoresis and color-producing detection. The present invention has the advantages of high detection speed, high specificity and high sensitivity.

Description

One pipe method half-nest type PCR detection reagent and detection method of Vibrio vulnificus
Technical field
The present invention relates to a kind of test kit that adopts a pipe method heminested PCR to detect Vibrio vulnificus, further relate to the method for using a pipe method half-nest type PCR detection reagent to detect Vibrio vulnificus.
Background technology
Vibrio vulnificus (Vibrio vulnificus) is the beastly cause of disease of suffering from altogether of a kind of people, mainly is distributed in all over the world water warm coastal waters and seawater, marine bottom sediment and the animal body in bay interior (Pfeffer et al, 2003; Zheng Guoxing etc., 1999), can cause infection by take food living or unseasoned seafood food or the skin contact seawater by breakage, can cause the mankind (Nascimento et al strongly, 2001) and multiple hydrocoles such as stone dish fish (Shen Zhiqiang etc., 2001), cabio (simple Ji Chang etc., 2003), (He Wei is virtuous for Portunus trituberculatus Miers, 2004) high septicemia and Sepsis such as mortality ratio, because the characteristics that this bacterium easily develops immunity to drugs, also do not treat at present the Perfected process of this serious disease, therefore, the rapid detection of Vibrio vulnificus will be to impel the key of human health and fishery products healthy aquaculture.The detection means of existing Vibrio vulnificus mainly contains traditional separation and Culture and biochemical identification, elisa technique, DNA hybridization technique, molecular probe hybridization technique, common double primer PCR technology etc., these methods have has time-consuming taking a lot of work and professional requirements height, have to have operation easier big, what have has shortcomings such as susceptibility is low, be difficult in the aquaculture process actual the popularization, obviously overslaugh the control of the great epidemic disease that causes because of Vibrio vulnificus.
Heminested PCR is realized by 3 Auele Specific Primers, wherein one is public primer, outside primer in two other is respectively, this method has obviously improved the specificity of PCR detection technique, have simple to operate, highly sensitive, quick and precisely wait advantage, be that domestic and international disease researcher carries out the prefered method that cause of disease detects, therefore, this technology is used widely in the detection of bacterial pathogen.
Summary of the invention
A but pipe method half-nest type PCR detection reagent that the purpose of this invention is to provide a kind of rapid detection Vibrio vulnificus, 3 kinds of Vibrio vulnificus Auele Specific Primer Vvg1, Vvg2, Vvg3 particularly are provided, and a kind of method of using a pipe method half-nest type PCR detection reagent to detect Vibrio vulnificus is provided.
The present invention utilizes 3 specificity sites of Vibrio vulnificus gyrB gene and has designed 3 oligonucleotide primers (Vvg1 of heminested PCR, Vvg2, Vvg3), in the PCR of high-fidelity polysaccharase system, at first with Vvg1 and Vvg2 several circulations of increasing, can produce the fragment of a certain amount of 700bp, and then adding primer Vvg3, at this moment, two couples of primers (Vvg1 and Vvg2, Vvg1 and Vvg3) all can the template of self effectively be increased, but the amplification efficiency of Vvg1 and this a pair of primer of Vvg3 is greater than last amplification efficiency to primer (Vvg1 and Vvg2), the whole output of so just having guaranteed 2 bar segment of 700bp and 350bp in the product is basic identical, reaches the highly sensitive and split hair caccuracy of detection with this.
The technical solution adopted in the present invention is: an a kind of pipe method heminested PCR detects the test kit of Vibrio vulnificus, comprising:
(1) DNA extraction reagent: contain buffer reagent A and cell cracking agent B; Reagent A contains NaCl 200～350mmol/L, Tutofusin tris 50～100mmol/L (ph=8.0), disodium ethylene diamine tetraacetate 50～100mmol/L; It is 5～10% sodium dodecyl sulfate aqueous solution that reagent B contains concentration;
(2) polymerase chain reaction reagent: comprise reaction premix reagent C and reaction primer reagent D and E; Reagent C comprises 10 * reaction buffer, 5 μ L, 500～1000 μ mol L -1DNTPs (four kinds of isocyatic mixtures of deoxynucleotide), 1～5mmol L -1MgCl 2, the hot polymerization synthase of 1～2 unit and an amount of sterilization ultrapure water; Reagent D comprises 2 kinds of primers, and concentration is 5～20 μ mol L -1, the primer here refers in particular to:
Vvg1(5’-CATCGTGGTGGTCATACTCATAGC-3’)
Vvg2(5’-GGTGCTCGGCTAAGAAGTCGTT-3’)
Reagent E comprises a kind of primer, and its concentration is 10～30 μ mol L -1, the primer here refers in particular to:
Vvg3(5’-AAGACGGGATTGCGGTTGAAGT-3’)
(3) electrophoresis and colouring reagents comprise reagent F, reagent G, reagent H and reagent I;
Reagent F: be 20-50 times of electrophoretic buffer, contain the trihydroxy methyl aminomethane---acetic acid and disodium ethylene diamine tetraacetate, concentration are respectively 0.01～0.04mol/L and 0.001mol/L;
Reagent G: contain 0.1～0.5g agarose;
Reagent H:Nucleic acid ClearDNA stain
Reagent I: standard molecular weight DL2000
The method that the test kit that uses an above-mentioned pipe method heminested PCR to detect Vibrio vulnificus detects Vibrio vulnificus comprises the following steps:
1, sample preparation: get 0.5～5 the gram sample (water sample: 1～5ml), with 50～1000 μ L reagent A suspended sample, and with the abundant mixing of sample, centrifugal 3～10 minutes of 1000～3000g gets the supernatant tube, centrifugal 3～10 minutes of 8000～15000g abandons supernatant;
2, DNA extraction: with 50～150 μ L reagent A precipitation that suspends once more, and add 5～10 μ L reagent B, mixing, room temperature was placed more than 5 minutes, was incubated 10～30 minutes in the boiling water bath; Centrifugal 10 minutes of 8000～15000g gets the supernatant tube, and adds the Virahol of supernatant 2/3 volume; Room temperature was placed 10 minutes behind the mixing, centrifugal 5 minutes of 8000～15000g; Remove supernatant, 75% washing with alcohol once allows ethanol thoroughly volatilize, and precipitation is dissolved in 10～50 μ L sterilization distilled water, has promptly made DNA extraction liquid;
3, polymerase chain reaction (PCR) amplification: get 0.5～2 μ L DNA extraction liquid and 40～45 μ L reagent C, the mixing of 1～2 μ L reagent D; On thermal cycler, increase by the following method: at first with 94 ℃ of pre-sex change 1～4min; Follow with 92～95 ℃ of sex change 0.5～2min, 62 ℃ of annealing 0.5～2min, 72 ℃ are extended 0.5～2min, 5～15 circulations of coamplification; Add 1～2 μ L reagent E, with 92～95 ℃ of sex change 0.5～2min, 62 ℃ of annealing 0.5～2min, 72 ℃ are extended 0.5～2min, 20～40 circulations of increasing again; Last 72 ℃ are extended 5～10min, 4 ℃ of preservations;
4, electrophoresis and color developing detection: reagent F is diluted to 1 times electrophoretic buffer, boil after getting the 1 times of electrophoretic buffer of 10～40mL and the abundant mixing of reagent G, nucleic acid level electrophoresis chamber glue with 10cm * 10cm, solidify the back and add 1 times of an amount of electrophoretic buffer, liquid level is higher than about glue face 0.5～2mm; The sample of getting after 5～20 μ L increase mixes on preservative film with 0.8～4 μ L reagent H, leaves standstill more than 5～10min, and this biased sample is added in the above-mentioned glue hole, simultaneously, adds the reagent I of 1～5 μ L in another glue hole; 50～100V electrophoresis, 20～30min; Detect under the UV-light, if one 700 nucleotide pair and one 's 350 nucleotide pair band is arranged simultaneously, interpret sample is infected or pollutes by Vibrio vulnificus; If have only one 350 nucleotide pair band in the sample, then need to do another amplified reaction experiment with primer Vvg1 and Vvg3, if can produce the band of one 700 nucleotide pair in the sample, same interpret sample has Vibrio vulnificus to infect or pollutes; Otherwise then do not have.
Advantage of the present invention:
1, present, at the PCR detection of Vibrio vulnificus, all be the regular-PCR of two primers, the present invention utilizes heminested PCR to realize the high specificity of this cause of disease, highly sensitive detection first.
2, the heminested PCR of standard needs to carry out respectively 2 amplifications in 2 different reaction systems, from collected specimens to reading detected result required detection time about 1 day.By condition optimizing, the present invention has realized heminested PCR through 1 amplification in 1 reaction system, to reading detected result required detection time within 4 hours, has improved the detection efficiency of heminested PCR from collected specimens.
Description of drawings
Fig. 1 detects Vibrio vulnificus sensitivity design sketch under the UV-light.M: reagent I; 1:10 6Individual thalline/ml sample; 2:10 5Individual thalline/ml sample; 3:10 4Individual thalline/ml sample; 4:10 3Individual thalline/ml sample; 5:10 2Individual thalline/ml sample; 6:10 1Individual thalline/ml sample; 7:10 0Individual thalline/ml sample; 8 and 9: blank sample.
Fig. 2 is the detection design sketch to Vibrio vulnificus in the environment seawater sample.M: reagent I; 1: seawater sample; 2:0.22 μ m membrane filtration seawater sample; 3: do not add the template amplification sample; 4:ddH 2The O sample.
Fig. 3 is the detection design sketch of Vibrio vulnificus in the prawn digestive tube.M: reagent I; 1: prawn digestive tube sample; 2: do not add the template amplification sample; 3:ddH 2The O sample.
Embodiment
The present invention is further described with indefiniteness embodiment below.
Embodiment one,
Detection follows these steps to carry out to the test of Vibrio vulnificus sensitivity:
1, determines that by microscopic counting the Vibrio vulnificus original liquid concentration is 10 6Individual thalline/ml;
2, be 10 with above-mentioned stoste as gradient dilution 5Individual thalline/ml, 10 4Individual thalline/ml, 10 3Individual thalline/ml, 10 2Individual thalline/ml, 10 1Individual thalline/ml and 10 0Individual thalline/ml;
3, each sample is got 1ml, and centrifugal 3 minutes of 10000g abandons supernatant;
4, with 60 μ L reagent A (NaCl 250mmol/L, Tutofusin tris 60mmol/L (ph=8.0), disodium ethylene diamine tetraacetate 50mmol/L) suspension precipitation, and add 8 μ L reagent B (8% sodium dodecyl sulfate aqueous solution), mixing, room temperature was placed more than 5 minutes, and insulation is 15 minutes in the boiling water bath; Centrifugal 10 minutes of 11000g gets the supernatant tube, and adds 50 μ L Virahols; Room temperature was placed 10 minutes behind the mixing, centrifugal 5 minutes of 12000g; Remove supernatant, 75% washing with alcohol once allows ethanol thoroughly volatilize, and precipitation is dissolved in 30 μ L sterilization distilled water, has promptly made DNA extraction liquid;
5, polymerase chain reaction (PCR) amplification: get 1 μ L DNA extraction liquid and 22 μ L reagent C (10 * reaction buffer, 5 μ L, 800 μ mol L -1DNTPs, 2mmol L -1MgCl 2, the hot polymerization synthase of 2 units and an amount of sterilization ultrapure water), 1 μ L reagent D (10 μ mol L -1Vvg1 and Vvg2) mix; On thermal cycler, increase by the following method: at first with 94 ℃ of pre-sex change 4min; Follow with 93 ℃ of sex change 1.5min, 62 ℃ of annealing 1.5min, 72 ℃ are extended 1.5min, 8 circulations of coamplification; Add 1 μ L reagent E (8 μ mol L -1Vvg3), with 93 ℃ of sex change 1min, 62 ℃ of annealing 1min, 72 ℃ are extended 1.5min, 30 circulations of increasing again; Last 72 ℃ are extended 7min, 4 ℃ of preservations;
6, electrophoresis and color developing detection: reagent F is diluted to 1 times electrophoretic buffer, the 1 times of electrophoretic buffer and the reagent G (0.3g agarose) that get 25mL fully boil behind the mixing, nucleic acid level electrophoresis chamber glue with 10cm * 10cm, solidify the back and add 1 times of an amount of electrophoretic buffer, liquid level is higher than about glue face 0.5mm; The sample of getting after 10 μ L increase mixes on preservative film with 2 μ L reagent H, leaves standstill 15min, and this biased sample is added in the above-mentioned glue hole, simultaneously, adds the reagent I of 3 μ L in another glue hole; 60V electrophoresis 25min; Detect under the UV-light, the results are shown in Figure 1, remove 10 1Outside two bands of individual thalline/ml sample (350bp and 700bp) are more weak, other 5 concentration gradients (10 6~ 10 2) two bands of tangible 700bp and 350bp are all arranged.
Can reach a conclusion from detected result: the limit of detection of the test kit of detection Vibrio vulnificus provided by the present invention can reach 10 thalline/ml.
Embodiment two,
Follow these steps to detection to Vibrio vulnificus in the environment seawater sample:
1, get seawater 3ml from the sea area, Haikou, centrifugal 10 minutes of 10000g abandons supernatant, with 50 μ L reagent A (NaCl 250mmol/L, Tutofusin tris 60mmol/L (ph=8.0), disodium ethylene diamine tetraacetate 50mmol/L) suspended sample, and with the abundant mixing of sample;
2, add 5 μ L reagent B (8% sodium dodecyl sulfate aqueous solution), mixing, room temperature was placed more than 8 minutes, and insulation is 20 minutes in the boiling water bath; Centrifugal 10 minutes of 12000g gets the supernatant tube, and adds 40 μ L Virahols; Room temperature was placed 10 minutes behind the mixing, centrifugal 5 minutes of 12000g; Remove supernatant, 75% washing with alcohol once allows ethanol thoroughly volatilize, and precipitation is dissolved in 20 μ L sterilization distilled water, has promptly made DNA extraction liquid;
3, polymerase chain reaction (PCR) amplification: get 3 μ L DNA extraction liquid and 43 μ L reagent C (10 * reaction buffer, 5 μ L, 1000 μ mol L -1DNTPs, 2.5mmol L -1MgCl 2, the hot polymerization synthase of 1.5 units and an amount of sterilization ultrapure water), 2 μ L reagent D (Vvg1 and the Vvg2 of 10 μ mol L-1) mix; On thermal cycler, increase by the following method: at first with 94 ℃ of pre-sex change 4min; Follow with 93 ℃ of sex change 1min, 61 ℃ of annealing 1min, 72 ℃ are extended 1.5min, 10 circulations of coamplification; Add 2 μ L reagent E (10 μ mol L -1Vvg3), with 93 ℃ of sex change 1min, 62 ℃ of annealing 0.5min, 72 ℃ are extended 1.5min, 25 circulations of increasing again; Last 72 ℃ are extended 10min, 4 ℃ of preservations;
4, electrophoresis and color developing detection: reagent F is diluted to 1 times electrophoretic buffer, the 1 times of electrophoretic buffer and the reagent G (0.3g agarose) that get 30mL fully boil behind the mixing, nucleic acid level electrophoresis chamber glue with 10cm * 10cm, solidify the back and add 1 times of an amount of electrophoretic buffer, liquid level is higher than about glue face 0.5mm; The sample of getting after 10 μ L increase mixes on preservative film with 2 μ L reagent H, leaves standstill more than the 10min, and this biased sample is added in the above-mentioned glue hole, simultaneously, adds the reagent I of 3 μ L in another glue hole; 60V electrophoresis 40min; Detect under the UV-light, obviously visible one 700 nucleotide pair and one 's 350 nucleotide pair band (see figure 2) shows in this waters sample and contains Vibrio vulnificus.
Embodiment three,
Follow these steps to the detection of Vibrio vulnificus in the prawn digestive tube:
1, buys breed Environment of Litopenaeus vannamei Low 500g (body is long for about 20cm) from market, get 2 tails at random as test sample, the digestive tube of anatomical isolation sample, change in the aseptic centrifuge tube of 5ml, add 1000 μ L reagent A suspended sample, and with the abundant mixing of sample, centrifugal 10 minutes of 1500g; Get the supernatant tube, centrifugal 8 minutes of 12000g abandons supernatant;
2, with 50 μ L reagent A (NaCl 250mmol/L, Tutofusin tris 60mmol/L (ph=8.0), disodium ethylene diamine tetraacetate 50mmol/L) suspended sample, and with the abundant mixing of sample, add 5 μ L reagent B (10% sodium dodecyl sulfate aqueous solution), mixing, room temperature were placed more than 5 minutes, and insulation is 25 minutes in the boiling water bath; Centrifugal 10 minutes of 10000g gets the supernatant tube, and adds 40 μ L Virahols; Room temperature was placed 10 minutes behind the mixing, centrifugal 5 minutes of 12000g; Remove supernatant, 75% washing with alcohol once allows ethanol thoroughly volatilize, and precipitation is dissolved in 30 μ L sterilization distilled water, has promptly made DNA extraction liquid;
3, polymerase chain reaction (PCR) amplification: get 2 μ L DNA extraction liquid and 50 μ L reagent C (10 * reaction buffer, 5 μ L, 1000 μ mol L -1DNTPs, 2.5mmol L -1MgCl 2, the hot polymerization synthase of 1.5 units and an amount of sterilization ultrapure water), 2 μ L reagent D (15 μ mol L -1Vvg1 and Vvg2) mix; On thermal cycler, increase by the following method: 94 ℃ of pre-sex change 3min; Follow with 93 ℃ of sex change 1min, 60 ℃ of annealing 1.5min, 72 ℃ are extended 1.5min, 10 circulations of coamplification; Add 2 μ L reagent E (10 μ mol L -1Vvg3), with 93 ℃ of sex change 1min, 60 ℃ of annealing 1min, 72 ℃ are extended 1.5min, 25 circulations of increasing again; Last 72 ℃ are extended 10min, 4 ℃ of preservations;
4, electrophoresis and color developing detection: reagent F is diluted to 1 times electrophoretic buffer, the 1 times of electrophoretic buffer and the reagent G (0.25g agarose) that get 25mL fully boil behind the mixing, nucleic acid level electrophoresis chamber glue with 10cm * 10cm, solidify the back and add 1 times of an amount of electrophoretic buffer, liquid level is higher than about glue face 0.5mm; The sample of getting after 10 μ L increase mixes on preservative film with 2 μ L reagent H, leaves standstill more than the 10min, and this biased sample is added in the above-mentioned glue hole, simultaneously, adds the reagent I of 3 μ L in another glue hole; 60V electrophoresis 40min; Detect under the UV-light, obvious visible one 700 nucleotide pair and one 's 350 nucleotide pair band (see figure 3), the result shows that this prawn sample is polluted by Vibrio vulnificus.

Claims (3)

1, an a kind of pipe method half-nest type PCR detection reagent comprises:
(1) DNA extraction reagent: contain buffer reagent A and cell cracking agent B; Reagent A contains NaCl 200～350mmol/L, Tutofusin tris 50～100mmol/L (ph=8.0), disodium ethylene diamine tetraacetate 50～100mmol/L; It is 5～10% sodium dodecyl sulfate aqueous solution that reagent B contains concentration;
(2) polymerase chain reaction reagent: comprise reaction premix reagent C and reaction primer reagent D and E; Reagent C comprises 10 * reaction buffer, 5 μ L, 500～1000 μ mol L -1DNTPs (four kinds of isocyatic mixtures of deoxynucleotide), 1～5mmol L -1MgCl 2, the hot polymerization synthase of 1～2 unit and an amount of sterilization ultrapure water; Reagent D comprises primer Vvg1 and Vvg2, and concentration is 5～20 μ mol L -1,
Vvg1 refers in particular to (5 '-CATCGTGGTGGTCATACTCATAGC-3 ');
Vvg2 refers in particular to (5 '-GGTGCTCGGCTAAGAAGTCGTT-3 ');
Reagent E comprises primer Vvg3, and its concentration is 10～30 μ mol L -1,
Vvg3 refers in particular to (5 '-AAGACGGGATTGCGGTTGAAGT-3 ');
(3) electrophoresis and colouring reagents comprise reagent F, reagent G, reagent H and reagent I; Reagent F: be 20-50 times of electrophoretic buffer, contain the trihydroxy methyl aminomethane--acetic acid and disodium ethylene diamine tetraacetate, concentration are respectively 0.01～0.04mol/L and 0.001mol/L;
Reagent G: contain 0.1～0.5g agarose;
Reagent H:Nucleic acid ClearDNA stain
Reagent I: standard molecular weight DL2000.
2, a kind of method of using the test kit detection Vibrio vulnificus in the claim 1 comprises the following steps:
1), sample preparation: get 0.5～5 gram sample, with 50～1000 μ L reagent A suspended sample, and with the abundant mixing of sample, centrifugal 3～10 minutes of 1000～3000g gets the supernatant tube, and centrifugal 3～10 minutes of 8000～15000g abandons supernatant;
2), DNA extraction: with 50～150 μ L reagent A precipitation that suspends once more, and add 5～10 μ L reagent B, mixing, room temperature was placed more than 5 minutes, was incubated 10～30 minutes in the boiling water bath; Centrifugal 10 minutes of 8000～15000g gets the supernatant tube, and adds the Virahol of supernatant 2/3 volume; Room temperature was placed 10 minutes behind the mixing, centrifugal 5 minutes of 8000～15000g; Remove supernatant, 75% washing with alcohol once allows ethanol thoroughly volatilize, and precipitation is dissolved in 10～50 μ L sterilization distilled water, has promptly made DNA extraction liquid;
3), polymerase chain reaction (PCR) amplification: get 0.5～2 μ L DNA extraction liquid and 40～45 μ L reagent C, the mixing of 1～2 μ L reagent D; On thermal cycler, increase by the following method: at first with 94 ℃ of pre-sex change 1～4min; Follow with 92～95 ℃ of sex change 0.5～2min, 62 ℃ of annealing 0.5～2min, 72 ℃ are extended 0.5～2min, 5～15 circulations of coamplification; Add 1～2 μ L reagent E, with 92～95 ℃ of sex change 0.5～2min, 62 ℃ of annealing 0.5～2min, 72 ℃ are extended 0.5～2min, 20～40 circulations of increasing again; Last 72 ℃ are extended 5～10min, 4 ℃ of preservations;
4), electrophoresis and color developing detection: reagent F is diluted to 1 times electrophoretic buffer, boil after getting the 1 times of electrophoretic buffer of 10～40mL and the abundant mixing of reagent G, nucleic acid level electrophoresis chamber glue with 10cm * 10cm, solidify the back and add 1 times of an amount of electrophoretic buffer, liquid level is higher than about glue face 0.5～2mm; The sample of getting after 5～20 μ L increase mixes on preservative film with 0.8～4 μ L reagent H, leaves standstill more than 5～10min, and this biased sample is added in the above-mentioned glue hole, simultaneously, adds the reagent I of 1～5 μ L in another glue hole; 50～100V electrophoresis, 20～30min; Detect under the UV-light, if one 700 nucleotide pair and one 's 350 nucleotide pair band is arranged simultaneously, interpret sample is infected or pollutes by Vibrio vulnificus; If have only one 350 nucleotide pair band in the sample, then need to do another amplified reaction experiment with primer Vvg1 and Vvg3, if can produce the band of one 700 nucleotide pair in the sample, same interpret sample has Vibrio vulnificus to infect or pollutes; Otherwise then do not have.
3, the method for detection Vibrio vulnificus according to claim 2 is characterized in that: described sample is 1～5ml water sample sample.
CNB2007101007582A 2007-04-17 2007-04-17 One pipe method half-nest type PCR detection reagent and detection method of Vibrio vulnificus Expired - Fee Related CN100572561C (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102329858A (en) * 2011-08-02 2012-01-25 广州甘蔗糖业研究所 Sugarcane smut bacteria nest type polymerase chain reaction (PCR) quick detection method
CN102653786A (en) * 2011-12-27 2012-09-05 杭州师范大学 Vibrio vulnificus PCR (polymerase chain reaction) detection kit and detection method

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102329858A (en) * 2011-08-02 2012-01-25 广州甘蔗糖业研究所 Sugarcane smut bacteria nest type polymerase chain reaction (PCR) quick detection method
CN102329858B (en) * 2011-08-02 2013-02-13 广州甘蔗糖业研究所 Sugarcane smut bacteria nest type polymerase chain reaction (PCR) quick detection method
CN102653786A (en) * 2011-12-27 2012-09-05 杭州师范大学 Vibrio vulnificus PCR (polymerase chain reaction) detection kit and detection method
CN102653786B (en) * 2011-12-27 2014-04-09 杭州师范大学 Vibrio vulnificus PCR (polymerase chain reaction) detection kit and detection method

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