CN113930528A - Method for detecting vibrio in coastal seawater - Google Patents
Method for detecting vibrio in coastal seawater Download PDFInfo
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- CN113930528A CN113930528A CN202010670320.3A CN202010670320A CN113930528A CN 113930528 A CN113930528 A CN 113930528A CN 202010670320 A CN202010670320 A CN 202010670320A CN 113930528 A CN113930528 A CN 113930528A
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- 241000607598 Vibrio Species 0.000 title claims abstract description 29
- 239000013535 sea water Substances 0.000 title claims abstract description 13
- 238000000034 method Methods 0.000 title abstract description 15
- 238000001190 Q-PCR Methods 0.000 claims abstract description 19
- 239000013612 plasmid Substances 0.000 claims abstract description 18
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 14
- 230000007613 environmental effect Effects 0.000 claims abstract description 12
- 238000001514 detection method Methods 0.000 claims abstract description 11
- 238000012408 PCR amplification Methods 0.000 claims abstract description 7
- 238000012163 sequencing technique Methods 0.000 claims abstract description 7
- 206010047400 Vibrio infections Diseases 0.000 claims abstract description 6
- 239000013641 positive control Substances 0.000 claims abstract description 6
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 11
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 9
- 229960000723 ampicillin Drugs 0.000 claims description 9
- 239000012154 double-distilled water Substances 0.000 claims description 9
- 239000012634 fragment Substances 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 8
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 7
- 238000000137 annealing Methods 0.000 claims description 7
- 238000004925 denaturation Methods 0.000 claims description 7
- 230000036425 denaturation Effects 0.000 claims description 7
- 238000012257 pre-denaturation Methods 0.000 claims description 7
- 230000001580 bacterial effect Effects 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 241000588724 Escherichia coli Species 0.000 claims description 5
- 239000000499 gel Substances 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 108020004707 nucleic acids Proteins 0.000 claims description 4
- 150000007523 nucleic acids Chemical class 0.000 claims description 4
- 102000039446 nucleic acids Human genes 0.000 claims description 4
- 235000010585 Ammi visnaga Nutrition 0.000 claims description 3
- 244000153158 Ammi visnaga Species 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 238000010276 construction Methods 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 239000000243 solution Substances 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 2
- 238000000576 coating method Methods 0.000 claims description 2
- 230000028744 lysogeny Effects 0.000 claims description 2
- 238000001962 electrophoresis Methods 0.000 abstract 1
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 241000607272 Vibrio parahaemolyticus Species 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000009360 aquaculture Methods 0.000 description 2
- 244000144974 aquaculture Species 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 235000020637 scallop Nutrition 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000143060 Americamysis bahia Species 0.000 description 1
- 206010003267 Arthritis reactive Diseases 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- 208000005577 Gastroenteritis Diseases 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 241000237509 Patinopecten sp. Species 0.000 description 1
- 241000237503 Pectinidae Species 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 208000012873 acute gastroenteritis Diseases 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
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- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
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- 208000013223 septicemia Diseases 0.000 description 1
- 235000015170 shellfish Nutrition 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention discloses a method for detecting vibrio in coastal seawater, which comprises the following steps: 1) designing upstream and downstream primers aiming at specific genes of vibrios; performing PCR amplification on positive control vibrios, performing electrophoresis detection on PCR amplification products, recovering gel from the presented target bands, and sequencing; 2) constructing a standard plasmid template; 3) establishing a Q-PCR quantitative standard curve: 4) and (3) environmental sample determination: the method shortens the detection time from 10-12 days of the original method to 1 day, only needs 3-4 hours under the condition of completing the standard curve, and secondly, overcomes the problem that the existing method can not determine the non-culturable vibrio, and improves the identification accuracy.
Description
Technical Field
The invention relates to the technical field of common bacteria detection, in particular to a method for detecting vibrio in coastal seawater.
Background
Spp is a gram-negative halophilic bacterium, which is mainly present in coastal sea water, sea water sediments, and marine products such as fish, shrimp, and shellfish. On the one hand, eating seafood contaminated with vibrio can cause diseases such as acute gastroenteritis, reactive arthritis, and the like, and even septicemia in severe cases. On the other hand, the vibrio can cause diseases of aquaculture products such as scallops, shrimps and the like, and cause aquaculture diseases. At present, the selective culture method consumes long time for detecting vibrios and large manpower and material resources. Therefore, there is a need for a method for detecting the number of non-culturable vibrios in a short time with high efficiency.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a high-efficiency short-detection-time detection method for vibrio in coastal seawater.
The technical scheme of the invention is summarized as follows:
a method for detecting vibrio in coastal seawater comprises the following steps:
1) designing an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2 aiming at a vibrio specific gene; performing PCR amplification on positive control vibrios, performing 1% agarose gel electrophoresis detection on PCR amplification products, cutting and recovering presented target bands, and sequencing;
2) construction of a standard plasmid template:
connecting the DNA fragment recovered by cutting the gel in the step 1) with a PGM-T carrier, introducing the DNA fragment into escherichia coli, and coating the escherichia coli with an LB (lysogeny broth) selection plate containing 50 mu g/ml ampicillin for overnight culture after enrichment culture; randomly picking a single colony by using a sterile toothpick, carrying out shake culture for 2h in an LB liquid selective culture medium containing 50 mu g/mL ampicillin, taking a bacterial liquid as a template, carrying out PCR screening on positive clones by using an upstream primer shown by SEQ ID NO.1 and a downstream primer shown by SEQ ID NO.2, detecting the positive clones by using 1% agarose gel electrophoresis, carrying out overnight culture on the bacterial liquid corresponding to the positive clones in the LB liquid selective culture medium containing 100 mu g/mL ampicillin, and extracting plasmid sequencing;
3) establishing a Q-PCR quantitative standard curve:
measuring the concentration of the plasmid obtained in the step 2) by using a nucleic acid content measuring instrument, and using online calculation software: http:// cells. uri. edu/gsc/cndna. html calculates the accurate copy number of the plasmid template, uses double distilled water to carry out 10 times gradient dilution, and takes the diluted plasmid template DNA water solution as the template to establish a reaction system: 2 × SYBR Green QPCR Mix 5 μ L, double distilled water 3.5 μ L, 10 μ M upstream primer 0.25 μ L, 10 μ M downstream primer 0.25 μ L, template 1 μ L; the Q-PCR program was run on a Q-PCR instrument: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1min, and 40 cycles; establishing a standard curve according to the Ct value read by the Q-PCR instrument after the reaction and the logarithm value of the copy number of the template aqueous solution;
4) and (3) environmental sample determination:
extracting DNA of an environment sample, and establishing a reaction system by taking the DNA of the environment sample as a template: 2 × SYBR Green QPCR Mix 5 μ L, double distilled water 3.5 μ L, 10 μ M upstream primer 0.25 μ L, 10 μ M downstream primer 0.25 μ L, template 1 μ L; the Q-PCR program was run on a Q-PCR instrument: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1min, and 40 cycles; and substituting the Ct value read by the Q-PCR instrument after the reaction is finished into the standard curve to calculate the vibrio concentration in the environmental sample.
The invention has the advantages that:
compared with the existing detection based on the selective culture method, the method shortens the detection time from 10-12 days of the original method to 1 day, only needs 3-4 hours under the condition of completing a standard curve, and secondly overcomes the problem that the existing method can not determine the non-culturable vibrio, thereby improving the identification accuracy.
Drawings
FIG. 1 is an agarose gel electrophoresis of a fragment of interest; the leftmost side is marker, the rightmost side + CON is positive control, and the rest are environmental samples;
FIG. 2 is a graph of Q-PCR amplification;
FIG. 3 is a graph of Q-PCR dissolution;
FIG. 4 is a Q-PCR standard curve.
Detailed Description
The positive control Vibrio is a purchased Vibrio parahaemolyticus strain Vibrio parahaemolyticus (ATCC17802), purchased from Taobao.com, Taobao, 5 months in 2018.
PGM-T vector (purchased Tiangen pGM-Simple-T Fast kit)
Nucleic acid content measuring apparatus (
ND-1000UV-VisSpectrophotometer)
The present invention will be further described with reference to the following examples.
Example 1
A method for detecting vibrio in coastal seawater comprises the following steps:
1) looking up related documents to obtain a specific gene of vibrio, wherein the size of a target fragment is about 113bp, and an upstream primer shown by SEQ ID NO.1 and a downstream primer shown by SEQ ID NO.2 are designed; the positive control vibrio (ATCC17802) was subjected to PCR amplification in the following system: 2 XPCR Master Mix 25. mu. L, ddH2O22. mu.L, forward primer (10. mu.M) 1. mu.L, and reverse primer (10. mu.M) 1. mu. L, DNA 1. mu.L. The PCR procedure used: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; final extension at 72 ℃ for 5 min. Carrying out 1% agarose gel electrophoresis detection on the PCR product, wherein a rightmost strip in the figure 1 shows that an environmental sample strip is a seawater sample collected from Qinhuang island city, and the environmental sample strip shows that the primer still can be singly amplified when the environmental sample DNA is taken as a template, and the presented target strip is cut and recovered with gel for sequencing;
an upstream primer: ggcgtaaagcgcatgcaggt SEQ ID NO. 1;
a downstream primer: gaaattctacccccctctacag SEQ ID NO. 2;
2) construction of a standard plasmid template:
purifying the DNA fragment recovered by cutting gel in the step 1) by using a Tiangen common agarose gel DNA recovery kit, connecting the purified DNA fragment with a PGM-T carrier at constant temperature of 16 ℃, adding the purified DNA fragment into a 1.5mL centrifuge tube containing 100 mu L of escherichia coli E.coli DH5 alpha competent cells in full quantity, slightly sucking, uniformly mixing, and carrying out ice bath for 30 minutes. After the heat shock at 42 ℃ for 180s, the ice bath is carried out again for 1 min. Adding about 1mL of LB liquid culture medium, oscillating at 37 ℃, and carrying out enrichment culture for 2 h; mixing, spreading about 100 μ L of the mixture on LB selective plate containing ampicillin 50 μ g/ml, sealing with sealing film, and culturing at 37 deg.C for overnight; 15 single colonies were picked at random with sterile toothpicks and shake-cultured for 2h in LB liquid selective medium containing ampicillin at a concentration of 50. mu.g/ml. And (3) carrying out PCR screening on positive clones by using the bacterial liquid as a template and using an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in ID NO.2, and detecting the positive clones by using 1% agarose gel electrophoresis. The bacterial suspension corresponding to the positive clone was cultured overnight in 5mL of LB liquid selection medium containing 100. mu.g/mL ampicillin. Plasmid extraction was performed with Tiangen brand plasmid extraction kit according to the instructions provided by the trade company, and sequencing was performed.
3) Establishing a Q-PCR quantitative standard curve:
the plasmid obtained in step 2) was produced by using the company Thermo Fisher Scientific, USA
Plasmid concentrations were determined with an ND-1000UV-Vis Spectrophotometer nucleic acid content meter and used in the on-line calculation software: http:// cells. uri. edu/gsc/cndna. html calculates the accurate copy number of the plasmid template, uses double distilled water to carry out 10 times gradient dilution, and takes the diluted plasmid template DNA water solution as the template to establish a reaction system: 2 × SYBR Green QPCR Mix 5 μ L, double distilled water 3.5 μ L, 10 μ M upstream primer 0.25 μ L, 10 μ M downstream primer 0.25 μ L, template 1 μ L; the Q-PCR program was run on a Q-PCR instrument: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1min, and 40 cycles;
the dissolution curve generated by the reaction is shown in fig. 3, which has only a single peak, indicating that the process is accurately feasible. As shown in FIG. 2, the amplification curves generated by the reaction,
establishing a standard curve according to the Ct value read by the Q-PCR instrument after the reaction and the logarithm value of the copy number of the template aqueous solution, and showing the standard curve in figure 4;
4) environmental sample assay
500mL of each seawater sample of 8 stations collected from Luan estuary scallop cultivation areas of Qinhuang island in 9 months of 2019 is intercepted on a microporous filter membrane with the aperture of 0.22 mu m by a suction filtration system, and DNA on the membrane is extracted by using an E.N.Z.A.TM. Water DNA Kit produced by American Omega company, and the concrete steps are referred to the Kit specification. Taking an environmental sample DNA as a template, establishing a reaction system: 2 × SYBR Green QPCR Mix 5 μ L, double distilled water 3.5 μ L, 10 μ M upstream primer 0.25 μ L, 10 μ M downstream primer 0.25 μ L, template 1 μ L; the Q-PCR program was run on a Q-PCR instrument: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1min, and 40 cycles; ct values read by the Q-PCR instrument after the reaction are substituted into the standard curve to calculate the vibrio concentration in the environmental sample, and the results are shown in Table 1.
TABLE 1 abundance of Vibrio bacteria at different sites in Kyue Qinhuang island
| Station number | Vibrio concentration (copies/mL) |
| 1 | 1.15E+06 |
| 2 | 9.90E+05 |
| 3 | 9.44E+05 |
| 4 | 2.73E+05 |
| 5 | 6.83E+05 |
| 6 | 4.50E+05 |
| 7 | 3.94E+05 |
| 8 | 2.55E+05 |
Sequence listing
<110> Tianjin university
<120> detection method of vibrio in coastal seawater
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gaaattctac ccccctctac ag 22
Claims (1)
1. A detection method of vibrio in coastal seawater is characterized by comprising the following steps:
1) designing an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2 aiming at a vibrio specific gene; performing PCR amplification on positive control vibrios, performing 1% agarose gel electrophoresis detection on PCR amplification products, cutting and recovering presented target bands, and sequencing;
2) construction of a standard plasmid template:
connecting the DNA fragment recovered by cutting the gel in the step 1) with a PGM-T carrier, introducing the DNA fragment into escherichia coli, and coating the escherichia coli with an LB (lysogeny broth) selection plate containing 50 mu g/ml ampicillin for overnight culture after enrichment culture; randomly picking a single colony by using a sterile toothpick, carrying out shake culture for 2h in an LB liquid selective culture medium containing 50 mu g/mL ampicillin, taking a bacterial liquid as a template, carrying out PCR screening on positive clones by using an upstream primer shown by SEQ ID NO.1 and a downstream primer shown by SEQ ID NO.2, detecting the positive clones by using 1% agarose gel electrophoresis, carrying out overnight culture on the bacterial liquid corresponding to the positive clones in the LB liquid selective culture medium containing 100 mu g/mL ampicillin, and extracting plasmid sequencing;
3) establishing a Q-PCR quantitative standard curve:
measuring the concentration of the plasmid obtained in the step 2) by using a nucleic acid content measuring instrument, and using online calculation software: http:// cells. uri. edu/gsc/cndna. html calculates the accurate copy number of the plasmid template, uses double distilled water to carry out 10 times gradient dilution, and takes the diluted plasmid template DNA water solution as the template to establish a reaction system: 2 × SYBR Green QPCR Mix 5 μ L, double distilled water 3.5 μ L, 10 μ M upstream primer 0.25 μ L, 10 μ M downstream primer 0.25 μ L, template 1 μ L; the Q-PCR program was run on a Q-PCR instrument: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1min, and 40 cycles; establishing a standard curve according to the Ct value read by the Q-PCR instrument after the reaction and the logarithm value of the copy number of the template aqueous solution;
4) and (3) environmental sample determination:
extracting DNA of an environment sample, and establishing a reaction system by taking the DNA of the environment sample as a template: 2 × SYBR Green QPCR Mix 5 μ L, double distilled water 3.5 μ L, 10 μ M upstream primer 0.25 μ L, 10 μ M downstream primer 0.25 μ L, template 1 μ L; the Q-PCR program was run on a Q-PCR instrument: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1min, and 40 cycles; and substituting the Ct value read by the Q-PCR instrument after the reaction is finished into the standard curve to calculate the vibrio concentration in the environmental sample.
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| CN109486971A (en) * | 2018-11-27 | 2019-03-19 | 天津大学 | The detection method of salmonella in a kind of coastal seawater |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109486971A (en) * | 2018-11-27 | 2019-03-19 | 天津大学 | The detection method of salmonella in a kind of coastal seawater |
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| Title |
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| 张亚豪等: ""基于实时荧光定量ＰＣＲ技术监测四川工业泡菜发酵过程中主要细菌的变化"", 《中国调味品》 * |
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