CN106939347B - Dual PCR (polymerase chain reaction) rapid detection kit and method for pathogenic vibrio natriegens of portunus trituberculatus - Google Patents
Dual PCR (polymerase chain reaction) rapid detection kit and method for pathogenic vibrio natriegens of portunus trituberculatus Download PDFInfo
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Abstract
The invention discloses a double PCR rapid detection kit for Portunus trituberculatus pathogeny vibrio natriegens, which comprises a negative control and vibrio natriegens (Vibrio natriegens) XA1 strain DNA as positive control, Mg-containing2+PCR buffer and primer pair of dNTP and DNA polymerase. The invention also discloses a double PCR rapid detection method for the pathogenic vibrio natriegens of the portunus trituberculatus, which comprises the following steps of extracting a DNA template: preparing a PCR premix and carrying out PCR amplification, and observing the result of electrophoresis under an ultraviolet analyzer after the amplification reaction is finished, wherein if the bands appear at 308bp and 526bp, the bands are positive, and if the bands appear at the other bands, the bands are negative. The method has higher sensitivity, can detect 13.2pg of vibrio natriegens genome DNA at the lowest, has simple operation, does not need special technical personnel, and has short whole process time and less time consumption.
Description
Technical Field
The invention relates to a detection kit and a detection method for pathogenic bacteria, in particular to a dual PCR rapid detection kit and a detection method for pathogenic vibrio natriegens of blue crabs.
Background
Vibrio natriegens are common facultative anaerobic bacteria in marine microorganisms, are widely present in seawater, seabed sediments and marine animals at estuaries and offshore banks, are conditional pathogenic bacteria, can cause aquatic animals to suffer from diseases and die, have the death rate of 87.5-100 percent, and seriously damage the healthy development of the marine aquaculture industry in China.
The current techniques for identifying vibrio include:
1. a classical physiological and biochemical identification method which is tedious, time-consuming and requires good professional literacy for operators;
2. a rapid automatic bacteria identification system, which requires expensive equipment and skilled operators;
3. nucleic acid-based detection technologies, including DNA probes and PCR (polymerase chain reaction) technologies, are fast, simple, and sensitive.
However, the research aiming at the vibrio natriegens mainly stays in the separation and laboratory identification of the pathogens at present, and a rapid detection method is not reported.
Disclosure of Invention
The invention aims to solve the technical problem of the prior art, provides a novel double PCR rapid detection kit for the pathogenic vibrio natriegens of the portunus trituberculatus, and realizes the double PCR rapid and accurate detection of the pathogenic vibrio natriegens of the portunus trituberculatus.
The invention also provides a method for carrying out double PCR (polymerase chain reaction) rapid detection on the pathogenic vibrio natriegens of the blue crabs by using the kit.
The technical problem to be solved by the present invention is achieved by the following technical means. The invention relates to a dual PCR (polymerase chain reaction) rapid detection kit for pathogenic vibrio natriegens of portunus trituberculatus, which is characterized by comprising the following components:
1) negative control, 1 count of 1m L sterilized double distilled water;
2) positive control, the reagent component is 100ng of vibrio natriegens dissolved in 1m L sterilized double distilled water (II)Vibrio natriegens) XA1 strain DNA 1;
3) containing Mg 2+1 PCR buffer solution of dNTP and DNA polymerase;
4) primer pair toxR-F/toxR-R
toxR-F is: 5'-AAT CCG CTT TCC TCT GTT-3' 1 branches;
toxR-R is: 5'-GGC GTT AGC ACA GGT ACA-3' 1 branches;
5) primer pair Vhh-F/Vhh-R
Vhh-F: 5'-AAT GTC ATC CGC CAA CGA-3' 1 branches;
Vhh-R: 5'-CCG TCA GGC GAA TCA ATG-3' 1 branches;
6) sterilizing 1 count of double distilled water;
7) the packing box is a foam plate, the size of the foam plate is the same as the bottom surface of the packing box, and the foam plate is arranged in the packing box; the foam board is provided with small holes with the number not less than that of the small tubes, and the small tubes are respectively and correspondingly placed in the small holes; storing the positive control separately; the kit was stored at-20 ℃.
The strain Vibrio natriegens involved in the method of the present inventionVibrio natriegens) XA1, has been disclosed in THE isolation and molecular characterization OF Portunus trituberculatus pathogen, Vibrio natriegens, and THE above publications are published in THE journal OF THE world SOCIETY for aquatic products (JOURNA L OF THE WOR L D AQUACU L TURE SOCIETY), 2016, 47(6):854 and 861.
Strain Vibrio natriegens (Vibrio natriegens) XA1 is a well-known public material, and is stored in laboratories of the Huai Hai institute of Industrial science, Marine Life and Aquaculture institute, and can be released to the outside by the public if necessary within twenty years from the application date of the patent.
The invention relates to a double PCR rapid detection kit for the pathogenic vibrio natriegens of blue crabs, which further adopts the preferable technical scheme that the design method of the primer design is as follows: two pairs of primers are designed according to a hemolysin vhh gene sequence DQ663483 of vibrio natriegens and a transmembrane transcription activator protein toxR gene sequence JF930637 recorded in GenBank.
The invention also discloses a double PCR rapid detection method for the pathogenic vibrio natriegens of the portunus trituberculatus, which is characterized in that the kit of the technical proposal is utilized to carry out the double PCR detection method for the pathogenic vibrio natriegens of the portunus trituberculatus; preparing a bacterial genome extraction kit before detection; the detection steps are as follows:
(1) extraction of DNA template Vibrio natriegens cultured overnight at 1m L (Vibrio natriegens) Centrifuging XA1 bacterial solution at 12,000g for 1min, discarding supernatant as much as possible, adding 100 μ L L B11 and 20 μ L proteinase K, shaking to thoroughly suspend thallus, incubating at 55 deg.C for 20min, adding 20 μ L RNaseA, mixing, standing for 2min, adding 400 μ L BB11, vortexing for 30s, adding all the liquid to centrifugal column, centrifuging for 30s at 12,000g, discarding eluate, adding 500 μ L CB11 and 12,000g, centrifuging for 30s, discarding eluate, adding 500 μ L CB11 and 12,000g, centrifuging for 30s, discarding eluate, adding 500 μ 36L WB11 and 12000g, centrifuging for 30s, discarding eluate, adding 500 μ L WB11 and 12,000g, centrifuging for 30s, discarding eluate, centrifuging for 2min at 12000g, thoroughly removing residual WB11, transferring the column to centrifugal columnAdding 100 μ L preheated eluent into the center of a new 1.5ml centrifuge tube, standing at room temperature for 5min, centrifuging at 12000g for 1min, eluting DNA, placing the eluent into a centrifugal column, standing at room temperature for 5min, centrifuging at 12000g for 1min to obtain DNA template, and storing at-20 deg.C;
(2) the preparation of PCR premix adopts the following methods respectively:
adding various components into the kit in the following way after centrifuging the reagent for several seconds before using, adding the extracted DNA template, positive control or negative control into the mixed reagent except the DNA template in advance, and separating the region added with the DNA template from the region added with other reagents, wherein the total volume is 25 mu L;
adding a PCR premix:
containing Mg2+PCR buffer 13. mu. L of dNTP and DNA polymerase
Primer pairs toxR-F/toxR-R each 2 mu L
Primer pairs vhh-F/vhh-R each 1 mu L
Sterilized double distilled water 4-5 mu L
DNA template or Positive control or negative control 1-2. mu. L
Total volume 25 mu L
(3) Loading the PCR premix into a PCR reaction tube, placing in a gene amplification instrument without a heat cover, and adding 1-2 drops of paraffin oil to seal the cover; amplification was performed by setting the PCR cycling parameters as follows:
after the pre-denaturation at 95 ℃ for 5min,
denaturation at 94 ℃ for 1min, annealing at 53.3 ℃ for 45s, extension at 72 ℃ for 1min, 30 cycles,
finally, extending for 10min at 72 ℃;
(4) and after the amplification reaction is finished, adding 2 mu L bromophenol blue and 3 mu L PCR mixed solution, uniformly mixing, performing 2% agarose gel electrophoresis, performing electrophoresis for 30-40min at a voltage of 5V/cm, and observing the result under an ultraviolet analyzer, wherein if bands appear at 308bp and 526bp, the vibrio natriegens are proved to be positive, which indicates that the positive control or the sample to be detected carries the vibrio natriegens, otherwise, the bands are negative, which indicates that the negative control or the sample to be detected does not carry the vibrio natriegens.
The above-mentioned method for the double PCR rapid detection of the pathogenic vibrio natriegens of the portunus trituberculatus further preferably adopts the technical scheme that in the extraction process of the DNA template: overnight cultured Vibrio natriegens: (Vibrio natriegens) The bacterial liquid XA1 culture method comprises adding filtered seawater into 2.6g 2216E solid culture medium to 100m L, adjusting pH to 7.2-7.4, sterilizing at 121 deg.C for 20min, pouring into flat plate, and inoculating Vibrio natriegens (or) (Vibrio natriegens) by streaking in ultra-clean benchVibrio natriegens) XA1, 24 hours at 28 ℃, single colonies were picked to 2m L2216E liquid medium and cultured overnight at 28 ℃.
In the process of culturing the shrimps and crabs, the kit and the detection method are adopted to regularly detect the culture objects and the culture water body, so that the dynamic monitoring of the pathogeny of the vibrio natriegens of the seawater crustacean is realized, once a certain animal carries the pathogeny, the corresponding medicine is used for early prevention and treatment in time, the spread and the propagation of the pathogeny among aquatic animals are avoided, the huge loss of the vibrio natriegens to the aquaculture industry is reduced, and the positive and effective effect is played on the healthy development of the aquaculture industry of the seawater crustacean. In the detection of aquatic animal offspring pathogen, the method can accurately detect the vibrio natriegens, ensure that the selected parents and the crabs/shrimp fries stocked do not carry the vibrio natriegens, and reduce the occurrence of vibrio natriegens in the culture process.
Compared with the prior art, the invention designs a rapid and specific dual PCR detection kit and a detection method, and realizes the accurate detection of vibrio natriegens. The advantages of the detection method are mainly embodied in the following aspects: 1) based on hemolysin (vhh) gene and transmembrane transcription activator protein (toxR) gene as target targets, a primer is designed for a gene specificity region to carry out PCR detection, the result shows positive only to vibrio natriegens, other vibrio and bacteria are negative, the occurrence of false positive in a 16S rRNA-PCR detection method is avoided, and the specificity is higher; 2) the detection method has higher sensitivity, and can detect 13.2pg of vibrio natriegens genome DNA at the lowest. 3) The optimized PCR system is used for detection, the operation is simple, special technical personnel are not needed, the whole process time is short, and the consumed time is short.
Drawings
FIG. 1 is a diagram showing the PCR specificity detection result of Vibrio natriens in example 4. in the diagram, M: D L2000 Marker, 1: the single PCR result of Vibrio natriens vhh gene, 2: the single PCR result of Vibrio natriens toxR gene, 3: the double PCR result of Vibrio natriens vhh gene and toxR gene, 4: the double PCR result of Vibrio cholerae, 5: the double PCR result of Vibrio parahaemolyticus, 6: the double PCR result of Vibrio estuarina, 7: the double PCR result of Vibrio anguillarum, 8: the double PCR result of Aeromonas hydrophila, 9: the double PCR result of Vibrio harveyi, 10: the double PCR result of Aeromonas salmonicida, 11: the double PCR result of Vibrio mermaitake, 12: the double PCR result of Aeromonas temperate, and 13: the double PCR result of Vi.
FIG. 2 is a graph showing the results of the double PCR sensitivity detection of Vibrio natriegens, wherein M is Marker at 1: 132. mu.g/ml, 2: 13.2. mu.g/ml, 3: 1.32. mu.g/ml, 4: 1.32 × 10-1μg/ml，5：1.32×10-2μg/ml,6：1.32×10-3μg/ml,7：1.32×10-4μg/ml,8：1.32×10-5μg/ml, 9：1.32×10-6μg/ml。
Detailed Description
The embodiments of the present invention will be further described with reference to the accompanying drawings so as to facilitate the further understanding of the present invention by those skilled in the art, and do not limit the right thereto.
Example 1, a dual PCR rapid detection kit for portunus trituberculatus pathogenic vibrio natriegens, which comprises the following components:
1) negative control, 1 count of 1m L sterilized double distilled water;
2) positive control, the reagent component is 100ng of vibrio natriegens dissolved in 1m L sterilized double distilled water (II)Vibrio natriegens) XA1 strain DNA 1;
3) containing Mg 2+1 PCR buffer solution of dNTP and DNA polymerase;
4) primer pair toxR-F/toxR-R
toxR-F is: 5'-AAT CCG CTT TCC TCT GTT-3' 1 branches;
toxR-R is: 5'-GGC GTT AGC ACA GGT ACA-3' 1 branches;
5) primer pair Vhh-F/Vhh-R
Vhh-F: 5'-AAT GTC ATC CGC CAA CGA-3' 1 branches;
Vhh-R: 5'-CCG TCA GGC GAA TCA ATG-3' 1 branches;
6) sterilizing 1 count of double distilled water;
7) the packing box is a foam plate, the size of the foam plate is the same as the bottom surface of the packing box, and the foam plate is arranged in the packing box; the foam board is provided with small holes with the number not less than that of the small tubes, and the small tubes are respectively and correspondingly placed in the small holes; storing the positive control separately; the kit was stored at-20 ℃.
Example 2, the kit of example 1 was used to perform a dual PCR detection method for vibrio natriegens of portunus trituberculatus; before detection, preparing a bacterial genome extraction kit provided by Beijing all-around gold biotechnology limited; the detection steps are as follows:
(1) extraction of DNA template Vibrio natriegens cultured overnight at 1m L (Vibrio natriegens) XA1 bacterial liquid, centrifuging for 1min at 12,000g, discarding supernatant as much as possible, adding 100 mu L L B11 and 20ul proteinase K, shaking until the thallus is completely suspended, incubating at 55 ℃ for 20min, adding 20 mu L RNaseA, mixing and standing for 2min, adding 400 mu L BB11, vortexing for 30s, adding all liquid into a centrifugal column, centrifuging for 30s at 12,000g, discarding eluate, adding 500 mu L CB11, 12,000g, centrifuging for 30s, discarding eluate, adding 11 at 500 mu L CB11, 12,000g, centrifuging for 30s, discarding eluate, adding 30s at 500 mu L WB11, 12000g, centrifuging for 30s, discarding eluate, adding L WB11, 12,000g, centrifuging for 30s, discarding eluate, centrifuging for 2min at 12000g, completely removing residual WB11, transferring the centrifugal column to a new centrifuge tube 1.5ml, adding 100 mu L g of preheated column in the center of the centrifuge tube, keeping the column for 30 min, discarding eluate, standing at room temperature, centrifuging for 1min, centrifuging at 12000 ℃, eluting at room temperature, and centrifuging for 20 ℃ again to obtain DNA eluate again, and standing for 20 ℃ again;
(2) the preparation of PCR premix adopts the following methods respectively:
adding various components into the kit in the following way after centrifuging the reagent for several seconds before using, adding the extracted DNA template, positive control or negative control into the mixed reagent except the DNA template in advance, and separating the region added with the DNA template from the region added with other reagents, wherein the total volume is 25 mu L;
adding a PCR premix:
containing Mg2+PCR buffer 13. mu. L of dNTP and DNA polymerase
Primer pairs toxR-F/toxR-R each 2 mu L
Primer pairs vhh-F/vhh-R each 1 mu L
Sterilized double distilled water 4-5 mu L
DNA template or Positive control or negative control 1-2. mu. L
Total volume 25 mu L
(3) Loading the PCR premix into a PCR reaction tube, placing in a gene amplification instrument without a heat cover, and adding 1-2 drops of paraffin oil to seal the cover; amplification was performed by setting the PCR cycling parameters as follows:
after the pre-denaturation at 95 ℃ for 5min,
denaturation at 94 ℃ for 1min, annealing at 53.3 ℃ for 45s, extension at 72 ℃ for 1min, 30 cycles,
finally, extending for 10min at 72 ℃;
(4) and after the amplification reaction is finished, adding 2 mu L bromophenol blue and 3 mu L PCR mixed solution, uniformly mixing, performing 2% agarose gel electrophoresis, performing electrophoresis for 30-40min at a voltage of 5V/cm, and observing the result under an ultraviolet analyzer, wherein if bands appear at 308bp and 526bp, the vibrio natriegens are proved to be positive, which indicates that the positive control or the sample to be detected carries the vibrio natriegens, otherwise, the bands are negative, which indicates that the negative control or the sample to be detected does not carry the vibrio natriegens.
Example 3, a dual PCR rapid detection kit for the pathogenic vibrio natriegens of portunus trituberculatus and a detection method application experiment. The kit of example 1 and the detection method described in example 2 were used:
the experimental contents comprise that the primers, experimental materials and experimental methods provided by Huaihai academy of industry are adopted to randomly sample seawater, portunus trituberculatus and prawns in 10 different shrimp and crab culture ponds in the Gancirus field of Lingyun harbor city, 30 portunus trituberculatus, 30 prawns and 30 water samples are collected in total, and then the detection is carried out by the methods of double PCR, 16S rRNA-PCR, in-vitro culture and the like of the research respectively.
The experimental result shows that the double PCR result of 18 swimming crabs, 9 prawns and 15 water samples is positive, the 16S rRNA-PCR result of 22 swimming crabs, 17 prawns and 20 water samples is positive, the samples which are respectively positive for the double PCR and the 16S rRNA-PCR detection and positive for the double PCR and the 16S rRNA-PCR respectively are subjected to vibrio in-vitro culture based on the PCR result, and the result shows that the vibrio can be separated from the sample with very bright double PCR and 16S rRNA-PCR results, and some samples with slightly weak PCR bands have vibrio growth, and the vibrio growth is not seen all the time even if the culture time is prolonged by 1 week. Therefore, by utilizing the high sensitivity of the method, in the detection of aquatic crustacean vibrio pathogeny, the double PCR method can detect whether the aquatic crustacean is in the initial stage of vibrio natriegens infection, while the isolated culture method can only detect the middle and later stages of infection. On the other hand, by utilizing the high specificity of the double PCR method, the double PCR method does not cause false detection like the 16S rRNA-PCR method, so that the double PCR detection method is more suitable for detecting trace vibrio natriegens and can carry out clinical detection on the early and middle stages of the infection of the seawater vibrio crustaceans.
Example 4, a double PCR rapid detection kit and a detection method for the pathogenic vibrio natriegens of the portunus trituberculatus research experiment:
designing a primer:
two pairs of primers were designed with reference to the gene sequence of hemolysin (vhh) (DQ 663483) and transmembrane transcription activator protein (toxR) (JF 930637) of Vibrio natriegens recorded in GenBank:
toxR-F: AAT CCG CTT TCC TCT GTT and toxR-R: GGC GTT AGC ACA GGT ACA (fragment size 308 bp)
Vhh-F: AAT GTC ATC CGC CAA CGA and Vhh-R: CCG TCA GGC GAA TCA ATG (fragment size 526 bp) was synthesized by Shanghai Jun Biotechnology, Inc.
Vibrio natriegens laboratory culture
Taking 2.6g of a 2216E solid culture medium produced by Hangzhou Tian and microbial agents Co., Ltd, adding filtered seawater to 100m L, adjusting the pH value to 7.2-7.4, sterilizing at 121 ℃ for 20 minutes, pouring the mixture into a flat plate, carrying out streak inoculation on vibrio natriegens in an ultra-clean bench, culturing at 28 ℃ for 24 hours, and picking out a single colony to a 2m L2216E liquid culture medium to be cultured overnight at 28 ℃.
Extraction of Vibrio natriegens genomic DNA
1m L bacterial suspension is taken and used for extracting the vibrio natriegens genome DNA by a bacterial genome extraction kit provided by Beijing all-type gold biotechnology limited company, and the vibrio natriegens genome DNA is stored at the temperature of-20 ℃ for later use.
Establishment and optimization of PCR system
Through a series of experiments, the reaction systems and parameters such as the concentration of each primer, the annealing temperature, the extension time and the like are adjusted and optimized, and finally the optimal system of the vibrio natriegens double PCR reaction is determined to be 13 mu L2 × Master Mix (containing Mg)2+dNTP and DNA polymerase), 10 mu mol/L primer vhh-F/vhh-R each 1 mu L, tox-F/tox-R each 2 mu L, genome DNA100ng, double distilled water to 25 mu L, PCR reaction conditions are that after pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 1min, annealing at 53.3 ℃ for 45s, extension at 72 ℃ for 1min, 30 cycles, and finally extension at 72 ℃ for 10 min.
PCR specificity detection
The control aquatic animal common pathogen: vibrio cholerae (Vibrio cholerae) Vibrio parahaemolyticus: (Vibrio parahaemolyticus) Vibrio estorum: (Vibrio aestuarianus) Vibrio anguillarum (V.anguillarum)Vibrio anguillarum) Aeromonas hydrophila (b) ((b))Aeromonas hydrophila) Vibrio harveyi: (Vibrio harveyi) Aeromonas salmonicida (Aeromonas salmonicida subsp) Vibrio mermaid: (Vibrio damsela) Aeromonas sobria: (Aeromonas sobria) And Vibrio vulnificus: (Vibrio vulnificus) The genomic DNA of (1) is respectively subjected to PCR system and condition optimization, and finally all PCR results are detected by using 2% agarose gel electrophoresis, and the results of single and double PCR of vibrio natriegens and double PCR of other bacterial pathogens are shown in figure 1. As can be seen from the figure, the Vibrio natriegens can amplify the target band regardless of single PCR or double PCR, while othersThe bacteria have no band, which shows that the PCR primer designed by the experiment has good specificity.
PCR sensitive detection
The Vibrio natriegens DNA of the original concentration of 132. mu.g/ml was subjected to 10-fold line gradient dilution, double PCR amplification using the above optimized PCR system and conditions, and then detection using agarose gel electrophoresis, and the results are shown in FIG. 2. As can be seen from the figure, the detection sensitivity of the hemolysin (vhh) gene primer is higher than that of the transmembrane transcription activator protein (toxR) gene by one order of magnitude, namely, vhh gene primer can amplify a band to 13.2pg of vibrio natriegens genome DNA, while the toxR gene can only amplify a band to 132pg of vibrio natriegens genome DNA, but the two genes are combined for use, so that the detection sensitivity is improved, the detection reliability is also enhanced, and the occurrence of false positive is avoided.
Claims (2)
1. A dual PCR rapid detection kit for pathogenic vibrio natriegens of portunus trituberculatus is characterized by comprising the following items:
1) negative control, 1 count of 1m L sterilized double distilled water;
2) positive control, the reagent component is 1 branch of DNA of 100ng vibrio natriegens XA1 strain dissolved in 1m L sterilized double distilled water;
3) containing Mg2+1 PCR buffer solution of dNTP and DNA polymerase;
4) primer pair toxR-F/toxR-R
toxR-F is: 5'-AAT CCG CTT TCC TCT GTT-3' 1 branches;
toxR-R is: 5'-GGC GTT AGC ACA GGT ACA-3' 1 branches;
5) primer pair Vhh-F/Vhh-R
Vhh-F: 5'-AAT GTC ATC CGC CAA CGA-3' 1 branches;
Vhh-R: 5'-CCG TCA GGC GAA TCA ATG-3' 1 branches;
6) sterilizing 1 count of double distilled water;
7) the packing box is a foam plate, the size of the foam plate is the same as the bottom surface of the packing box, and the foam plate is arranged in the packing box; the foam board is provided with small holes with the number not less than that of the small tubes, and the small tubes are respectively and correspondingly placed in the small holes; storing the positive control separately; the kit was stored at-20 ℃.
2. The dual PCR rapid detection kit for the pathogenic vibrio natriegens of the portunus trituberculatus according to claim 1, which is characterized in that the design method of the primer design is as follows: two pairs of primers are designed according to a hemolysin vhh gene sequence DQ663483 of vibrio natriegens and a transmembrane transcription activator protein toxR gene sequence JF930637 recorded in GenBank.
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