CN111304347A - PCR reaction system for rapidly detecting vibrio rotifer in culture site - Google Patents
PCR reaction system for rapidly detecting vibrio rotifer in culture site Download PDFInfo
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Abstract
The invention belongs to the technical field of disease control in mariculture, and particularly relates to a PCR reaction system for rapidly detecting vibrio rotifer in a culture site. And carrying out fluorescent quantitative PCR reaction by using the designed specific primer, qualitatively detecting the vibrio rotifer according to whether fluorescence appears, and quantitatively calculating the number of the vibrio rotifer in the detected sample according to the excitation intensity of the fluorescence. The invention can realize the accurate and rapid detection of the vibrio rotifer in the mariculture field, thereby providing a guarantee technology for effectively preventing and controlling the infection of the vibrio rotifer in the culture production.
Description
Technical Field
The invention belongs to the technical field of disease control in mariculture, and particularly relates to a PCR reaction system for rapidly detecting vibrio rotifer in a culture site.
Background
Vibrio rotifer (Vibrio rotiferanus) is a new marine culture pathogen and can infect various marine culture organisms such as fish, shrimp and the like to cause diseases. The earliest reports on Vibrio rotifer were isolated in Rotifera plicatilis by Gomez-Gil et al (2003) in 2003. Thereafter, foreign schwdhury (2011) and labbat (2011) and other foreign scholars have successively conducted researches on pathogenic mechanisms, physiological and biochemical characteristics and other aspects of vibrio rotifer. In recent years, scholars in China respectively isolated vibrio rotifer from cultured cynoglossus semilaevis (Chengqiang et al, 2012), penaeus vannamei (golden spring english et al, 2013), hippocampus kelloggi et al (Yang Quihua et al, 2017) and sebastes schlegeli (Wangkai et al, 2018). The main symptoms of cynoglossus semilaevis infected with vibrio rotifer are body surface ulceration and tail bleeding; the main symptoms of the penaeus vannamei boone are red body surface, white turbid muscle, yellow and ulcerated gill part; the main symptoms of sebastes schlegeli hilgendorf are traumatic ulcer, fin ray ulceration, eyeball protrusion and the like on the body surface. Because of the lack of host specificity, the vibrio rotifer has become one of the new pathogens which endanger the healthy development of the mariculture industry.
It is worth mentioning that the colony of the vibrio rotifer growing on the TCBS medium is green, which is different from the yellow colony of most vibrio, which indicates that sucrose is not fermented and cannot be qualitatively identified by the TCBS medium. Therefore, the detection method for researching the specificity of the vibrio rotifer is one of the key technologies which need to be broken through for effectively preventing and controlling the vibrio rotifer infection.
Disclosure of Invention
The invention aims to solve the technical problem that the qualitative identification of the vibrio rotifer cannot be carried out by adopting a TCBS culture medium because the vibrio rotifer has no host specificity and does not ferment sucrose. Therefore, a specific detection method aiming at the vibrio rotifer in a culture field is lacked at present.
In order to solve the problems, a specific primer for accurately identifying the vibrio rotifer and an application method thereof are provided, so that the vibrio rotifer can be accurately and quickly detected in a mariculture field, and a guarantee technology is provided for effectively preventing and controlling the infection of the vibrio rotifer in culture production.
In order to achieve the purpose, the invention is realized by the following technical scheme: a PCR reaction system for quickly detecting vibrio rotifer in culture site features that the specific primer is used to perform fluorescent quantitative PCR reaction, and the quantitative detection of vibrio rotifer is based on the existence of fluorescence and the quantitative calculation of the amount of vibrio rotifer in specimen.
Further, the specific primers are designed based on the vibrio rotifer toxR gene and are respectively DNA forward sequence VRF:5'-CAAAATCCCCCGAGTTTGTA-3' and reverse sequence VRR: 5'-TTGACCTCCAGTAGCGATAA-3'; the length of the target fragment of the toxR gene amplified by using the specific primer is 500 bp.
The two primer sequences are used for screening a toxR gene sequence which is 897bp in total length and highly conserved on the chromosome of the vibrio rotifer as a target sequence by analyzing genome data after the whole genome sequence of the vibrio rotifer is determined. the toxR gene is widely distributed in the vibriaceae bacteria and is an important virulence expression regulation gene. The gene was first found in Vibrio cholerae (Vibrio cholerae), and then confirmed in various Vibrionaceae bacteria such as Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio anguillarum, Vibrio mimicus, Vibrio alginolyticus (Vibrio. The nucleotide sequence similarity of the toxR gene between heterogeneous bacteria is low, so the toxR gene is also an ideal molecular target for identification between species in the family Vibrionaceae.
The target sequences are as follows:
blast alignment of the sequences on NCBI revealed that the first 8 sequences similar thereto were all from vibrio rotifer and the sequence similarity was all around 99% (see fig. 2, where E value ═ 0), which is a good proof that the toxR gene sequence is a suitable target sequence for distinguishing vibrio rotifer from other bacteria. We designed the above two pairs of primers for this sequence, and amplified the sequence with a length of 500bp (the underlined part of the above toxR gene sequence, wherein the bold is the forward primer and the reverse primer respectively), which is about 57.8% of the total length sequence of the toxR gene, and is sufficient for the detection and identification of Vibrio rotifer, which is the technical basis for the implementation of the present invention.
Meanwhile, the designed Primer sequence is subjected to Primer-universal Blast comparison in Primer-Blast of NCBI, and the result shows that the Primer sequence can be completely matched with the genome of the vibrio rotifer in the NCBI database (figure 3), thereby proving that the Primer sequence has good specificity to the vibrio rotifer.
Further, the PCR reaction system for detection is 8 μ L of micro premix PCR reaction system lyophilized and packaged in advance by a freeze vacuum drying method, and comprises 5 μ L of 2 XBuffer Buffer solution, 0.5 μ L of 5 μ M DNA forward sequence primer, 0.5 μ L of 5 μ M DNA reverse sequence primer, 0.5 μ L of 5 μ L SYBR GREEN fluorescent dye, 0.5 μ L of 5U DNA polymerase, and 1 μ L of RNAseH-free solution2O。
The above components are mixed to form a PCR reaction system, 2 XChamQ Universal SYBRqPCR Master Mix comprises rapid reaction enzyme, dNTP, fluorescent dye and the like, and the primer is used for amplification. The PCR reaction system is well prepared in advance, can be stored for a long time after freeze-drying, can be directly used for PCR reaction after being carried to a field and sterile water and a DNA template are added during detection, simplifies the procedure and is more convenient to operate.
Further, the preparation method of the premixed PCR reaction system comprises the following steps:
(1-1) first, an 8. mu.L PCR microreaction system comprising 5. mu.L of 2 XBuffer Buffer, 0.5. mu.L of 5. mu.M DNA forward sequence primer, 0.5. mu.L of 5. mu.M DNA reverse sequence primer, 0.5. mu.L of 5. mu.L LSYBR GREEN fluorescent dye, 0.5. mu.L of 5U DNA polymerase, and 1. mu.L of RNAseH-free solution was prepared in a 200. mu.L PCR reaction tube2O, 8 mu L in total; sequentially adding into a reaction tube and mixing;
(1-2) pre-freezing the PCR tube containing the 8 mu L micro reaction system in an ultra-low temperature refrigerator at-80 ℃ for 1 hour, then freeze-drying by a freeze vacuum drying method to form a pre-mixed reaction system, covering the PCR tube, and then packaging with a sealing film for later use. The purpose of freeze-drying is to store a pre-prepared PCR reaction system for a long time, so that the storage and carrying are convenient, the operation procedure in use is simplified, and the PCR operation is convenient to be carried out on site. The prepared reaction system can be quickly cooled to a eutectic point by pre-freezing at an ultralow temperature of-80 ℃, so that active ingredients in the system can be effectively protected.
Further, when the PCR reaction system for rapidly detecting the vibrio rotifer in the culture site is applied, the method comprises the following steps:
(2-1) shearing 0.2-0.3g of the tissue of the part to be detected of the cultured animal, placing the sheared animal in a 1.5mL centrifuge tube, grinding the sheared animal by a grinding rod, adding 200 mu L of 2 xLysis Buffer tissue lysate of Shanghai Chunkun biological technology Co., Ltd and 300 mu L of non-RNAse H2O。
(2-2) placing the centrifuge tube filled with the sample in the palm center, holding a fist for 5-10 min, fully cracking the tissue under the condition of human body temperature (about 37 ℃), and centrifuging for 2min at 8000rpm of a palm centrifuge to obtain supernatant.
(2-3) pipette 10. mu.L of supernatant into a new sterile centrifuge tube and add 90. mu.L of RNAse-free H2And O, diluting the supernatant, shaking and uniformly mixing to obtain the nucleic acid template to be detected.
(2-4) adding 2. mu.L (2-3) of the prepared nucleic acid template and 8. mu.L of RNAse-free H to the pre-mixed reaction system in which the pre-lyophilized package is carried out2And O, fully and uniformly shaking to form a PCR reaction system.
(2-5) carrying out fluorescent quantitative PCR reaction on the PCR reaction system obtained in the step (2-4); and after the PCR program is finished, performing fluorescence detection on the product, wherein the length of the target fragment is 500 bp.
(2-6) the product in the reaction chip has fluorescence, which proves that the sample to be detected contains the vibrio rotifer, and the amount of the vibrio rotifer in the sample to be detected can be quantitatively calculated according to the excitation intensity of the fluorescence.
Further, the step (2-5) is to transfer the PCR reaction system obtained in the step (2-4) into a Chipfor GeneChecker reaction chip by using a pipette and place the chip into a GeneChecker UF-150 portable ultra-fast PCR instrument for carrying out fluorescent quantitative PCR reaction.
Further, the reaction procedure of the step (2-5) is that the denaturation at ① 95 ℃ is carried out for 30s, the denaturation at ② 95 ℃ is carried out for 10s, the annealing at 3595 ℃ is carried out for 40s, the annealing at 60 ℃ is carried out for 72 ℃ and the extension is carried out for 10s, and ② is repeated for 30 cycles.
The invention has the beneficial effects that:
1. the invention adopts a pair of specific primers of the vibrio rotifer toxR gene, has higher conservation and better interspecific identification compared with sequences such as 16s rDNA gene, gyrB gene and the like, and can accurately identify the vibrio rotifer.
2. The invention adopts a premixed fluorescent quantitative PCR reaction system, freeze-dried and packaged for standby, and is matched with a GeneChecker UF-150 portable ultra-fast PCR instrument, so that the requirement of an experimental field of fluorescent quantitative PCR reaction is reduced, the operation procedure of PCR reaction in field application is obviously simplified, the kit is suitable for a field simple and crude use environment, has the condition of detecting vibrio rotifer in a culture field, can obtain an accurate detection result in a short time, and has obvious progress compared with the prior art. Meanwhile, the method can also carry out quantitative detection on the vibrio rotifer, and is favorable for carrying out quick diagnosis and high-efficiency prevention and control on mariculture.
3. The micro-quantitative PCR reaction system has small volume and simple procedure, shortens the whole detection time to about 40min, greatly improves the detection efficiency, and is suitable for field rapid detection.
4. According to the invention, through a special experimental method, the lysis solution is added, and the lysis is carried out by depending on the body temperature of a human body, so that the pretreatment process of the PCR reaction is greatly simplified, the condition requirement of sample pretreatment is reduced, and the convenience of application in a culture site is further improved.
Drawings
FIG. 1 shows the results of the detection of different experimental groups by a GeneChecker UF-150 portable ultra-fast PCR instrument;
note: the GeneChecker UF-150 reflects the detection result of the chip. The chip comprises 10 reaction channels, wherein the channel 1 and the channel 2 are sebastes schlegeli muscle tissue samples injected with rotifer vibrio; channel 3 and channel 4 are sebastes schlegeli muscle tissue samples injected with vibrio anguillarum; channel 5 and channel 6 are muscle tissue samples injected with edwardsiella tarda; channel 7 and channel 8 are sebastes schlegeli muscle tissue samples injected with 1.5% sterile NaCl solution; channel 9 and channel 10 are RNAseH-free2O。
FIG. 2 Blast alignment of the sequences of the toxR gene used in the present invention at NCBI.
FIG. 3 shows that the primer pair of the present invention realizes specific matching with the genomic data of Vibrio rotifer at NCBI.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
(1) firstly, an 8 mu L PCR micro reaction system is prepared in a 200 mu L PCR reaction tube, and comprises 5 mu L2 Xbuffer Buffer solution, 0.5 mu L5 mu M DNA forward sequence primer, 0.5 mu L5 mu M DNA reverse sequence primer, 0.5 mu L5 mu L LSYBR GREEN fluorescent dye, 0.5 mu L5U DNA polymerase and 1 mu L RNAseH-free solution2O, 8 mu L in total;
(2) pre-freezing the PCR tube containing the 8 μ L micro reaction system in an ultra-low temperature refrigerator at-80 deg.C for 1 hr, freeze-drying by vacuum freeze-drying to form a pre-mixed reaction system, covering the PCR tube, and packaging with sealing film.
(3) In this example, 23 strains of bacteria were selected and tested for the specificity of vibrio rotifer using the specific primers and methods of the present invention, and the selected strains and their sources are shown in table 1.
TABLE 1 reference strains for the specific detection of Vibrio rotifer
The frozen bacterial liquid of each strain listed in the table above is taken out from a laboratory at-80 ℃ ultra-low temperature refrigerator, placed at 4 ℃ to slowly melt the bacterial liquid, 30 mu L of the frozen bacterial liquid is sucked and streaked on a TSB solid culture medium, and the TSB solid culture medium is inverted and cultured for 24-36 hours at 28 ℃. After colonies were formed, single colonies were picked and streaked again on a fresh solid TSB medium for purification at 28 ℃. After 3 times of purification and culture, a single colony was scraped and suspended in 0.5mL of a sterile 1.5% NaCl solution to prepare a bacterial suspension.
(4) Adding 100 μ L of 2 × Lysis Buffer tissue lysate of Shanghai Chungkun biological technology, Inc., into the above bacterial suspension, sealing, holding in palm core for 10min, centrifuging at 8000rpm for 2min with a palm centrifuge, and transferring the supernatant to a sterile centrifuge tube as a nucleic acid template of bacteria to be detected.
(5) In a pre-freeze-packaged PCR reaction tube, 2. mu.L of the above bacterial suspension and 8. mu.L of RNAseH-free solution were added2And O, fully and uniformly shaking. Then transferring 10 mu L of PCR reaction system to PCR, namely a Chip for GeneChecker reaction Chip by using a pipette, and placing the Chip in a GeneChecker UF-150 portable ultra-fast PCR instrument for carrying out fluorescent quantitative PCR reaction. The reaction program was set as: pre-denaturation at 95 ℃ for 30 s; (denaturation at 95 ℃ for 10s + annealing at 60 ℃ for 40s + extension at 72 ℃ for 10s) for 30 cycles.
(6) The PCR reaction lasted for 30 min. After the reaction is finished, the fluorescence detection is carried out on the reaction chip of 23 strains of bacteria in the experiment, and the result shows that only the reaction chip of 3 strains of vibrio rotifer generates green fluorescence, so that the detection method provided by the invention has better specificity on the vibrio rotifer, has shorter detection time and is suitable for rapid detection of the vibrio rotifer.
Example 2:
in the aquarium system of the laboratory, 4 experimental groups are respectively arranged, and each experimental group is used for culturing 30 sebastes schlegeli hilgendorf with the weight of 40 +/-5 g/tail. After 5 days of temporary rearing, the groups were labeled as test group 1, 2, 3 and control group, respectively, wherein the test group 1 had an injection of 0.05mL per fish at a concentration of 1.0X 104cfu/mL live bacteria solution of vibrio rotifer, 0.05mL concentration of 1.0 × 10 is injected into each fish of experiment group 24cfu/mL Vibrio anguillarum viable bacteria solution, 0.05mL concentration of 1.0 × 10 is injected into each fish of experiment group 34cfu/mL Edwardsiella tarda viable bacteria solution, and control group injected with 0.05mL of 1.5% sterile NaCl solution per fish. The injection sites were all in the back muscle of the fish.
On day 3 after injection, five fishes were randomly selected from each group, 0.2g of the muscle tissue of the injection site was cut and placed in 5 sterilized 1.5mL centrifuge tubes, and the tissue was ground with different sterile grinding rods, 200. mu.L of 2 × lysine buffer tissue lysate and 300. mu.L of RNAse H from Shanghai Chunkun Biotech Co., Ltd2And O. The centrifuge tube cover is covered, the centrifuge tube is sealed by a sealing film, and a plurality of centrifuge tubes are placed in water temperature of 37 ℃ (simulating palm temperature) for 10min to ensure that the tissues are fully cracked.
Centrifuging the above lysed tissue fluid with a palm centrifuge at 8000rpm for 2min, sucking 10 μ L of supernatant from each centrifuge tube into a new sterile centrifuge tube, and adding 90 μ L of non-RNAse H2And O, diluting the supernatant, shaking and uniformly mixing to obtain the nucleic acid template for later use.
Adding 2 μ L of the above nucleic acid template and8 uL of RNAseH-free2And O, fully and uniformly shaking to form a PCR reaction system. Transferring the mixture into a PCR Chip for GeneChecker reaction by using a pipette, and placing the Chip in a GeneChecker UF-150 portable ultra-fast PCR instrument to perform fluorescent quantitative PCR reaction. The reaction program was set as: pre-denaturation at 95 ℃ for 30 s; (denaturation at 95 ℃ for 10s + annealing at 60 ℃ for 40s + extension at 72 ℃ for 10s) for 30 cycles.
After 30min, the PCR reaction program is finished, and the fluorescence detection of the product is carried out. As shown in FIG. 1, 5 reaction systems of experimental group 1 all showed green fluorescence, while 5 reaction systems of experimental groups 2, 3 and control group all showed no fluorescence signal, which proves that the method of the present invention has excellent specificity for detecting vibrio verticillatus infected in vivo with sebastes schlegeli hilgendorf.
Wherein the PCR reaction system was prepared in the same manner as in example 1.
Finally, it should be noted that the above embodiments describe specific embodiments of the present invention, but do not limit the present invention; it will be understood by those skilled in the art that these are by way of example only and that the scope of the invention is defined by the appended claims. All changes, modifications and equivalents that come within the meaning and range of equivalency of the claims are to be embraced within their scope.
Sequence listing
<110> research institute for aquatic products in yellow sea of China institute for aquatic science
<120> PCR reaction system for rapidly detecting vibrio rotifer in culture site
<130> research institute for aquatic products in yellow sea of China institute for aquatic science
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aattctctcg ctgaccaaca aagtggcaat gaagttgttc gcttaggaag caatgaaagt 120
cgaattctgc ttatgctttc agagagacca aatgaagtat taacccgcca tgagctccat 180
gagtttgttt ggcgagatca aggctttgag gtggatgact caagcctaac tcaagcgatt 240
tctacattgc gcaaaatgtt gaaggactca acaaaatccc ccgagtttgt aaaaacggta 300
ccaaaacgcg gctatcagtt gatttgtagt gtcgaaagaa tgagcactcc aacctcagag 360
ccaaatgccg agcttgaaga tatcgacgct gaagaaagca cttttgaaag tgttaccgaa 420
gagattcaag cgcaaccgca ggttgaagac tctgtatcaa cagatccaag ctcagtaagc 480
gacaacgcaa caccagtttc agcggcaaaa gtaagttcaa aaaacaactg gttcacgcgt 540
gccattttat tcgttgctgt actgctgcca atcatcgtct ttaccctcac taaaccagct 600
gaatctcaat ttcgccaaat tgcagagttt agcggtgtac ccgttatgac tccggcaaac 660
caccctcaat tgatgcagtg gatgccttcc atcgagcaat gtgttgctcg atacgtagaa 720
aatcatacaa acgatgtgat gccagtgaaa gttatcgcta ctggaggtca aggtaataag 780
cttgtattga actacattca cgatacggac cattcatatg aaaatgtgac attgcgtatt 840
tttgctggtc aaaacgaccc aactgacatc tgcaaataa 879
Claims (6)
1. A PCR reaction system for rapidly detecting vibrio rotifer in a culture site is characterized in that: the specific primers are designed based on the vibrio rotifer toxR gene and are respectively DNA forward sequence VRF:5'-CAAAATCCCCCGAGTTTGTA-3' and reverse sequence VRR: 5'-TTGACCTCCAGTAGCGATAA-3'; the length of the target fragment of the toxR gene amplified by using the specific primer is 500 bp.
2. The PCR reaction system for rapidly detecting Vibrio rotifer at a culture site as claimed in claim 1, wherein: the PCR reaction system for detection is 8 muL of micro-premix PCR reaction system which is freeze-dried and packaged in advance by a freeze vacuum drying method, and contains 5 muL of 2 XBuffer Buffer solution, 0.5 muL of 5 muM DNA forward sequence primer, 0.5 muL of 5 muM DNA reverse sequence primer, 0.5 muL of 5 muL of SYBR GREEN fluorescent dye, 0.5 muL of 5U DNA polymerase and 1 muL of RNAseH-free2O。
3. The PCR reaction system for rapidly detecting Vibrio rotifer at a culture site as claimed in claim 2, wherein: the preparation method of the premixed PCR reaction system comprises the following steps:
(1-1) first, an 8. mu.L PCR miniprep reaction system was prepared in a 200. mu.L PCR reaction tube, containing 5. mu.L of 2 XBuffer Buffer, 0.5. mu.L of 5. mu.M DNA forward sequence primer, 0.5. mu.L of 5. mu.M DNA reverse sequence primer, 0.5. mu.L of 5. mu.L SYBRGREEN fluorescent dye, 0.5. mu.L of 5U DNA polymerase, and 1. mu.L of no-PCR miniprep reaction systemRNAseH2O, 8 mu L in total;
(1-2) pre-freezing the PCR tube containing the 8 mu L micro reaction system in an ultra-low temperature refrigerator at-80 ℃ for 1 hour, then freeze-drying by a freeze vacuum drying method to form a pre-mixed reaction system, covering the PCR tube, and then packaging with a sealing film for later use.
4. The PCR reaction system for rapidly detecting Vibrio rotifer at a culture site as claimed in claim 1, wherein: when the PCR reaction system for rapidly detecting the vibrio rotifer in the culture site is applied, the method comprises the following steps:
(2-1) shearing 0.2-0.3g of part tissue to be detected of the cultured animal, placing the part tissue in a 1.5mL centrifuge tube, grinding by using a grinding rod, adding 200 mu L of tissue lysate and 300 mu L of non-RNAse H2O；
(2-2) placing the centrifugal tube filled with the sample in the palm center and making a fist, fully cracking the tissue under the condition of human body temperature, and then centrifuging by using a palm centrifuge to obtain a supernatant;
(2-3) pipette 10. mu.L of supernatant into a new sterile centrifuge tube and add 90. mu.L of RNAse-free H2Diluting the supernatant, and shaking and uniformly mixing to obtain the nucleic acid template to be detected;
(2-4) adding 2. mu.L (2-3) of the prepared nucleic acid template and 8. mu.L of RNAse-free H to the pre-mixed reaction system in which the pre-lyophilized package is carried out2O, fully and uniformly shaking to form a PCR reaction system;
(2-5) carrying out fluorescent quantitative PCR reaction on the PCR reaction system obtained in the step (2-4); carrying out fluorescence detection on the product after the PCR program is finished;
(2-6) the product in the reaction chip has fluorescence, which proves that the sample to be detected contains the vibrio rotifer, and the amount of the vibrio rotifer in the sample to be detected can be quantitatively calculated according to the excitation intensity of the fluorescence.
5. The PCR reaction system for rapidly detecting Vibrio rotifer in culture sites as claimed in claim 4, wherein: and (2-5) transferring the PCR reaction system obtained in (2-4) into a PCR (polymerase chain reaction) Chip for GeneChecker reaction by using a pipette, and placing the Chip in a GeneChecker UF-150 portable ultra-fast PCR instrument for carrying out fluorescent quantitative PCR reaction.
6. The PCR reaction system for rapidly detecting Vibrio rotifer at a culture site as claimed in claim 4, wherein the reaction procedure in the step (2-5) is pre-denaturation at ① 95 ℃ for 30s, denaturation at ② 95 ℃ for 10s + annealing at 60 ℃ for 40s +72 ℃ for 10s, and ② repetition for 30 cycles.
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| GENBANK: "Genbank Accession:AP019798.1", 《GENBANK》 * |
| KIM ET AL.: "Detection of Vibrio and ten Vibiro species in cage-cultured fish by multiplex polymerase chain reaction using house-keeping genes", 《AQUACULTURE》 * |
| PASCUAL ET AL.: "Multilocus sequence analysis of the central clade of the genus Vibrio using the 16S rRNA, recA, pyrH, rpoD, gyrB, rctB, and toxR genes", 《INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY》 * |
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