CN105838803A - Specific molecular genetic marker for pathogenic vibrio harveyi and application of specific molecular genetic marker - Google Patents

Specific molecular genetic marker for pathogenic vibrio harveyi and application of specific molecular genetic marker Download PDF

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CN105838803A
CN105838803A CN201610310289.6A CN201610310289A CN105838803A CN 105838803 A CN105838803 A CN 105838803A CN 201610310289 A CN201610310289 A CN 201610310289A CN 105838803 A CN105838803 A CN 105838803A
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vibrio harveyi
specific molecular
genetic marker
molecular genetic
disease
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徐先栋
周永灿
谢珍玉
王世锋
蔡岩
刘开放
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Hainan University
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

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Abstract

The invention relates to the field of molecular biology, in particular to a specific molecular genetic marker for pathogenic vibrio harveyi and application of the specific molecular genetic marker. Particularly, the genetic marker for the pathogenic vibrio harveyi has a base sequence shown in a sequence table SEQ ID No.1 and can be used for specifically detecting the pathogenic vibrio harveyi. Accordingly, the pathogenic vibrio harveyi can be quickly and accurately detected on the basis of the marker, the marker is wide in application range, simple and easy to operate, and the steps of bacterium culturing, DNA extracting and the like are not needed.

Description

A kind of cause a disease vibrio harveyi specific molecular genetic marker and application thereof
Technical field
The present invention relates to biology field, especially a kind of cause a disease vibrio harveyi specific molecular genetic marker and Application.
Background technology
Vibrio harveyi (Vibrio harveyi) is distributed widely in briny environment, is that marine cultured animal is most important carefully One of bacterium sexually transmitted disease (STD) is former, it is possible to infect vertebra and spinal animal, including fish, shrimp, shellfish, spiral shell, Stichopus japonicus etc..As infecting with other, The early diagnosis that the vibrio harveyi that causes a disease infects is beneficial to the prevention and control of disease.But, same to vibrio cholera, vibrio parahaemolytious and eel arc The important Aquatic product infectiousness cause of disease such as bacterium is the same, i.e. only has the virulent strain energy of a few molecules epidemiologic feature in same vibrio Cause the generation of explosive disease, and most bacterial strain is non-virulent strain, and do not have pathogenic causing a disease, Vibro harveyi Virulence between different strains differs greatly, and many bacterial strains are the normal bacteria of marine environment (including marine animal internal milieu) Strain；Some bacterial strain is then the encountered pathogenic of marine cultured animal, all causes Global Seawater cultured fishes, Shrimp waste and shellfish Extremely serious harm.At present, existing several methods utilizing molecular biology method detection vibrio harveyi, including passing through Pcr analysis hemolysin gene vhh, regulatory protein gene toxR, gyrase gene gyrB, vhhP2 gene and 16S rRNA base Cause.Owing to these genes all cannot distinguish between toxic strain and avirulent strain, it is impossible to Accurate Diagnosis vibrio harveyi disease, and some method needs Want antibacterial culturing and DNA extraction, operationally have the regular hour to require and technology requirement.
Summary of the invention
Present invention aim at providing the pathogenic vibrio harveyi specific molecular genetic marker of one and application thereof.
The technical solution used in the present invention is:
Cause a disease vibrio harveyi specific molecular genetic marker: the base sequence in sequence table SEQ ID No.1.
The application of specific molecular genetic marker: described genetic marker can cause a disease vibrio harveyi in specific detection.
1, according to the pathogenic vibrio harveyi specific molecular heredity mark shown in the base sequence in sequence table SEQ ID No.1 Note, the primer related to is: primer 3858P1F (5 '-TACATCTACCATTTCCGTAAACA-3 ') and 3858P1R (5 '- ATTGAGGTAAAAGTTTCAGCACATC-3’)。
, the preparation of fish tissues sample to be measured: under aseptic condition, take fish brain, liver, spleen, kidney, by brain, liver, spleen Dirty, kidney adds 1ml physiological saline solution and is homogenized, and is then diluted, stand-by.
, the preparation of environmental sample: in environment, take 20ml water body, be 80 ~ 120 μm fast quantitative analysis filter paper mistakes with aperture Filter, removes large granular impurity, and filtrate is centrifuged 10min through 12000rpm, removes supernatant, precipitates resuspended, in boiling with 100 μ l sterilized water Water boils 5 min.
, 3858P1F and 3858P1R that take 10 μMs be primer, with the sample in 2 μ l steps 2 or 3 as template, carry out PCR expansion Increase, if obtaining 656 bp PCR primer, i.e. can determine that fish tissues sample to be measured caused a disease vibrio harveyi infect.
The tissue homogenate sterilized water of described step 2 carries out 10 × dilution, and diluent then boils in boiling water 5 min.The precipitation 0.5ml sterilized water of described step 3 is resuspended.The PCR reaction system of described step 4 is 20 μ l；PCR process is: 94 DEG C of 5min denaturation template DNAs, then 94 DEG C of 45s, 60 DEG C of 45s, 72 DEG C of 1min, again 72 DEG C of extensions after 20-25 circulation Reaction 10min.
Present invention have the advantage that
1, accuracy is higher, and the genetic marker used by the present invention is protected in being present in pathogenic vibrio harveyi and having certain genus Keeping property, thus the least with the probability of other vibrio cross reaction；
2, quickly and easily.The pathogenic vibrio harveyi detection method implementation process of the present invention is simple, does not need antibacterial culturing, DNA extraction Etc. step, only need conventional PCR, within 2 ~ 3 hours, result can be obtained；
3, applied range, sample without particular/special requirement, can be detected environment and biogenic sample by the detection method of the present invention；
4, the present invention is present in pathogenic Kazakhstan arc by pathogenic vibrio harveyi gene specific molecular marker fragment 385-8, this fragment In bacterium, but it is not present in non-pathogenic vibrio harveyi and other vibrio, including the molten algae the nearest with vibrio harveyi evolutionary relationship In vibrio, vibrio parahaemolytious, thus this genetic fragment is a species specific pathogenic vibrio harveyi genetic marker, with it as PCR Probe can detect the vibrio harveyi that causes a disease accurately and rapidly from multiple biology and environmental sample.
Accompanying drawing explanation
Fig. 1 is electrophoresis pattern (the wherein swimming lane M:DNA marker that the present invention detects the vibrio harveyi that causes a disease from fish tissues DL2000；Swimming lane 1,2,3,4 is respectively with the cabrilla brain injecting GDH11385, liver, spleen, kidney for template gained PCR primer；Swimming lane 5 is positive control；Swimming lane 6,7 is negative control, is respectively with the liver injecting PBS, kidney as template The PCR primer of gained).
Fig. 2 is electrophoresis pattern (the wherein swimming lane M:DNA marker DL2000 of the pathogenic vibrio harveyi of detection in sea water body； Swimming lane T is for adding interference bacterium and positive bacteria GDH11385 sea water sample, and swimming lane C is not added with positive bacteria sea water for only adding interference bacterium Sample).
Detailed description of the invention
Experimental technique routinely involved in the embodiment of the present invention is all adopted with the following method:
1, plasmid extraction, DNA (PCR) product purification all use the corresponding reagent of " precious biological engineering (Dalian) company limited " Box；
2, the various components of PCR reaction are all purchased from " precious biological engineering (Dalian) company limited "；
Embodiment 1
The vibrio harveyi specific molecular genetic marker that causes a disease is made up of the 385-8 base sequence in sequence table SEQ ID No.1.
Sequence table SEQ ID No.1 is:
TACATCTACCATTTCCGTAAACATGAAGTTGAACACCGACCCGAATGCGGCGCTGTCTGACATTCTGGCAAAA ACCAACTCCGTTCGCTCACAACTTCCAAAAGAAGCGGAAGACCCAACAGTAACGATGTCGACTGGCTCGACTACCGC GGTACTGTACATCGGTTTCACCAGTGACGAGTTGTCTTCTAGCCAGATCACCGACTATCTTGAGCGTGTAATCAACC CGCTGCTATTTACGGTAAATGGTGTATCGAAAGTTGACCTATACGGTGGTATGAAATACGCGCTACGCGTATGGCTA GATCCAGCGAAAATGGGCGCACTTAAGCTGACAGCAACCGACGTGATGACGGTACTAAACGCCAACAACTATCAGTC GGCAACCGGTCAGGCGACAGGTGAATTCGTACTTTACAACGGTAGCGCAGATACTCAAGTATCCAACGTTGCTGAAC TCGAAGCGCTGGTTGTTAAAACTGGTGAAGGTGACGTCATTCGTCTTGGCGACATTGCGAAAGTGACCTTAGAGAAG AGCCACGACGTTTACCGCGCAAGTGCGAACGGTCAAGAGGCTGTAGTTGCAGCGATCAACGCGGCACCAAGTGCTAA CCCAATCAACATCGCGGCAGATGTGCTGAAACTTTTACCTCAAT
(a) sequence signature:
Length: 656bp
Type: base sequence
Chain: strand
Topological structure: linear
(b) molecule type: double-stranded DNA
C () is assumed: no
(d) antisense: no
E () is initially originated: vibrio harveyi GDH11385
(f) specificity title: 3858P1
Causing a disease the application of vibrio harveyi specific molecular genetic marker, its genetic marker can cause a disease Kazakhstan arc in specific detection Bacterium.
The detection of pathogenic vibrio harveyi in fish tissues:
1) artificial challenge of cabrilla
In liquid 2216E culture medium, cultivate vibrio harveyi GDH11385 (be stored in the torrid zone, Hainan Province aquatile technology emphasis Laboratory strains preservation center, deposit number is: PBVH33111), 30 DEG C, shaken cultivation to OD600Be 0.6,4 DEG C, 8000g from Heart 5min, collects thalline, is suspended in normal saline to final concentration of 1 × 106 cfμ/ml.By 10 tail cabrilla (body weight 15 ~ 20g/ tail) it is randomly divided into 2 groups, often organize 5 tails, one group is experimental group (T group), lumbar injection above-mentioned vibrio harveyi suspension (100 μ l/ tail), another group is matched group (C group), every tail fish lumbar injection 100 μ l normal saline respectively.
Wherein the constituent of 2216E fluid medium is by weight percentage: 0.5% peptone, 0.1% yeast extract, 0.01%, 3%NaCl, 96.4%H2O, adjusts pH value to 7.4；Normal saline constituent is by weight percentage: 0.85% NaCl；
2) prepared by cabrilla tissue sample
In above-mentioned steps 1) after cabrilla infects 48 h, under aseptic condition, take fish brain, liver, spleen, kidney, be placed in glass even In slurry device, add 1ml normal saline and be homogenized.Tissue homogenate sterilized water is carried out 10 × dilution.By diluent in Boiling water boils 5 min.
3) PCR detection
In 0.2ml PCR pipe, add following ingredients: 2 μ l above-mentioned steps 2) serum of tissue diluent after high-temperature process is dense Spend 10 μMs of primer 3858P1F (5 '-TACATCTACCATTTCCGTAAACA-3 ') and 3858P1R (5 '- ATTGAGGTAAAAGTTTCAGCACATC-3 ') each 1 μ l, 2 μ l 10 × PCR b μ ffer [200mM Tris-NCl (pH 8.4), 200mM KCl, 15mM MgCl2], 2 μ l 10mM dNTPs (each 2.5 mmol/L), 0.25 μ l Taq archaeal dna polymerase, Adding water to cumulative volume is 20 μ l.
Wherein primer 3858P1F and 3858P1R is the vibrio harveyi fragment 385-8 specific primer that causes a disease, and its PCR primer is big Little for 656bp.
PCR:94 DEG C of 5min denaturation template DNA, then 94 DEG C of 45s is carried out by following condition, 60 DEG C of 45s, 72 DEG C of 1min, Again at 72 DEG C of extension 10min after 20-25 circulation.Take 5 μ l PCR primer to observe in 1% agarose gel electrophoresis.Result Show (see accompanying drawing 1), be only the PCR of masterplate with the cabrilla tissue homogenate of injection vibrio harveyi and amplify a 656bp Positive band, be the PCR of masterplate then without any band with the cabrilla tissue homogenate of injecting normal saline.
Embodiment 2
The detection of pathogenic vibrio harveyi in water body
1) prepared by water sample
Preparing two parts of sterilizing sea water (named C and T), take 20ml water body respectively, C sea water liquid adds final concentration of respectively 104 The nontoxic vibrio harveyi of cfu/ml, vibrio alginolyticus V. alginolyticus ATCC 33787, vibrio parahaemolytious V. Parahemolyticus ATCC17802, vibrio mimicus V. mimicus ATCC 33653, Freund vibrio V. furnissii ATCC 33813 and Vibrio natriegen V. natriegens ATCC 33788, T sea water liquid, in addition to adding above-mentioned antibacterial, adds end Concentration is 104 The pathogenic vibrio harveyi GDH11385 of cfu/ml.C, T sea water liquid is that 80 ~ 120 μm fast quantifications divide with aperture respectively Analysis filter paper filtering, removes large granular impurity, and filtrate is centrifuged 10min through 12000rpm, removes supernatant, and precipitation uses 0.1ml sterilized water In resuspended, in boiling water, boil 5 min.
2) PCR detection
In 0.2ml PCR pipe, add following ingredients: 2 μ l above-mentioned steps 1) C or the T re-suspension liquid crossed of high-temperature process, concentration 10 μ M primer 3858P1F (5 '-TACATCTACCATTTCCGTAAACA-3 ') and 3858P1R (5 '- ATTGAGGTAAAAGTTTCAGCACATC-3 ') each 1 μ l, 2 μ l 10 × PCR b μ ffer [200mM Tris-NCl (pH 8.4), 200mM KCl, 15mM MgCl2], 2 μ l 10mM dNTPs (each 2.5 mmol/L), 0.25 μ l Taq archaeal dna polymerase, Adding water to cumulative volume is 20 μ l.
PCR reaction condition is with embodiment 1 step 3).Result shows (see accompanying drawing 2), only with the addition of pathogenic vibrio harveyi The T group sea water sample of GDH11385 amplifies the positive band of a 656bp, and C group sea water sample, without any PCR band, illustrates T group water Sample contains pathogenic vibrio harveyi, and C group water sample does not contains pathogenic vibrio harveyi.
Result above shows, the pathogenic vibrio harveyi detection method of the present invention can detect rapidly and accurately tissue and Pathogenic vibrio harveyi in seawater sample.
SEQUENCE LISTING
＜ 110 ＞ University Of Hainan
＜ 120 ＞ pathogenic vibrio harveyi specific molecular genetic marker and application thereof
〈160〉1
〈210〉1
〈211〉656
〈212〉DNA
〈213〉Vibrio harveyi GDH11385
〈400〉1
TACATCTACC ATTTCCGTAA ACATGAAGTT GAACACCGAC CCGAATGCGG CGCTGTCTGA 60
CATTCTGGCA AAAACCAACT CCGTTCGCTC ACAACTTCCA AAAGAAGCGG AAGACCCAAC 120
AGTAACGATG TCGACTGGCT CGACTACCGC GGTACTGTAC ATCGGTTTCA CCAGTGACGA 180
GTTGTCTTCT AGCCAGATCA CCGACTATCT TGAGCGTGTA ATCAACCCGC TGCTATTTAC 240
GGTAAATGGT GTATCGAAAG TTGACCTATA CGGTGGTATG AAATACGCGC TACGCGTATG 300
GCTAGATCCA GCGAAAATGG GCGCACTTAA GCTGACAGCA ACCGACGTGA TGACGGTACT 360
AAACGCCAAC AACTATCAGT CGGCAACCGG TCAGGCGACA GGTGAATTCG TACTTTACAA 420
CGGTAGCGCA GATACTCAAG TATCCAACGT TGCTGAACTC GAAGCGCTGG TTGTTAAAAC 480
TGGTGAAGGT GACGTCATTC GTCTTGGCGA CATTGCGAAA GTGACCTTAG AGAAGAGCCA 540
CGACGTTTAC CGCGCAAGTG CGAACGGTCA AGAGGCTGTA GTTGCAGCGA TCAACGCGGC 600
ACCAAGTGCT AACCCAATCA ACATCGCGGC AGATGTGCTG AAACTTTTAC CTCAAT 656

Claims (6)

1. a pathogenic vibrio harveyi specific molecular genetic marker, it is characterised in that: the vibrio harveyi specific molecular that causes a disease is lost Pass shown in the base sequence being labeled as in sequence table SEQ ID No.1.
2. the application of the vibrio harveyi specific molecular genetic marker that causes a disease as claimed in claim 1, it is characterised in that: described something lost Pass labelling can specific detection cause a disease vibrio harveyi.
3. the application of the vibrio harveyi specific molecular genetic marker that causes a disease as claimed in claim 2, it is characterised in that:
(1), according to the pathogenic vibrio harveyi specific molecular heredity mark shown in the base sequence in sequence table SEQ ID No.1 Note, the primer related to is: primer 3858P1F (5 '-TACATCTACCATTTCCGTAAACA-3 ') and 3858P1R (5 '- ATTGAGGTAAAAGTTTCAGCACATC-3’)；
(2), the preparation of tissue sample to be measured: under aseptic condition, take fish brain, liver, spleen, kidney, by brain, liver, spleen and Kidney adds 1ml physiological saline solution and is homogenized, and is then diluted, stand-by；
(3), the preparation of environmental sample: take 20ml water body in environment, be 80 ~ 120 μm fast quantitative analysis filter paper mistakes with aperture Filter, removes large granular impurity, and filtrate is centrifuged 10min through 12000rpm, removes supernatant, precipitates resuspended, in boiling with 100 μ l sterilized water Water boils 5 min；
(4), 3858P1F and 3858P1R that take 10 μMs be primer, with the sample in 2 μ l step (2) or (3) as template, carry out PCR expands, if obtaining 656bp PCR primer, i.e. can determine that fish tissues sample to be measured and seawater sample are infected by vibrio harveyi.
4. the application of the vibrio harveyi specific molecular genetic marker that causes a disease as claimed in claim 3, it is characterised in that: described step Suddenly the tissue homogenate sterilized water of (2) carries out 10 × dilution, and diluent then boils in boiling water 5 min.
The application of pathogenic vibrio harveyi specific molecular genetic marker the most as claimed in claim 3, it is characterised in that: described The precipitation 0.1ml sterilized water of step (3) is resuspended.
6. the application of the vibrio harveyi specific molecular genetic marker that causes a disease as claimed in claim 3, it is characterised in that: described step Suddenly the PCR reaction system of (4) is 20 μ l；PCR process is: 94 DEG C of 5min denaturation template DNAs, then 94 DEG C of 45s, 60 DEG C of 45s, 72 DEG C of 1min, again at 72 DEG C of extension 10min after 20-25 circulation.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109022602A (en) * 2018-08-27 2018-12-18 海南大学 A kind of primer pair, kit and detection method detecting coral bleaching cause of disease vibrio alginolyticus XSBZ03 and XSBZ14
CN116732204A (en) * 2023-05-29 2023-09-12 海南大学 Multiplex LAMP primer group for simultaneously detecting multiple pathogens, detection method and kit

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Publication number Priority date Publication date Assignee Title
CN101818191A (en) * 2009-02-27 2010-09-01 中国科学院海洋研究所 Vibrio harveyi specific molecular genetic marker and application thereof

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Publication number Priority date Publication date Assignee Title
CN101818191A (en) * 2009-02-27 2010-09-01 中国科学院海洋研究所 Vibrio harveyi specific molecular genetic marker and application thereof

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张晓君 等: "中国对虾糠虾幼体病原哈氏弧菌的鉴定及毒力基因检测", 《渔业科学进展》 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109022602A (en) * 2018-08-27 2018-12-18 海南大学 A kind of primer pair, kit and detection method detecting coral bleaching cause of disease vibrio alginolyticus XSBZ03 and XSBZ14
CN109022602B (en) * 2018-08-27 2021-10-26 海南大学 Primer pair, kit and detection method for detecting coral albino pathogenic vibrio alginolyticus XSBZ03 and XSBZ14
CN116732204A (en) * 2023-05-29 2023-09-12 海南大学 Multiplex LAMP primer group for simultaneously detecting multiple pathogens, detection method and kit
CN116732204B (en) * 2023-05-29 2024-02-13 海南大学 Multiplex LAMP primer group for simultaneously detecting multiple pathogens, detection method and kit

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