CN109022602B - Primer pair, kit and detection method for detecting coral albino pathogenic vibrio alginolyticus XSBZ03 and XSBZ14 - Google Patents
Primer pair, kit and detection method for detecting coral albino pathogenic vibrio alginolyticus XSBZ03 and XSBZ14 Download PDFInfo
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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
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention relates to a primer pair, a kit and a detection method for detecting coral albino pathogenic vibrio alginolyticus XSBZ03 and XSBZ 14. The primer pair comprises an upstream primer F1 and a downstream primer R1; the sequences of the upstream primer F1 and the downstream primer R1 are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The kit comprises an upstream primer F1, a downstream primer R1, 2 XPCR Master Mix, ultrapure water, a positive control and a negative control. The kit can be used for simultaneously and rapidly detecting coral albino pathogenic vibrio alginolyticus XSBZ03 and XSBZ14, and has the advantages of strong specificity, high sensitivity, accurate detection and short time consumption, so that the kit has higher accuracy for diagnosing coral albino diseases caused by XSBZ03 and XSBZ14, and has guiding significance for evaluating vibrio diseases of sea coral in China and formulating effective disease control measures.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a primer pair, a kit and a detection method for detecting coral albino pathogenic vibrio alginolyticus, in particular to a primer pair, a kit and a detection method for simultaneously and rapidly detecting coral pathogenic strains coral albino pathogenic vibrio XSBZ03 and XSBZ 14.
Background
Coral reefs are extremely special ecosystems in the ocean, keep high biodiversity and primary productivity, are known as tropical rainforests in the ocean and oases in blue deserts, and are always important life support systems. However, coral diseases caused by bacterial pathogens occur frequently, resulting in the continuous disruption of coral reef ecosystem structures, and two thirds of coral reefs are now at risk of extinction.
The platypodium fasciolium albinism syndrome caused by Vibrio algebralyticus strains XSBZ03 and XSBZ14 poses a serious threat to the coral reef ecosystem in the south China sea. In order to deal with the harm of the pathogen to China sea coral, firstly, XSBZ03 and XSBZ14 need to be accurately diagnosed, so that the establishment of a rapid detection method of coral pathogen strains XSBZ03 and XSBZ14 is particularly important, and the method is a primary premise for carrying out the diagnosis of the coral diseases in China, is an important basis for explaining the prevalence rules, transfer mechanisms and pathogenic mechanisms of the pathogen, and is a necessary way for evaluating the harm degree of the pathogen to the south China sea coral and formulating corresponding disease control measures.
For the above reasons, the present application has been made.
Disclosure of Invention
The invention aims to solve the problems pointed out in the background art and the defects in the prior art, and aims to provide a primer pair for simultaneously and rapidly detecting coral albino pathogenic vibrio alginolyticus XSBZ03 and XSBZ14, a kit and a detection method thereof.
The first purpose of the invention is to provide a PCR detection primer pair for simultaneously and rapidly detecting coral albino pathogenic vibrio alginolyticus XSBZ03 and XSBZ14, which has strong specificity and can distinguish the coral pathogenic strains XSBZ03 and XSBZ14 from other common marine strains.
In order to achieve the first object, the technical scheme of the invention is as follows:
the primer pair for simultaneously and rapidly detecting the XSBZ03 strain and the XSBZ14 strain comprises an upstream primer F1 and a downstream primer R1, wherein the sequence of the upstream primer F1 is shown as SEQ ID NO: 1, the sequence of the downstream primer R1 is shown as SEQ ID NO: 2, respectively.
The strong specificity primer designed by the invention is a key factor for realizing the purpose, and the applicant of the invention screens out the GenBank accession number of the target gene detected by PCR of the invention through bioinformatics analysis and a large number of experiments, wherein the GenBank accession number is as follows: MH702378, and the primer set F1/R1 described above was designed based on the target gene. 39 Vibrio corallina albidus, 16 Vibrio harveyi (Vibrio harveyi), 3 Vibrio Parahaemolyticus (Vibrio Parahaemolyticus), 1 Vibrio rotifer (Vibrio rotiferaticus), 1 Vibrio natriegens (Vibrio natriegens), 1 Vibrio vulnificus (Vibrio vulnifica), and the primers are used for amplifying ultrapure water, and only XSBZ03 and XSBZ14 can generate amplification products through agarose gel electrophoresis detection.
The second purpose of the invention is to provide a kit for simultaneously and rapidly detecting the coral albino pathogenic vibrio alginolyticus XSBZ03 and XSBZ14, wherein the kit is a PCR detection kit prepared based on the primer pair, and has the advantages of strong specificity, high sensitivity, accurate detection, short time consumption and effectiveness.
In order to achieve the second object, the technical scheme of the invention is as follows:
the kit comprises: an upstream primer F1, a downstream primer R1, 2 XPCR Master Mix, ultrapure water, a positive control and a negative control.
Preferably, the forward primer F1 and the reverse primer R1 are 2.0. mu.L each, 2 XPCR Master Mix 25. mu.L, 17. mu.L of ultrapure water, a positive control, and a negative control, respectively.
As a preferred embodiment, the 2 XPCR Master Mix consists of 3mmol/LMgCl2100mmol/L KCl, 500. mu. mol/L dNTP each, 20mmol/L LTris-HCl (pH 8.3) and 0.4U/. mu.L AceTaq DNA Polymerase.
Preferably, the positive control is mixed inactivated lyophilized powder of XSBZ03 and XSBZ14 or mixed PCR product of XSBZ03 and XSBZ 14.
The negative control was ultrapure water.
The third purpose of the invention is to provide a detection method for simultaneously and rapidly detecting the coral albino pathogenic vibrio alginolyticus XSBZ03 and XSBZ14, wherein the method is based on the detection method of the PCR detection kit of the XSBZ03 and XSBZ14, and the method is simple to operate, short in PCR time consumption and high in accuracy.
In order to achieve the third object of the invention, the technical scheme of the invention is as follows:
a detection method for simultaneously and rapidly detecting Vibrio corallina Hance XSBZ03 and XSBZ14, the method comprising the following steps:
1) PCR template: extracting DNA of a sample to be detected, or directly using a bacterial liquid as a template;
2) gene amplification: adding the DNA extracted in the step 1) and a positive control into a detection kit for PCR amplification;
3) and (3) detection: and carrying out agarose gel electrophoresis detection on the PCR amplification product.
As a preferred scheme, the sample to be detected in the step 1) is a pure bacterial culture or an environmental water sample.
Preferably, the kit for PCR amplification comprises 17. mu.L of ultrapure water, 25. mu.L of 2 XPCR Master Mix, 2.0. mu.L of each of the upstream and downstream primers, and 4.0. mu.L of sample DNA to be detected.
Preferably, the PCR amplification process in step 2) specifically includes:
5min at 94 ℃; 30s at 94 ℃, 30s at 60 ℃, 60s at 72 ℃ and 35 cycles; 5min at 72 ℃.
Compared with the prior art, the primer pair or the kit for detecting XSBZ03 and XSBZ14 provided by the invention has the following beneficial effects:
(1) instruments and conditions of a basic routine epidemic disease diagnosis laboratory can meet the detection requirements; (2) the primer pair can simultaneously amplify XSBZ03 and XSBZ14 strains and can distinguish the XSBZ03 and the XSBZ14 strains from other related strains, (3) the kit has high detection sensitivity, the genome detection limit of the kit to XSBZ03 is 170 fg/mu L, and the genome detection limit of the kit to XSBZ03 bacteria is 6.4 multiplied by 103CFU/mL; genomic detection limit for XSBZ14 of 20 fg/. mu.L and for XSBZ14 bacteria of 8.5X 102CFU/mL; (4) the kit can simultaneously and quickly detect the coral albino pathogenic vibrio alginolyticus XSBZ03 and XSBZ14, has strong specificity, high sensitivity, accurate detection, short time consumption and high accuracy in coral albino disease diagnosis caused by XSBZ03 and XSBZ14, and can evaluate the harm degree of the two pathogens to the sea coral in China and provide guiding significance for formulating control measures of the disease.
Drawings
FIG. 1 is a gel electrophoresis image in a specificity evaluation experiment: wherein lane M is a DNA Marker DL500bp molecular weight standard; XSBZ03 and XSBZ14 in lanes 1 and 2, respectively, and numbers 3-32 are Vibrio alginolyticus HN07014, Vibrio alginolyticus HN07011, Vibrio alginolyticus HN08809, Vibrio alginolyticus HN08304, Vibrio alginolyticus HN08307, Vibrio alginolyticus HN08805, Vibrio alginolyticus HN07005, Vibrio alginolyticus XSE381, Vibrio alginolyticus HN08803, Vibrio alginolyticus HN08810, Vibrio alginolyticus 30031, Vibrio alginolyticus HN08203, Vibrio alginolyticus HN07006, Vibrio alginolyticus HN08811, Vibrio alginolyticus HN08155, Vibrio HN07002, Vibrio alginolyticus HN 06003, Vibrio alginolyticus HN08306, Vibrio HN alginolyticus HN07009, Vibrio alginolyticus HN 0841108335, Vibrio alginolyticus HN 083083801, HN alginolyticus HN08801, HN 01, Vibrio HN E167, Vibrio HN08335, HN alginolyticus HN 07019, HN07002, HN 07018, HN081 0709, HN 18, HN 0709, HN 08319, HN 18, HN 18, HN 18, HN 18, HN 66, HN 66, HN 16, HN HB 0839, HN 18, HN 66, HN 18, HN 66, HN Y179, HN 18, HN Y082 18, HN Y082, HN 18, HN Y082 18, HN Y082, HN 18, HN Y082 18, HN Y082 18, HN 9, HN 18, HN Y082, HN 18, HN Y0829, HN 18, HN 9, HN 18, HN 9, HN Y082, HN 18, HN 9, HN 18, HN 9, HN 18, HN Y082 18, HN 9, HN 18, HN, vibrio harveyi NS131241, Vibrio harveyi NS131751, Vibrio harveyi NS131251, Vibrio harveyi NS131651, Vibrio harveyi NS131451, Vibrio harveyi NS131632, Vibrio harveyi NS131631, Vibrio harveyi WC13D121, Vibrio harveyi HNH11011, Vibrio harveyi HNH11001, Vibrio harveyi WC13DH21, Vibrio harveyi WC13H252, Vibrio harveyi HNH11009, Vibrio harveyi HNH11013, Vibrio harveyi WC13DH52, Vibrio harveyi PBVH78461, Vibrio parahaemolyticus PBVPY07150, Vibrio parahaemolyticus PBVPY06106, Vibrio parahaemolyticus ATCC 076, Vibrio vulnificus standard ATCC 27562, Vibrio natrii ATCC 33788, and 17171752.
FIG. 2 is a gel electrophoresis of the XSBZ03 genome concentration in a sensitivity assessment experiment, Lane M DNA Marker DL500bp molecular weight standard, Lane 1-7: the DNA concentration of XSBZ03 in the template was 1.7 ng/. mu.L, 170 pg/. mu.L, 17 pg/. mu.L, 1.7 pg/. mu.L, 170 fg/. mu.L, 17 fg/. mu.L, 1.7 fg/. mu.L, lane 8: ultrapure water.
FIG. 3 shows the gel electrophoresis of the XSBZ14 genome concentration in the sensitivity evaluation experiment, Lane M DNA Marker DL500bp molecular weight standard lanes 1-7: the DNA concentrations of XSBZ03 in the template were 2.0 ng/. mu.L, 200 pg/. mu.L, 20 pg/. mu.L, 2.0 pg/. mu.L, 200 fg/. mu.L, 20 fg/. mu.L, 2.0 fg/. mu.L, respectively.
FIG. 4 is a gel electrophoresis of XSBZ03 bacterial concentration in a sensitivity assessment experiment, lane M: DNA Marker DL500bp molecular weight Standard, lanes 1-8: the bacterial concentrations of XSBZ03 in the template were 6.4X 10, respectively7CFU/mL， 6.4×106CFU/mL，6.4×105CFU/mL，6.4×104CFU/mL，6.4×103CFU/mL，6.4×102CFU/mL， 6.4×10CFU/mL，6.4CFU/mL。
FIG. 5 shows the gel electrophoresis of XSBZ14 bacterial concentration in a sensitivity assessment experiment, lane M DNA Marker DL500bp molecular weight standard, lanes 1-8: the bacterial concentrations of XSBZ03 in the template were 8.5X 10, respectively7CFU/mL， 8.5×106CFU/mL，8.5×105CFU/M，8.5×104CFU/mL，8.5×103CFU/mL，8.5×102CFU/mL， 8.5×10CFU/mL，8.5CFU/mL。
Detailed Description
The present invention will be described in further detail with reference to the following embodiments and accompanying drawings. The present invention is implemented on the premise of the technology of the present invention, and the detailed embodiments and specific procedures are given to illustrate the inventive aspects of the present invention, but the scope of the present invention is not limited to the following embodiments.
Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.
For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Example 1
1. Primer design
In order to distinguish the coral pathogenic strains XSBZ03 and XSBZ14 from other related strains and improve the accuracy of disease diagnosis, the applicant screens a target gene detected by PCR according to the invention through bioinformatics analysis and a large number of experiments, and designs a pair of proper primers according to the target gene, wherein the GenBank accession number of the target gene is as follows: MH702378 is a sequence in the whole genome of Vibrio alginolyticus XSBZ14, and primers designed according to the sequence can amplify XSBZ03 and XSBZ14, but have no amplification effect on other strains mentioned in the application.
Primer F1(SEQ ID NO: 1) 5'-GCGCACTCAAAAAACCTATCAA-3',
primer R1(SEQ ID NO: 2): 5'-GCACCAGTCTCACCACGC-3'.
2. Template preparation
The Vibrio species such as XSBZ03 and XSBZ14 were isolated and purified from the TCBS medium, and then cultured in 2216E medium at 37 ℃ for 12 hours, and the bacterial DNA was extracted using TaKaRa's bacterial genome Extraction Kit (TaKaRa MiniBEST bacterial Genomic DNA Extraction Kit Ver.3.0) and stored at-20 ℃ for further use.
3. PCR amplification
The extracted DNA template was subjected to PCR amplification using the primers, and the PCR reaction system was 50. mu.L, which is shown in Table 1.
TABLE 1 PCR reaction System Table
The PCR reaction parameters are as follows:
5min at 94 ℃; 30s at 94 ℃, 30s at 60 ℃, 15s at 72 ℃ and 35 cycles; 5min at 72 ℃.
Determining whether the sample contains XSBZ03 or XSBZ14 according to the following judgment criteria: and (3) taking 6 mu L of the PCR amplification product, performing 2.5% agarose gel electrophoresis, and observing under the irradiation of an ultraviolet lamp, wherein when only one specific band appears after the electrophoresis of the PCR amplification product, and the size of the band is about 122bp, the sample to be detected is XSBZ03 or XSBZ 14. The negative control has no PCR amplification band, and the positive control only has a specific band with the size of about 122 bp.
Example 2
Sensitivity test of detection kit
XSBZ03 genomic detection limit (fig. 2): DNA was extracted and its concentration was determined for the purpose of the assay using the nucleic acid as described in example 1, heading 2, DNA was extracted and its concentration was determined using a nucleic acid analyzer at 17 ng/. mu.L for XSBZ03 genome, and a ten-fold gradient dilution with ultrapure water was performed for 7 gradients, 1.7 ng/. mu.L (lane 1), 170 pg/. mu.L (lane 2), 17 pg/. mu.L (lane 3), 1.7 pg/. mu.L (lane 4), 170 fg/. mu.L (lane 5), 17 fg/. mu.L (lane 6), 1.7 fg/. mu.L (lane 7), ultrapure water (lane 8), and DNA marker 500 (lane 9) showing that the detection limit of the primer pair XSBZ03 genome was 170 fg/. mu.L.
XSBZ14 genomic detection limit (fig. 3): XSBZ14 was initially at a concentration of 20 ng/. mu.L and was diluted in a tenfold gradient with ultrapure water for a total dilution of 7 gradients, 2 ng/. mu.L (lane 1), 200 pg/. mu.L (lane 2), 20 pg/. mu.L (lane 3), 2 pg/. mu.L (lane 4), 200 fg/. mu.L (lane 5), 20 fg/. mu.L (lane 6), 2 fg/. mu.L (lane 7). Ultrapure water (lane 8), DNA marker 500 (lane 9). The result shows that the genome detection limit of the primer pair XSBZ14 is 20 fg/. mu.L.
XSBZ03 bacterial liquid detection limit (fig. 4): the initial bacterial liquid concentration was measured by ten-fold dilution with PBS buffer and plate coating, and the result showed that the initial bacterial liquid concentration of XSBZ03 was 6.4X 108CFU/mL, take 7 dilution gradients, 6.4X 107CFU/mL (lane 1), 6.4X 106CFU/mL (lane 2), 6.4X 105CFU/mL (lane 3), 6.4X 104CFU/mL (lane 4), 6.4X 103CFU/mL (lane 5), 6.4X 102CFU/mL (lane 6), 64CFU/mL (lane 7) ultrapure water (lane 8), DNA marker 500 (lane 9). The result shows that the detection limit of the primer pair XSBZ03 bacterial liquid is 6.4 multiplied by 103CFU/mL。
XSBZ14 bacterial liquid detection limit (fig. 5): the initial concentration of the bacterial liquid was measured by ten-fold dilution with PBS buffer and plate coating, and the result showed that the initial concentration of XSBZ14 bacterial liquid was 8.5X 108CFU/mL, take 7 dilution gradients, 8.5X 107CFU/mL，8.5×107CFU/mL，8.5×106CFU/mL，8.5×105CFU/mL，8.5× 104CFU/mL，8.5×103CFU/mL，8.5×102CFU/mL, 85CFU/mL, the result shows that the detection limit of the primer pair XSBZ03 bacterial liquid is 8.5X 102CFU/mL。
With reference to examples 1 and 2, it is demonstrated that the kit has high specificity and sensitivity for the detection of the coral pathogenic strains XSBZ03 and XSBZ 14.
Sequence listing
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atgcgttcag ctaaccatag cgaacttagc aacgccctac ttgcgatcag tcaatctttg 60
gctgatcgca gccagcttac ccaaaccttg gatgctgtgt tgaccgctgc gagacaaatg 120
accctcgcaa agcacggcat catttatgtt ttggaccaga ctggtcaggc gttgattcca 180
agtaccgctc accataacga taaaacgatt gtttcccacc cttgggaggc attgcagatt 240
gattccgcaa gtgagaacga cccatttaac tttgccattc gcaatggtga agtggtattg 300
attaacgagt tgtataagta caacggctat gactgtgaca gcatctatca aactgagcag 360
acgctaggac taaaaagcga gaacttgctg gcttggcctt tgattgatag cgaaagtaag 420
accattggac tcttggtctt gctcgattta agtgttatcg acaatgaggc tgcattgact 480
gagttttgtc gcatggccgc aagtaatatt cgccaagctg tttggttaga gcaatacggg 540
caagtgatta aaagcctcag tgccgataat caagccctcg ttcgtgagaa tacgcaactc 600
aaaaagcgca ctcaaaaaac ctatcaaggc cctattgcag agagcgaaga aatgctcaat 660
gtcttgaatc gtcttgataa agtgctgtct cttcctgtcg acgttctgtt gcgtggtgag 720
actggtgccg gtaaagaggt gatcgcgaaa tacattcacg aaaactctaa ccgttcagaa 780
cagcctttga tcgtacaaaa ctgtgcggca attcccgaac agttgttaga gtctgaactg 840
tttgggcata agaaaggttc attcacgggc gcggacaaag acaaagtcgg tttgtttgaa 900
gcagccaacg gcggcacgct tttcctagat gaaattggcg atatgccaat gctactacaa 960
gcgaagttac tgcgtgtact gcaagagcgt aaagttcgcc ctatcggtac cagcaaagag 1020
attgaagtgg acgtgcgcgt tatcgctgca acccactgta atcttatgca gcaaatcaaa 1080
gatggcggat tccgtgccga cttgttctat cgcttaaatg tcttccctat tacattgccg 1140
cccctgagag cccgtaagtc ggacattatt ccccttgctg aacatttcgt gcaacacaca 1200
accaacacgc taggtttgcc gcaagcgcca gggttaagtg ccaatgtacg taaacagctg 1260
cttgcctacc aatacccagg gaacgtgcgt gagcttaaaa acatcatcga gcgctcggta 1320
ctactgtccg attttgaaac cattactcac attgagtttg gcgaacaaat cccagaagat 1380
gtaccaagta tcgacatgaa agcagcttca cctaccccag agcaacctgc ttacgagcag 1440
caagcacaag cggatttaga agacgtttca aagagcctta aagacgtcgt tagccaatac 1500
gagcgtacag taatcatcga ttgcttaaat gcctgcaatt ggcacaccaa gaaggccgcc 1560
gagcaattgg cacttccaat gagcactctc aaccataaga tgaagaaata cgatatttct 1620
gcagcgggtt ga 1632
Claims (10)
1. A primer pair for simultaneously and rapidly detecting coral albino pathogenic vibrio alginolyticus XSBZ03 and XSBZ14 is characterized in that: the primer pair comprises an upstream primer F1 and a downstream primer R1, wherein the sequence of the upstream primer F1 is shown in SEQ ID NO: 1, the sequence of the downstream primer R1 is shown as SEQ ID NO: 2, respectively.
2. A kit for simultaneously and rapidly detecting coral albino pathogenic vibrio alginolyticus XSBZ03 and XSBZ14 is characterized in that: the kit comprises: the upstream primer F1, the downstream primer R1 and the 2 XPCR Master Mix of claim 1, ultrapure water, a positive control and a negative control.
3. The kit of claim 2, wherein: 2.0. mu.L of each of the forward primer F1 and the reverse primer R1, 25. mu.L of 2 XPCR Master Mix, 17. mu.L of ultrapure water, a positive control, and a negative control.
4. The kit according to claim 2 or 3, characterized in that: the 2 XPCR Master Mix consists of 3mmol/L MgCl2100mmol/L KCl, 500. mu. mol/L dNTP, 20mmol/L Tris-HCl and 0.4U/. mu.L AceTaq DNA Polymerase.
5. The kit of claim 2, wherein: the positive control is mixed inactivated freeze-dried powder of XSBZ03 and XSBZ14 or mixed PCR products of XSBZ03 and XSBZ 14.
6. The kit of claim 2, wherein: the negative control was ultrapure water.
7. A detection method for simultaneously and rapidly detecting coral albino pathogenic vibrio alginolyticus XSBZ03 and XSBZ14 for non-disease diagnosis is characterized in that: the detection method is based on the detection method of the kit for simultaneously and rapidly detecting the coral albino pathogenic vibrio alginolyticus XSBZ03 and XSBZ14 as claimed in claim 2; the method comprises the following steps:
1) PCR template: extracting DNA of a sample to be detected;
2) gene amplification: adding the DNA extracted in the step 1) and a positive control into a detection kit for PCR amplification;
3) and (3) detection: and carrying out agarose gel electrophoresis detection on the PCR amplification product.
8. The detection method according to claim 7, characterized in that: in the step 1), the sample to be detected is a pure bacterial culture or an environmental water sample.
9. The detection method according to claim 7, characterized in that: the PCR reaction system comprises 17 mu L of ultrapure water, 25 mu L of 2 XPCR Master Mix, 2.0 mu L of each downstream primer and 4.0 mu L of sample DNA to be detected.
10. The detection method according to claim 7, characterized in that: the PCR amplification process of the step 2) specifically comprises the following steps: 5min at 94 ℃; 30s at 94 ℃, 30s at 60 ℃, 60s at 72 ℃ and 35 cycles; 5min at 72 ℃.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101691613A (en) * | 2009-09-09 | 2010-04-07 | 中国水产科学研究院南海水产研究所 | Primer group for detecting vibrio coralliilyticus by using LAMP, quick diagnosis kit and detecting method |
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| TW200819540A (en) * | 2006-07-11 | 2008-05-01 | Genelux Corp | Methods and compositions for detection of microorganisms and cells and treatment of diseases and disorders |
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