CN102719551B - Loop-mediated isothermal amplification detection primer of vibrio shilonii, detection kit and detection method - Google Patents
Loop-mediated isothermal amplification detection primer of vibrio shilonii, detection kit and detection method Download PDFInfo
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Abstract
The invention discloses a loop-mediated isothermal amplification detection primer of vibrio shilonii, a detection kit and a detection method. The detection primers are that: a front outer primer F3:5'-CAAACGGTTGGCGGTGTC-3'; a rear outer primer B3:5'-CCGCAAGATGCTGAAGGATC-3'; a front inner primer FIP:5'-CTGCAAGAGCCAAACTCTGCGTCTTTTGGAGCTGGTGGGAT-3', and a rear inner primer BIP:5'-GCTTCTGATGAAGCGACAGGCAGTAAAGACGGTGCCTAAGCG-3'. The detection technique of the vibrio shilonii achieves the international advanced level; the detection ability is greatly improved; the detection limit of a loop-mediated isothermal amplification detection method of the vibrio shilonii is 100-200 times higher than that of the traditional detection method. On the other hand, the loop-mediated isothermal amplification detection method of the vibrio shilonii disclosed by the invention has the characteristics that the requirement on an apparatus device is simple, the detection time (0.5-1.0 hours) is short, and the specificity is high (6/4 specific primers are designed as to 8/6 areas of a target gene); the disadvantages that the requirement of a classic PCR (polymerase chain reaction) amplification technology on the apparatus device is high and the technology is complicated are compensated, and the loop-mediated isothermal amplification detection method of the vibrio shilonii has the advantages that the detection capability is strong, the detection time is short, and the requirement on the apparatus device is simple, and has wide application prospect.
Description
Technical field:
The invention belongs to microorganism detection field, be specifically related to a kind of ring mediated isothermal amplification of executing Roche vibrios (Vibrio shilonii) of developing based on ring mediated isothermal amplification (1oop-mediated isothermal ampification, LAMP) know-why and detect primer, detection kit and detection method.
Background technology:
Executing Roche vibrios (Vibrio shilonii) is a kind of vibrio marinopraesens, can infect coral, causes the bacillary albefaction of coral.At present, the detection method of executing Roche vibrios is mainly traditional microorganism classification authentication method and the detection method that depends on round pcr.Traditional microorganism classification and authentication method are mainly enough to the characteristics such as the morphology of microorganism and Physiology and biochemistry as basis for estimation, although credible result degree is high, loaded down with trivial details and time-consuming.(PCR) is highly sensitive in polymerase chain reaction, can reach 98%～100%, but specificity is poor, and needs the relevant expensive equipments such as pcr amplification instrument, gel-electrophoretic apparatus, is unfavorable for basic unit's experiment centre and field quick detection.
Ring mediated isothermal amplification (1oop-mediated isothermal amplification, LAMP) technology is a kind of novel isothermal nucleic acid amplification method that Notomi equals 2000 noon paper roads, this method is for 4 special primers of 6 zone design of target gene, utilize a kind of archaeal dna polymerase (Bst archaeal dna polymerase) with strand displacement activity, at constant temperature (65 ℃ of left and right), hatch about 60min, can complete nucleic acid amplification reaction, produce the white magnesium pyrophosphate precipitation of macroscopic byproduct of reaction one.By reversed transcriptive enzyme, also can carry out RT-LAMP and complete the amplification for RNA.This technology has does not need the PCR instrument, naked eyes can judged result and the advantage such as the reaction times is short.Amplification is the fluorescence dye SYBR Green I dyeing of available combination double-stranded DNA also, under ultraviolet lamp or daylight, by naked eyes, judges, if contain amplified production, it is green that reaction mixture turns; Otherwise, keep that SYBR Green I's is orange constant.At present LAMP technology has successfully the report for other pathogen detection.
Summary of the invention:
The object of this invention is to provide a kind of detectivity strong, detect limit for height, detection time is short, specificity is high, ring mediated isothermal amplification (LAMP) that plant and instrument requirement is simply executed to Roche vibrios (Vibrio shilonii) detects primer, detection kit and detection method.
The present invention utilizes loop-mediated isothermal amplification technique, the toxR gene (Genebank accession number is EU727208.1) of executing Roche vibrios of take designs Auele Specific Primer for target gene, carry out loop-mediated isothermal amplification, and optimize reaction conditions, in ring mediated isothermal amplification testing process, add positive control quality control product and negative control quality control product, finally by electrophoretic analysis or SYBR Green I fluorescent dye development process, detect the amplified production of positive control quality control product, the amplified production of the amplified production of negative control quality control product and target sample group, thereby judging whether target sample contains executes Roche vibrios, reach simple, economical, quick and sensitive effect, thereby realized object of the present invention.
The ring mediated isothermal amplification of executing Roche vibrios of the present invention detects primer, it is characterized in that, described detection primer is as follows:
Front outer primer F3:5 ,-CAAACGGTTGGCGGTGTC-3; (as shown in SEQ ID NO.1)
Rear outer primer B3:5 ,-CCGCAAGATGCTGAAGGATC-3; (as shown in SEQ ID NO.2)
Front inner primer FIP:5 ,-CTGCAAGAGCCAAACTCTGCGTCTTTTGGAGCTGGTGGGAT-3; (as shown in SEQ ID NO.3)
Rear inner primer BIP:5 ,-GCTTCTGATGAAGCGACAGGCAGTAAAGACGGTGCCTAAGCG-3; (as shown in SEQ ID NO.4).
The loop-mediated isothermal amplification detection kit of executing Roche vibrios of the present invention, comprises loop-mediated isothermal amplification liquid, BstDNA polysaccharase, positive control quality control product, negative control quality control product and detects primer, and it is characterized in that, described detection primer comprises:
Front outer primer F3:5 ,-CAAACGGTTGGCGGTGTC-3; (as shown in SEQ ID NO.1)
Rear outer primer B3:5 ,-CCGCAAGATGCTGAAGGATC-3; (as shown in SEQ ID NO.2)
Front inner primer FIP:5 ,-CTGCAAGAGCCAAACTCTGCGTCTTTTGGAGCTGGTGGGAT-3; (as shown in SEQ ID NO.3)
Rear inner primer BIP:5 ,-GCTTCTGATGAAGCGACAGGCAGTAAAGACGGTGCCTAAGCG-3; (as shown in SEQ IDNO.4).
Described positive control quality control product is preferably the plasmid DNA that contains the toxR gene (Genebank accession number is EU727208.1) of executing Roche vibrios, can build by artificial method, as the method for PCR obtains toxR gene from executing the genomic dna amplification of Roche vibrios.
Described negative control quality control product is the DNA sequence dna that is different from the toxR gene (Genebank accession number is EU727208.1) of executing Roche vibrios.
Whether effectively positive control quality control product of the present invention and negative control quality control product are to weigh laboratory test results important symbol.In every batch of experiment, must introduce the detection of the positive control quality control product of a Vibrio shilonii, to guarantee that reaction system there will not be false negative reaction; Same, in every batch of experiment, must introduce the detection of a negative control quality control product, to guarantee that reaction system there will not be false positive results.
The loop-mediated isothermal amplification detection method of executing Roche vibrios of non-diagnostic purpose of the present invention, is characterized in that, comprises the following steps:
(1) sample is increased to bacterium and cultivate, extract the genomic dna of thalline in enrichment liquid as template;
(2) use the above-mentioned ring mediated isothermal amplification of executing Roche vibrios to detect primer, be mixed to form amplification reaction system with the genomic dna of loop-mediated isothermal amplification liquid, BstDNA polysaccharase and sample thalline, carry out loop-mediated isothermal amplification, using positive control quality control product and negative control quality control product respectively as positive control and negative control;
(3) after amplified reaction completes, by contrasting with positive control and negative control, in judgement sample amplification reaction system, whether increase and obtain amplified production, come whether to contain and execute Roche vibrios in judgement sample.
Described passing through contrasts with positive control and negative control, in judgement sample amplification reaction system, whether increase and obtain amplified production, come whether to contain in judgement sample and execute Roche vibrios and be specifically preferably the amplified production that simultaneously detects positive control, negative control and sample amplification reaction system by electrophoretic analysis or SYBR Green I fluorescent dye development process, according to the color that whether has scalariform electrophoretic band or reaction system, carry out whether to execute in judgement sample Roche vibrios.
SYBR Green I dyeing: green if reaction mixture turns, illustrate and contain amplified production, in sample, contain and execute Roche vibrios (Vibrio shilonii); Otherwise reaction mixture keeps the orange constant of SYBR Green I, does not contain in sample and executes Roche vibrios (Vibrio shilonii).
Agarose gel electrophoresis detects: if the electrophoretic band of the amplified production of sample amplification reaction system presents specific scalariform band, illustrate in reaction mixture and contain object product, contain and execute Roche vibrios (Vibrio shilonii) in sample; If produce without scalariform band, do not contain in interpret sample and execute Roche vibrios (Vibrio shilonii).
The amplification reaction system of described step (2) is preferably: 8mmol/L MgSO
4, 1.0mmol/L dNTPs, 0.8mol/Lbetaine(trimethyl-glycine), outer primer B3,4 * 10 after outer primer F3,0.2 μ mol/L before inner primer BIP, 0.2 μ mol/L after inner primer FIP, 1.2 μ mol/L before 1.2 μ mol/L
4u/L Bst archaeal dna polymerase, 1 * ThermoPol buffer, appropriate template DNA and water.
Described step (2) carry out loop-mediated isothermal amplification, its reaction parameter is preferably: 65 ℃ of incubations 1 hour, 80 ℃ of deactivation 10min then, last 10 ℃ of preservations.
Described positive control quality control product is preferably the plasmid DNA that contains the toxR gene (Genebank accession number is EU727208.1) of executing Roche vibrios, can build by artificial method, as the method for PCR obtains toxR gene from executing the genomic dna amplification of Roche vibrios.
Described negative control quality control product is the DNA sequence dna that is different from the toxR gene (Genebank accession number is EU727208.1) of executing Roche vibrios.
One aspect of the present invention is the blank of filling up executing aspect Roche vibrios (Vibrio shilonii) ring mediated isothermal amplification (LAMP) detection technique, making to execute Roche vibrios (Vibrio shilonii) detection technique reaches advanced world standards, detectivity significantly improves, and executes the detectability of ring mediated isothermal amplification (LAMP) detection method of Roche vibrios (Vibrio shilonii) higher than 100 ~ 200 times of traditional detection methods, ring mediated isothermal amplification (LAMP) detection method of executing Roche vibrios (Vibrio shilonii) of the present invention has plant and instrument requirement simple (thermostat container that only needs simple structure) on the other hand, detection time short (0.5 ~ 1.0 hour), the features such as specificity high (for 6/4 kind of special primer of 8/6 zone design of target gene), having made up classical polymerase chain reaction (PCR) amplification technique requires high (as pcr amplification instrument to plant and instrument, agargel electrophoresis system etc.), many drawbacks such as technology is comparatively complicated, there is detectivity strong, detection time is short, plant and instrument is required to simple advantage, have broad application prospects.
Accompanying drawing explanation:
Fig. 1 is the electrophorogram of the amplified production after the loop-mediated isothermal amplification of sample, positive control quality control product and negative control quality control product in embodiment 3, wherein M:marker; 1,2 positive contrasts; 3,4 negative contrasts; 5 is sample;
Fig. 2 is amplified production after the loop-mediated isothermal amplification of sample, positive control quality control product and negative control quality control product in the embodiment 3 result figure after fluorescent dye, wherein three of 1,2,3 positive contrasts repetitions, 4, the 5th, two repetitions of sample, three repetitions of 6,7,8 negative contrasts.
Embodiment:
Following examples are to further illustrate of the present invention, rather than limitation of the present invention.
Embodiment 1: the design of primer and synthetic
According to the principle of design of loop-mediated isothermal amplification (LAMP) primer, utilize Genebank database lookup to execute the gene order of applicable ring mediated isothermal amplification in Roche vibrios, selected toxR gene (Genebank accession number is EU727208.1) is target gene, for the toxR gene design loop-mediated isothermal amplification (LAMP) primer of executing Roche vibrios (Vibrio shilonii).According to LAMP design of primers principle, utilize the LAMP gene order of Genebank database lookup Vibrio shilonii, choose its specific sequence, utilize PrimerExplorer IV software to be further analyzed, for the target gene of executing Roche vibrios, design a set of primer.
The ring mediated isothermal amplification detection primer of executing Roche vibrios of design is thus:
Front outer primer F3:5 ,-CAAACGGTTGGCGGTGTC-3;
Rear outer primer B3:5 ,-CCGCAAGATGCTGAAGGATC-3;
Front inner primer FIP:5 ,-CTGCAAGAGCCAAACTCTGCGTCTTTTGGAGCTGGTGGGAT-3;
Rear inner primer BIP:5 ,-GCTTCTGATGAAGCGACAGGCAGTAAAGACGGTGCCTAAGCG-3.
Embodiment 2: nucleic acid extraction
From 37 ℃ of shaking tables, cultivate 10 hours execute and Roche vibrios bacteria suspension, draw bacterium liquid 1mL, the centrifugal 5min of 12000r/min, remove supernatant, in precipitation, add 100 μ L sterilized waters, after mixing, in 100 ℃ of water-bath 10min, ice bath 2min again, the centrifugal 5min of 12000r/min, supernatant liquor is executes Roche vibrios genomic dna, is used for loop-mediated isothermal amplification using it as DNA profiling.
Embodiment 3: loop-mediated isothermal amplification
The loop-mediated isothermal amplification system of sample, reaction system 25 μ L, comprising: the DNA profiling 2 μ L of embodiment 2,1 * ThermoPol damping fluid (reaction system final concentration), 8mmol/L MgSO
4(reaction system final concentration), 1.0mmol/LdNTPs(reaction system final concentration), 0.8mol/L betaine(reaction system final concentration), inner primer FIP(reaction system final concentration before 1.2 μ mol/L), inner primer BIP(reaction system final concentration after 1.2 μ mol/L), outer primer F3(reaction system final concentration before 0.2 μ mol/L), outer primer B3(reaction system final concentration after 0.2 μ mol/L), 1UBstDNA polysaccharase, with sterilizing deionized water polishing to 25 μ L.
The loop-mediated isothermal amplification system of positive control quality control product, reaction system 25 μ L, comprising: the plasmid DNA 2 μ L of Roche vibrios toxR gene, 1 * ThermoPol damping fluid (reaction system final concentration), 8mmol/LMgSO are executed in artificial constructed containing
4(reaction system final concentration), 1.0mmol/L dNTPs(reaction system final concentration), 0.8mol/L betaine(reaction system final concentration), inner primer FIP(reaction system final concentration before 1.2 μ mol/L), inner primer BIP(reaction system final concentration after 1.2 μ mol/L), outer primer F3(reaction system final concentration before 0.2 μ mol/L), outer primer B3(reaction system final concentration after 0.2 μ mol/L), 1U Bst archaeal dna polymerase, with sterilizing deionized water polishing to 25 μ L.
The preparation method that the plasmid DNA of Roche vibrios toxR gene is executed in described artificial constructed containing is: using pUC19 plasmid as carrier, according to the gene order synthetic toxR gene sheet degree in Genebank, as target DNA fragment, using E.coliDH5 ɑ as competent cell.After pUC19 plasmid is cut with Hind III enzyme, be connected under the catalysis of T4 ligase enzyme with target DNA fragment, Transformed E .coli DH5 ɑ competent cell, amoxicillin flat screen is selected positive colony, with alkaline lysis method of extracting recombinant plasmid, through the exactness of restriction enzyme digestion and electrophoresis and PCR electrophoresis detection plasmid construction.
The loop-mediated isothermal amplification system of negative control quality control product, reaction system 25 μ L, comprise: negative control quality control product (usining intestinal bacteria (E.coli) genomic dna as negative control quality control product) 2 μ L, 1 * ThermoPol damping fluid (reaction system final concentration), 8mmol/L MgSO
4(reaction system final concentration), 1.0mmol/L dNTPs(reaction system final concentration), 0.8mol/Lbetaine(reaction system final concentration), inner primer FIP(reaction system final concentration before 1.2 μ mol/L), inner primer BIP(reaction system final concentration after 1.2 μ mol/L), outer primer F3(reaction system final concentration before 0.2 μ mol/L), outer primer B3(reaction system final concentration after 0.2 μ mol/L), 1UBstDNA polysaccharase, with sterilizing deionized water polishing to 25 μ L.
After respectively above-mentioned three kinds of loop-mediated isothermal amplification systems being mixed, put into constant water bath box, set reaction conditions: 65 ℃, 60min; 80 ℃, 10min, termination reaction; 10 ℃ of preservations.
After completing, gets loop-mediated isothermal amplification the amplified production of the above-mentioned three kinds of amplification reaction systems of 5 μ L, on 2% sepharose, carry out electrophoresis detection, as shown in Figure 1, electrophoretic analysis detected result shows: the electrophoretic band of the loop-mediated isothermal amplification system of sample and the loop-mediated isothermal amplification system of positive control quality control product presents specific scalariform band, and the loop-mediated isothermal amplification system of negative control quality control product is without specific band, the DNA profiling of interpret sample contains the specificity toxR gene of executing Roche vibrios, in interpret sample, contain and execute Roche vibrios (Vibrio shilonii).
Get respectively 1 μ L SYBR Green I fluorescence dye, mix with the above-mentioned three kinds of amplification systems after having reacted, whether color changes observing response system.As shown in Figure 2, SYBR Green I fluorescent dye development process detected result shows: the loop-mediated isothermal amplification system of sample and the loop-mediated isothermal amplification system of positive control quality control product become green, and the loop-mediated isothermal amplification system of negative control quality control product is still orange, the DNA profiling of interpret sample contains the specificity toxR gene of executing Roche vibrios, in interpret sample, contains and executes Roche vibrios (Vibrio shilonii).
In sum, utilize detection primer of the present invention and detection method, can be simply, economical, detect and execute Roche vibrios fast, delicately, and detected result is accurately and reliably.
Claims (10)
1. the ring mediated isothermal amplification of executing Roche vibrios detects a primer, it is characterized in that, described detection primer is as follows:
Front outer primer F3:5 ,-CAAACGGTTGGCGGTGTC-3;
Rear outer primer B3:5 ,-CCGCAAGATGCTGAAGGATC-3;
Front inner primer FIP:5 ,-CTGCAAGAGCCAAACTCTGCGTCTTTTGGAGCTGGTGGGAT-3;
Rear inner primer BIP:5 ,-GCTTCTGATGAAGCGACAGGCAGTAAAGACGGTGCCTAAGCG-3.
2. execute a loop-mediated isothermal amplification detection kit for Roche vibrios, comprise loop-mediated isothermal amplification liquid, Bst archaeal dna polymerase, positive control quality control product, negative control quality control product and detect primer, it is characterized in that, described detection primer comprises:
Front outer primer F3:5 ,-CAAACGGTTGGCGGTGTC-3;
Rear outer primer B3:5 ,-CCGCAAGATGCTGAAGGATC-3;
Front inner primer FIP:5 ,-CTGCAAGAGCCAAACTCTGCGTCTTTTGGAGCTGGTGGGAT-3;
Rear inner primer BIP:5 ,-GCTTCTGATGAAGCGACAGGCAGTAAAGACGGTGCCTAAGCG-3.
3. the loop-mediated isothermal amplification detection kit of executing Roche vibrios according to claim 2, is characterized in that, described positive control quality control product is the plasmid DNA that contains the toxR gene of executing Roche vibrios.
4. the loop-mediated isothermal amplification detection kit of executing Roche vibrios according to claim 2, is characterized in that, described negative control quality control product is the DNA sequence dna that is different from the toxR gene of executing Roche vibrios.
5. a loop-mediated isothermal amplification detection method of executing Roche vibrios for non-diagnostic purpose, is characterized in that, comprises the following steps:
(1) sample is increased to bacterium and cultivate, extract the genomic dna of thalline in enrichment liquid as template;
(2) right to use requires the ring mediated isothermal amplification of executing Roche vibrios described in 1 to detect primer, be mixed to form amplification reaction system with the genomic dna of loop-mediated isothermal amplification liquid, Bst archaeal dna polymerase and sample thalline, carry out loop-mediated isothermal amplification, using positive control quality control product and negative control quality control product respectively as positive control and negative control;
(3) after amplified reaction completes, by contrasting with positive control and negative control, in judgement sample amplification reaction system, whether increase and obtain amplified production, come whether to contain and execute Roche vibrios in judgement sample.
6. the loop-mediated isothermal amplification detection method of executing Roche vibrios of non-diagnostic purpose according to claim 5, it is characterized in that, by contrasting with positive control and negative control, in judgement sample amplification reaction system, whether increase and obtain amplified production, carrying out whether to contain in judgement sample to execute Roche vibrios is specially by electrophoretic analysis or SYBR Green I fluorescent dye development process and detects positive control simultaneously, the amplified production of negative control and sample amplification reaction system, according to the color that whether has scalariform electrophoretic band or reaction system, carry out whether to execute in judgement sample Roche vibrios.
7. the loop-mediated isothermal amplification detection method of executing Roche vibrios of non-diagnostic purpose according to claim 5, is characterized in that, the amplification reaction system of described step (2) is: 8mmol/L MgSO
4, outer primer B3,4 * 10 after outer primer F3,0.2 μ mol/L before inner primer BIP, 0.2 μ mol/L after inner primer FIP, 1.2 μ mol/L before 1.0mmol/L dNTPs, 0.8mol/L betaine, 1.2 μ mol/L
4u/LBst archaeal dna polymerase, 1 * ThermoPol buffer, appropriate template DNA and water.
8. the loop-mediated isothermal amplification detection method of executing Roche vibrios of non-diagnostic purpose according to claim 5, it is characterized in that, described step (2) carry out loop-mediated isothermal amplification, its reaction parameter is: 65 ℃ of incubations 1 hour, then 80 ℃ of deactivation 10min, last 10 ℃ of preservations.
9. the loop-mediated isothermal amplification detection method of executing Roche vibrios of non-diagnostic purpose according to claim 5, is characterized in that, described positive control quality control product is the plasmid DNA that contains the toxR gene of executing Roche vibrios.
10. the loop-mediated isothermal amplification detection method of executing Roche vibrios of non-diagnostic purpose according to claim 5, is characterized in that, described negative control quality control product is the DNA sequence dna that is different from the toxR gene of executing Roche vibrios.
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