CN104988232A - Primers and probe sequence for edwardsiella tarda LAMP-LFD detection and application of primers and probe - Google Patents

Primers and probe sequence for edwardsiella tarda LAMP-LFD detection and application of primers and probe Download PDF

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CN104988232A
CN104988232A CN201510409824.9A CN201510409824A CN104988232A CN 104988232 A CN104988232 A CN 104988232A CN 201510409824 A CN201510409824 A CN 201510409824A CN 104988232 A CN104988232 A CN 104988232A
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etaompa
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陈炯
周前进
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Ningbo University
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Abstract

The invention discloses primers and a probe for edwardsiella tarda LAMP-LFD (Loop-Mediated Isothermal Amplification-Lateral Flow Dipstick) detection and application of the primers and the probe. The primers and the probe are characterized by comprising three pairs of primers, namely EtaompA-F3, EtaompA-B3; EtaompA-FIP, EtaompA-BIP; EtaompA-LF and EtaompA-LB, of LAMP, and a probe EtaompA-HP, wherein the nucleotide sequence is shown as SEQ NO1-NO7; the visible detection of edwardsiella tarda is realized by utilizing the primers and the probe, and an amplification step of an LAMP reaction system, a hybridization step of the probe and an LAMP reaction product, and an LFD detecting step; the primers and the probe have the advantages that the rapidness, specificity and sensitivity are higher, the instrument requirement is simple, the primers and the probe are favourable for the early diagnosis and detection of the edwardsiella tarda, and the requirements on the detection of a primary detecting mechanism and an on-site epidemic focus can be met.

Description

The primer detected for Edwardsiella tarda LAMP-LFD and the application of probe sequence and primer and probe
Technical field
The present invention relates to a kind of primer for detecting Edwardsiella tarda and probe sequence, especially relating to the application of a kind of primer of detecting for Edwardsiella tarda LAMP-LFD and probe sequence and primer and probe.
Background technology
Edwardsiella tarda ( edwardsiella tarda) be a kind of Gram-negative tyrothricin, belong to enterobacteriaceae, Edwardsiella, host range is also very extensive, can infect fish, batrachians, reptiles, and comprise the Mammals etc. of the mankind.This bacterium is prevalent in fresh water and briny environment, and water-borne transmission is an important route of transmission of Edwardsiella tarda, and the deterioration of water body environment easily causes the outburst of this disease.Fish are the modal hosts of Edwardsiella tarda, more than the 20 kind of seawater and freshwater fish that comprise lefteye flounder, turbot, common eel, carp, tilapia etc. can be infected, cause occurring the symptoms such as the dense ulcer of body surface, skin ulceration, abdominal cavity flatulence, ascites, liver tumour, huge to the harm of culturing economic fish.At present, for Edwardsiella tarda identify still based on the Pathogen Biology means of routine in conjunction with physiological and biochemical analysis etc., these class methods are consuming time, effort, usually incur loss through delay the best prevention and control time of epidemic disease, far can not meet the needs of actual production.Therefore, set up the novel method of quick, accurate, sensitive detection Edwardsiella tarda, for the early diagnosis of disease and early warning, Real-Time Monitoring, treatment in time has great importance.
The detection method of Edwardsiella tarda has the qualification of conventional separation and Culture, Physiology and biochemistry, and polymerase chain reaction and 16S rDNA check order.Ordinary method has shortcomings such as growing detection time, take time and effort; PCR, polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) technology, although the highly sensitive instrument cost of the correlation techniques such as quantitative fluorescent PCR is expensive, is not suitable for basic unit; Though Enzyme-linked Immunosorbent Assay technology has the advantages such as simple and quick, its sensitivity is not high, and operation is comparatively complicated, easily causes higher false positive; The operation of polygene sequential analysis detection technique is equally comparatively complicated, and sense cycle is longer, is not suitable for basic unit and detects.
LAMP is as the emerging nucleic acid amplification technologies of one, because its reaction can complete under the constant temperature of 61-65 DEG C, avoid many temperature changing processes such as the sex change of similar pcr amplification, annealing, extension, significantly reduce the production cost of conversion unit, simultaneously it still can retain the advantages such as the highly sensitive that is equal to round pcr and specificity, illustrates huge vigor in field of fast detection.By transverse flow Lateral Flow Strip (Lateral flow dipstick, LFD) detection (i.e. LAMP-LFD technology) of LAMP product is applied to, nucleic acid amplification product directly can pass through naked eyes interpretation experimental result by LFD, detection departs from the dependence for specific equipment completely, thus reduce the demand of whole testing process for equipment to the full extent, be particularly useful for the spot inspection of the clinical sample of basic unit.At present, this technology Successful utilization in Taura syndrome ( taura syndrome virus, TSV), infectivity muscle necrosis virus ( infectious myonecrosis virus, IMNV), infectious spleen and kidney necrosis virus ( infectious spleen and kidney necrosis virus, ISKNV), Vibrio vulnificus ( vibrio vulnificus), and Streptococcus iniae ( strepstococcus iniae) etc. the detection of aquatic products pathogenic micro-organism, have not been reported the diagnosis and detection this technology being applied to Edwardsiella tarda both at home and abroad.
Summary of the invention
Technical problem to be solved by this invention is to provide that a kind of detection speed is fast, and testing cost is low, the application of the primer that detection sensitivity and the high LAMP-LFD for Edwardsiella tarda of accuracy detect and probe sequence and primer and probe.
The present invention solves the problems of the technologies described above adopted technical scheme:
1, the primer that detects of a kind of LAMP-LFD for Edwardsiella tarda and probe sequence, according to Edwardsiella tarda outer membrane protein A(GenBank accession number: EF528483) encoding sequence design three pairs of primer sequences of LAMP and a probe sequence, primer sequence is specific as follows:
EtaompA-F3：5’-CATTAGCAGTGGCACTGG-3’
EtaompA-B3：5’-TGCCTTGAACTTACCGTTC-3’，
EtaompA-FIP：5’-TCGGATGAGACTTCGTGGAGTttttGTAGGTGGTAAACTGGGTTG-3’，
EtaompA-BIP：5’-CGGTGCTTTCTTCGGTTACCAttttCCAGTCGTAGCCCATTTC-3’，
EtaompA-LF：5’-AAGCTGTTACCGATGTAGTGG-3’，
EtaompA-LB：5’-CTAATCCGTACCTGGGCTTC-3’，
Probe EtaompA-HP:5 '-ATACGAATCAGCTGGGCGC-3 ',
Wherein, the 5 ' end of EtaompA-FIP is biotin labeling; 5 ' the end of probe EtaompA-HP is marked by fluorescein isothiocyanate.
2, the primer detected for the LAMP-LFD of Edwardsiella tarda and the application of probe sequence in Edwardsiella tarda LAMP-LFD visible detection method, concrete detection method step is as follows:
1) LAMP reaction system is configured: the final concentration of each composition of reaction system is respectively: each 0.2 μm of ol/L of outer primer EtaompA-F3 and EtaompA-B3, the each 1.6 μm of ol/L of inner primer EtaompA-FIP and EtaompA-BIP, the each 0.4 μm of ol/L of ring primer EtaompA-LF and EtaompA-LB, dNTPs 1.4mmol/L, Tris-HCl(pH 8.8) 20mmol/L, KCl 10mmol/L, MgSO 46.5mmol/L, (NH4) 2sO 410mmol/L, Triton X-100 0.1%, 8U Bst archaeal dna polymerase large fragment (New England Biolabs) and 2 μ L sample masterplates, add distilled water and make reaction system cumulative volume be 25 μ l;
2) LAMP reaction system amplification: above-mentioned reaction system is carried out amplified reaction, and amplified reaction temperature is 63 DEG C, and the amplified reaction time is 25 min;
3) probe hybridization and LFD detect: add in reaction system by the probe EtaompA-HP of 20 pmol after amplification, 63 DEG C of incubation 5 min, hybridize, get 5 μ L hybridization solutions to add in 100 μ L damping fluids and mix, then LFD test strip is immersed to add in the damping fluid of hybridization solution and develop the color, judge the result that transverse flow ELISA test strip LAMP increases.
3, the application in Edwardsiella tarda LAMP-LFD Visual retrieval test kit prepared by the primer detected for the LAMP-LFD of Edwardsiella tarda and probe, this test kit comprises LAMP reaction system: the final concentration of each composition of reaction system is respectively each 0.2 μm of ol/L of outer primer EtaompA-F3 and EtaompA-B3, the each 1.6 μm of ol/L of inner primer EtaompA-FIP and EtaompA-BIP, the each 0.4 μm of ol/L of ring primer EtaompA-LF and EtaompA-LB, dNTPs 1.4mmol/L, Tris-HCl(pH 8.8) 20mmol/L, KCl 10mmol/L, MgSO 46.5mmol/L, (NH4) 2sO 410mmol/L, Triton X-100 0.1%, 8U Bst archaeal dna polymerase large fragment (New England Biolabs) and 2 μ L sample masterplates, add distilled water and make reaction system cumulative volume be 25 μ l.
Compared with prior art, the invention has the advantages that:
1, detection sensitivity is high, and the detection sensitivity of present method is 3.50 × 10 2cfu/mL is 100 times that Standard PCR detects;
2, detection time is short, and amplified reaction only needs 25 min, and judge from the result of having extracted of sample gene group DNA, whole testing process only needs 70 min, shortens more than 2h, increase substantially detection speed than Standard PCR detection technique;
3, high specificity, Auele Specific Primer used is according to the different zones design of eight in the outer membrane protein gene of Edwardsiella tarda, and also have the specific probe of DNA, effectively can avoid the false positive issue utilizing the method such as agarose gel electrophoresis, fluorescence dye to cause;
4, plant and instrument requires low, without the need to Standard PCR PCR instrument used, gel electrophoresis and imaging system etc., only needs a water-bath can complete detection;
5, simple to operate, result is obvious, and whole testing process does not relate to complex instrument and equipment, and the personnel of molecular biology mechanism of slightly having get final product complete operation; Detected result is obviously clear, and visual inspection can judge;
6, to the person and environment safer, do not relate to the toxic reagents such as EB in testing process.
In sum, primer of the present invention and probe is used to adopt LAMP-LFD method to detect Edwardsiella tarda, there is higher agility, specificity and sensitivity, instrument demand is simple, be conducive to early diagnosis and the detection of Edwardsiella tarda, the needs of feeler mechanism of basic unit and the detection of on-the-spot plague area can be met.
Accompanying drawing explanation
Fig. 1 is LAMP specificity experiments result; M:100 bp Plus DNA ladder (Fermentas, the U.S.); NC: using aseptic deionized water as template; Eta: with the genomic dna of Edwardsiella tarda for template; Asa, Ahy, Ppu, Pae, Sin, Val, Vfl, Vha, Van, Vro, Vvu, Lmo: respectively with the genomic dna of aeromonas salmonicida, Aeromonas hydrophila, pseudomonas putida, Pseudomonas aeruginosa, Streptococcus iniae, vibrio alginolyticus, vibrio fluvialis, Vibrio harveyi, Vibrio anguillarum, wheel animalcule vibrios, Vibrio vulnificus, Listeria monocytogenes for template;
Fig. 2 is LAMP-LFD specificity experiments result; NC: using aseptic deionized water as template; Eta: with the genomic dna of Edwardsiella tarda for template; Asa, Ahy, Ppu, Pae, Sin, Val, Vfl, Vha, Van, Vro, Vvu, Lmo: respectively with the genomic dna of aeromonas salmonicida, Aeromonas hydrophila, pseudomonas putida, Pseudomonas aeruginosa, Streptococcus iniae, vibrio alginolyticus, vibrio fluvialis, Vibrio harveyi, Vibrio anguillarum, wheel animalcule vibrios, Vibrio vulnificus, Listeria monocytogenes for template;
Fig. 3 is the sensitivity results that LAMP detects; M:100 bp Plus DNA ladder (Fermentas, the U.S.); NC: using aseptic deionized water as template; 10 7, 10 6, 10 5, 10 4, 10 3, 10 2, 10 1: the bacterial concentration (a=3.50, unit: cfu/mL) of Edwardsiella tarda, the genomic dna utilizing it to extract is as template;
Fig. 4 is the sensitivity results that LAMP-LFD detects; NC: using aseptic deionized water as template; 10 7, 10 6, 10 5, 10 4, 10 3, 10 2, 10 1: the bacterial concentration (a=3.50, unit: cfu/mL) of Edwardsiella tarda, the genomic dna utilizing it to extract is as template;
Fig. 5 is the sensitivity results that PCR detects; M:100 bp Plus DNA ladder (Fermentas, the U.S.); NC: using aseptic deionized water as template; 10 7, 10 6, 10 5, 10 4, 10 3, 10 2, 10 1: the bacterial concentration (a=3.50, unit: cfu/mL) of Edwardsiella tarda, the genomic dna utilizing it to extract is as template.
Embodiment
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Embodiment 1
The foundation of the method for LAMP-LFD technology for detection Edwardsiella tarda
1. design of primers: the Edwardsiella tarda outer membrane protein A(GenBank accession number according to having delivered in NCBI: EF528483) encoding sequence design, wherein, primer sequence is specific as follows:
EtaompA-F3：5’-CATTAGCAGTGGCACTGG-3’
EtaompA-B3：5’-TGCCTTGAACTTACCGTTC-3’，
EtaompA-FIP：5’-TCGGATGAGACTTCGTGGAGTttttGTAGGTGGTAAACTGGGTTG-3’，
EtaompA-BIP：5’-CGGTGCTTTCTTCGGTTACCAttttCCAGTCGTAGCCCATTTC-3’，
EtaompA-LF：5’-AAGCTGTTACCGATGTAGTGG-3’，
EtaompA-LB：5’-CTAATCCGTACCTGGGCTTC-3’，
Probe EtaompA-HP:5 '-ATACGAATCAGCTGGGCGC-3 ',
Wherein, the 5 ' end of EtaompA-FIP is biotin labeling; 5 ' the end of probe EtaompA-HP is marked by fluorescein isothiocyanate.
2. sample DNA extracts: the Edwardsiella tarda bacterial classification (E. tarda MCCC235) that-70 DEG C are preserved for a long time is lined LB solid medium, agar powder 1.5 %, 30 DEG C of overnight incubation, picking mono-clonal is in 5 mL LB liquid nutrient mediums, 30 DEG C, 10 hours cultivated by 165 r/min shaking tables, and it is 3.50 × 10 that counting obtains bacterium liquid initial concentration 9cfu/mL.After dilution, choose 3.50 × 10 7, 3.50 × 10 6, 3.50 × 10 5, 3.50 × 10 4, 3.50 × 10 3, 3.50 × 10 2, 3.50 × 10 1each 1 mL of bacterium liquid of 7 concentration gradients such as cfu/mL, adopts commercial test kit to extract genomic dna, is dissolved in the aseptic ddH of 50 μ L 2o is used for LAMP and PCR checking.
3. Edwardsiella tarda LAMP reacts
Utilize the Auele Specific Primer that step 1 designs, with Edwardsiella tarda genomic dna for masterplate carries out LAMP amplification.
3.1 LAMP reaction systems, the final concentration of each composition is respectively: each 0.2 μm of ol/L of outer primer EtaompA-F3 and EtaompA-B3, the each 1.6 μm of ol/L of inner primer EtaompA-FIP and EtaompA-BIP, the each 0.4 μm of ol/L of ring primer EtaompA-LF and EtaompA-LB, dNTPs 1.4mmol/L, Tris-HCl(pH 8.8) 20mmol/L, KCl 10mmol/L, MgSO 46.5mmol/L, (NH4) 2sO 410mmol/L, Triton X-100 0.1%, 8U Bst archaeal dna polymerase large fragment (New England Biolabs) and 2 μ L sample masterplates, add distilled water and make reaction system cumulative volume be 25 μ L;
3.2 LAMP reaction conditionss: above-mentioned reaction system is carried out amplified reaction, amplified reaction temperature is 63 DEG C, and the amplified reaction time is 25 min.
4. probe hybridization and LFD detect: add in reaction system by the EtaompA-HP probe of 20 pmol after amplification, 63 DEG C of incubation 5 min, hybridize, get 5 μ L hybridization solutions to add in 100 μ L buffer and mix, then LFD test strip is immersed to add in the buffer of hybridization solution and develop the color, judge the amplification situation of LAMP.
Embodiment 2
Primer of the present invention and probe is used to carry out the specific assay of Edwardsiella tarda LAMP-LFD detection
Auele Specific Primer designed by utilization and probe, respectively with Edwardsiella tarda MCCC235, aeromonas salmonicida ATCC 33658, Aeromonas hydrophila ATCC 7966, Vibrio vulnificus ATCC 27562, Vibrio harveyi ATCC 33866, vibrio fluvialis ATCC 33809, pseudomonas putida MCCC 1A01082, Streptococcus iniae ATCC 29178, vibrio alginolyticus ATCC 33787, Vibrio anguillarum ayu-H080701, wheel animalcule vibrios DSM 17186T, Listeria monocytogenes ATCC 19115, the genomic dna of Pseudomonas aeruginosa ATCC 9027 grade is masterplate, LAMP-LFD reaction is carried out by the step 3 of above-described embodiment 1 and step 4, the specificity of checking primer and probe, distilled water is as negative control.Result as depicted in figs. 1 and 2, utilize electrophoretic method (Fig. 1) and LFD(Fig. 2) all can only increase from the genome DNA sample of Edwardsiella tarda obtains object band, other sample is without amplified band, illustrate and use primer provided by the invention and probe to carry out LAMP-LFD detection, there is good specificity.
Embodiment 3
Primer of the present invention and probe is used to carry out the sensitivity determination of Edwardsiella tarda LAMP-LFD detection
Adopt the method for the step 2 of above-described embodiment 1 to extract the genomic dna of Edwardsiella tarda, carry out 10 times of gradient dilutions, select 3.50 × 10 7, 3.50 × 10 6, 3.50 × 10 5, 3.50 × 10 4, 3.50 × 10 3, 3.50 × 10 2, 3.50 × 10 1cfu/mL, as template, carries out LAMP-LFD reaction by the step 3 of above-described embodiment 1 and step 4, and the sensitivity of checking primer and probe, distilled water is as negative control.Result is as shown in Fig. 3, Fig. 4 and Fig. 5, and the sensitivity that the LAMP-LFD using primer provided by the invention and probe to carry out detects is 3.50 × 10 2cfu/mL(Fig. 4), utilizing agarose gel electrophoresis to detect the sensitivity obtained consistent (Fig. 3) with the amplified production of LAMP, is the Standard PCR detection method that EtaompA-F3 and EtaompA-B3 sets up as primer 100 times (Fig. 5).
Embodiment 4
The Edwardsiella tarda in the crucian tissue of artificial contamination is specifically detected by LAMP-LFD technology of the present invention.
1. Edwardsiella tarda artificial contamination and detected sample extracting genome DNA
Liver organization 100 mg getting some parts of healthy crucians adds a small amount of sterilized water, after abundant homogenate, is settled to 1 mL.Get the Edwardsiella tarda bacterium liquid (about 3.50 × 10 of 1 mL fresh culture 8cfu/mL) carry out 10 times of concentration gradient dilutions, getting concentration is respectively 3.50 × 10 5, 3.50 × 10 4, 3.50 × 10 3, 3.50 × 10 2, 3.50 × 10 1each 1 mL of bacterium liquid of cfu/mL and crucian liver tissue homogenate liquid equal-volume mix.The parallel preparation of each concentration three samples.The tissue sample getting 1 mL bacterial contamination extracts genomic dna by method described in embodiment 1 step 2.Respectively get 2 μ L for LAMP-LFD and pcr amplification.The hepatic homogenate liquid of healthy crucian makes negative control.
2. the preparation of LAMP reaction system and reaction conditions, carry out according to embodiment 1 step 3.
3. LFD colour developing, detected result judges, carries out according to embodiment 1 step 4.
Result is presented at after crucian organizes medium body contamination to contaminate the Edwardsiella tarda of different concns, and LAMP-LFD can from 3.50 × 10 3in the liver organization that cfu/mL pollutes, stable detection is to cause of disease, and detection sensitivity is 70 CFU/ reactions.PCR can only from 3.50 × 10 4in the liver organization that cfu/mL pollutes, stable detection is to cause of disease; The detected result of LAMP-LFD to unpolluted healthy Carassius auratus liver tissue is negative (table 1), and specificity is good.
Table 1 is LAMP-LFD detection of the present invention and PCR detected result after utilizing Edwardsiella tarda artificial contamination crucian tissue sample
Note: "+" represents that detected result is positive; "-" represents that detected result is negative.
Embodiment 5
With the Edwardsiella tarda in LAMP-LFD Visual retrieval morbidity crucian sample of the present invention
1. crucian actual sample extracting genome DNA
Collect the doubtful crucian 20 suffering from Edwardsiella tarda disease, the method first utilizing traditional bacteria distribution to cultivate is separated cause of disease, identifies.Get appropriate liver organization simultaneously, extract DNA through kit method.
2. the preparation of LAMP reaction system and reaction conditions, carry out according to embodiment 1 step 3.
3. LFD colour developing, detected result judges, carries out according to embodiment 1 step 4.
Result shows, traditional bacteria distribution cultural method detects in the sick fish sample of discovery 20 has 3 Edwardsiella tardas to detect the positive.Utilize LAMP-LFD and PCR all from the sick fish hepatic tissue of this infection Edwardsiella tarda, all can obtain positive amplification, be all feminine gender to the detected result of other 17 sick fish hepatic tissues, detected result is consistent with traditional bacteria distribution cultural method result.
Above-mentioned explanation is not limitation of the present invention, and the present invention is also not limited to above-mentioned citing.Those skilled in the art are in essential scope of the present invention, and the change made, remodeling, interpolation or replacement, also should belong to protection scope of the present invention.

Claims (5)

1. the primer detected for the LAMP-LFD of Edwardsiella tarda and a probe sequence, it is characterized in that three pairs of primer sequences according to the encoding sequence design LAMP of Edwardsiella tarda outer membrane protein A and a probe sequence, primer sequence is specific as follows:
EtaompA-F3：5’-CATTAGCAGTGGCACTGG-3’
EtaompA-B3：5’-TGCCTTGAACTTACCGTTC-3’，
EtaompA-FIP：5’-TCGGATGAGACTTCGTGGAGTttttGTAGGTGGTAAACTGGGTTG-3’，
EtaompA-BIP：5’-CGGTGCTTTCTTCGGTTACCAttttCCAGTCGTAGCCCATTTC-3’，
EtaompA-LF：5’-AAGCTGTTACCGATGTAGTGG-3’，
EtaompA-LB：5’-CTAATCCGTACCTGGGCTTC-3’，
Probe EtaompA-HP:5 '-ATACGAATCAGCTGGGCGC-3 ',
Wherein, the 5 ' end of EtaompA-FIP is biotin labeling; 5 ' the end of probe EtaompA-HP is marked by fluorescein isothiocyanate.
2. the primer that detects of the LAMP-LFD for Edwardsiella tarda according to claim 1 and the application of probe sequence in Edwardsiella tarda LAMP-LFD visible detection method.
3. the primer that detects of the LAMP-LFD for Edwardsiella tarda according to claim 2 and the application of probe sequence in Edwardsiella tarda LAMP-LFD visible detection method, is characterized in that concrete detection method step is as follows:
1) LAMP reaction system is configured: the final concentration of each composition of reaction system is respectively: each 0.2 μm of ol/L of outer primer EtaompA-F3 and EtaompA-B3, the each 1.6 μm of ol/L of inner primer EtaompA-FIP and EtaompA-BIP, the each 0.4 μm of ol/L of ring primer EtaompA-LF and EtaompA-LB, dNTPs 1.4mmol/L, Tris-HCl(pH 8.8) 20mmol/L, KCl 10mmol/L, MgSO 46.5mmol/L, (NH4) 2sO 410mmol/L, Triton X-100 0.1%, 8U Bst archaeal dna polymerase large fragment (New England Biolabs) and 2 μ L sample masterplates, add distilled water and make reaction system cumulative volume be 25 μ l;
2) LAMP reaction system amplification: above-mentioned reaction system is carried out amplified reaction, and amplified reaction temperature is 63 DEG C, and the amplified reaction time is 25 min;
3) probe hybridization and LFD detect: add in reaction system by the probe EtaompA-HP of 20 pmol after amplification, 63 DEG C of incubation 5 min, hybridize, get 5 μ L hybridization solutions to add in 100 μ L damping fluids and mix, then LFD test strip is immersed to add in the damping fluid of hybridization solution and develop the color, judge the result that transverse flow ELISA test strip LAMP increases.
4. the application in Edwardsiella tarda LAMP-LFD Visual retrieval test kit prepared by the primer that detects of the LAMP-LFD for Edwardsiella tarda according to claim 1 and probe.
5. Edwardsiella tarda LAMP-LFD Visual retrieval test kit according to claim 4, it is characterized in that this test kit comprises LAMP reaction system: the final concentration of each composition of reaction system is respectively each 0.2 μm of ol/L of outer primer EtaompA-F3 and EtaompA-B3, the each 1.6 μm of ol/L of inner primer EtaompA-FIP and EtaompA-BIP, the each 0.4 μm of ol/L of ring primer EtaompA-LF and EtaompA-LB, dNTPs 1.4mmol/L, pH 8.8 Tris-HCl 20mmol/L, KCl 10mmol/L, MgSO 46.5mmol/L, (NH4) 2sO 410mmol/L, Triton X-100 0.1%, 8U Bst archaeal dna polymerase large fragment and 2 μ L sample masterplates, add distilled water and make reaction system cumulative volume be 25 μ l.
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CN107245521A (en) * 2017-07-05 2017-10-13 宁波大学 Primer and probe sequence for anisakis simplex/Pai Shi Anisakid nematode LAMP LFD detections
CN112481221A (en) * 2019-09-10 2021-03-12 宁波大学 Edwardsiella tarda efficient lytic phage vB _ EtaM-IME523 and application thereof
CN114854887A (en) * 2022-06-30 2022-08-05 南开大学 Loop-mediated isothermal amplification detection method for Edwardsiella tarda and application thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105925683A (en) * 2016-05-13 2016-09-07 中国科学院水生生物研究所 Detection kit of edwardsiella tarda and application of detection kit
CN105925683B (en) * 2016-05-13 2019-08-06 中国科学院水生生物研究所 The detection kit and its application of Edwardsiella tarda
CN107245521A (en) * 2017-07-05 2017-10-13 宁波大学 Primer and probe sequence for anisakis simplex/Pai Shi Anisakid nematode LAMP LFD detections
CN107245521B (en) * 2017-07-05 2020-11-03 宁波大学 Primer and probe sequence for LAMP-LFD detection of heterodera simplicissima/heterodera pekinensis
CN112481221A (en) * 2019-09-10 2021-03-12 宁波大学 Edwardsiella tarda efficient lytic phage vB _ EtaM-IME523 and application thereof
CN114854887A (en) * 2022-06-30 2022-08-05 南开大学 Loop-mediated isothermal amplification detection method for Edwardsiella tarda and application thereof

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