CN104988232B - Application for the primer and probe sequence and primer and probe of Edwardsiella tarda LAMP LFD detections - Google Patents

Application for the primer and probe sequence and primer and probe of Edwardsiella tarda LAMP LFD detections Download PDF

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CN104988232B
CN104988232B CN201510409824.9A CN201510409824A CN104988232B CN 104988232 B CN104988232 B CN 104988232B CN 201510409824 A CN201510409824 A CN 201510409824A CN 104988232 B CN104988232 B CN 104988232B
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陈炯
周前进
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Ningbo University
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Abstract

The invention discloses the application of the primer and probe and primer and probe detected for Edwardsiella tarda LAMP LFD, feature is to include three couples of LAMP primer EtaompA F3, EtaompA B3；EtaompA‑FIP、EtaompA‑BIP；EtaompA‑LF、EtaompA‑LB；With a probe EtaompA HP, nucleotide sequence is as shown in SEQ NO1 NO7, utilize above-mentioned primer and probe, by LAMP reaction system amplification steps, probe hybridizes LAMP reaction product steps, and the Visual retrieval of Edwardsiella tarda is realized using LFD detecting steps, advantage is that have higher agility, specificity and sensitivity, instrument demand is simple, is advantageous to the early diagnosis and detection of Edwardsiella tarda, can meet the needs of testing agency of basic unit and the detection of live plague area.

Description

Primer and probe sequence and primer for Edwardsiella tarda LAMP-LFD detections With the application of probe
Technical field
The present invention relates to a kind of primer and probe sequence for being used to detect Edwardsiella tarda, used more particularly, to one kind In the application of the primer and probe sequence and primer and probe of Edwardsiella tarda LAMP-LFD detections.
Background technology
Edwardsiella tarda (Edwardsiella tarda) it is a kind of Gram-negative brevibacterium, belong to enterobacteria Section, Edwardsiella, host range is also quite varied, can infect fish, amphibian animal, reptiles, and including humans Mammal etc..The bacterium is prevalent in fresh water and briny environment, and water-borne transmission is a weight of Edwardsiella tarda Route of transmission is wanted, the deterioration of water body environment easily causes the sick outburst.Fish are the most common hosts of Edwardsiella tarda, More than 20 kinds of seawater and the freshwater fish including lefteye flounder, turbot, common eel, carp, Tilapia mossambica etc. can be infected, causes body occur The symptoms such as the dense ulcer of table, exanthemv, abdominal cavity flatulence, ascites, liver tumour, culturing economic fish are endangered huge.At present, for The identification of Edwardsiella tarda is still based on Pathogen Biology means combination physiological and biochemical analysis of routine etc., such method consumption When, it is laborious, be usually delayed the optimal prevention and control time of epidemic disease, far from the needs for meeting actual production.Therefore, establish quick, accurate Really, the new method of Edwardsiella tarda is sensitively detected, early diagnosis and early warning, monitoring in real time for disease, is controlled in time Treatment has great importance.
The detection method of Edwardsiella tarda have it is conventional be separately cultured, Physiology and biochemistry identification, PCR And 16S rDNA sequencings etc..Conventional method has the shortcomings that detection time is long, taken time and effort；PCR, PCR limit Property fragment length polymorphism (PCR-RFLP) technology processed, although the correlation technique high sensitivity instrument such as quantitative fluorescent PCR into This costliness, basic unit is not suitable for；Though Enzyme-linked Immunosorbent Assay technology has the advantages that simple and quick, its sensitivity is not high, behaviour Make complex, easily cause higher false positive；The operation of polygenes sequence analysis detection technique is same complex, detection week Phase is longer, is not suitable for basic unit's detection.
LAMP, because its reaction can be completed under 61-65 DEG C of constant temperature, is kept away as a kind of emerging nucleic acid amplification technologies Exempt from the multi-temperature change procedures such as the denaturation, annealing, extension of similar PCR amplifications, significantly reduce being produced into for consersion unit This, at the same its remain to retain be equal to round pcr high sensitivity and it is specific the advantages that, illustrated in field of fast detection Huge vigor.By lateral flow Lateral Flow Strip（Lateral flow dipstick, LFD）Inspection applied to LAMP products Survey（That is LAMP-LFD technologies）, nucleic acid amplification product can be complete directly by naked eyes interpretation experimental result, detection by LFD Depart from the dependence for special equipment, it is especially suitable so as to reduce demand of the whole testing process for equipment to the full extent Field test for the clinical sample of basic unit.At present, the technology Successful utilization in Taura syndrome（Taura syndrome virus, TSV）, infectivity muscle necrosis virus（Infectious myonecrosis virus, IMNV）, infectious spleen and kidney it is bad Dead virus（Infectious spleen and kidney necrosis virus, ISKNV）, Vibrio vulnificus（Vibrio vulnificus）, and Streptococcus iniae（Strepstococcus iniae）Deng the detection of aquatic products pathogenic microorganism, both at home and abroad Have not been reported the diagnosis and detection that the technology is applied to Edwardsiella tarda.
The content of the invention
The technical problems to be solved by the invention are to provide that a kind of detection speed is fast, and testing cost is low, detection sensitivity and The application of primer and probe sequence and primer and probe that the high LAMP-LFD for Edwardsiella tarda of accuracy is detected.
Technical scheme is used by the present invention solves above-mentioned technical problem：
1st, the primer and probe sequence that a kind of LAMP-LFD for Edwardsiella tarda is detected, according to slow Edward Salmonella outer membrane protein A（GenBank accession number：EF528483）Coded sequence design LAMP three pairs of primer sequences and one spy Pin sequence, primer sequence are 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, EtaompA-FIP 5 ' ends are biotin labeling；Probe EtaompA-HP 5 ' ends are isosulfocyanic acid fluorescence Element mark.
2nd, for Edwardsiella tarda LAMP-LFD detect primer and probe sequence in Edwardsiella tarda Application in LAMP-LFD visible detection methods, specific detection method step are as follows：
1）Configure LAMP reaction systems：The final concentration of each composition of reaction system is respectively：Outer primer EtaompA-F3 and EtaompA-B3 each 1.6 μm of ol/L of each 0.2 μm of ol/L, inner primer EtaompA-FIP and EtaompA-BIP, ring primer EtaompA-LF and EtaompA-LB each 0.4 μm of ol/L, dNTPs 1.4mmol/L, Tris-HCl（pH 8.8）20mmol/L, KCl 10mmol/L, MgSO46.5mmol/L, (NH4)2SO410mmol/L, Triton X-100 0.1%, 8U Bst DNA gather Synthase large fragment (New England Biolabs) and 2 μ L sample masterplates, distilled water is added to make reaction system cumulative volume be 25 μ l；
2）LAMP reaction systems expand：Above-mentioned reaction system is subjected to amplified reaction, amplified reaction temperature is 63 DEG C, is expanded The increasing reaction time is 25 min；
3）Probe hybridizes and LFD detections：20 pmol probe EtaompA-HP is added in reaction system after amplification, 63 DEG C incubate 5 min, hybridized, take 5 μ L hybridization solutions add 100 μ L buffer solutions in mix, then LFD test strips are immersed Add in the buffer solution of hybridization solution and develop the color, judge the result of lateral flow ELISA test strip LAMP amplifications.
3rd, Edwardsiella tarda is being prepared for the LAMP-LFD of the Edwardsiella tarda primer and probes detected Application in LAMP-LFD Visual retrieval kits, the kit include LAMP reaction systems：The end of each composition of reaction system Concentration be respectively outer primer EtaompA-F3 and EtaompA-B3 each 0.2 μm of ol/L, inner primer EtaompA-FIP and EtaompA-BIP each 1.6 μm of ol/L, ring primer EtaompA-LF and EtaompA-LB each 0.4 μm of ol/L, dNTPs 1.4mmol/L, Tris-HCl（pH 8.8）20mmol/L, KCl 10mmol/L, MgSO46.5mmol/L, (NH4)2SO4 10mmol/L, Triton X-100 0.1%, 8U Bst archaeal dna polymerases large fragment (New England Biolabs) and 2 μ L Sample masterplate, distilled water is added to make reaction system cumulative volume be 25 μ l.
Compared with prior art, the advantage of the invention is that：
1st, detection sensitivity is high, and the detection sensitivity of this method is 3.50 × 102Cfu/mL, it is the 100 of Standard PCR detection Times；
2nd, detection time is short, and amplified reaction only needs 25 min, and result judgement is completed from extracting for sample gene group DNA, Whole testing process only needs 70 min, shortens than Standard PCR detection technique more than 2h, increases substantially detection speed；
3rd, high specificity, specific primer used according to eight in the outer membrane protein gene of Edwardsiella tarda not Designed with region, and also DNA specific probe, it can effectively avoid utilizing the side such as agarose gel electrophoresis, fluorescent dye False positive issue caused by method；
4th, instrument and equipment requires low, without the PCR instrument used in Standard PCR, gel electrophoresis and imaging system etc., only needs one Water-bath can be completed to detect；
5th, simple to operate, as a result substantially, whole detection process is not related to complex instrument and equipment, slightly has a molecular biology base The personnel of plinth can complete to operate；Testing result is clearly obvious, visually observes and can determine whether；
6th, it is safer to the person and environment, it is not related to the toxic reagents such as EB in detection process.
In summary, Edwardsiella tarda is examined using LAMP-LFD methods using the primer and probe of the present invention Survey, there is higher agility, specificity and sensitivity, instrument demand is simple, and the early stage for being advantageous to Edwardsiella tarda examines Disconnected and detection, the needs of testing agency of basic unit and the detection of live plague area can be met.
Brief description of the drawings
Fig. 1 is LAMP specificity experiments results；M：100 bp Plus DNA ladder (Fermentas, the U.S.)； NC：Template is used as using aseptic deionized water；Eta：Using the genomic DNA of Edwardsiella tarda as template；Asa、Ahy、Ppu、 Pae、Sin、Val、Vfl、Vha、Van、Vro、Vvu、Lmo：It is false single with aeromonas salmonicida, Aeromonas hydrophila, stench respectively Born of the same parents bacterium, pseudomonas aeruginosa, Streptococcus iniae, vibrio alginolyticus, vibrio fluvialis, Vibrio harveyi, Vibrio anguillarum, wheel animalcule vibrios, wound Hinder vibrios, the genomic DNA of Listeria monocytogenes is template；
Fig. 2 is LAMP-LFD specificity experiments results；NC：Template is used as using aseptic deionized water；Eta：With slow love moral The genomic DNA of Fahrenheit bacterium is template；Asa、Ahy、Ppu、Pae、Sin、Val、Vfl、Vha、Van、Vro、Vvu、Lmo：Respectively With aeromonas salmonicida, Aeromonas hydrophila, pseudomonas putida, pseudomonas aeruginosa, Streptococcus iniae, vibrio alginolyticus, river It is template to flow vibrios, Vibrio harveyi, Vibrio anguillarum, wheel animalcule vibrios, Vibrio vulnificus, the genomic DNA of Listeria monocytogenes；
Fig. 3 is the sensitivity results of LAMP detections；M：100 bp Plus DNA ladder (Fermentas, the U.S.)； NC：Template is used as using aseptic deionized water；107、106、105、104、103、102、101：The bacterial concentration of Edwardsiella tarda （A=3.50, unit：cfu/mL）, by the use of its extraction genomic DNA be used as template；
Fig. 4 is the sensitivity results of LAMP-LFD detections；NC：Template is used as using aseptic deionized water；107、106、105、 104、103、102、101：The bacterial concentration of Edwardsiella tarda（A=3.50, unit：cfu/mL）, utilize the gene of its extraction DNA is as template for group；
Fig. 5 is the sensitivity results of PCR detections；M：100 bp Plus DNA ladder (Fermentas, the U.S.)； NC：Template is used as using aseptic deionized water；107、106、105、104、103、102、101：The bacterial concentration of Edwardsiella tarda （A=3.50, unit：cfu/mL）, by the use of its extraction genomic DNA be used as template.
Embodiment
The present invention is described in further detail below in conjunction with accompanying drawing embodiment.
Embodiment 1
The foundation of the method for LAMP-LFD technology for detection Edwardsiella tardas
1. design of primers：According to the Edwardsiella tarda outer membrane protein A delivered in NCBI（GenBank is logged in Number：EF528483）Coded sequence be designed, 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, EtaompA-FIP 5 ' ends are biotin labeling；Probe EtaompA-HP 5 ' ends are isosulfocyanic acid fluorescence Element mark.
2. sample DNA extracts：By -70 DEG C of Edwardsiella tarda preserved for a long time strains（E. tarda MCCC235） Line LB solid mediums, the % of agar powder 1.5,30 DEG C of overnight incubations, picking monoclonal in 5 mL LB fluid nutrient mediums, 30 DEG C, 165 r/min shaking table cultures 10 hours or so, it is 3.50 × 10 to count and obtain bacterium solution initial concentration9cfu/mL.Dilution Afterwards, 3.50 × 10 are chosen7、3.50×106、3.50×105、3.50×104、3.50×103、3.50×102、3.50×101 Each 1 mL of bacterium solution of 7 concentration gradients such as cfu/mL, using commercialization kit extract genomic DNA, be dissolved in 50 μ L without Bacterium ddH2O is used for LAMP and PCR and verified.
3. Edwardsiella tarda LAMP reacts
The specific primer designed using step 1, LAMP expansions are carried out by masterplate of Edwardsiella tarda genomic DNA Increase.
3.1 LAMP reaction systems, the final concentration of each composition are respectively：Outer primer EtaompA-F3 and EtaompA-B3 is each 0.2 μm of ol/L, inner primer EtaompA-FIP and EtaompA-BIP each 1.6 μm of ol/L, ring primer EtaompA-LF and EtaompA-LB each 0.4 μm of ol/L, dNTPs 1.4mmol/L, Tris-HCl（pH 8.8）20mmol/L, KCl 10mmol/L, MgSO46.5mmol/L, (NH4)2SO410mmol/L, Triton X-100 0.1%, 8U Bst archaeal dna polymerase large fragments (New England Biolabs) and 2 μ L sample masterplates, distilled water is added to make reaction system cumulative volume be 25 μ L；
3.2 LAMP reaction conditions：Above-mentioned reaction system is subjected to amplified reaction, amplified reaction temperature is 63 DEG C, and amplification is anti- It is 25 min between seasonable.
4. probe hybridizes and LFD detections：20 pmol EtaompA-HP probes are added in reaction system after amplification, 63 DEG C incubate 5 min, hybridized, take 5 μ L hybridization solutions add 100 μ L buffer in mix, then LFD test strips are immersed Add in the buffer of hybridization solution and develop the color, judge LAMP amplification situation.
Embodiment 2
The specific assay of Edwardsiella tarda LAMP-LFD detections is carried out using the primer and probe of the present invention
Using designed specific primer and probe, respectively with Edwardsiella tarda MCCC235, aeromonas salmonicida ATCC 33658, Aeromonas hydrophila ATCC 7966, Vibrio vulnificus ATCC 27562, Vibrio harveyi ATCC 33866, river Vibrios ATCC 33809, pseudomonas putida MCCC 1A01082, Streptococcus iniae ATCC 29178, vibrio alginolyticus ATCC 33787th, Vibrio anguillarum ayu-H080701, wheel animalcule vibrios DSM 17186T, Listeria monocytogenes ATCC 19115, P. aeruginosa The genomic DNA of the grades of bacterium ATCC 9027 is masterplate, carries out LAMP-LFD reactions by the step 3 and step 4 of above-described embodiment 1, tests The specificity of primer and probe is demonstrate,proved, distilled water is as negative control.As a result as depicted in figs. 1 and 2, electrophoresis is utilized（Fig. 1）With LFD（Fig. 2）It can only all be expanded from the genome DNA sample of Edwardsiella tarda and obtain purpose band, other samples are without expansion Increase band, illustrate to carry out LAMP-LFD detections using primer and probe provided by the invention, there is good specificity.
Embodiment 3
The sensitivity determination of Edwardsiella tarda LAMP-LFD detections is carried out using the primer and probe of the present invention
Using the genomic DNA of the method extraction Edwardsiella tarda of the step 2 of above-described embodiment 1,10 times of ladders are carried out Degree dilution, selects 3.50 × 107、3.50×106、3.50×105、3.50×104、3.50×103、3.50×102、3.50× 101Cfu/mL carries out LAMP-LFD reactions by the step 3 and step 4 of above-described embodiment 1, verifies primer and probe as template Sensitivity, distilled water is as negative control.As a result as shown in Fig. 3, Fig. 4 and Fig. 5, primer and probe provided by the invention is used The sensitivity of the LAMP-LFD detections of progress is 3.50 × 102cfu/mL（Fig. 4）, agarose is utilized with LAMP amplified production The sensitivity that detected through gel electrophoresis obtains is consistent（Fig. 3）, it is the routine that EtaompA-F3 and EtaompA-B3 establish as primer 100 times of PCR detection method（Fig. 5）.
Embodiment 4
The Edwardsiella tarda in the crucian tissue of artificial contamination is specifically detected with LAMP-LFD technologies of the present invention.
1. Edwardsiella tarda artificial contamination and detected sample extracting genome DNA
Take the mg of liver organization 100 of several pieces health crucian to add a small amount of sterilized water, fully after homogenate, be settled to 1 mL. Take the Edwardsiella tarda bacterium solution of 1 mL fresh cultureds（About 3.50 × 108cfu/mL）10 times of concentration gradient dilutions are carried out, point It is 3.50 × 10 not take concentration5、3.50×104、3.50×103、3.50×102、3.50×101Cfu/mL each 1 mL of bacterium solution Mixed in equal volume with crucian liver tissue homogenate liquid.Each concentration is parallel to prepare three samples.Take the tissue sample of 1 mL germ contaminations Product are by step 2 methods described of embodiment 1 extraction genomic DNA.Respectively take 2 μ L to be used for LAMP-LFD and PCR to expand.Healthy crucian Hepatic homogenate liquid makees negative control.
2. LAMP reaction systems are prepared and reaction condition, carried out according to the step 3 of embodiment 1.
3. LFD develops the color, testing result judges, is carried out according to the step 4 of embodiment 1.
As a result after being shown in the Edwardsiella tarda of crucian tissue medium body contamination dye various concentrations, LAMP-LFD can be with From with 3.50 × 103Stable detection is reacted to cause of disease, detection sensitivity for 70 CFU/ in the liver organization of cfu/mL pollutions. PCR can only be from 3.50 × 104 Stable detection is to cause of disease in the liver organization of cfu/mL pollutions；LAMP-LFD is to unpolluted The testing result of healthy Carassius auratus liver tissue is feminine gender（Table 1）, specificity is well.
Table 1 utilizes LAMP-LFD detections of the present invention and PCR inspections after Edwardsiella tarda artificial contamination's crucian tissue sample Survey result
Note："+" represents that testing result is positive；"-" represents that testing result is negative.
Embodiment 5
With the Edwardsiella tarda in the LAMP-LFD Visual retrievals morbidity crucian sample of the present invention
1. crucian actual sample extracting genome DNA
The doubtful crucian 20 with Edwardsiella tarda disease is collected, first with the side of traditional bacteria distribution culture Method is separated to cause of disease, identified.Appropriate liver organization is taken simultaneously, DNA is extracted through kit method.
2. LAMP reaction systems are prepared and reaction condition, carried out according to the step 3 of embodiment 1.
3. LFD develops the color, testing result judges, is carried out according to the step 4 of embodiment 1.
As a result show, the detection of traditional bacteria distribution cultural method finds there are 3 slow Edwards in 20 sick fish samples Salmonella detection is positive.It can be infected in the sick fish hepatic tissue of Edwardsiella tarda from this using LAMP-LFD and PCR and obtained Positive amplification is obtained, the testing result to other 17 sick fish hepatic tissues is all negative, and testing result is trained with traditional bacteria distribution It is consistent to support methods and resultses.
Described above is not limitation of the present invention, and the present invention is also not limited to the example above.The art it is common Technical staff is in the essential scope of the present invention, the variations, modifications, additions or substitutions made, should also belong to the protection of the present invention Scope.

Claims (3)

1. the primer and probe sequence that a kind of LAMP-LFD for Edwardsiella tarda is detected, it is characterised in that according to slow Tarda outer membrane protein A coded sequence design LAMP three pairs of primer sequences and a probe sequence, primer sequence tool Body is 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, EtaompA-FIP 5 ' ends are biotin labeling；Probe EtaompA-HP 5 ' ends are fluorescein isothiocynate mark Note.
2. the primer and probe that a kind of LAMP-LFD according to claim 1 for Edwardsiella tarda is detected is being made Application in standby Edwardsiella tarda LAMP-LFD Visual retrieval kits.
3. the primer and probe that the LAMP-LFD according to claim 2 for Edwardsiella tarda is detected is being prepared late Application in slow tarda LAMP-LFD Visual retrieval kits, it is characterised in that the kit includes LAMP reactants 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, inner primer EtaompA-FIP and EtaompA-BIP each 0.4 μm of ol/L of each 1.6 μm of ol/L, ring primer EtaompA-LF and EtaompA-LB, DNTPs 1.4mmol/L, pH 8.8Tris-HCl 20mmol/L, KCl 10mmol/L, MgSO46.5mmol/L, (NH4)2SO4 10mmol/L, Triton X-100 0.1%, 8U Bst archaeal dna polymerases large fragment and 2 μ L sample masterplates, add distilled water to make instead It is 25 μ l to answer system cumulative volume.
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specific and rapid diagnosis of Edwardsiella tarda by a novel loop-mediated isothermal amplification targeting the upstream region of hlyb gene;Guo-Si Xie等;《journal of aquatic animal health》;20130502;摘要、第111页左栏第1、3段、右栏第4段-第113页左栏第1段、第112页表2、第113页跨栏段、第115页左栏第3段 *
病原迟钝爱德华菌毒力基因及双重PCR与LAMP检测方法的建立;张晓君等;《水产学报》;20130731;全文 *

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