CN104726567A - Streptococcus agalactiae loop-mediated isothermal amplification kit and application thereof - Google Patents
Streptococcus agalactiae loop-mediated isothermal amplification kit and application thereof Download PDFInfo
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Abstract
The invention discloses a streptococcus agalactiae loop-mediated isothermal amplification kit and application thereof. The kit comprises an LAMP primer, a 2*reaction buffer solution, Bst DNA polymerase, a fluorescent visual detection reagent, ultrapure water and a streptococcus agalactiae DNA template, wherein the LAMP primer comprises outer primerS F3 and B3 and inner primers FIP and BIP. Specificity detection and sensitivity detection prove that the LAMP detection kit disclosed by the invention can be used for monitoring reaction in real time, quantitatively detecting the copy number of the streptococcus agalactiae, rapidly and accurately obtaining detection results and providing convenience for simply and rapidly detecting the streptococcus agalactiae.
Description
Technical field
The present invention relates to technical field of microbial detection, relate to a kind of quick, visual and can the loop-mediated isothermal amplification kit of Real_time quantitative detection streptococcus agalactiae and application thereof specifically.
Background technology
Fish streptococcicosis is a kind of disease caused by streptococcal infection, and pathogenic bacteria belongs to 3 and belongs to and 7 kinds.The pathogenic bacteria of streptococcus have Streptococcus iniae (
streptococcus iniae), streptococcus agalactiae (
streptococcus agalactme), the scorching suis of accessory breast
(Streptococcus parauberis) and streptococcus dysgalactiae (
streptococcus dysgalactiae); Lactococcus have Lactococcus garvieae (
lactococcus garvieae) and fish galactococcus (
lactococcus piscium); Roaming Pseudomonas have salmon roam bacterium (
vagococcus salmoninarum).Wherein, Streptococcus iniae, streptococcus agalactiae, the scorching suis of accessory breast, streptococcus dysgalactiae and Lactococcus garvieae are the pathogenic bacteria of warm water fish streptococcicosis, and fish galactococcus and salmon roaming Pseudomonas are in the pathogenic bacteria of cold water fishes streptococcicosis.
Current discovery Streptococcus iniae and streptococcus agalactiae are the main two kinds of cause of diseases causing multiple wild fish and economic cultured fishes streptococcicosis.Wherein, the streptococcicosis caused by streptococcus agalactiae can cause multiple fresh water in worldwide and seawater fish big area infects, and has very high infection rate and lethality rate.This bacterium can infect and comprise rainbow trout, Sea Bream, tilapia, Yellow Tail, catfish, spends 17 kinds of fish of fish, catfish, grey mullet etc. in vain.
Tilapia streptococcicosis is Global prevalence, and annual world's tilapia industry of giving causes great economic loss.In China, there is streptococcicosis in tilapia cultivation in 2001 ~ 2006 years, and causes the cumulative mortality of 5 ~ 15% in indivedual plant successively.2009 ~ 2012 continuous 4 years, this interior syndrome state tilapia main culture zone big area outbreak of epidemic, cumulative mortality was between 15 ~ 95%, and the financial loss caused to China's tilapia aquaculture is accumulative up to 1,600,000,000 dollars.According to investigations, in China, the main pathogen of this disease also changes the streptococcus agalactiae after 2008 into by the Streptococcus iniae before 2008.Therefore, how fast, to diagnose streptococcus agalactiae disease just to seem especially important efficiently and accurately.
At present, the detection technique of tilapia streptococcus agalactiae mainly contains ordinary method and molecular biology method.Ordinary method is by identifying the phenotypic characteristic of bacterium and biochemical indicator.The biochemical indicator of qualification streptococcus agalactiae mainly comprises: the experiment of gramstaining, oxydase, the experiment of peroxidation oxygenase, CAMP experiment, V-P react the experiment of (Fu Gesi-Puli's Squall) product, aggegation experiment, ELISA experiment; LancefieldShi group specific antigen group qualification etc.But there is complicated operation in general survey method, the shortcoming that required time is long and specificity is not strong.Some automatic quick bacteria identification systems are had to identify for suis at present, as RAPID Strep strip, API rapid strep 32, API 20E Vitek system, ATB rapid detection system etc., but the automatic identification equipment of bacterium detects the unstable result of streptococcus agalactiae, and due to type strain limited amount in the database of bacterium automatization qualification, still under study for action, therefore the application of the method is also restricted streptococcic critical data.
The specific PCR method of genome 16SrRNA Data mining is utilized to be usually used in identifying suis.In addition, 16S-23SrDNA sequence, Lactate Oxidase (lctO) and the CAMP factor etc. is utilized to carry out fast PCR qualification to suis in addition.In addition, the dual-PCR method of streptococcus agalactiae and nested PCR method is also established for detecting streptococcus agalactiae.Although PCR method comparatively ordinary method quick and precisely, need complicated plant and instrument, cost is higher, is not suitable for basic unit and Site Detection.
Summary of the invention
The object of this invention is to provide a kind of for basic unit is easy, fast detect the method for streptococcus agalactiae exactly, disclose the loop-mediated isothermal amplification kit of a kind of detection by quantitative streptococcus agalactiae of quick, real-time quantitative.The technical scheme used for realizing the object of the invention is:
A kind of streptococcus agalactiae loop-mediated isothermal amplification kit, this test kit comprises LAMP primer, 2 × reaction buffer, Bst archaeal dna polymerase, fluorescence visual detection reagent, ultrapure water and streptococcus agalactiae DNA profiling; Described LAMP primer comprises outer primer F3(SEQ ID NO:1) with B3(SEQ ID NO:2) and inner primer be FIP(SEQ ID NO:3) and BIP(SEQ ID NO:4);
Wherein the sequence of primer is respectively:
F3 GTAGCAGCCCCTAGAGTG
B3 GTTGTGCTACCGGTGTTG
FIP AGCTGATACATGCTCTGGTGATGGCAAGTGTTAAAGTAGTCACTC
BIP GTTCCTGTGACTACGACTTCAACTTTTGTGCTACCGGAAGG
2 described × reaction buffer comprises Tris-HCL, KCL, MgSO
4, (NH
4)
2sO
4, Tween20, Betaine and dNTPs.
Above-described streptococcus agalactiae DNA profiling is the streptococcus agalactiae DNA using bacterial genomes DNA extraction kit to extract.
Above-described fluorescence visual detection reagent adopts fluorexon fluorescent reagent, and fluorescent reagent adds before the reaction.
Above-described 2 × reaction buffer comprises Tris-HCL 35-55mM, KCL 18-32mM, MgSO
415-25mM, (NH
4)
2sO
422-28mM, Tween20 0.3-0.6 ℅, Betaine 1.5-3 M and dNTPs3-4 .5mM, above-mentioned solvent, under pH is 9.0 conditions, evenly obtains by its compound method.
An application for streptococcus agalactiae loop-mediated isothermal amplification kit, for detecting doubtful streptococcus agalactiae, concrete detecting step comprises:
(1) Design and synthesis of LAMP primer
(2) extraction of streptococcus agalactiae DNA profiling
(3) LAMP reaction system is set up
(4) LAMP detection method.
Above-described LAMP reaction system sets up LAMP reaction system in 25 μ l,
2 × reaction buffer 12.5 μ L
Bst archaeal dna polymerase 1 μ L
FIP 40 pmol
BIP 40 pmol
F3 5 pmol
B3 5 pmol
Streptococcus agalactiae DNA 2 μ L
Ultrapure water supplies 25 μ L.
Above-described LAMP detection method is the method adopting specific detection, sensitivity Detection and fluorescent visual to detect.
Above-described LAMP detection method adopts the real-time turbidimeter of Loopamp LA-320C to carry out airtight complete monitoring, and temperature of reaction is 63 DEG C, reacts appearance amplification in 30-40 minute.
Substantive distinguishing features of the present invention and marked improvement are:
1) high specificity
The negative control virus detected, negative control bacterium and all no positive result of water contrast are out, consistent with PCR detected result.And easy and simple to handle, obtain detected result fast, without the need to instrument costly.
2) highly sensitive
The sensitivity of common PCR detection method is 3.55 × 10
-3ng/ μ L number level, and use detection method, detectability is about 3.55 × 10
-5ng/ μ L is about 100 times of regular-PCR.
3) obtain a result rapidly
The whole process of common PCR just can be obtained a result at 24 hours, the LAMP reaction method that current majority is set up after the completion of reaction, the video picture of agarose gel electrophoresis ultraviolet analysis must be adopted to carry out result of determination, from bacterial genomes DNA extraction to acquisition test-results, need 4-5 hours.Amplification is there is in LAMP detection method reaction provided by the invention between 30-40 minute, amplification can be completed in 60 minutes, and result decision procedure easy-amplification terminate to get final product judged result, not needing to carry out the video picture of agarose gel electrophoresis ultraviolet analysis again and carry out result of determination, can complete in 2-3 hour to obtaining net result from plasmid extraction.
4) do not pollute
The fluorescence dye that current LAMP method is used for directly observing is for adding after reaction, but development process can only be reaction terminate after uncap and add fluorescence dye and carry out color reaction, whether observe has colour developing to carry out interpretation test-results, or the method for being swum by leakage of electricity carries out result judgement, specific amplification and non-specific amplification cannot be distinguished, because this increasing the probability of false positive diagnoses result; And for weak positive reaction, be probably mistaken for feminine gender by the mode of artificial naked eyes interpretation; In addition, carried out the mode of sentence read result by the method for electrophoresis, add experimentation cost, time-consuming and easily cause laboratory pollution.And LAMP fluorescence detection method of the present invention, fluorescence dye adds before the reaction, do not need to uncap, and effectively can avoid Aerosol Pollution.In addition, LAMP detection method of the present invention, on result judges, directly can be carried out result of determination by the turbidity value of turbidimeter detection reaction pipe, can not carry out fluorescent dye determination detected result or carry out agarose gel electrophoresis detected result, do not need to uncap, can effectively avoid polluting.
5) can real-time quantitative
The present invention utilizes Tubidimeter real-time LA-320 turbidimeter to carry out the result of real-time analysis LAMP reaction, the typical curve that time of the turbidity value that the concentration of different standard models is corresponding is depicted as, for people's typical curve equation, the streptococcus agalactiae copy number of each time can be obtained, reach the object of detection by quantitative product.
Accompanying drawing explanation
Fig. 1 is LAMP method specific detection result of the present invention; There is the upcurve of turbidity in 1 strain fish streptococcus agalactiae reaction tubes, 8 strain negative control bacterium reaction tubess and water control reaction are all without amplification.
Fig. 2 and Fig. 3 is the sensitivity Detection result of LAMP method of the present invention and traditional PCR method; Wherein A:3.55 × 10
1ng/ Μ l; B:3.55 × 10
0ng/ μ L; C:3.55 × 10
-1ng/ μ L; D:3.55 × 10
-2ng/ μ L; E:3.55 × 10
-3ng/ μ L; F:3.55 × 10
-4ng/ μ L; G:3.55 × 10
-5ng/ μ L; H:3.55 × 10
-6ng/ μ L; I:3.55 × 10
-7ng/ μ L; J:water.Recombinant plasmid pMD18-T-
sipinitial concentration be 3.55 × 10ng/ μ L, after 10 times of doubling dilutions, carry out LAMP and pcr amplification, result display LAMP method detectability is about 3.55 × 10
-5ng/ μ L, and the detection of PCR method is limited to 3.55 × 10
-3ng/ μ L.
Fig. 4 is visual results after adding fluorescence dye: the response situation that right pipe is is template with streptococcus agalactiae genomic dna is positive findings, and left pipe is the response situation of negative control, is negative findings.
Fig. 5 is the typical curve of detection by quantitative streptococcus agalactiae LAMP method of the present invention: utilize the typical curve that turbidity value corresponding to the concentration of different standard models was depicted as the time, substitutes into typical curve equation, can obtain the streptococcus agalactiae copy number of each time.
Embodiment
1, the preparation of material
Streptococcus agalactiae, intestinal bacteria, Aeromonas hydrophila, Aeromonas veronii, Channel-catfish Ai Dehuashi Zymomonas mobilis, the lonely bacterium of Kazakhstan, the lonely bacterium of wound, Streptococcus iniae are all separated from tilapia, preserve for Guangxi Zhuang Autonomous Region aquatic products research institute is separated.LAMP method DNA cloning test kit is purchased from Beijing Lanpu Biological Technology Co., Ltd., and article No. SLP204, it is century bio tech ltd purchased from health that bacterial genomes extracts test kit, article No. CW0522.
2, the Design and synthesis of LAMP primer
According to the streptococcus agalactiae Sip sequence in GenBank, utilize a set of LAMP primer of LAMP method primer Autocad PrimerExplorer V4 software design, wherein F3, B3 are outer primer, and FIP, BIP are inner primer, wherein F3, B3 are that streptococcus agalactiae PCR detects primer, wherein
F3 GTAGCAGCCCCTAGAGTG
B3 GTTGTGCTACCGGTGTTG
FIP AGCTGATACATGCTCTGGTGATGGCAAGTGTTAAAGTAGTCACTC
BIP GTTCCTGTGACTACGACTTCAACTTTTGTGCTACCGGAAGG
3, bacterial genomes DNA extraction
The bacterial genomes DNA extraction kit using health to produce for century bio tech ltd extracts bacterial genomes DNA.
4, LAMP reaction system is set up
According to test kit specification sheets, by 25 μ l system configurations:
2 × reaction buffer 12.5 μ L
BstDNA polysaccharase 1 μ L
FIP 40 pmol
BIP 40 pmol
F3 5 pmol
B3 5 pmol
Streptococcus agalactiae DNA 2 μ L
Ultrapure water supplies 25 μ L.
LAMP reaction is with real-time turbidimeter (LA-320C, Rong Yan company of Japan) form of carrying out airtight complete monitoring monitor present method detect situation, turbidimeter monitors amplification situation in real time, can drawing standard curve, time value corresponding to 0.1 turbidity value is reached by obtaining unknown sample, can calculate the starting copy number of this sample from typical curve, temperature of reaction recommend with test kit 63 DEG C is as temperature of reaction.
5, LAMP detection method
1) specific detection
Extract the genomic dna template of react as LAMP of tilapia streptococcus agalactiae, intestinal bacteria, Aeromonas hydrophila, Aeromonas veronii, Channel-catfish Ai Dehuashi Zymomonas mobilis, the lonely bacterium of Kazakhstan, the lonely bacterium of wound, Streptococcus iniae respectively, check the specificity of LAMP method.
2) sensitivity Detection
With the tilapia streptococcus agalactiae genomic dna extracted for template, carry out pcr amplification with outer primer F3 and B3, by goal gene connection carrier pMD-18T, clone, extract the plasmid of mono-clonal bacterium, measure its concentration, through sequence verification.Become 9 extent of dilution with the continuous 10 times of doubling dilutions of RNA-Free Water, get each extent of dilution plasmid 2 μ L and carry out LAMP amplification as template.
3) fluorescent visual detects
According to the condition that turbidimeter monitoring is optimized, add fluorescence dye, fluorescence dye adds before the reaction, the dyestuff added is fluorexon commercial dyes, react at 63 DEG C after 60 minutes, observe under ultraviolet lamp, do not adopt the video picture of agarose gel electrophoresis ultraviolet analysis, avoid uncapping and run the Aerosol Pollution that electrophoresis observation causes.
The specific outcome of embodiment 1 LAMP detection method
LAMP amplification is carried out to 1 strain streptococcus agalactiae, 8 strain negative control bacterium and water contrast, result as shown in Figure 1, the upcurve of turbidity is there is in streptococcus agalactiae reaction tubes at about 37 minutes, for positive findings, 8 strain negative control bacterium reaction tubess and water control reaction pipe curve all occur without amplification situation, are negative findings.
The susceptibility results of embodiment 2 LAMP detection method
Recombinant plasmid pMD18-T-
sipinitial concentration be 3.55 × 10
1ng/ μ L, carries out LAMP and pcr amplification after 10 times of doubling dilutions, and as shown in Figures 2 and 3, result display LAMP method detectability is about 3.55 × 10 to result
-5ng/ μ L, and the detection of PCR method is limited to 3.55 × 10
-3ng/ μ L.
The fluorescent visual detected result of embodiment 3 LAMP detection method
According to the condition that turbidimeter monitoring is optimized, reactor adds fluorescence dye, and 63 DEG C of reactions are after 60 minutes, observe under ultraviolet lamp, Fig. 4 is observations, and left pipe is negative control, right pipe is take streptococcus agalactiae as the response situation of template, show that the method set up can facilitate basic unit to use, the LAMP primer that only test kit need be used to coordinate present method to design, after adding sample, with cheap water-bath keep 63 DEG C 60 minutes, get final product rapid examination result, and without the need to uncapping, avoid pollution.
The drafting of embodiment 4 streptococcus agalactiae quantitation curves
Contrast is set: concentration is 3.55 × 10
1ng/ μ L, 3.55 × 10
0ng/ μ L, 3.55 × 10
-1ng/ μ L, 3.55 × 10
-2ng/ μ L, 3.55 × 10
-3ng/ μ L, 3.55 × 10
-4ng/ μ L, 3.55 × 10
-5the recombinant plasmid pMD18-T-of ng/ μ L
sipeach one of standard model, because the negative logarithm of concentration and turbidity value be 0.1 time value linear, so the value that turbidimeter can be captured and time (as table 1) make typical curve, obtain typical curve equation, y=0.3559x-12.621, as shown in Figure 5.From typical curve equation coefficient R
2be 0.9951, in good linear relationship.Take time as X value, can obtain the negative time number formulary of Y value and concentration, then concentration is 10-
y, then be multiplied by radix 3.55, be 3.55 x10
-yng/ μ L.According to copy number reduction formula copies/ μ L=(6.02 x 10
23x (ng/ul x 10
-9))/(DNA length x 660), DNA length is that carrier sequence size adds goal gene sequence size, is 2693+220=2913 bp, is converted into copy number (copies/ μ L): 6.02 x 10
23x(3.35 x10
-yx 10
-9)/(2913 x 660), be reduced to: 1.05 x 10
9x10
-y.As certain test sample reach turbidity value be 0.1 time be 36 minutes time, bring set up typical curve equation into, obtain Y and equal 0.1916, then concentration is 10
-0.1916, then be multiplied by radix 3.55, be the concentration 3.35 × 10 of this sample
0.1916ng/ μ L, copy number is 1.05 x 10
9x10
0.1916, be 1.05 x 10
9.1916copies/ μ L, thus reach quantitative effect.
Table 1
| Time (min) | 33 | 35.7 | 38.1 | 40.7 | 43.6 | 46.3 | 50.2 |
| Standard value (-LOG) | -1 | 0 | 1 | 2 | 3 | 4 | 5 |
Claims (8)
1. a streptococcus agalactiae loop-mediated isothermal amplification kit, is characterized in that, this test kit comprises LAMP primer, 2 × reaction buffer, Bst archaeal dna polymerase, fluorescence visual detection reagent, ultrapure water and streptococcus agalactiae DNA profiling; Described LAMP primer comprises outer primer F3(SEQ ID NO:1) with B3(SEQ ID NO:2) and inner primer be FIP(SEQ ID NO:3) and BIP(SEQ ID NO:4);
Wherein the sequence of primer is respectively:
F3 GTAGCAGCCCCTAGAGTG
B3 GTTGTGCTACCGGTGTTG
FIP AGCTGATACATGCTCTGGTGATGGCAAGTGTTAAAGTAGTCACTC
BIP GTTCCTGTGACTACGACTTCAACTTTTGTGCTACCGGAAGG；
2 described × reaction buffer comprises Tris-HCL, KCL, MgSO
4, (NH
4)
2sO
4, Tween20, Betaine and dNTPs.
2. streptococcus agalactiae loop-mediated isothermal amplification kit according to claim 1, is characterized in that, described streptococcus agalactiae DNA profiling is the genomic dna using bacterial genomes DNA extraction kit to extract streptococcus agalactiae culture.
3. streptococcus agalactiae loop-mediated isothermal amplification kit according to claim 1, is characterized in that, described fluorescence visual detection reagent adopts fluorexon fluorescent reagent, and fluorescent reagent adds before the reaction.
4. streptococcus agalactiae loop-mediated isothermal amplification kit according to claim 1, is characterized in that, 2 described × reaction buffer comprises Tris-HCL 35-55mM, KCL 18-32mM, MgSO
415-25mM, (NH
4)
2sO
422-28mM, Tween20 0.3-0.6 ℅, Betaine 1.5-3 M and dNTPs3-4 .5mM.
5. an application for streptococcus agalactiae loop-mediated isothermal amplification kit, is characterized in that, for detecting doubtful streptococcus agalactiae, concrete detecting step comprises:
(1) Design and synthesis of LAMP primer
(2) extraction of streptococcus agalactiae DNA profiling
(3) LAMP reaction system is set up
(4) LAMP detection method.
6. the application of streptococcus agalactiae loop-mediated isothermal amplification kit according to claim 5, is characterized in that, described LAMP reaction system sets up LAMP reaction system in 25 μ l,
2 × reaction buffer 12.5 μ L
Bst archaeal dna polymerase 1 μ L
FIP 40 pmol
BIP 40 pmol
F3 5 pmol
B3 5 pmol
Streptococcus agalactiae DNA 2 μ L
Ultrapure water supplies 25 μ L.
7. the application of streptococcus agalactiae loop-mediated isothermal amplification kit according to claim 5, is characterized in that, described LAMP detection method is the method adopting specific detection, sensitivity Detection and fluorescent visual to detect.
8. the application of streptococcus agalactiae loop-mediated isothermal amplification kit according to claim 5, is characterized in that, described LAMP detection method adopts real-time turbidimeter to carry out airtight complete monitoring.
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| CN111187849A (en) * | 2020-02-17 | 2020-05-22 | 深圳麦科田生物医疗技术有限公司 | Primer group, kit and detection method for detecting group B streptococcus based on loop-mediated isothermal amplification technology |
| CN112608310A (en) * | 2020-12-17 | 2021-04-06 | 北京丹大生物技术有限公司 | Risperidone and 9-hydroxy risperidone hapten, antigen and antibody and application thereof |
| CN112730841A (en) * | 2020-12-17 | 2021-04-30 | 北京丹大生物技术有限公司 | Immunological detection method of risperidone and/or 9-hydroxy risperidone |
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