CN102747165B - Reagent kit and method for quickly detecting tilapia streptococcus agalactiae by using loop-mediated isothermal amplification technology - Google Patents
Reagent kit and method for quickly detecting tilapia streptococcus agalactiae by using loop-mediated isothermal amplification technology Download PDFInfo
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- CN102747165B CN102747165B CN 201210260204 CN201210260204A CN102747165B CN 102747165 B CN102747165 B CN 102747165B CN 201210260204 CN201210260204 CN 201210260204 CN 201210260204 A CN201210260204 A CN 201210260204A CN 102747165 B CN102747165 B CN 102747165B
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Abstract
The invention relates to a reagent kit for quickly detecting tilapia streptococcus agalactiae by using the loop-mediated isothermal amplification technology, which can be used for quickly detecting and real-time monitoring for streptococcus agalactiae in fish body and aquatic water. The invention discloses two pairs of specific primers for LAMP detection of streptococcus agalactiae and a reagent kit used for fish farms. The reagent comprises a streptococcus agalactiae genome extraction reagent and an LAMP reaction liquid, and the reaction liquid comprises 10*LAMP Buffer (200 mM Tris-Hcl, pH8.8; 100 mM potassium chloride; 100 mM ammonium sulfate; 20 mM magnesium sulfate and 1% TritonX-100), BstDNA polymerase, dNTP, internal primers GBS-F3 and GBS-B3, external primers GBS-FIP and GBS-BIP, betaine, ultrapure water and fluorescent dye. The reagent kit has the characteristics of short detection time, simplicity for operation, obvious results, high sensitivity, strong specificity, safety to human and environment and the like.
Description
Technical field:
The present invention relates to aquatic living things cause of disease Fast Detection Technique, specifically be utilize ring mediated isothermal amplification (LAMP) technology rapid detection tilapia streptococcus agalactiae (
Streptococcus agalactiae, GBS) detection kit of cause of disease and detection method thereof.
Background technology:
Streptococcus agalactiae (
S.
Agalactiae) also claim B group streptococcus (GBS), be that a class extensively is present in natural gram-positive microorganism, host range is wide, all can work the mischief to mammals, reptiles, batrachians and fish.From 1958 Japan rainbow trout (
Oncorhychus mikiss) in obtain the suis cause of disease first after, the fish streptococcicosis is popular in a plurality of countries in succession.Be mainly in the tilapia of breed in recent years in China, it is infectious strong, and mortality ratio height, and treatment difficulty cause various places, China south cultivating tilapia explosive dead, have caused enormous economic loss for the aquatic products aquaculture.The technology that the relevant bacteria pathogeny of having set up at present detects is more, as the biochemistry detection method of routine, mainly is based upon quick proprietary enzyme reaction in the biological chemistry and the detection technique of opsonigenous substances; Based on the detection method of immunology such as the radioimmunoassay in the immunology (RIA), enzyme immunoassay (EIA), fluoroimmunoassay (FIA), time resolved fluoro-immunoassay (TrFIA), chemiluminescence immune assay (CIA), (BLIA) bioluminescence immunoassay (BIA) etc., be enough to detect the microbial antigen of trace in the clinical samples; And analyze based on the nucleic acid probe of molecular biology and polymerase chain reaction (PCR).But these methods or length consuming time, or susceptibility is poor or specificity is not strong.The PCR detection method of GBS, though under laboratory condition, can carry out relative accurate detection to GBS, but because detecting, conventional PCR needs expensive PCR instrument, gel electrophoresis and imaging system etc., and this has limited PCR detection method applying in production reality greatly.
Therefore develop easy fast, the accurate new technology of sensitive detection GBS, strengthen seed and the quarantine of different size fingerling and the detection of aquaculture water, to the forecast prediction, the prevention epidemic situation infects, cut off transmission of pathogen, ensure that the healthy and sustainable development of China's tilapia industrial chain is significant.
Summary of the invention:
The present invention seeks to, a kind of tilapia streptococcus agalactiae loop-mediated isothermal amplification detection kit is provided, to realize quick, sensitive, special, the easy rapid detection to tilapia streptococcus agalactiae disease.
Ring mediated isothermal amplification method (LAMP) is a kind of novel nucleic acid amplification method, this method is to six Auele Specific Primers of eight zone design on the cause of disease conservative gene, utilize the Bst archaeal dna polymerase to carry out constant-temperature amplification 30-60min at 60-65 ℃, by observing response thing turbidity or combination dye colour-change result of determination, this method sensitivity, quick, accurate, easy, do not need expensive instrument, cost is low, can be used for sample field rapid detection on the spot.
The present invention specifically comprises three parts: 1. the chelex-100 resin-bonded water-boiling method of legal medical expert's Molecular Identification being used is used for rapid extraction tilapia streptococcus agalactiae genomic dna, simplifies greatly and when shortening the operating time, has guaranteed the quality of the DNA that extracts; 2. two couples of primer sequences that are used for streptococcus agalactiae LAMP rapid detection, called after GBS-F3 successively are provided; GBS-B3; GBS-FIP; GBS-BIP, length is respectively 21bp, 18 bp, 51bp, 50 bp; 3. the LAMP method for quick of a kind of tilapia streptococcus agalactiae is provided, it at first is fast and convenient preparation feedback masterplate, and configuration LAMP reaction system, by the LAMP response procedures reaction masterplate is increased then, the color that shows according to reaction mixture is carried out interpretation to detected result at last, represent then that as the reactant demonstration is green the GBS detected result of this sample is positive, detect negative if show the orange-yellow GBS of this sample that then represents.
Above-mentioned two couples of LAMP primer sequence (GBS-F3; GBS-B3; GBS-FIP; GBS-BIP) identical or complementary with the nucleotide sequence of corresponding position on the short hemolytic factor gene of tilapia streptococcus agalactiae respectively, its sequence is as follows:
GBS-F3: 5’-AGTAATAGCCTCATTAACCGG-3’;
GBS-B3: 5’-CTAGTGGCTGGTGCATTG-3’;
GBS-FIP:
5’-GCTCAAAAGCTTGATCAAGATAGCTTTTCTGTTCCCTGAACATTATCTTTG-3’;
GBS-BIP:
5’- TTACTTGATTTACCACTTGTGGAGTTTTTTTCACCAGCTGTATTAGAAGT-3’;
Streptococcus agalactiae LAMP quick detection kit of the present invention is formed by extracting streptococcus agalactiae genome reagent and LAMP reaction reagent:
Described extraction streptococcus agalactiae genome reagent is formed:Chelex-100, Tris, EDTA, Tritonx-100;
Described LAMP reaction solution is made up of reaction solution A and reaction solution B:Reaction solution A contains 10 * LAMP Buffer (200mM Tris-Hcl, pH8.8; 100 mM Repone K; 100 mM ammonium sulfate; 20mM sal epsom and 1%TritonX-100), Bst archaeal dna polymerase, dNTP, interior outer primer (GBS-FIP; GBS-BIP; GBS-F3; GBS-B3), trimethyl-glycine, ultrapure water.
GBS-F3: AGTAATAGCCTCATTAACCGG;
GBS-B3: CTAGTGGCTGGTGCATTG;
GBS-FIP: GCTCAAAAGCTTGATCAAGATAGCTTTTCTGTTCCCTGAACATTATCTTTG;
GBS-BIP: TTACTTGATTTACCACTTGTGGAGTTTTTTTCACCAGCTGTATTAGAAGT;
Reaction solution B is fluorescence dye.
Said extracted streptococcus agalactiae genome reagent 5%chelex-100(pH=10.4) comprising:5%chelex-100,10mmol/L Tris (PH=8.0), l mmol/L EDTA, 1% Tritonx-100.
In the above-mentioned LAMP reaction reagent:Bst archaeal dna polymerase concentration is that 8U/ μ L, dNTP concentration are 2.5 mM, inner primer (GBS-FIP; GBS-BIP) concentration is 20 μ M, outer primer (GBS-F3; GBS-B3) concentration is 20 μ M, trimethyl-glycine concentration is 5M.
Described fluorescence dye is:1000 * SYBR Green I.
The method that test kit of the present invention detects streptococcus agalactiae comprises the steps:
(1) gets 1ml streptococcus agalactiae bacterium liquid, abandon supernatant behind the centrifugal 5min of 10000rpm, be diluted to 50 μ L with sterilized water again;
(2) add 5% chelex-100 complex liquid to cumulative volume 200ul, hatch 15-30min for 56 ℃, concuss 10s;
(3) change in 100 ℃ of boiling water and hatch 8-10min, again concuss 10s;
(4) ice bath 2min, under 4 ℃ of conditions, the centrifugal 3min of 12000 rpm, it is standby as template to draw supernatant liquor;
(5) in the reaction tubes of two LAMP reaction reagents that 23 μ L are housed, add the above-mentioned template of 2 μ L and positive control nucleic acid respectively;
(6) above-mentioned reaction tubes is placed 65 ℃ of insulation 60min, place 80 ℃ of insulation 10min to carry out the LAMP reaction then;
(7) after the end, in each reaction tubes, add 2 μ L1000 * SYBR Green I, the LAMP reaction product of visual inspection sample represents then that as the reactant demonstration is green the GBS detected result of this sample is positive then, detects negative if show the orange-yellow GBS of this sample that then represents.
Detection method provided by the present invention has following advantage:
(1) detection time is short: 1.5-2h can obtain detected result, saves 4-6h than the fastest existing PCR detection method;
(2) simple to operate, the result is obvious: whole testing process does not relate to instrument or the equipment of complex and expensive, only needs a water-bath can finish detection, and detected result can directly detect by an unaided eye;
(3) highly sensitive: the limit of detection to the tilapia streptococcus agalactiae can be low to moderate 9 bacteriums, and is more highly sensitive more than 10 times than regular-PCR.
(4) high specificity: used Auele Specific Primer is that specificity surpasses conventional PCR according to 6 different zones designs in the short hemolytic factor gene of tilapia streptococcus agalactiae.
(5) safety: testing process does not relate to toxic reagent, can not work the mischief to human and environment.
In a word, this method has solved long, the defectives such as sensitivity is lower, complicated operation, rig-site utilization difficulty of required cycle of method that detect streptococcus agalactiae in the prior art, can be widely used in fields such as animal doctor, aquatic products, entry and exit quarantine.
Specific implementation method:
Case study on implementation:
Make the loop-mediated isothermal amplification detection kit of streptococcus agalactiae by following prescription:
1. configuration streptococcus agalactiae genome extracts reagent:
(1) TEX
（10mmol/L Tris [pH=8.0]，1mmol/L EDTA，1%TritonX-100
）
(2)Utilize the chelex-100(pH=10.4 of TEX preparation 5%).
Extract the streptococcus agalactiae genome:
(1) gets 1ml streptococcus agalactiae bacterium liquid, abandon supernatant behind the centrifugal 5min of 10000rpm, be diluted to 50 μ L with sterilized water again;
(2) add 5% chelex-100 complex liquid to cumulative volume 200 μ L, hatch 15-30min for 56 ℃, concuss 10s;
(3) change in 100 ℃ of boiling water and hatch 8-10min, again concuss 10s;
(4) ice bath 2min, under 4 ℃ of conditions, the centrifugal 3min of 12000 rpm, it is standby as template to draw supernatant liquor;
3. dispose the LAMP reaction reagent:
(1)
Reaction solution A:2.5 μ L 10 * LAMP Buffer, 1.5 μ L Bst archaeal dna polymerases (8U/ μ L), 4 μ L dNTP(2.5 mM), 0.1 μ L outer primer GBS-F3(20 μ M), 0.1 μ L outer primer GBS-B3(20 μ M), 0.8 0.8 μ L inner primer GBS-BIP(20 μ M μ L inner primer GBS-FIP(20 μ M)), 2.5 μ L trimethyl-glycines (5M), 10.7 μ L ultrapure waters.
Two pairs of LAMP primer sequences of the present invention (GBS-F3; GBS-B3; GBS-FIP; GBS-BIP) identical or complementary with the nucleotide sequence of corresponding position on the short hemolytic factor gene of tilapia streptococcus agalactiae respectively, its sequence is as follows:
GBS-F3:5’-AGTAATAGCCTCATTAACCGG-3’(21bp)
GBS-B3:5’-CTAGTGGCTGGTGCATTG-3’(18bp)
GBS-FIP:
5’-GCTCAAAAGCTTGATCAAGATAGCTTTTCTGTTCCCTGAACATTATCTTTG-3’,
(51bp)
GBS-BIP:
5’-TTACTTGATTTACCACTTGTGGAGTTTTTTTCACCAGCTGTATTAGAAGT-3’
(50bp)
(2
) reaction solution B: 1000 * SYBR Green I.
The ring mediated isothermal amplification of streptococcus agalactiae and color reaction:
(1) in the reaction tubes of two LAMP reaction reagents that 23 μ L are housed, adds the above-mentioned template of 2 μ L and positive control nucleic acid respectively;
(2) above-mentioned reaction tubes is placed 65 ℃ of insulation 60min, place 80 ℃ of insulation 10min to carry out the LAMP reaction then;
(3) after the end, in each reaction tubes, add 2 μ L, 1000 * SYBR Green I, the LAMP reaction product of visual inspection sample then, represent then that as the reactant demonstration is green the GBS detected result of this sample is positive, namely contain streptococcus agalactiae, represent that then the GBS detection of this sample is negative if demonstration is orange-yellow, namely do not contain streptococcus agalactiae.
Embodiment 1: the susceptibility of the effect of test kit of the present invention in detecting streptococcus agalactiae:
(1)
Sample:(original concentration is the tilapia streptococcus agalactiae: 9.1 * 10
8Cfu/mL).
(2)
Sample preparation:(original concentration is: 27.0 μ g/mL), and it is done 10 to extract tilapia streptococcus agalactiae DNA
-1-10
-8Doubling dilution.
(3)
Streptococcus agalactiae LAMP detection kit reaction solution A is formulated as follows:2.5 μ L 10 * LAMP Buffer, 1.5 μ L Bst archaeal dna polymerases (8U/ μ L), 4 μ L dNTP(2.5 mM), 0.1 μ L outer primer GBS-F3(20 μ M), 0.1 μ L outer primer GBS-B3(20 μ M), 0.8 0.8 μ L inner primer GBS-BIP(20 μ M μ L inner primer GBS-FIP(20 μ M)), 2.5 μ L trimethyl-glycines (5M), 10.7 μ L ultrapure waters
(4) ring of streptococcus agalactiae mediation amplified reaction:
In the reaction tubes of two LAMP reaction reagents that 23 μ L are housed, add the above-mentioned template of 2 μ L and positive control nucleic acid respectively, above-mentioned reaction tubes is placed 65 ℃ of insulation 60min, place 80 ℃ of insulation 10min to carry out the LAMP reaction then;
(5) color reaction of amplified production:
After the end, in each reaction tubes, add 2 μ L, 1000 * SYBR Green I, the LAMP reaction product of visual inspection sample represents then that as the reactant demonstration is green the GBS detected result of this sample is positive then, detects negative if show the orange-yellow GBS of this sample that then represents.
Susceptibility result:
Test-results shows: adopt the low energy of test kit of the present invention to detect 9 bacterium/pipes, show that this detection method has very high susceptibility.
Embodiment 2: clinical detection
(1) sample: tilapia streptococcus agalactiae positive 5 strains that the tilapia onset site is gathered and negative sample 12 strains (comprise other pathogenic bacterias that ill tilapia body separates as: Streptococcus iniae, Aeromonas hydrophila, Aeromonas sobria, Edwardsiella tarda, light green aerococcus, streptococcus aureus, proteus vulgaris, lactobacillus, point-like Aeromonas etc.).
Utilize 5% chelex-100 water-boiling method to extract the DNA of each sample:
1. get 1ml streptococcus agalactiae bacterium liquid, abandon supernatant behind the centrifugal 5min of 10000rpm, be diluted to 50 μ L with sterilized water again;
2. add 5% chelex-100 complex liquid to cumulative volume 200 μ L, hatch 15-30min for 56 ℃, concuss 10s;
3. change in 100 ℃ of boiling water and hatch 8-10min, again concuss 10s;
4. ice bath 2min, under 4 ℃ of conditions, the centrifugal 3min of 12000 rpm, it is standby as template to draw supernatant liquor;
(3) each sample is carried out LAMP reaction and the color developing detection concrete steps as follows:
1. in the reaction tubes of the LAMP reaction A reagent that 23 μ L are housed, add above-mentioned each the sample template of 2 μ L and positive control nucleic acid respectively;
2. above-mentioned reaction tubes is placed 65 ℃ of insulation 60min, place 80 ℃ of insulation 10min to carry out the LAMP reaction then;
3. after finishing, in each reaction tubes, add the i.e. 1000 * SYBR Green I of 2 μ L reaction solution B, the LAMP reaction product of visual inspection sample then, represent then that as the reactant demonstration is green the GBS detected result of this sample is positive, namely contain streptococcus agalactiae, represent that then the GBS detection of this sample is negative if demonstration is orange-yellow, namely do not contain streptococcus agalactiae.
Experimental result shows:
More than detect through LAMP and be shown as the positive, all can detect the existence of streptococcus agalactiae by other Physiology and biochemistries and molecular biology method; Negative through the LAMP detection, detect affirmation by Physiology and biochemistry and molecular biology method and all do not contain streptococcus agalactiae.Above result shows this detection method high specificity, and detected result accurately and reliably.
Claims (3)
1. two pairs of primers that are used for streptococcus agalactiae LAMP rapid detection, two pairs of Auele Specific Primers are: GBS-F3 and GBS-B3, GBS-FIP and GBS-BIP, above-mentioned two pairs of LAMP primers are identical or complementary with the nucleotide sequence of corresponding position on the short hemolytic factor gene of tilapia streptococcus agalactiae respectively, above-mentioned two pairs of primer sequences composed as follows:
GBS-F3:5’-AGTAATAGCCTCATTAACCGG-3’；
GBS-B3: 5’-CTAGTGGCTGGTGCATTG-3’；
GBS-FIP:
5’-GCTCAAAAGCTTGATCAAGATAGCTTTTCTGTTCCCTGAACATTATCTTTG-3’；
GBS-BIP:
5’-TTACTTGATTTACCACTTGTGGAGTTTTTTTCACCAGCTGTATTAGAAGT-3’。
2. test kit that utilizes loop-mediated isothermal amplification technique rapid detection tilapia streptococcus agalactiae is characterized in that reagent comprises:
1. extract streptococcus agalactiae genome reagent: 5% chelex-100 complex liquid; Sterile distilled water;
Above-mentioned 5% chelex-100 complex liquid is made of in proportion following component: 5% chelex-100, and formulated by TEX, final pH is adjusted to 10.4; TEX consists of: 10mmol/L Tris, pH=8.0; 1mmol/LEDTA; 1%TritonX-100;
2. the LAMP reaction reagent is made up of reaction solution A and reaction solution B:
Reaction solution A includes 10 * LAMP Buffer, Bst archaeal dna polymerase, dNTP, trimethyl-glycine, ultrapure water, inner primer GBS-F3 and GBS-B3, and outer primer GBS-FIP and GBS-BIP, above-mentioned two pairs of primer sequences composed as follows:
GBS-F3:5’-AGTAATAGCCTCATTAACCGG-3’；
GBS-B3: 5’-CTAGTGGCTGGTGCATTG-3’；
GBS-FIP:
5’-GCTCAAAAGCTTGATCAAGATAGCTTTTCTGTTCCCTGAACATTATCTTTG-3’；
GBS-BIP:
5’-TTACTTGATTTACCACTTGTGGAGTTTTTTTCACCAGCTGTATTAGAAGT-3’;
Above-mentioned 10 * LAMP Buffer consists of: 200mM Tris-Hcl, pH8.8; 100 mM Repone K; 100 mM ammonium sulfate; 20mM sal epsom; 1%TritonX-100;
Reaction solution B is fluorescence dye.
3. a kind of test kit that utilizes loop-mediated isothermal amplification technique rapid detection tilapia streptococcus agalactiae according to claim 2 is characterized in that described fluorescence dye is 1000 * SYBR Green I.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103789410A (en) * | 2013-12-11 | 2014-05-14 | 广西大学 | Visual LAMP detection kit for streptococcic mastitis pathogenic bacteria |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102888458A (en) * | 2012-09-27 | 2013-01-23 | 广西工学院 | Method for quickly detecting (Streptococcus agalactiae)sip with high sensitivity |
| CN103255090B (en) * | 2013-05-06 | 2015-01-07 | 中国农业科学院兰州畜牧与兽药研究所 | Rapid separation and identification kit for streptococcus agalactiae and application for same |
| CN103695552B (en) * | 2013-12-25 | 2017-05-17 | 广西科技大学 | Culture-independent semiquantitative detection kit of tilapia streptococcus agalactiae |
| CN104263842B (en) * | 2014-10-05 | 2016-08-03 | 中山大学 | A kind of fluorescent quantitative PCR detection method of source of fish streptococcus agalactiae |
| CN107541509B (en) * | 2016-06-29 | 2020-10-09 | 博奥生物集团有限公司 | LAMP primer combination for detecting 6 infectious pathogens of cow mastitis and application thereof |
| CN108018377B (en) * | 2017-12-07 | 2021-07-20 | 中国水产科学研究院珠江水产研究所 | RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection primer group, kit and method for Luo lake virus |
| CN108531630A (en) * | 2018-06-06 | 2018-09-14 | 上海速创诊断产品有限公司 | The streptococcic primer sets of detection B races include the primer liquid and kit of the primer sets and its application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101890159A (en) * | 2010-06-23 | 2010-11-24 | 广西壮族自治区水产研究所 | Method for preparing bivalent inactivated vaccine of tilapia streptococcus |
-
2012
- 2012-07-26 CN CN 201210260204 patent/CN102747165B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101890159A (en) * | 2010-06-23 | 2010-11-24 | 广西壮族自治区水产研究所 | Method for preparing bivalent inactivated vaccine of tilapia streptococcus |
Non-Patent Citations (4)
| Title |
|---|
| LAMP检测无乳链球菌方法的建立及应用;王永等;《华北农学报》;20091231;第24卷(第5期);第234-238页 * |
| 广东省罗非鱼主养区无乳链球菌的分离、鉴定与致病性;柯剑;《广东海洋大学学报》;20100630;第30卷(第3期);第22-27页 * |
| 柯剑.广东省罗非鱼主养区无乳链球菌的分离、鉴定与致病性.《广东海洋大学学报》.2010,第30卷(第3期),第22-27页. |
| 王永等.LAMP检测无乳链球菌方法的建立及应用.《华北农学报》.2009,第24卷(第5期),第234-238页. |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103789410A (en) * | 2013-12-11 | 2014-05-14 | 广西大学 | Visual LAMP detection kit for streptococcic mastitis pathogenic bacteria |
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