CN102864226A - LAMP (loop-mediated isothermal amplification) kit for rapid detection of staphylococcus aureus - Google Patents
LAMP (loop-mediated isothermal amplification) kit for rapid detection of staphylococcus aureus Download PDFInfo
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Abstract
The invention discloses an LAMP (loop-mediated isothermal amplification) kit for rapid detection of staphylococcus aureus. The LAMP kit for rapid detection of staphylococcus aureus comprises LAMP reaction fluid, a standard positive template and a negative quality control standard. The LAMP reaction fluid contains large fragments in Bst DNA (deoxyribonucleic acid) polymerase, primers, LAMP 10Xbuffer, dNTPs (deoxyribonucleotide triphosphates) solution, MgSO4 solution and betaine. The primers include forward primers and reverse primers. The LAMP kit for rapid detection of staphylococcus aureus has the advantages that the LAMP kit is high in specificity, high in sensitivity, high in speed, simple and high in reliability, results are recognizable to naked eyes, and the like. The LAMP kit is applicable to rapid qualitative detection of staphylococcus aureus in industrial foods, and can be a substitute for commonly used traditional culture methods and serodiagnosis methods.
Description
Technical field
The present invention relates to a kind of LAMP test kit of fast detecting Staphylococcus aureus, belong to the food inspection field.
Background technology
Streptococcus aureus (Staphylococcus aureus) is a kind of Gram-positive spherical bacteria.Microscopically is arranged in thyrsiform, and streptococcus aureus is without gemma, flagellum, and is most of without pod membrane.The common pathogenic bacterium that cause food poisoning.Be common in skin surface and upper respiratory tract mucous membrane.Streptococcus aureus is modal pathogenic bacteria in the human suppurative infection, can cause local suppurative infection, also can cause pneumonia, pseudomembranous enteritis, pericarditis etc., even the systemic infection such as septicemia, Sepsis.
The ring mediated isothermal amplification that development in recent years is got up (loop-mediated isothermal amplification, the advantages such as LAMP) technology is highly sensitive with it, speed fast, high specificity are being used widely aspect the gene qualitative detection, and have become one of important method of current viral nucleic acid qualitative detection.
Ring mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) technology is a kind of novel isothermal nucleic acid amplification method, this method is for 4 special primers of 6 zone design of target gene, utilize a kind of archaeal dna polymerase (Bst archaeal dna polymerase) with strand displacement activity, at the about 60min of constant temperature (about 65 ℃) insulation, can finish nucleic acid amplification reaction, produce macroscopic byproduct of reaction-white magnesium pyrophosphate precipitation.This technology has does not need PCR instrument, amplified production not to need to uncap, and it is short with the naked eye to get final product judged result and reaction times, high specificity, the advantages such as sensitivity height.
The cultural method of traditional detection streptococcus aureus needs separation, screening and biochemical identification, also needs in case of necessity serological identification, is generally 4～6d, wastes time and energy, and has the shortcomings such as sensitivity is low, poor specificity, false positive, time and effort consuming.Various conventional PCR method have the advantages such as susceptibility is strong, specificity is high, easy, quick, the report that has more application to detect in streptococcus aureus, but owing to exist the PCR aftertreatment to produce the problem such as false positive that pollution causes and the special plant and instrument of needs has limited its application in grass-roots unit.Yin Ci Qi accurate, sensitive, quick, free of contamination Clinical Laboratory method to be developed.
Summary of the invention
Technical problem to be solved by this invention provides a kind of LAMP test kit and using method thereof of fast detecting Staphylococcus aureus.
For achieving the above object, the present invention by the following technical solutions:
A kind of LAMP test kit of fast detecting Staphylococcus aureus, this test kit comprises: a) LAMP reaction solution, b) standard positive template, c) negative quality control standard product; The LAMP reaction solution contains primer, and primer is any a group in following 6 group-specific primerses:
(1) upstream outer primer F3:5 '-GCGATTGATGGTGATACGGTTA-3 '; (SEQ ID No.1) downstream outer primer B3:5 '-GCCACGTCCATATTTATCAGTTCT-3 '; (SEQ ID No.2)
Upstream inner primer FIP:5 '-GGATGCTTTGTTTCAGGTGTATCATGTACAAAGGTCAACCAATGAC-3 '; (SEQ ID No.3)
Downstream inner primer BIP:5 '-TATGGTCCTGAAGCAAGTGCAGACCTTTGTCAAACTCGACTTC-3 '; (SEQ ID No.4)
(2) upstream outer primer F3:5 '-ACATAAAGAACCTGCGACA-3 '; (SEQ ID No.5)
Downstream outer primer B3:5 '-CTTTGTCAAACTCGACTTCA-3 '; (SEQ ID No.6)
Upstream inner primer FIP:5 '-CTGAATGTCATTGGTTGACCTTTGTAATTAAAGCGATTGATGGTGAT-3 '; (SEQ ID No.7)
Downstream inner primer BIP:
5′-CACCTGAAACAAAGCATCCTAAAAAGCATTTTCTACCATCTTTTTCGTA-3′（SEQ ID No.8）
(3) upstream outer primer F3:5 '-GCGACATTAATTAAAGCGATTG-3 '; (SEQ ID No.9)
Downstream outer primer B3:5 '-CTTTGTCAAACTCGACTTCAA-3 '; (SEQ ID No.10)
Upstream inner primer FIP:
5′-GCTTTGTTTCAGGTGTATCAACCAAATTAATGTACAAAGGTCAACCAATG-3′；（SEQ ID No.11）
Downstream inner primer BIP:5 '-AAGGTGTAGAGAAATATGGTCCTGATTCTTTGCATTTTCTACCATCT-3 '; (SEQ ID No.12)
(4) upstream outer primer F3:5 '-CGACATTAATTAAAGCGATTGATG-3 '; (SEQ ID No.13)
Downstream outer primer B3:5 '-CTTTGTCAAACTCGACTTCA-3 '; (SEQ ID No.14)
Upstream inner primer FIP:5 '-GGATGCTTTGTTTCAGGTGTATCATGTACAAAGGTCAACCAATG-3 '; (SEQ ID No.15)
Downstream inner primer BIP:5 '-AAGGTGTAGAGAAATATGGTCCTGATTCTTTGCATTTTCTACCATCT-3 '; (SEQ ID No.16)
(5) upstream outer primer F3:5 '-GCGATTGATGGTGATACTGTT-3 '; (SEQ ID No.17)
Downstream outer primer B3:5 '-CACGTCCATATTTATCAGTTCTT-3 '; (SEQ ID No.18)
Upstream inner primer FIP:5 '-AGGATGCTTTGTTTCAGGTGTATTACAAAGGTCAACCAATGACAT-3 '; (SEQ ID No.19)
Downstream inner primer BIP:5 '-AAGGTGTAGAGAAATATGGTCCTGATCGACTTCAATTTTCTTTGCA-3 '; (SEQ ID No.20)
(6) upstream outer primer F3:5 '-AGGTCAACCAATGACATTCA-3 '; (SEQ ID No.21)
Downstream outer primer B3:5 '-AATATACGCTAAGCCACGT-3 '; (SEQ ID No.22)
Upstream inner primer FIP:5 '-CAGGACCATATTTCTCTACACCTTTACTATTATTGGTTGATACACCTGA-3 '; (SEQ ID No.23)
Downstream inner primer BIP:5 '-AGCAAGTGCATTTACGAAAAAGATGTTTATCAGTTCTTTGACCTTTGTC-3 '; (SEQ ID No.24)
Specifically,
A) LAMP reaction solution
The LAMP reaction solution is by outer primer, the inner primer of 10 μ mol/L, 5mol/L trimethyl-glycine, the 50mmol/L MgSO of Bst archaeal dna polymerase large fragment, LAMP10 * buffer, 10 μ mol/L
4Form with 10mmol/L dNTPs.
In a concrete scheme of the present invention, the LAMP reaction solution is by LAMP 10 * buffer 2.5 μ l, and 10 μ mol/L inner primer FIP, BIP be 2.0 μ l respectively, each 1.0 μ l of 10 μ mol/L outer primer F3, B3,10mmol/L dNTPs3.0 μ l, 50mmol/L MgSO
43.0 μ l, 5.0mol/L trimethyl-glycine 4.5 μ l, Bst archaeal dna polymerase large fragment 1.0 μ l form, reaction solution cumulative volume 20 μ l.
B) standard positive template
Standard positive template is the pMD18-T-nuc carrier that contains the nucleotide fragments formation of 349 base pairs of streptococcus aureus high conservative gene nuc gene, and this carrier can be bred in bacillus coli DH 5 alpha.
The nucleotides sequence of nuc gene is classified as:
5′-TACATAAAGAACCTGCGACATTAATTAAAGCGATTGATGGTGATACTGTTAAATTAATGTACAAAGGTCAACCAATGACATTCAGACTATTATTGGTTGATACACCTGAAACAAAGCATCCTAAAAAAGGTGTAGAGAAATATGGTCCTGAAGCAAGTGCATTTACGAAAAAGATGGTAGAAAATGCAAAGAAAATTGAAGTCGAGTTTGACAAAGGTCAAAGAACTGATAAATATGGACGTGGCTTAGCGTATATTTATGCTGATGGAAAAATGGTAAACGAAGCTTTAGTTCGTCAAGGCTTGGCTAAAGTTGCTTATGTTTATAAACCTAACAATACACATGAACA-3′。
（SEQ ID No.25）
C) negative quality control standard product
Negative quality control standard product are aseptic double-distilled water.
The principle of work of this test kit:
Use quick visualization provided by the invention to detect streptococcus aureus LAMP test kit, the about 60min of (about 65 ℃) insulation under constant temperature, can finish nucleic acid amplification reaction, and supervene macroscopic byproduct of reaction-white magnesium pyrophosphate precipitation.After reaction finishes, the centrifugal 30s of 12000r/min, pipe end white precipitate can detect by an unaided eye.In fast detecting Staphylococcus aureus LAMP test kit provided by the invention, for the singularity in the streptococcus aureus detection, different target fragments is carried out reaction system, such as primer, dNTPs, Mg
2+The optimization of concentration, trimethyl-glycine etc., pass through prioritization scheme, repeatedly experiment, set up the loop-mediated isothermal amplification method that detects streptococcus aureus, and develop the LAMP test kit that detects streptococcus aureus, the sensitivity of this test kit can detect 10 copies in each reaction system, can satisfy the requirement of fast detecting Staphylococcus aureus.
Use LAMP test kit of the present invention to detect the method for streptococcus aureus, may further comprise the steps:
A) cultivate sample 1-4h to be checked with buffered peptone water (BPW) according to National Standard Method;
B) from testing sample, extract DNA with water-boiling method;
C) get b) step in sample DNA join in the LAMP reaction solution, 65 ℃ the insulation 1h; Do respectively positive control and negative control with standard positive template and negative quality control standard product simultaneously;
D) after reaction finished, centrifugal, the visual inspection precipitation was carried out qualitative analysis to sample.
The fast detecting Staphylococcus aureus LAMP test kit that provides among the present invention can carry out qualitative detection to streptococcus aureus, and alternative traditional culture method of always continuing to use and serum method detection method.Compared with prior art have the following advantages and effect:
1, compare with culture-based method, detection speed is fast, and only 1h adds the extraction preparation of sample DNA, altogether is no more than 5h.
2, specificity is good, and is highly sensitive;
3, step is simple, and is repeatable high;
4, can carry out simultaneously a plurality of sample detection.
Description of drawings
Fig. 1 is the centrifugal rear observation white precipitate result of streptococcus aureus among the embodiment 1, among the figure:
1-standard positive template, 2-negative control, 3-streptococcus aureus positive sample, 4-streptococcus aureus negative sample, 5-streptococcus aureus positive sample, 6-streptococcus aureus negative sample, 7-streptococcus aureus positive sample, 8-streptococcus aureus negative sample;
Fig. 2 is streptococcus aureus 1.5% agarose gel electrophoresis detected result figure among the embodiment 1, among the figure:
M-DNA Marker, 1-standard positive template, 2-negative control, 3-streptococcus aureus positive sample, 4-streptococcus aureus positive sample, 5-streptococcus aureus positive sample, 6-streptococcus aureus negative sample, 7-streptococcus aureus negative sample, 8-streptococcus aureus negative sample;
Fig. 3 is the centrifugal rear observation white precipitate result of streptococcus aureus among the embodiment 2, among the figure:
1-standard positive template, 2-negative control, 3-streptococcus aureus positive sample, 4-streptococcus aureus negative sample, 5-streptococcus aureus positive sample, 6-streptococcus aureus negative sample, 7-streptococcus aureus positive sample, 8-streptococcus aureus negative sample;
Fig. 4 is the centrifugal rear observation white precipitate result of streptococcus aureus among the embodiment 3, among the figure:
1-standard positive template, 2-negative control, 3-streptococcus aureus positive sample, 4-streptococcus aureus negative sample, 5-streptococcus aureus positive sample, 6-streptococcus aureus negative sample, 7-streptococcus aureus positive sample, 8-streptococcus aureus negative sample;
Fig. 5 is the centrifugal rear observation white precipitate result of streptococcus aureus among the embodiment 4, among the figure:
1-standard positive template, 2-negative control, 3-streptococcus aureus positive sample, 4-streptococcus aureus negative sample, 5-streptococcus aureus positive sample, 6-streptococcus aureus negative sample, 7-streptococcus aureus positive sample, 8-streptococcus aureus negative sample;
Fig. 6 is the centrifugal rear observation white precipitate result of streptococcus aureus among the embodiment 5, among the figure:
1-standard positive template, 2-negative control, 3-streptococcus aureus positive sample, 4-streptococcus aureus negative sample, 5-streptococcus aureus positive sample, 6-streptococcus aureus negative sample, 7-streptococcus aureus positive sample, 8-streptococcus aureus negative sample;
Fig. 7 is the centrifugal rear observation white precipitate result of streptococcus aureus among the embodiment 6, among the figure:
1-standard positive template, 2-negative control, 3-streptococcus aureus positive sample, 4-streptococcus aureus negative sample, 5-streptococcus aureus positive sample, 6-streptococcus aureus negative sample, 7-streptococcus aureus positive sample, 8-streptococcus aureus negative sample;
Fig. 8 is the centrifugal rear observation white precipitate result of streptococcus aureus among the embodiment 7, among the figure:
1-standard positive template, 2-negative control, 3-streptococcus aureus sample, 4-Salmonella paratyphi A sample, 5-Salmonella typhimurium sample, 6-intestinal bacteria sample, 7-shigella dysenteriae sample, 8-Shigella sonnei sample;
Fig. 9 is that streptococcus aureus LAMP test kit sensitivity experiment is observed the white precipitate result among the embodiment 8, among the figure:
1-dilution 10
-1Doubly, 2-dilution 10
-2Doubly, 3-dilution 10
-3Doubly, 4-dilution 10
-4Doubly, 5-dilution 10
-5Doubly, 6-dilution 10
-6Doubly, 7-dilution 10
-7Doubly, 8-dilution 10
-8Doubly, 9-dilution 10
-9Doubly, 10-dilution 10
-10Doubly.
Embodiment
Below in conjunction with the implementation example, further set forth the present invention.Be to be understood that, these embodiments only are used for explanation the present invention and are not used in restriction the scope of protection of present invention, unreceipted concrete experiment condition and method in the following example, usually according to normal condition such as chief editors such as J. Pehanorm Brookers, Science Press, 1992, molecular cloning experiment guide (second edition); D.L. Spector etc., Science Press, 2001, cell experiment guide; Lv Hongsheng, Science Press, 1982, or connect the condition of advising according to manufacturer.
Embodiment 1:
1. novel quick visualization detects streptococcus aureus LAMP test kit composition and preparation, and reagent forms:
A) LAMP reaction mixture
The LAMP reaction mixture is by LAMP 10 * buffer 2.5 μ l, and 10 μ mol/L inner primer FIP, BIP be 2.0 μ l respectively, each 1.0 μ l of 10 μ mol/L outer primer F3, B3,10mmol/L dNTPs 3.0 μ l, 50mmol/LMgSO
43.0 μ l, 5.0mol/L trimethyl-glycine 4.5 μ l, Bst archaeal dna polymerase large fragment 1.0 μ l form, reaction solution cumulative volume 20 μ l.
Wherein primer sequence is as follows:
Upstream outer primer F3:5 '-GCGATTGATGGTGATACGGTTA-3 ';
Downstream outer primer B3:5 '-GCCACGTCCATATTTATCAGTTCT-3 ';
Upstream inner primer FIP:5 '-GGATGCTTTGTTTCAGGTGTATCATGTACAAAGGTCAACCAATGAC-3 ';
Downstream inner primer BIP:5 '-TATGGTCCTGAAGCAAGTGCAGACCTTTGTCAAACTCGACTTC-3 ';
B) standard positive template
Standard positive template pMD18-T-nuc is the pMD18-T-nuc carrier that contains the nucleotide fragments formation of 349 base pairs of streptococcus aureus high conservative gene nuc gene, and this carrier can be bred in bacillus coli DH 5 alpha; Testing used standard substance is the plasmid pMD18-T-invA that contains the purpose amplified fragments, uses alkaline lysis method of extracting after this plasmid transformation escherichia coli DH5 α propagation, through DNA purification kit purifying, and-20 ℃ of preservations.
C) negative quality control standard product
Negative quality control standard product are aseptic double-distilled water.
2. use described PCR test kit based on streptococcus aureus in the loop-mediated isothermal amplification technique rapid detection industrial food to detect the method for streptococcus aureus, may further comprise the steps:
A, foodstuff samples pre-treatment: get 3 portions of positive food of streptococcus aureus of identifying through culture-based method, 3 portions of negative food of streptococcus aureus, taking by weighing respectively 25g(mL) sample puts into the aseptic homogeneous cup that fills 225mL BPW, with 8000r/min～10000r/min homogeneous 1min～2min, or place the aseptic homogenizing bag that fills 225mL BPW, pat 1min～2min with slap type homogenizer.If sample is liquid, do not need homogeneous, the vibration mixing gets final product.Aseptic technique goes to sample in the 500mL Erlenmeyer flask, as using homogenizing bag, can directly cultivate, and cultivates 1-4h in 36 ℃ ± 1 ℃.As be frozen prods, should thaw being no more than 15min below 45 ℃.
The preparation of b, bacterium template DNA: get respectively 100 μ L bacterial culturess, the centrifugal 2min of 12000r/min removes supernatant, adds 50 μ L sterilized waters, abundant mixing, and 100 ℃ of water-bath 10min, the centrifugal 2min of 12000r/min, supernatant is template DNA.
C, LAMP reaction: get respectively 5 μ l b) the sample supernatant joins in the 20 μ l LAMP reaction solutions 65 ℃ of constant-temperature amplification 60min in the step.Do respectively positive control and negative control with standard positive template and negative quality control standard product simultaneously.
D, result judge: the centrifugal 30S of 12000r/min after question response finishes, observe to have or not to precipitate producing (seeing accompanying drawing 1).Get 5 μ l LAMP products and detect with 1.5% agarose gel electrophoresis, positive sample has band, and negative sample is then without band (seeing accompanying drawing 2).Accompanying drawing 1 is centrifugal rear observation white precipitate result, and accompanying drawing 2 is 1.5% agarose gel electrophoresis detected result.
E, conclusion: revision test is 3 times repeatedly, and the gained detected result is identical, and experimental group all has precipitation or electrophoretic band, and control group is all without precipitation or electrophoretic band, and is consistent with expected results.It is respond well to illustrate that this test kit detects streptococcus aureus, and the precipitation observation is consistent with electrophoresis detection method effect, and the detected result between the different batches has comparability, has good repeatability.
Embodiment 2:
1. novel quick visualization detects streptococcus aureus LAMP test kit composition and preparation, is that from the difference of embodiment 1 described test kit specific primer sequence is different, and the primer sequence of this test kit is as follows:
Upstream outer primer F3:5 '-ACATAAAGAACCTGCGACA-3 ';
Downstream outer primer B3:5 '-CTTTGTCAAACTCGACTTCA-3 ';
Upstream inner primer FIP:5 '-CTGAATGTCATTGGTTGACCTTTGTAATTAAAGCGATTGATGGTGAT-3 ';
Downstream inner primer BIP:
5′-CACCTGAAACAAAGCATCCTAAAAAGCATTTTCTACCATCTTTTTCGTA-3′
2. method and the embodiment 1 described method of using described PCR test kit based on streptococcus aureus in the loop-mediated isothermal amplification technique rapid detection industrial food to detect streptococcus aureus are identical, revision test is 3 times repeatedly, the gained detected result is identical, experimental group all has precipitation, control group is all without precipitation (seeing accompanying drawing 3), and is consistent with expected results.It is respond well to illustrate that this test kit detects streptococcus aureus, and the detected result between the different batches has comparability, has good repeatability.
Embodiment 3:
1. novel quick visualization detects streptococcus aureus LAMP test kit composition and preparation, is that from the difference of embodiment 1 described test kit specific primer sequence is different, and the primer sequence of this test kit is as follows:
Upstream outer primer F3:5 '-GCGACATTAATTAAAGCGATTG-3 ';
Downstream outer primer B3:5 '-CTTTGTCAAACTCGACTTCAA-3 ';
Upstream inner primer FIP:
5′-GCTTTGTTTCAGGTGTATCAACCAAATTAATGTACAAAGGTCAACCAATG-3′；
Downstream inner primer BIP:5 '-AAGGTGTAGAGAAATATGGTCCTGATTCTTTGCATTTTCTACCATCT-3 ';
2. method and the embodiment 1 described method of using described PCR test kit based on streptococcus aureus in the loop-mediated isothermal amplification technique rapid detection industrial food to detect streptococcus aureus are identical, revision test is 3 times repeatedly, the gained detected result is identical, experimental group all has precipitation, control group is all without precipitation (seeing accompanying drawing 4), and is consistent with expected results.It is respond well to illustrate that this test kit detects streptococcus aureus, and the detected result between the different batches has comparability, has good repeatability.
Embodiment 4:
1. novel quick visualization detects streptococcus aureus LAMP test kit composition and preparation, is that from the difference of embodiment 1 described test kit specific primer sequence is different, and the primer sequence of this test kit is as follows:
Upstream outer primer F3:5 '-CGACATTAATTAAAGCGATTGATG-3 ';
Downstream outer primer B3:5 '-CTTTGTCAAACTCGACTTCA-3 ';
Upstream inner primer FIP:5 '-GGATGCTTTGTTTCAGGTGTATCATGTACAAAGGTCAACCAATG-3 ';
Downstream inner primer BIP:5 '-AAGGTGTAGAGAAATATGGTCCTGATTCTTTGCATTTTCTACCATCT-3 ';
2. method and the embodiment 1 described method of using described PCR test kit based on streptococcus aureus in the loop-mediated isothermal amplification technique rapid detection industrial food to detect streptococcus aureus are identical, revision test is 3 times repeatedly, the gained detected result is identical, experimental group all has precipitation, control group is all without precipitation (seeing accompanying drawing 5), and is consistent with expected results.It is respond well to illustrate that this test kit detects streptococcus aureus, and the detected result between the different batches has comparability, has good repeatability.
Embodiment 5:
1. novel quick visualization detects streptococcus aureus LAMP test kit composition and preparation, is that from the difference of embodiment 1 described test kit specific primer sequence is different, and the primer sequence of this test kit is as follows:
Upstream outer primer F3:5 '-GCGATTGATGGTGATACTGTT-3 ';
Downstream outer primer B3:5 '-CACGTCCATATTTATCAGTTCTT-3 ';
Upstream inner primer FIP:5 '-AGGATGCTTTGTTTCAGGTGTATTACAAAGGTCAACCAATGACAT-3 ';
Downstream inner primer BIP:5 '-AAGGTGTAGAGAAATATGGTCCTGATCGACTTCAATTTTCTTTGCA-3 ';
2. method and the embodiment 1 described method of using described PCR test kit based on streptococcus aureus in the loop-mediated isothermal amplification technique rapid detection industrial food to detect streptococcus aureus are identical, revision test is 3 times repeatedly, the gained detected result is identical, experimental group all has precipitation, control group is all without precipitation (seeing accompanying drawing 6), and is consistent with expected results.It is respond well to illustrate that this test kit detects streptococcus aureus, and the detected result between the different batches has comparability, has good repeatability.
Embodiment 6:
1. novel quick visualization detects streptococcus aureus LAMP test kit composition and preparation, is that from the difference of embodiment 1 described test kit specific primer sequence is different, and the primer sequence of this test kit is as follows:
Upstream outer primer F3:5 '-AGGTCAACCAATGACATTCA-3 ';
Downstream outer primer B3:5 '-AATATACGCTAAGCCACGT-3 ';
Upstream inner primer FIP:5 '-CAGGACCATATTTCTCTACACCTTTACTATTATTGGTTGATACACCTGA-3 ';
Downstream inner primer BIP:5 '-AGCAAGTGCATTTACGAAAAAGATGTTTATCAGTTCTTTGACCTTTGTC-3 ';
2. method and the embodiment 1 described method of using described PCR test kit based on streptococcus aureus in the loop-mediated isothermal amplification technique rapid detection industrial food to detect streptococcus aureus are identical, revision test is 3 times repeatedly, the gained detected result is identical, experimental group all has precipitation, control group is all without precipitation (seeing accompanying drawing 7), and is consistent with expected results.It is respond well to illustrate that this test kit detects streptococcus aureus, and the detected result between the different batches has comparability, has good repeatability.
Embodiment 7:
The experiment of rapid detection Shigellae LAMP test kit specificity
The preparation of a, dna profiling: the streptococcus aureus of the DNA validation verification of learning from else's experience respectively, shigella dysenteriae, Shigella sonnei, Salmonella paratyphi A, Salmonella typhimurium, colibacillary bacterium liquid 10 μ l, the centrifugal 2min of 12000r/min, remove supernatant, add 50 μ L sterilized waters, abundant mixing, 100 ℃ of water-bath 10min, the centrifugal 2min of 12000r/min, supernatant is template DNA.
B, LAMP reaction: get 51 μ la) the sample supernatant joins in the 20 μ l LAMP reaction solutions 65 ℃ of constant-temperature amplification 60min in the step.Do respectively positive control and negative control with standard positive template and negative quality control standard product simultaneously.
C, result judge: the centrifugal 30S of 12000r/min after question response finishes, observe to have or not to precipitate producing (seeing accompanying drawing 8).Accompanying drawing 8 is streptococcus aureus LAMP test kit specificity experimental observation white precipitate result.
D, conclusion: result's demonstration only has positive controls, streptococcus aureus adularescent precipitation, and Salmonella paratyphi A, Salmonella typhimurium, intestinal bacteria, shigella dysenteriae, Shigella sonnei and negative control do not have white precipitate.
Above-mentioned this test kit of description of test has good specificity.
Embodiment 8: fast detecting Staphylococcus aureus LAMP test kit sensitivity experiment
A, the streptococcus aureus nutrient solution of getting the 1ml incubated overnight are done 10 times of doubling dilutions, are diluted to 10
-10Get each dilution bacterium liquid 10 μ l, the centrifugal 2min of 12000r/min removes supernatant, adds 50 μ L sterilized waters, abundant mixing, and 100 ℃ of water-bath 10min, the centrifugal 2min of 12000r/min, supernatant is template DNA.
B, LAMP the reaction: get 5 μ l a) go on foot in the sample supernatant join in the 20 μ l LAMP reaction solutions 65 ℃ of constant-temperature amplification 60min.
C, result judge: after reaction finished, whether the centrifugal 30s of 12000r/min produced white precipitate at the bottom of the observation tube, and the result shows 10
-1-10
-8Equal adularescent precipitation at the bottom of the sample tube, 10
-9-10
-10Then there is not white precipitate (seeing accompanying drawing 9) at the bottom of the sample tube.Accompanying drawing 9 is that streptococcus aureus LAMP test kit sensitivity experiment is observed the white precipitate result.
D, conclusion: the result shows that working as bacterium liquid is diluted to 10
-8In time, still can detect, and the lowest detection limit is 50CFU/ml.
Above-mentioned this test kit of description of test has good sensitivity.
Can illustrate from above-mentioned example, LAMP detection method specificity is good, highly sensitive, good reproducibility, easy and simple to handle, and the LAMP detection kit only needs just can finish less than 5 hours to the detection of sample, and traditional culture method just can be finished about 1 week of need approximately, therefore, use this test kit greatly to shorten detection time.
The operation of this test kit only needs 1 people can finish whole operating process, once can detect a plurality of (being needed to determine by the reality detection) sample, has so also reduced the waste of manpower.
SEQUENCE LISTING
＜110〉the true good fortune medical sci-tech in Wuhan Development Co., Ltd
＜120〉the LAMP test kit of fast detecting Staphylococcus aureus
<130>
<160> 25
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cgacattaat taaagcgatt gatg 24
<210> 14
<211> 20
<212> DNA
＜213〉artificial sequence
<400> 14
ctttgtcaaa ctcgacttca 20
<210> 15
<211> 44
<212> DNA
＜213〉artificial sequence
<400> 15
ggatgctttg tttcaggtgt atcatgtaca aaggtcaacc aatg 44
<210> 16
<211> 47
<212> DNA
＜213〉artificial sequence
<400> 16
aaggtgtaga gaaatatggt cctgattctt tgcattttct accatct 47
<210> 17
<211> 21
<212> DNA
＜213〉artificial sequence
<400> 17
gcgattgatg gtgatactgt t 21
<210> 18
<211> 23
<212> DNA
＜213〉artificial sequence
<400> 18
cacgtccata tttatcagtt ctt 23
<210> 19
<211> 45
<212> DNA
＜213〉artificial sequence
<400> 19
aggatgcttt gtttcaggtg tattacaaag gtcaaccaat gacat 45
<210> 20
<211> 46
<212> DNA
＜213〉artificial sequence
<400> 20
aaggtgtaga gaaatatggt cctgatcgac ttcaattttc tttgca 46
<210> 21
<211> 20
<212> DNA
＜213〉artificial sequence
<400> 21
aggtcaacca atgacattca 20
<210> 22
<211> 19
<212> DNA
＜213〉artificial sequence
<400> 22
aatatacgct aagccacgt 19
<210> 23
<211> 49
<212> DNA
＜213〉artificial sequence
<400> 23
caggaccata tttctctaca cctttactat tattggttga tacacctga 49
<210> 24
<211> 49
<212> DNA
＜213〉artificial sequence
<400> 24
agcaagtgca tttacgaaaa agatgtttat cagttctttg acctttgtc 49
<210> 25
<211> 349
<212> DNA
＜213〉streptococcus aureus
<400> 25
tacataaaga acctgcgaca ttaattaaag cgattgatgg tgatactgtt aaattaatgt 60
acaaaggtca accaatgaca ttcagactat tattggttga tacacctgaa acaaagcatc 120
ctaaaaaagg tgtagagaaa tatggtcctg aagcaagtgc atttacgaaa aagatggtag 180
aaaatgcaaa gaaaattgaa gtcgagtttg acaaaggtca aagaactgat aaatatggac 240
gtggcttagc gtatatttat gctgatggaa aaatggtaaa cgaagcttta gttcgtcaag 300
gcttggctaa agttgcttat gtttataaac ctaacaatac acatgaaca 349
Claims (4)
1. the LAMP test kit of a fast detecting Staphylococcus aureus is characterized in that: be comprised of LAMP reaction solution, standard positive template, negative quality control standard product; The LAMP reaction solution contains primer, and primer is any a group in following 6 group-specific primerses:
(1) upstream outer primer F3:5 '-GCGATTGATGGTGATACGGTTA-3 ';
Downstream outer primer B3:5 '-GCCACGTCCATATTTATCAGTTCT-3 ';
Upstream inner primer FIP:5 '-GGATGCTTTGTTTCAGGTGTATCATGTACAAAGGTCAACCAATGAC-3 ';
Downstream inner primer BIP:5 '-TATGGTCCTGAAGCAAGTGCAGACCTTTGTCAAACTCGACTTC-3 ';
(2) upstream outer primer F3:5 '-ACATAAAGAACCTGCGACA-3 ';
Downstream outer primer B3:5 '-CTTTGTCAAACTCGACTTCA-3 ';
Upstream inner primer FIP:5 '-CTGAATGTCATTGGTTGACCTTTGTAATTAAAGCGATTGATGGTGAT-3 ';
Downstream inner primer BIP:
5′-CACCTGAAACAAAGCATCCTAAAAAGCATTTTCTACCATCTTTTTCGTA-3′
(3) upstream outer primer F3:5 '-GCGACATTAATTAAAGCGATTG-3 ';
Downstream outer primer B3:5 '-CTTTGTCAAACTCGACTTCAA-3 ';
Upstream inner primer FIP:
5′-GCTTTGTTTCAGGTGTATCAACCAAATTAATGTACAAAGGTCAACCAATG-3′；
Downstream inner primer BIP:5 '-AAGGTGTAGAGAAATATGGTCCTGATTCTTTGCATTTTCTACCATCT-3 ';
(4) upstream outer primer F3:5 '-CGACATTAATTAAAGCGATTGATG-3 ';
Downstream outer primer B3:5 '-CTTTGTCAAACTCGACTTCA-3 ';
Upstream inner primer FIP:5 '-GGATGCTTTGTTTCAGGTGTATCATGTACAAAGGTCAACCAATG-3 ';
Downstream inner primer BIP:5 '-AAGGTGTAGAGAAATATGGTCCTGATTCTTTGCATTTTCTACCATCT-3 ';
(5) upstream outer primer F3:5 '-GCGATTGATGGTGATACTGTT-3 ';
Downstream outer primer B3:5 '-CACGTCCATATTTATCAGTTCTT-3 ';
Upstream inner primer FIP:5 '-AGGATGCTTTGTTTCAGGTGTATTACAAAGGTCAACCAATGACAT-3 ';
Downstream inner primer BIP:5 '-AAGGTGTAGAGAAATATGGTCCTGATCGACTTCAATTTTCTTTGCA-3 ';
(6) upstream outer primer F3:5 '-AGGTCAACCAATGACATTCA-3 ';
Downstream outer primer B3:5 '-AATATACGCTAAGCCACGT-3 ';
Upstream inner primer FIP:5 '-CAGGACCATATTTCTCTACACCTTTACTATTATTGGTTGATACACCTGA-3 ';
Downstream inner primer BIP:5 '-AGCAAGTGCATTTACGAAAAAGATGTTTATCAGTTCTTTGACCTTTGTC-3 '.
2. LAMP test kit according to claim 1 is characterized in that, the LAMP reaction solution is by outer primer, the inner primer of 10 μ mol/L, 5mol/L trimethyl-glycine, the 50mmol/L MgSO of Bst archaeal dna polymerase large fragment, LAMP10 * buffer, 10 μ mol/L
4Form with 10mmol/L dNTPs.
3. LAMP test kit according to claim 1 and 2, it is characterized in that, standard positive template is the pMD18-T-nuc carrier that contains the nucleotide fragments formation of 349 base pairs of streptococcus aureus high conservative gene nuc gene, and the nucleotide sequence of nuc gene is shown in SEQ ID No.25.
4. right to use requires 1～3 described LAMP test kit to detect the method for streptococcus aureus, it is characterized in that, may further comprise the steps:
1. cultivate sample 4h to be checked with buffered peptone water according to National Standard Method;
2. from testing sample, extract DNA with water-boiling method;
3. DNA is joined in the reaction system of LAMP test kit 65 ℃ of insulation 1h; Do respectively positive control and negative control with standard positive template and negative quality control standard product simultaneously;
4. after reaction finished, centrifugal, the visual inspection precipitation was carried out qualitative analysis to sample.
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| CN201210355602XA CN102864226A (en) | 2012-09-21 | 2012-09-21 | LAMP (loop-mediated isothermal amplification) kit for rapid detection of staphylococcus aureus |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210355602XA CN102864226A (en) | 2012-09-21 | 2012-09-21 | LAMP (loop-mediated isothermal amplification) kit for rapid detection of staphylococcus aureus |
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Cited By (9)
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| CN103305623A (en) * | 2013-07-01 | 2013-09-18 | 浙江省质量检测科学研究院 | Multiple rapid detection method of two food-borne pathogenic bacteria and detection primer groups as well as kit |
| CN104946780A (en) * | 2015-07-14 | 2015-09-30 | 东北农业大学 | Kit capable of rapidly detecting staphylococcus aureus in meat and application |
| CN105063220A (en) * | 2015-08-21 | 2015-11-18 | 上海市计量测试技术研究院 | Polynucleotide for staphylococcus aureus detection, method and kit |
| CN106434917A (en) * | 2016-09-24 | 2017-02-22 | 中华人民共和国广州机场出入境检验检疫局 | LAMP primer group and detection kit for staphylococcus aureus and use method of detection kit |
| WO2017067942A1 (en) * | 2015-10-19 | 2017-04-27 | Institut Pasteur | Detection of microbial pathogens related to bacterial infections through amplification especially by rt-lamp |
| CN105063220B (en) * | 2015-08-21 | 2018-08-31 | 上海市计量测试技术研究院 | A set of polynucleotides, method and kit for staphylococcus aureus detection |
| CN108546768A (en) * | 2018-05-11 | 2018-09-18 | 重庆出入境检验检疫局检验检疫技术中心 | The sandwich DNA hybridization of staphylococcus aureus, which quickly detects, uses probe, kit and detection method |
| CN110904256A (en) * | 2019-12-19 | 2020-03-24 | 内蒙古自治区农牧业科学院 | Primer group for simultaneously detecting salmonella, pasteurella multocida, staphylococcus aureus and avibacterium paragallinarum and application |
| CN111004855A (en) * | 2020-01-03 | 2020-04-14 | 上海海关动植物与食品检验检疫技术中心 | Primer combination and kit for detecting staphylococcus aureus |
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| CN103305623A (en) * | 2013-07-01 | 2013-09-18 | 浙江省质量检测科学研究院 | Multiple rapid detection method of two food-borne pathogenic bacteria and detection primer groups as well as kit |
| CN104946780A (en) * | 2015-07-14 | 2015-09-30 | 东北农业大学 | Kit capable of rapidly detecting staphylococcus aureus in meat and application |
| CN105063220A (en) * | 2015-08-21 | 2015-11-18 | 上海市计量测试技术研究院 | Polynucleotide for staphylococcus aureus detection, method and kit |
| CN105063220B (en) * | 2015-08-21 | 2018-08-31 | 上海市计量测试技术研究院 | A set of polynucleotides, method and kit for staphylococcus aureus detection |
| WO2017067942A1 (en) * | 2015-10-19 | 2017-04-27 | Institut Pasteur | Detection of microbial pathogens related to bacterial infections through amplification especially by rt-lamp |
| CN106434917A (en) * | 2016-09-24 | 2017-02-22 | 中华人民共和国广州机场出入境检验检疫局 | LAMP primer group and detection kit for staphylococcus aureus and use method of detection kit |
| CN108546768A (en) * | 2018-05-11 | 2018-09-18 | 重庆出入境检验检疫局检验检疫技术中心 | The sandwich DNA hybridization of staphylococcus aureus, which quickly detects, uses probe, kit and detection method |
| CN110904256A (en) * | 2019-12-19 | 2020-03-24 | 内蒙古自治区农牧业科学院 | Primer group for simultaneously detecting salmonella, pasteurella multocida, staphylococcus aureus and avibacterium paragallinarum and application |
| CN111004855A (en) * | 2020-01-03 | 2020-04-14 | 上海海关动植物与食品检验检疫技术中心 | Primer combination and kit for detecting staphylococcus aureus |
| WO2021135495A1 (en) * | 2020-01-03 | 2021-07-08 | 上海海关动植物与食品检验检疫技术中心 | Primer combination and kit for detecting staphylococcus aureus |
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