CN105349672A - Mycoplasma hyopneumoniae loop-mediated isothermal amplification kit and application thereof - Google Patents
Mycoplasma hyopneumoniae loop-mediated isothermal amplification kit and application thereof Download PDFInfo
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Abstract
The invention discloses a mycoplasma hyopneumoniae loop-mediated isothermal amplification kit and the application thereof. The kit comprises LAMP primers, 2x reaction buffer, Bst DNA polymerase, a fluorescence visual detection reagent, ultrapure water and a mycoplasma hyopneumoniae DNA template, wherein the LAMP primers include outer primers F3 and B3, inner primers FIP and BIP, and loop primers LF and LB. The kit can be applied to detection of lesion tissue of suspected mycoplasma pneumoniae of swine and a mycoplasma hyopneumoniae culture. Specificity detection and sensitivity detection prove that by the adoption of the LAMP detection method, reaction can be monitored in real time, the mycoplasma hyopneumoniae copy number can be detected, a detection result can be obtained quickly and accurately, and convenience is brought to easy, quick and reliable detection of mycoplasma hyopneumoniae.
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 mycoplasma hyopneumoniae and application thereof specifically.
Background technology
Mycoplasma hyopneumoniae (Mycoplasmahyopneumoniae, Mhp) be cause porcine mycoplasmal pneumonia (mycoplasmalpneumoniaofswine, MPS), also a kind of important cause of disease of swine enzootic pneumonia is claimed, by Goodwin(1964) isolate from cell-free medium first, Shanghai Academy of Agricultural Sciences's animal and veterinary institute was separated to pathogenic strain first in 1973 subsequently, the Chronic exposure respiratory infectious disease that this cause of disease mainly causes pig to be cardinal symptom with cough and asthma.
Mhp, by brone infection, is difficult to isolation and blocks its route of transmission, causing swine enzootic pneumonia sickness rate high, and Epidemic Scope is wide, in wide-scale distribution all over the world, with contact, hyperinfection, chronic, high incidence and low case fatality rate for feature.The dysplasia of sick pig long term growth, growth rate and feed conversion rate reduce, pig immunity is caused to reduce after infection, the often polyinfection such as viral with pasteurella multocida, actinobacillus pleuropneumoniae, porcine reproductive and respiratory syndrome clinically, porcine respiratory syndrome (porcinechronicrespiratoryinfectiousdisease, PRDC) a most important cause of disease, very large to the harm of pig industry, cause serious financial loss to pig industry.
To accurate, the rapid detection of mycoplasma hyopneumoniae, be not only conducive to plant and correct treatment measures are made to mycoplasma pneumonia of swine, and be conducive to the control of other porcine respiratory diseases.Existing in the Methods of Detection of Pathogens of mycoplasma hyopneumoniae, pathogen separation is the method the most accurately that porcine mycoplasmal pneumonia is made a definite diagnosis, but the Isolation and culture of current mycoplasma hyopneumoniae and qualification, the equipment that difficulty is large, sensitivity is low, consuming time, needs are complicated and culture condition, so in developing country and the limited area of China's clinical condition, set up one simply, fast diagnostic method just seem particularly important.Polymerase chain reaction (PCR) although spy's property led such as method and susceptibility all higher, need the expensive precision instrument such as PCR instrument, electrophoresis chamber, be not suitable for development at the basic level.
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 mycoplasma hyopneumoniae exactly, disclose a kind of detection mycoplasma hyopneumoniae loop-mediated isothermal amplification kit of quick, real-time quantitative.The technical scheme used for realizing the object of the invention is: a kind of mycoplasma hyopneumoniae loop-mediated isothermal amplification kit, this test kit comprises LAMP primer, 2 × reaction buffer, BstDNA polysaccharase, fluorescence visual detection reagent, ultrapure water and mycoplasma hyopneumoniae DNA profiling, and described LAMP primer comprises outer primer F3(SEQIDNO:1) and B3(SEQIDNO:2), inner primer FIP(SEQIDNO:3) and BIP(SEQIDNO:4) and ring primer LF(SEQIDNO:5) and LB(SEQIDNO:6); Wherein the sequence of primer is respectively:
F3AGGCAGCAACTTCAACAA
B3ATCAGATCCAGTAATTAGTCGA
FIPGGCGCTGGACTTAAATTAGCTCAAAAAACCAGGATGCACAA
BIPATTGCCCCTGAAAATGGAAGTTCATAGGCAACAATCGGAAT
LFGCTTGCTGAGTGAGTCAGTTAT
LBAGTTGGAACTGCTGTTAATACA
2 described × reaction buffer comprises Tris-HCL, KCL, MgSO4, (NH4) 2SO4, Tween20, Betaine and dNTPs.
Described mycoplasma hyopneumoniae DNA profiling is the genomic dna of the clinical diseased lung tissue of mycoplasma hyopneumoniae culture or the suspected infection mycoplasma hyopneumoniae using bacterial genomes DNA extraction kit to extract.
Described fluorescence visual detection reagent adopts fluorexon fluorescent reagent, and fluorescent reagent adds before the reaction.
2 described × reaction buffer comprises Tris-HCL25-60mM, KCL15-40mM, MgSO410-20mM, (NH4) 2SO415-25mM, Tween200.3-0.8 ℅, Betaine1.5-4M and dNTPs2.5-5mM, above-mentioned solvent, under pH is about 8.5 conditions, evenly obtains by its compound method.
An application for mycoplasma hyopneumoniae loop-mediated isothermal amplification kit, whether there is mycoplasma hyopneumoniae for the clinical diseased lung tissue detecting doubtful porcine mycoplasmal pneumonia or detection suspected infection mycoplasma hyopneumoniae, concrete detecting step comprises:
(1) Design and synthesis of LAMP primer
(2) extraction of mycoplasma hyopneumoniae DNA profiling
(3) LAMP reaction system is set up
(4) LAMP detection method.
Described LAMP reaction system is set up in 25 μ L,
2 × reaction buffer 12.5 μ L
BstDNA polysaccharase 1 μ L
FIP40pmol
BIP40pmol
F35pmol
LF20pmol
LB20pmol
Mycoplasma hyopneumoniae DNA2 μ L
Ultrapure water supplies 25 μ L.
Described LAMP detection method is the method adopting specific detection, sensitivity Detection and fluorescent visual to detect.
Described LAMP detection method adopts the real-time turbidimeter of LoopampLA-320C to carry out airtight complete monitoring.
Substantive distinguishing features of the present invention and significant progress are:
1) high specificity
LAMP method specific detection of the present invention goes out mycoplasma hyopneumoniae, and the negative control mycoplasma detected, bacterium and all no positive result of water contrast are out, consistent with PCR detected result.
2) highly sensitive
The sensitivity of regular-PCR detection method is 1.50 × 10-5ng/ μ L, and uses LAMP detection method of the present invention, and detectability is about 1.50 × 10-7ng/ μ L, is 100 times of regular-PCR.
3) result is obtained 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, agarose gel electrophoresis ultraviolet Imaging Analysis must be adopted to carry out sentence read result, from extracting genome DNA to acquisition test-results, need 4-5 hours.Amplification is there is in LAMP detection method reaction provided by the invention at about 13 minutes, can complete amplification in 60 minutes, and result interpretation mode is easy, under visible light positive and negative pipe is compared, obviously can see that positive reaction is obvious muddiness by naked eyes, negative reaction pipe is transparent; Of short duration centrifugal after, have obvious white magnesium pyrophosphate throw out bottom positive reaction pipe, and a sediment-free bottom negative reaction pipe; Or add fluorescence dye, positive reaction pipe under ultraviolet light in green, with the naked eye just observable experimental result.Not needing to carry out the video picture of agarose gel electrophoresis ultraviolet analysis again or uncap to add fluorescence dye and carry out carrying out sentence read result, can complete in 2-3 hour to obtaining net result from extracting genome DNA.
4) do not pollute
The fluorescence dye that current LAMP method is used for directly observing is for adding after reaction, and fluorescence dye of the present invention is the fluorexon commercial dyes (non-syber-green) added before the reaction, and testing process does not need to uncap, and effectively avoids test for contamination environment.In addition, LAMP detection method of the present invention, in result interpretation, directly carrys 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 Tubidimeterreal-timeLA-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, substitute into typical curve equation, the mycoplasma hyopneumoniae copy number of each time can be obtained, reach the object of detection by quantitative product.
Accompanying drawing explanation
Fig. 1 is the specific outcome of mycoplasma hyopneumoniae LAMP detection method of the present invention; Wherein 1: mycoplasma hyopneumoniae; 2: mycoplasma ovine pneumoniae; 3: acholeplasma modicum; 4: thread mycoplasma; 5: intestinal bacteria; 6:2 type suis; 7: Salmonellas; 8: bordetella bacilli; 9: blank (water).There is the upcurve of turbidity in result display mycoplasma hyopneumoniae reaction tubes, is positive findings, and 3 strain contrast mycoplasma reaction tubess, 4 strain contrast bacterial reaction pipes and water control reaction Guan Junwu increase, and are negative findings.
Fig. 2 and Fig. 3 is the result of the mycoplasma hyopneumoniae sensitivity Detection using LAMP method of the present invention and regular-PCR method to carry out respectively, wherein M:Mark; 1:1.50 × 10ng/ μ L; 2:1.50ng/ μ L; 3:1.50 × 10-1ng/ μ L; 4:1.50 × 10-2ng/ μ L; 5:1.50 × 10-3ng/ μ L; 6:1.50 × 10-4ng/ μ L; 7:1.50 × 10-5ng/ μ L; 8:1.50 × 10-6ng/ μ L; 9:1.50 × 10-7ng/ μ L;
10：water。The initial concentration of mycoplasma hyopneumoniae original DNA is 1.50 × 10ng/ μ L, after 10 times of multiple proportions serial dilutions, carry out LAMP and pcr amplification, result shows LAMP method detectability of the present invention and is about 1.50 × 10-7ng/ μ L, and regular-PCR method detectability is about 1.50 × 10-5ng/ μ L.
Fig. 4 is the fluorescent visual detected result of mycoplasma hyopneumoniae LAMP detection method of the present invention: the response situation that Zuo Guanwei is template with mycoplasma hyopneumoniae genomic dna, is positive findings, and right pipe is the response situation of negative control, is negative findings.
Fig. 5 is mycoplasma hyopneumoniae LAMP detection method quantitation curves of the present invention; Utilize the typical curve that turbidity value corresponding to the concentration of different standard models was depicted as the time, substitute into typical curve equation, the mycoplasma hyopneumoniae copy number of each time can be obtained.
Embodiment
1, the preparation of material
Mycoplasma hyopneumoniae, mycoplasma ovine pneumoniae, acholeplasma modicum, thread mycoplasma, intestinal bacteria, suis 2 type, Salmonellas and bordetella bacilli are Guangxi veterinary institute isolation identification and preservation.LAMPDNA amplification kit purchased from Beijing Lanpu Biological Technology Co., Ltd., article No. LMP204; Bacterial genomes DNA extraction kit is century bio tech ltd purchased from health, article No. CW0552.
2, the Design and synthesis of LAMP primer
According to the mycoplasma hyopneumoniae P46 gene order in GenBank, utilize a set of LAMP primer of LAMP method primer Autocad PrimerExplorerV4 software design, wherein F3, B3 are outer primer, and FIP, BIP are inner primer, wherein F3, B3 are that mycoplasma hyopneumoniae PCR detects primer, wherein
F3AGGCAGCAACTTCAACAA
B3ATCAGATCCAGTAATTAGTCGA
FIPGGCGCTGGACTTAAATTAGCTCAAAAAACCAGGATGCACAA
BIPATTGCCCCTGAAAATGGAAGTTCATAGGCAACAATCGGAAT
LFGCTTGCTGAGTGAGTCAGTTAT
LBAGTTGGAACTGCTGTTAATACA
3, bacterial genomes DNA extraction
The bacterial genomes DNA extraction kit using health to produce for century bio tech ltd, extracts the genomic dna of the lung tissue of mycoplasma hyopneumoniae DNA or doubtful mycoplasma hyopneumoniae infection, and the genomic dna of control strain.
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
FIP40pmol
BIP40pmol
F35pmol
LF20pmol
LB20pmol
Mycoplasma hyopneumoniae DNA2 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, the starting copy number of this sample can be calculated from typical curve, temperature of reaction with 63 DEG C as temperature of reaction.
5, LAMP detection method
1) specific detection
Bacterial genomes DNA extraction kit is used to extract the genomic dna of mycoplasma hyopneumoniae, mycoplasma ovine pneumoniae, acholeplasma modicum, thread mycoplasma, intestinal bacteria, 2 type suis, Salmonellas and bordetella bacilli, as the template of LAMP reaction, carry out each LAMP amplification, simultaneously using water as blank, the specificity of inspection LAMP method.
2) sensitivity Detection
The mycoplasma hyopneumoniae genomic dna extracted, measure its concentration, 9 extent of dilution are become with the continuous 10 times of doubling dilutions of RNA-FreeWater, using each DNA extent of dilution as template, carry out LAMP method amplification of the present invention and standard PCR amplification, contrast LAMP method of the present invention and regular-PCR to the susceptibility detecting mycoplasma hyopneumoniae.
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 laboratory that electrophoresis observation causes.
The specific outcome of embodiment 1LAMP detection method
LAMP amplification is carried out to 1 strain mycoplasma hyopneumoniae, 3 strain contrast mycoplasmas, 4 strain contrast bacteriums and water contrast, result as shown in Figure 1, the upcurve of turbidity is there is in mycoplasma hyopneumoniae reaction tubes at about 13 minutes, for positive findings, 3 strain contrast mycoplasma reaction tubess, 4 strain contrast bacterial reaction pipes and water control reaction pipe curve all occur without amplification situation, are negative findings.
The susceptibility results of embodiment 2LAMP detection method
The initial concentration of mycoplasma hyopneumoniae original DNA is 1.50 × 10ng/ μ L, after 10 times of multiple proportions serial dilutions, carry out LAMP and pcr amplification, result as shown in Figures 2 and 3, result shows LAMP method detectability of the present invention and is about 1.50 × 10-7ng/ μ L, and the detection of Standard PCR method is limited to 1.50 × 10-5ng/ μ L.
The fluorescent visual detected result of embodiment 3LAMP 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, the response situation that Zuo Guanwei is template with mycoplasma hyopneumoniae genomic dna, for positive findings, right pipe is negative control, is negative findings.Test-results shows, the LAMP method of foundation 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, can rapid examination result, and without the need to uncapping, avoid pollution.
The drafting of embodiment 4 mycoplasma hyopneumoniae quantitation curves
With mycoplasma hyopneumoniae DNA for template, outer primer F3 and B3 designed with this LAMP method is pcr amplification primer, the goal gene fragment obtained by pcr amplification is connected to carrier pMD18-T, transformation of E. coli recipient cell DH5a, ampicillin resistant screening obtains mono-clonal bacterium, extract the plasmid DNA of restructuring, after order-checking confirms, measure the initial concentration of recombinant plasmid pMD18-T-P46, as standard model, carry out continuous 10 times of doubling dilutions 9 extent of dilution with RNA-FreeWater, get each extent of dilution 2 μ L and carry out LAMP amplification as template
Contrast is set: concentration is 1.50 × 10ng/ μ L, 1.50ng/ μ L, 1.50 × 10-1ng/ μ L, 1.50 × 10-2ng/ μ L, 1.50 × 10-3ng/ μ L, 1.50 × 10-4ng/ μ L, 1.50 × 10-5ng/ μ L, each one of the recombinant plasmid pMD18-T-P46 standard model of 1.50 × 10-6ng/ μ L and 1.50 × 10-7ng/ μ L, because the negative logarithm of sample concentration and its amplification 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=0.7112x-9.9726, as shown in Figure 5.Be 0.9907 from typical curve equation relation conefficient R2, 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 is multiplied by radix 1.50, is 1.50x10-yng/ μ L.According to copy number reduction formula copies/ μ L=(6.02x1023x (ng/ulx10-9))/(DNAlengthx660), DNAlength is that carrier sequence size adds goal gene sequence size, for 2693+179=2872bp, be converted into copy number (copies/ μ L): 6.02x1023x(1.50x10-yx10-9)/(2872x660), after simplification is: 4.76x108x10-y.As certain test sample reach turbidity value be 0.1 time be 17 minutes time, bring set up typical curve equation into, obtain Y and equal 2.1178, then concentration is 10-2.1178, be multiplied by radix 1.50 again, be concentration 1.50 × 10-2.1178ng/ μ L of this test sample, then its copy number is: 4.76x108x10-2.1178=4.76x105.8822copies/ μ L, thus reaches quantitative effect.
Table 1 sample concentration negative logarithm and MHP-LAMP turbidity value linearly relation table
| Time | 13 | 14 | 15 | 18 | 20 | 21 |
| Concentration bears logarithm | -1 | 0 | 1 | 3 | 4 | 5 |
sequence table
<110> Veterinary Institute of Guangxi Zhuang Autonomous Region
<120> mycoplasma hyopneumoniae loop-mediated isothermal amplification kit and application thereof
<160>6
<210>1
<211>18
<212>DNA
<213> artificial sequence
<400>1
AGGCAGCAACTTCAACAA
<210>2
<211>22
<212>DNA
<213> artificial sequence
<400>2
ATCAGATCCAGTAATTAGTCGA
<210>3
<211>41
<212>DNA
<213> artificial sequence
<400>3
GGCGCTGGACTTAAATTAGCTCAAAAAACCAGGATGCACAA
<210>4
<211>41
<212>DNA
<213> artificial sequence
<400>4
ATTGCCCCTGAAAATGGAAGTTCATAGGCAACAATCGGAAT
<210>5
<211>22
<212>DNA
<213> artificial sequence
<400>5
GCTTGCTGAGTGAGTCAGTTAT
<210>6
<211>22
<212>DNA
<213> artificial sequence
<400>6
AGTTGGAACTGCTGTTAATACA
Claims (8)
1. a mycoplasma hyopneumoniae loop-mediated isothermal amplification kit, it is characterized in that, this test kit comprises LAMP primer, 2 × reaction buffer, BstDNA polysaccharase, fluorescence visual detection reagent, ultrapure water and mycoplasma hyopneumoniae DNA profiling, and described LAMP primer comprises outer primer F3(SEQIDNO:1) and B3(SEQIDNO:2), inner primer FIP(SEQIDNO:3) and BIP(SEQIDNO:4) and ring primer LF(SEQIDNO:5) and LB(SEQIDNO:6);
Wherein the sequence of primer is respectively:
F3AGGCAGCAACTTCAACAA
B3ATCAGATCCAGTAATTAGTCGA
FIPGGCGCTGGACTTAAATTAGCTCAAAAAACCAGGATGCACAA
BIPATTGCCCCTGAAAATGGAAGTTCATAGGCAACAATCGGAAT
LFGCTTGCTGAGTGAGTCAGTTAT
LBAGTTGGAACTGCTGTTAATACA
2 described × reaction buffer comprises Tris-HCL, KCL, MgSO
4, (NH
4)
2sO
4, Tween20, Betaine and dNTPs.
2. mycoplasma hyopneumoniae loop-mediated isothermal amplification kit according to claim 1, it is characterized in that, described mycoplasma hyopneumoniae DNA profiling is the genomic dna of the clinical diseased lung tissue of mycoplasma hyopneumoniae culture or the suspected infection mycoplasma hyopneumoniae using bacterial genomes DNA extraction kit to extract.
3. mycoplasma hyopneumoniae 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. mycoplasma hyopneumoniae loop-mediated isothermal amplification kit according to claim 1, is characterized in that, 2 described × reaction buffer comprises Tris-HCL25-60mM, KCL15-40mM, MgSO
410-20mM, (NH
4)
2sO
415-25mM, Tween200.3-0.8 ℅, Betaine1.5-4M and dNTPs2.5-5mM.
5. the application of a mycoplasma hyopneumoniae loop-mediated isothermal amplification kit, it is characterized in that, whether the clinical diseased lung tissue etc. for detecting doubtful mycoplasma hyopneumoniae or detection suspected infection mycoplasma hyopneumoniae exists mycoplasma hyopneumoniae, and concrete detecting step comprises:
(1) Design and synthesis of LAMP primer
(2) extraction of mycoplasma hyopneumoniae DNA profiling
(3) LAMP reaction system is set up
(4) LAMP detection method.
6. the application of mycoplasma hyopneumoniae loop-mediated isothermal amplification kit according to claim 5, is characterized in that, described LAMP reaction system is set up in 25 μ L,
2 × reaction buffer 12.5 μ L
BstDNA polysaccharase 1 μ L
FIP40pmol
BIP40pmol
F35pmol
LF20pmol
LB20pmol
Mycoplasma hyopneumoniae DNA2 μ L
Ultrapure water supplies 25 μ L.
7. the application of mycoplasma hyopneumoniae 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 mycoplasma hyopneumoniae 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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| CN110218819A (en) * | 2019-06-14 | 2019-09-10 | 广西壮族自治区兽医研究所 | A kind of high-throughput micro-fluidic chip and detection method detecting porcine respiratory epidemic disease cause of disease |
| CN112662793A (en) * | 2021-01-15 | 2021-04-16 | 首都医科大学附属北京友谊医院 | Primer and kit for detecting mycoplasma pneumoniae |
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