CN105779655A - Porcine torque teno sus virus type 1 loop-mediated isothermal amplification kit and application thereof - Google Patents
Porcine torque teno sus virus type 1 loop-mediated isothermal amplification kit and application thereof Download PDFInfo
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- CN105779655A CN105779655A CN201610259272.2A CN201610259272A CN105779655A CN 105779655 A CN105779655 A CN 105779655A CN 201610259272 A CN201610259272 A CN 201610259272A CN 105779655 A CN105779655 A CN 105779655A
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Abstract
The invention discloses a porcine torque teno sus virus type 1 loop-mediated isothermal amplification kit and application thereof. The kit comprises an LAMP primer, a 2*reaction buffer solution, a BstDNA (BstDeoxyribonucleic Acid) polymerase, a fluorescent visual inspection reagent, ultra-pure water and a porcine torque teno sus virus type 1 DNA template, wherein the LAMP primer comprises outer primers F3 and B3, inner primers FIP and BIP and loop primers LF and LB. The application of the porcine torque teno sus virus type 1 loop-mediated isothermal amplification kit comprises the following steps: detecting a porcine torque teno sus virus type 1 diseased tissue sample by using the kit and performing porcine torque teno sus virus type 1 pathogen qualitative research. Specificity detection and sensitivity detection verify that an LAMP detection method provided by the invention can monitor the reaction in real time, quantitatively detect the copy number of a porcine torque teno sus virus type 1 and quickly and accurately obtain detection results, thereby bringing convenience to simple, convenient, quick and reliable detection of the porcine torque teno sus virus type 1.
Description
Technical field
The present invention relates to technical field of microbial detection, particularly relate to a kind of quickly, visualization and can the loop-mediated isothermal amplification kit of Real_time quantitative detection pig thin circovirus virus 1 type and application thereof.
Background technology
Thin circovirus virus (the Torquetenosusvirus of pig, TTSuV) it is a kind of without cyst membrane, the annular DNA virus of sub-thread minus strand, according to its nucleotide sequence difference, TTSuV can be divided into two hypotypes and TTSuV1 and TTSuV2, broadly fall into finger ring Viraceae (Anelloviridae), in type finger ring Tobamovirus in the ninth of the ten Heavenly Stems (Iotatorquevirus), found by the Japanese scholars Nishizawa serum equal to hepatitis after transfusion in 1997.But TTV host range is widely, except infecting common domestic birds and animals, also can infect and include people and other primates.The swinery of different age group is all to the thin circovirus virus (Torquetenosusvirus of pig, TTSuV) susceptible, China's swinery infection rate is substantially 24%～100%, wherein show from the morbidity pig of 6 provinces and cities of China and the testing result of health pig sample equal to acquiring between 2008～2009 years according to king Meng Meng, TTSuV1 infection rate is about 37.6%, TTSuV2 infection rate is about 82.6%, and the two mixed infection rate is 38.4%.From now on, people still do not know that TTSuV's is pathogenic, current research only recognizes that TTSuV exists certain synergism with the porcine reproductive respiratory syndrome virus virus such as (PRRSV), pig circular ring virus (PCV), can increase the weight of pmws (PMWS) and the symptom of the Corii Sus domestica inflammation nephrotic syndrome when there is mixed infection.
Monitoring and quick diagnosis closely to pig thin circovirus virus 1 type, is conducive to this disease and the preventing and treating of other coinfection diseases.Currently, the existence of TTSuV1 can be detected by technological means such as Standard PCR, quantitative fluorescent PCR and sleeve type PCRs, but these technology still also exist some shortcomings on using, and hardware aspect is had higher requirements, also need to carry out agarose gel electrophoresis on result judges, easily causing laboratory pollution and cause false positive results occur, the used time is also longer, is not suitable for basic unit and Site Detection.Hence set up a kind of simple and sensitive TTSuV1 detection method, there is certain clinical meaning.
Summary of the invention
It is an object of the invention to provide a kind of method detecting pig thin circovirus virus 1 type exactly easy for basic unit, quick, disclose the thin circovirus virus 1 type loop-mediated isothermal amplification kit of detection pig of a kind of quick, real-time quantitative.The technical scheme used for realizing the object of the invention is: a thin circovirus virus 1 type loop-mediated isothermal amplification kit of boar, this test kit includes TTSuV1-LAMP primer, 2 × reaction buffer, BstDNA polymerase, fluorescence visual detection reagent, ultra-pure water and TTSuV1-DNA template, and described TTSuV1-LAMP primer includes 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 as follows:
F3AAGACCCCGCCAAGGTAC
B3CACTGCTCGTGCTTGAGAT
FIPTGTGGGGAGTTCGAGCAGTCTTCAGTCCTCAACCCTTGGGA
BIPGGAGGAGACGGAGAAGGCGTGCTCTCTTCGTCGGAGTCT
LFGTTCTAACAATCCTGTCACAGTCAT
LBACCCACTCCTTGGACAAAAGA
2 described × reaction buffer includes Tris-HCL, KCL, MgSO4、(NH4)2SO4, Tween20, Betaine and dNTPs.
The thin circovirus virus 1 type DNA profiling of described pig, is use virus genom DNA/RNA genomic DNA extracted in the clinical pathological tissues of suspected infection pig thin circovirus virus 1 type that test kit extracts.
Described fluorescence visual detection reagent adopts calcein fluorometric reagent, and fluorometric reagent adds before the reaction.
2 described × reaction buffer includes Tris-HCL35-55mM, KCL10-30mM, MgSO415-28mM、(NH4)2SO418-28mM, Tween200.5-1.5, Betaine1.3-3.5M and dNTPs2.4-4.8mM, its compound method is under about 8.6 conditions at pH, is obtained by above-mentioned solvent mix homogeneously.
The application of the one thin circovirus virus 1 type loop-mediated isothermal amplification kit of boar, veterinary clinic is used for detect in doubtful pig thin circovirus virus 1 type pathological tissues or culture and whether contains pig thin circovirus virus 1 type, and concrete detecting step includes:
(1) design of LAMP primer and synthesis
(2) extraction of the thin circovirus virus 1 type DNA profiling of pig
(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 polymerase 1 μ L
FIP40pmol
BIP40pmol
F35pmol
B35pmol
LF20pmol
LP20pmol
Pig thin circovirus virus 1 type DNA2 μ L
Ultra-pure water supplies 25 μ L.
Described LAMP detection method is the method adopting specific detection, sensitivity Detection and fluorescent visual detection.
Described LAMP detection method adopts the real-time transmissometer of LoopampLA-320C to carry out airtight complete monitoring, and reaction temperature is 63 DEG C, reaction appearance amplification between 18-25 minute.
The substantive distinguishing features of the present invention with significant progress is:
1) high specificity
The LAMP method specific detection of the present invention goes out pig thin circovirus virus 1 type, the negative control virus detected and water and compares all no positive result out, consistent with PCR testing result.And easy and simple to handle, quickly obtain testing result, without instrument costly.
2) highly sensitive
The sensitivity of regular-PCR detection method is 9.25 × 10-6Ng/ μ L, and use the LAMP detection method of the present invention, detection limit is about 9.25 × 10-8Ng/ 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, most LAMP reaction methods set up are after the completion of reaction at present, agarose gel electrophoresis ultraviolet Imaging Analysis must be adopted to carry out sentence read result, from extracting genome DNA to obtaining result of the test, it is necessary to 4-5 hours.There is amplification at about 19 minutes in LAMP detection method provided by the invention reaction, amplification can be completed in 60 minutes, and result interpretation mode is easy, is compared by positive and negative pipe under visible light, can being clearly visible positive reaction in substantially muddy by naked eyes, negative reaction pipe is transparent；Of short duration centrifugal after, have significantly white magnesium pyrophosphate precipitate bottom positive reaction pipe, and a deposit-free bottom negative reaction pipe；Or addition fluorescent dye, positive reaction pipe under ultraviolet light in green, with the naked eye just observable experimental result.Agarose gel electrophoresis ultraviolet analysis development need not be carried out again or addition fluorescent dye of uncapping carries out carrying out sentence read result, can complete in 2-3 hour from extracting genome DNA to obtaining final result.
4) do not pollute
Current LAMP method is addition after reaction for the fluorescent dye directly observed, and the fluorescent dye of the present invention is the calcein commercial dyes (non-syber-green) added before the reaction, and detection process need not be uncapped.In addition, the LAMP detection method of the present invention is in result interpretation, and the turbidity value detecting reaction tube either directly through transmissometer carrys out result of determination, can not carry out fluorescent dye determination testing result or carry out agarose gel electrophoresis testing result, need not uncap, pollution can be prevented effectively from.
5) can real-time quantitative
The present invention utilizes Tubidimeterreal-timeLA-320 transmissometer to analyze the result of LAMP reaction in real time, the standard curve that the time of the turbidity value that the concentration of different standard sample is corresponding is depicted as, substitute into standard curve equation, the thin circovirus virus 1 type copy number of pig of each time can be obtained, reach the purpose of detection by quantitative product.
Accompanying drawing explanation
Fig. 1 is LAMP method specific detection result of the present invention；Wherein 1: pig thin circovirus virus 1 type；2: pig thin circovirus virus 2 type；3: PRV (Pseudorabies virus)；4: swine fever virus；5: PRRS virus；6: pig parvoviral；7: pig gyrate virus II type；8: epidemic diarrhea virus；9: blank (water).There is the ascending curve of turbidity in the thin circovirus virus 1 type reaction tube of pig, and for positive findings, 7 strain comparison virus reaction tube and water control reaction Guan Junwu amplifications, for negative findings.
Fig. 2 and Fig. 3 is the result of the thin circovirus virus 1 type sensitivity Detection of pig using LAMP method of the present invention and regular-PCR method to carry out respectively, wherein 1:9.25 × 10ng/ μ L；2:9.25ng/ μ L；3:9.25 × 10-1ng/μL；4:9.25 × 10- 2ng/μL；5:9.25 × 10-3ng/μL；6:9.25 × 10-4ng/μL；7:9.25 × 10-5ng/μL；8:9.25 × 10-6ng/μL；9:9.25 × 10-7ng/μL；10:9.25 × 10-8ng/μL；11:water.The initial concentration of the thin circovirus virus 1 type original DNA of pig is 9.25 × 10ng/ μ L, after 10 times of multiple proportions serial dilutions, carries out LAMP and pcr amplification, and the LAMP method detection limit of the result display present invention is about 9.25 × 10-8Ng/ μ L, and regular-PCR method detection limit is about 9.25 × 10-6ng/μL。
Fig. 4 is the response situation that after addition fluorescent dye, visual results: Zuo Guanwei is template with the thin circovirus virus 1 type genomic DNA of pig, and for positive findings, right pipe is the response situation of negative control, for negative findings.
Fig. 5 is the thin circovirus virus 1 type quantitation curves of pig of the present invention；The standard curve that time is depicted as by the turbidity value utilizing the concentration of different standard sample corresponding, substitutes into standard curve equation, can obtain the thin circovirus virus 1 type copy number of pig of each time.
Detailed description of the invention
1, the preparation of material
Pig thin circovirus virus 1 type, pig thin circovirus virus 2 type, PRV (Pseudorabies virus), swine fever virus, PRRS virus, pig parvoviral, pig gyrate virus II type and epidemic diarrhea virus；Malicious or Guangxi veterinary institute isolation identification and preservation for commercial available vaccines.LAMPDNA amplification kit is purchased from Beijing Lanpu Biological Technology Co., Ltd., article No. LMP204；It is century bio tech ltd purchased from health that virus genom DNA/RNA extracts test kit, article No. CW0552.
2, the design of LAMP primer and synthesis
According to the thin circovirus virus 1 type ORF2-ORF3 gene order of the pig in GenBank, utilizing the LAMP method primer Autocad a set of LAMP primer of PrimerExplorerV4 software design, wherein F3, B3 are outer primer, and FIP, BIP are inner primer, and LF and LB is ring primer；Wherein F3, B3 are the thin circovirus virus 1 type PCR detection primer of pig, wherein
F3AAGACCCCGCCAAGGTAC
B3CACTGCTCGTGCTTGAGAT
FIPTGTGGGGAGTTCGAGCAGTCTTCAGTCCTCAACCCTTGGGA
BIPGGAGGAGACGGAGAAGGCGTGCTCTCTTCGTCGGAGTCT
LFGTTCTAACAATCCTGTCACAGTCAT
LBACCCACTCCTTGGACAAAAGA
3, virus genom DNA extracts
Use health is the century virus genom DNA/RNA of bio tech ltd's production extract test kit, extracts the genomic DNA of the pathological tissues of pig thin circovirus virus 1 type DNA or the infection of doubtful pig thin circovirus virus 1 type, and compare viral genomic DNA.
4, LAMP reaction system is set up
According to test kit description, by 25 μ L system configurations:
2 × reaction buffer 12.5 μ L
BstDNA polymerase 1 μ L
FIP40pmol
BIP40pmol
F35pmol
B35pmol
LF20pmol
LP20pmol
Pig thin circovirus virus 1 type DNA2 μ L
Ultra-pure water supplies 25 μ L
LAMP reacts with real-time transmissometer (LA-320C, Rong Yan company of Japan) carry out the form of airtight complete monitoring and monitor the detection situation of this method, transmissometer monitor in real time amplification situation, can drawing standard curve, the time value that 0.1 turbidity value is corresponding is reached by obtaining unknown sample, the starting copy number of this sample can be calculated from standard curve, reaction temperature with 63 DEG C as reaction temperature.
5, LAMP detection method
1) specific detection
Use virus genom DNA/RNA to extract test kit and extract the genomic DNA of pig thin circovirus virus 1 type, pig thin circovirus virus 2 type, PRV (Pseudorabies virus), swine fever virus, PRRS virus, pig parvoviral, pig gyrate virus II type and epidemic diarrhea virus, template as LAMP reaction, carry out each LAMP amplification, simultaneously using water as blank, check the specificity of LAMP method.
2) sensitivity Detection
The thin circovirus virus 1 type genomic DNA of pig extracted, measure its concentration, 10 dilution factors are become with the continuous 10 times of doubling dilutions of RNA-FreeWater, using each DNA dilution factor as template, carry out LAMP method amplification and the standard PCR amplification of the present invention, the LAMP method of the contrast present invention and the regular-PCR sensitivity to detection pig thin circovirus virus 1 type.
3) fluorescent visual detection
According to the condition that transmissometer monitoring optimizes, add fluorescent dye, fluorescent dye adds before the reaction, the dyestuff added is calcein commercial dyes, after reacting 60 minutes at 63 DEG C, observe under uviol lamp, do not adopt agarose gel electrophoresis ultraviolet analysis to develop, it is to avoid uncap and run the Aerosol Pollution laboratory that electrophoresis observation causes.
The specific outcome of embodiment 1LAMP detection method
1 strain pig thin circovirus virus 1 type, 7 strain comparison viruses and water comparison are carried out LAMP amplification, result is as shown in Figure 1, there is the ascending curve of turbidity at about 19 minutes in the thin circovirus virus 1 type reaction tube of pig, for positive findings, 7 strain comparison virus reaction tubes and water control reaction pipe curve all occur without amplification situation, for negative findings.
The susceptibility results of embodiment 2LAMP detection method
The initial concentration of the thin circovirus virus 1 type original DNA of pig is 9.25 × 10ng/ μ L, after 10 times of multiple proportions serial dilutions, carries out LAMP and pcr amplification, and as shown in Figures 2 and 3, the LAMP method detection limit of the result display present invention is about 9.25 × 10 to result-8Ng/ μ L, and the detection of Standard PCR method is limited to 9.25 × 10-6ng/μL。
The fluorescent visual testing result of embodiment 3LAMP detection method
According to transmissometer monitoring optimize condition, reactor add fluorescent dye, 63 DEG C reaction 60 minutes after, observing under uviol lamp, Fig. 4 is observed result, the response situation that Zuo Guanwei is template with the thin circovirus virus 1 type genomic DNA of pig, for positive findings, right pipe is negative control, for negative findings.Result of the test shows, the LAMP method of foundation can facilitate basic unit to use, and only need to use the LAMP primer that test kit coordinates this method to design, after adding sample, keep 63 DEG C 60 minutes with cheap water-bath, get final product rapid examination result, and without uncapping, it is to avoid pollution.
The drafting of the thin circovirus virus 1 type quantitation curves of embodiment 4 pig
With pig thin circovirus virus 1 type DNA for template, outer primer F3 and the B3 designed with this LAMP method is for pcr amplification primer, the genes of interest fragment obtained by pcr amplification is connected to carrier pMD18-T, convert escherichia coli receptor cell DH5a, ampicillin resistant screening obtains monoclonal bacterium, extract the plasmid DNA of restructuring, after order-checking confirms, measure the initial concentration of recombiant plasmid pMD18-T-ORF2-ORF3, as standard sample, carry out continuous 10 dilution factors of 10 times of doubling dilutions with RNA-FreeWater, take each dilution factor 2 μ L and carry out LAMP amplification as template
Comparison is set: concentration is 9.25 × 101ng/μL、9.25ng/μL、9.25×10-1ng/μL、9.25×10-2ng/μL、9.25×10-3ng/μL、9.25×10-4ng/μL、9.25×10-5ng/μL、9.25×10-6ng/μL、9.25×10-7Ng/ μ L and 9.25 × 10-8Each one of the recombiant plasmid pMD18-T-ORF2-ORF3 standard sample of ng/ μ L, because it is linear that the negative logarithm of sample concentration expands the time value that turbidity value is 0.1 with it, so the value that transmissometer can be captured and time (such as table 1) make standard curve, obtain standard curve equation y=0.5249x-11.019, as shown in Figure 5.From standard curve equation correlation coefficient R2=0.9972, in good linear relationship.With the time for X value, can obtain the negative time number formulary of Y value and concentration, then concentration is 10-y, then it is multiplied by radix 9.26, it is 9.25x10-yng/μL.According to copy number reduction formula copies/ μ L=(6.02 × 1023×(ng/ul×10-9))/(DNAlength × 660), DNAlength is carrier sequence size plus genes of interest sequence size, for 2693+207=2900bp, is converted into copy number (copies/ μ L): 6.02 × 1023× (9.25 × 10-y×10-9)/(2900 × 660), after simplification be: 2.9 × 109×10-y.As certain test specimen reach the time that turbidity value is 0.1 be 20 minutes time, bring the standard curve equation set up into, obtain Y equal to-0.521, then concentration is 100.521, then it is multiplied by radix 9.25, it is the concentration 9.25 × 10 of this test specimen0.521Ng/ μ L, then its copy number is: 2.9 × 109×100.521=2.9×109.521Copies/ μ L, thus reaching quantitative effect.
Table 1 sample concentration negative logarithm and TTSuV1-LAMP turbidity value linearly relation table
| Time (divides) | 19 | 21 | 23 | 25 | 26.5 |
| Concentration bears logarithm | -1 | 0 | 1 | 2 | 3 |
Sequence table
<110>Veterinary Institute of Guangxi Zhuang Autonomous Region
<120>the one thin circovirus virus 1 type loop-mediated isothermal amplification kit of boar and application thereof
<160>6
<210>1
<211>18
<212>DNA
<213>artificial sequence
<400>1
AAGACCCCGCCAAGGTAC
<210>2
<211>19
<212>DNA
<213>artificial sequence
<400>2
CACTGCTCGTGCTTGAGAT
<210>3
<211>41
<212>DNA
<213>artificial sequence
<400>3
TGTGGGGAGTTCGAGCAGTCTTCAGTCCTCAACCCTTGGGA
<210>4
<211>39
<212>DNA
<213>artificial sequence
<400>4
GGAGGAGACGGAGAAGGCGTGCTCTCTTCGTCGGAGTCT
<210>5
<211>25
<212>DNA
<213>artificial sequence
<400>5
GTTCTAACAATCCTGTCACAGTCAT
<210>6
<211>21
<212>DNA
<213>artificial sequence
<400>6
ACCCACTCCTTGGACAAAAGA
Claims (9)
1. a thin circovirus virus 1 type loop-mediated isothermal amplification kit of boar, it is characterized in that, this test kit includes LAMP primer, and its primer includes 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 as follows:
F3AAGACCCCGCCAAGGTAC
B3CACTGCTCGTGCTTGAGAT
FIPTGTGGGGAGTTCGAGCAGTCTTCAGTCCTCAACCCTTGGGA
BIPGGAGGAGACGGAGAAGGCGTGCTCTCTTCGTCGGAGTCT
LFGTTCTAACAATCCTGTCACAGTCAT
LBACCCACTCCTTGGACAAAAGA。
2. the thin circovirus virus 1 type loop-mediated isothermal amplification kit of pig according to claim 1, it is characterised in that described test kit also includes the thin circovirus virus 1 type DNA profiling of 2 × reaction buffer, BstDNA polymerase, fluorescence visual detection reagent, ultra-pure water and pig；2 described × reaction buffer includes Tris-HCL, KCL, MgSO4、(NH4)2SO4, Tween20, Betaine and dNTPs.
3. the thin circovirus virus 1 type loop-mediated isothermal amplification kit of pig according to claim 2, it is characterised in that described fluorescence visual detection reagent adopts calcein fluorometric reagent, and fluorometric reagent adds before the reaction.
4. the thin circovirus virus 1 type loop-mediated isothermal amplification kit of pig according to claim 2, it is characterised in that 2 described × reaction buffer includes Tris-HCL35-55mM, KCL10-30mM, MgSO415-28mM、(NH4)2SO418-28mM, Tween200.5-1.5, Betaine1.3-3.5M and dNTPs2.4-4.8mM.
5. the application of the thin circovirus virus 1 type loop-mediated isothermal amplification kit of pig according to claim 2, it is characterised in that concrete operation step includes:
(1) design of LAMP primer and synthesis
(2) extraction of the thin circovirus virus 1 type DNA profiling of pig
(3) LAMP reaction system is set up
(4) LAMP detection method.
6. the application of the thin circovirus virus 1 type loop-mediated isothermal amplification kit of pig according to claim 5, it is characterised in that described LAMP reaction system is set up in 25 μ L,
2 × reaction buffer 12.5 μ L
BstDNA polymerase 1 μ L
FIP40pmol
BIP40pmol
F35pmol
B35pmol
LF20pmol
LP20pmol
Pig thin circovirus virus 1 type DNA2 μ L
Ultra-pure water supplies 25 μ L.
7. the application of the thin circovirus virus 1 type loop-mediated isothermal amplification kit of pig according to claim 5, it is characterised in that described LAMP detection method is the method adopting specific detection, sensitivity Detection and fluorescent visual detection.
8. the application of the thin circovirus virus 1 type loop-mediated isothermal amplification kit of pig according to claim 5, it is characterised in that described LAMP detection method adopts real-time transmissometer to carry out airtight complete monitoring.
9. the application of the thin circovirus virus 1 type loop-mediated isothermal amplification kit of pig according to claim 5, it is characterised in that described LAMP detection method reaction temperature is 63 DEG C, reacts and amplification occurred at 18-25 minute.
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| CN107119121A (en) * | 2017-05-05 | 2017-09-01 | 广西壮族自治区兽医研究所 | A kind of real-time quantitative LAMP primer, kit and method for detecting bacillus rhusiopathiae suis |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107119121A (en) * | 2017-05-05 | 2017-09-01 | 广西壮族自治区兽医研究所 | A kind of real-time quantitative LAMP primer, kit and method for detecting bacillus rhusiopathiae suis |
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