CN102876808B - Muscovy duckling parvovirus LAMP (loop-mediated isothermal amplification) detection reagent kit - Google Patents
Muscovy duckling parvovirus LAMP (loop-mediated isothermal amplification) detection reagent kit Download PDFInfo
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Abstract
The invention discloses a muscovy duckling parvovirus LAMP (loop-mediated isothermal amplification) detection reagent kit. A loop-mediated isothermal amplification primer group for detecting muscovy duckling parvoviruses is provided and comprises a primer 1, a primer 2, a primer 3 and a primer 4, and nucleotide sequences of the primer 1, the primer 2, the primer 3 and the primer 4 are sequence 1, sequence 2, sequence 3 and sequence 4 in a sequence table sequentially. Experiments prove that six primers aiming at eight specific areas are designed by the aid of conserved sequence design of MDPV (muscovy duckling parvoviruses), the muscovy duckling parvovirus LAMP detection reagent kit is more specific and sensitive than a conventional detection method when used for LAMP detection, only one temperature-controllable water bath kettle needs to be used, and results can be judged by naked eyes without apparatuses. Therefore, the muscovy duckling parvovirus LAMP detection reagent kit is suitable for rapid detection of grass-root veterinary stations and livestock farms and has good application prospect.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of kind Duck parvovirus LAMP detection kit.
Background technology
Kind Duck parvovirus (Muscovy duckling parvovirus, MDPV), is an important pathogen for harm kind duck cultivation, can cause that age in 1 week age-3 week, young bird kind duck fell ill, and lethality rate can reach 50%-80%.This virus is also very high virus of the susceptible of duckling and lethality rate.
Traditional detection method, need to be used expensive instrument and reagent etc.And loop-mediated isothermal amplification technique (loop-mediated isothermal amplification, LAMP) be to equal by Notomi a kind of novel nucleic acids amplification technique of setting up for 2000, that this technology has is easy and simple to handle, react the advantages such as quick, with low cost and result visualization, is widely used at present in the detection of some pathogenic micro-organisms.
At present, be showed no the relevant report of setting up the visual detection kit of MDPV LAMP and method both at home and abroad.
Summary of the invention
An object of the present invention is to provide a kind of loop-mediated isothermal amplification (LAMP) primer group of detection kind Duck parvovirus.
Primer sets provided by the invention, comprises primer 1, primer 2, primer 3 and primer 4;
The nucleotide sequence of described primer 1, described primer 2, described primer 3 and described primer 4 is followed successively by respectively sequence 1, sequence 2, sequence 3 and the sequence 4 in sequence table.
In above-mentioned primer sets, the mol ratio of described primer 1, described primer 2, described primer 3 and described primer 4 is 8:8:1:1.
Above-mentioned primer sets also comprises primer 5 and primer 6; The nucleotide sequence of described primer 5 and described primer 6 is followed successively by respectively sequence 5 and the sequence 6 in sequence table;
Above-mentioned primer sets is specifically made up of described primer 1, described primer 2, described primer 3, described primer 4, described primer 5 and described primer 6.
In above-mentioned primer sets, the mol ratio of described primer 1, described primer 2, described primer 3, described primer 4, described primer 5 and described primer 6 is 8:8:1:1:4:4.
The ring mediated isothermal amplification reagent of the detection that contains above-mentioned primer sets kind Duck parvovirus is also the scope of protection of the invention.
Above-mentioned ring mediated isothermal amplification reagent is by above-mentioned primer sets, ring mediated isothermal amplification damping fluid, archaeal dna polymerase, dNTPs, magnesium sulfate, fluorexon, trimethyl-glycine, MnCl
2form with water.
In above-mentioned ring mediated isothermal amplification reagent, the primer 1 in described primer sets and the described primer 2 final concentration in described ring mediated isothermal amplification reagent is 1.6umol/L;
Primer 3 in described primer sets and described primer 4 final concentration in described ring mediated isothermal amplification reagent is 0.2umol/L;
Primer 5 in described primer sets and described primer 6 final concentration in described ring mediated isothermal amplification reagent is 0.8umol/L.
Second object of the present invention is to provide a kind of loop-mediated isothermal amplification kit of detection kind Duck parvovirus.
Test kit provided by the invention, comprises above-mentioned primer sets or above-mentioned ring mediated isothermal amplification reagent.
Above-mentioned primer sets, above-mentioned ring mediated isothermal amplification reagent or above-mentioned loop-mediated isothermal amplification kit are in the application of preparing in the product that whether contains kind Duck parvovirus in detection and/or auxiliary detection testing sample.
Above-mentioned application adopts ring mediated isothermal amplification, and the condition of described ring mediated isothermal amplification is 63 DEG C of reaction 50h, 80 DEG C of effect 5min deactivation.Above-mentioned application is specially and utilizes respectively above-mentioned primer sets, above-mentioned reagent or mentioned reagent box to carry out loop-mediated isothermal amplification to described testing sample.
The conserved sequence that experiment showed, MDPV of the present invention of the present invention has designed 6 primers for 8 special regions, makes amplified reaction have very high specificity, increases by two ring primers and improved amplification efficiency between inner primer, has shortened the reaction times of LAMP.By optimizing after reaction conditions, set up the LAMP detection method of MDPV.The MDPV LAMP detection method of setting up only need to be reacted and can complete for 50 minutes in the water-bath of 62-64 DEG C, detection MDPV that can be special, and the sensitivity detecting is very high, the MDPV DNA sample of 10fg can be detected, and sensitivity is 10 times of conventional PCR.In a word, primer of the present invention and method, there is the feature more special, more responsive than conventional sense method, only need to use the water-bath of an energy temperature control, and result can only be judged by naked eyes without instrument, be adapted at carrying out rapid detection in veterinary station of basic unit and plant, there is good application prospect.
Brief description of the drawings
Fig. 1 is the specificity that LAMP method detects MDPV
Fig. 2 is the susceptibility that LAMP method detects MDPV
Fig. 3 is the susceptibility that PCR method detects MDPV
Fig. 4 is the partial results that clinical sample detects
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
In following embodiment, part material is as follows: Bst archaeal dna polymerase (large fragment) is purchased from New England Biolabs company; PCR test kit is purchased from Dalian precious biotechnology company limited; EasyPure viral DNA/RNA kit is purchased from Beijing Quanshijin Biotechnology Co., Ltd.
Duck I Hepatitis virus AV2111 strain (DHV AV2111) and kind Duck parvovirus MDPV(AV238) purchased from China Veterinery Drug Inspection Office;
Kind Duck parvovirus (A17F4) is documented in " separation and the qualification of Guangxi kind Duck parvovirus ", Guangxi animal and veterinary, and 2002,18 (6): 5-7, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
Duck circovirus (DuCV) is documented in " investigation of some areas of Guangxi duck circovirus infection conditions ", Chinese animal and veterinary, 2010,37(11): 156-158, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
Goose Parvovirus (GPV) is documented in " foundation of Goose Parvovirus fluorescent quantitative PCR detection method ", Shanghai animal and veterinary communication, and 2008,160 (06): 30-31, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
Avian pneumo-encephalitis virus is documented in " research of newcastle disease vegetables oil emulsion seedling ", Chinese Preventive Veterinary Medicine report, and 2000, (04): 23-27, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
Duck plague virus AV1221 strain is purchased from China Veterinery Drug Inspection Office;
H9 subtype avian influenza virus (AIV H9) is documented in " foundation that multiple reverse transcriptional PCR rapid detection is differentiated H9 subtype avian influenza virus method ", China Amphixenosis journal, 2006, (09): 858-860, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region.
The design of embodiment 1, primer
According to the gene conserved sequence of in GenBank kind of Duck parvovirus MDPV, use online software PrimerExplorer V4 (http://primerexplorer.jp/e/v4-manual/index.html) design LAMP primer.Primer is synthetic by Guangzhou Invitrogen company.Article 6, primer sequence is in table 1.
Table 1 is LAMP primer sequence
LF and LB primer do not add passable yet, add the susceptibility that can improve detection.
Embodiment 2, primer detect the application in kind Duck parvovirus at LAMP
One, the extraction of nucleic acid
Extract test kit specification sheets with reference to EasyPure viral DNA/RNA kit, extract RNA, kind Duck parvovirus MDPV(AV238 of duck I Hepatitis virus AV2111 strain) DNA, DNA, the DNA of duck circovirus of kind Duck parvovirus (A17F4), DNA, the RNA of H9 subtype avian influenza virus (AIV H9) of Goose Parvovirus, the RNA of Avian pneumo-encephalitis virus, the DNA of duck plague virus.Respectively above-mentioned RNA reverse transcription is obtained to cDNA.
Two, optimize LAMP reaction system and reaction conditions
25 μ L LAMP reaction systems: 1-4 μ L dNTPs (10mmol/L, final concentration is 0.4mmol/L-1.6mmol/L), 2.5 μ L10 × Bst buffer, 1ul Bst archaeal dna polymerase 8U(final concentration are 320U/L), 4-7 μ L trimethyl-glycine Betaine (5mmol/L, final concentration is 0.8mmol/L-1.4mmol/L), 2-9 μ LMgSO
4(25mmol/L, final concentration is 2mmol/L-9mmol/L), 2 μ L primers (FIP 20 μ mol/L, BIP 20 μ mol/L, LF 10 μ mol/L, LB10 μ mol/L, B3 7.5 μ mol/L, F3 7.5mol/L; Final concentration is FIP 1.6 μ mol/L, BIP 1.6 μ mol/L, LF 0.8 μ mol/L, LB 0.8 μ mol/L, B30.2 μ mol/L, F3 0.2mol/L), 1ul fluorexon Calcein (625 μ mol/L, final concentration is 25 μ mol/L), 1ul MnCl
2the DNA of (12.5mmol/L, final concentration is 0.5mmol/L) and 1 μ L template (kind Duck parvovirus MDPV(AV238)), add water to 25 μ L.
Temperature is increased progressively successively by 60 DEG C, 61 DEG C, 62 DEG C, 63 DEG C, 64 DEG C, 65 DEG C, repeatedly after revision test, determine optimum annealing temperature.
Optimum response system is as follows: 25 μ L LAMP reaction systems: 3.5 μ L dNTPs (10mmol/L, final concentration is 1.4mmol/L), 2.5 μ L10 × Bst buffer, 1ulBst archaeal dna polymerase 8U(final concentration are 320U/L), 5 μ L trimethyl-glycine Betaine (5mmol/L, final concentration is 1mmol/L), 7 μ L MgSO
4(25mmol/L, final concentration is 7mmol/L), 2 μ L primers (FIP 20 μ mol/L, BIP 20 μ mol/L, LF 10 μ mol/L, LF10 μ mol/L, B3 7.5 μ mol/L, F3 7.5mol/L, wherein the final concentration of each primer is FIP 1.6 μ mol/L, BIP 1.6 μ mol/L, LF 0.8 μ mol/L, LB 0.8 μ mol/L, B3 0.2 μ mol/L, F3 0.2mol/L), 1ul fluorexon Calcein (625 μ mol/L, final concentration is 25 μ mol/L), 1ul MnCl
2(12.5mmol/L, final concentration is 0.5mmol/L), 1 μ L template DNA, add water to 25 μ L.
Optimum reaction condition: 63 DEG C of reaction 50h, 80 DEG C of effect 5min deactivation.
Kind Duck parvovirus LAMP detection kit comprises 6 primers or the above-mentioned reaction system (removing template) of 4 primers of FIP, BIP, F3, B3 or FIP, BIP, F3, B3, LF, LB.
Three, specific detection
Respectively taking above-mentioned one cDNA, kind Duck parvovirus MDPV(AV238 that obtains duck I Hepatitis virus AV2111 strain) the DNA of cDNA, duck plague virus of cDNA, Avian pneumo-encephalitis virus of DNA, H9 subtype avian influenza virus (AIV H9) of DNA, Goose Parvovirus of DNA, duck circovirus of DNA, kind Duck parvovirus (A17F4) as template, carry out LAMP reaction according to optimum response system and optimum reaction condition in above-mentioned two.
The judgement of LAMP reaction result has 2 kinds:
1) colour-change that directly detects by an unaided eye: if reaction product color becomes green (emerald green), in interpret sample, contain a kind Duck parvovirus, otherwise, in interpret sample, do not contain a kind Duck parvovirus;
2) electrophoresis observation: LAMP reaction product is carried out agarose electrophoresis detection, and under gel imaging instrument observations; If reaction product has typical scalariform band continuously, in interpret sample, contain a kind Duck parvovirus; Otherwise, in sample, do not contain a kind Duck parvovirus;
Result as shown in Figure 1, A figure is electrophoresis detection result, B figure is visual inspection result, and wherein M is that 100bp DNAladder, 1 is that MDPV AV238,2 is that kind Duck parvovirus (A17F4), 3 is that AIV H9,4 is that duck circovirus, 5 is that duck plague virus, 6 is that Avian pneumo-encephalitis virus, 7 is that Goose Parvovirus, 8 is duck I Hepatitis virus AV2111,9 negative contrasts (water); Can find out, only have MDPV AV238(1) and the test positive result of kind Duck parvovirus (A17F4) (2) (visual inspection is emerald green as seen, agarose gel electrophoresis presents typical scalariform band continuously), and to all negative (visual inspection is orange-yellow as seen, does not all have characteristic trapezoid-shaped strips to occur) of the detection of DHV AV2111, H9 subtype avian influenza virus, duck circovirus, Avian pneumo-encephalitis virus, Goose Parvovirus and duck plague virus DNA/cDNA.
Four, sensitivity detects
By a kind Duck parvovirus MDPV(AV238) DNA by 10 times of doubling dilutions to 1ng/ul, 100pg/ul, 10pg/ul, 1pg/ul, 100fg/ul, 10fg/ul, 1fg/ul, 0.1fg/ul and 0.01fg/ul be template, carry out LAMP reaction according to optimum response system and optimum reaction condition in above-mentioned two.
Result as shown in Figure 2, A figure is electrophoresis detection result, B figure is visual inspection result, and wherein M is that 100bp DNAladder, 1 is that 1ng, 2 is that 100pg, 3 is that 10pg, 4 is that 1pg, 5 is that 100fg, 6 is that 10fg, 7 is that 1fg, 8 is that 0.1fg, 9 is 0.01fg, 10 negative contrasts (water); Can find out, it is emerald green as seen that LAMP is limited to 10fg(visual inspection to the minimum detection of MDPV AV238DNA, agarose gel electrophoresis expression characteristics trapezoid-shaped strips).
Control group: by a kind Duck parvovirus MDPV(AV238) DNA by 10 times of doubling dilutions to 1ng/ul, 100pg/ul, 10pg/ul, 1pg/ul, 100fg/ul, 10fg/ul, 1fg/ul, 0.1fg/ul and 0.01fg/ul be template, with conventional pcr amplification (conventional pcr amplification primer M1:CAT CAC AAG CGG AAC ATT G, M2:GCA TTC CTG CCA GGT TAC).
As shown in Figure 3, M is that 100bp DNA ladder, 1 is that 1ng/ul, 2 is that 100pg/ul, 3 is that 10pg/ul, 4 is that 1pg/ul, 5 is that 100fg/ul, 6 is that 10fg/ul, 7 is that 1fg/ul, 8 is that 0.1fg/ul, 9 is 0.01fg/ul, 10 negative contrasts (being water) to control group 2% agarose gel electrophoresis observations; Obtain PCR and expect that amplified fragments size is about 590bp(and opens big, Ji Yanju, Chen Fangyan, Deng. foundation and the Preliminary Applications [J] of kind Duck parvovirus type " ichthyophthiriasis " PCR detection method. Guangdong animal and veterinary science and technology, 2008,33 (6): 18-23.), conventional PCR method minimum detection is limited to 100fg.
As can be seen from the above, the LAMP susceptibility that primer of the present invention is set up is higher 10 times than conventional PCR.
The detection of embodiment 3, clinical sample
To carrying out Virus Isolation, and extract duck pathological material of disease and carry out LAMP detection.
Sample to be tested is the 50 parts of duck samples (being numbered 1-50) that gather from Guangxi Ya Chang in 2012, extracts respectively the DNA of duck pathological material of disease (lungs of the liver of sick duck, the spleen of sick duck or sick duck).
Respectively using the DNA that is numbered each sample of 1-50 as template, according to embodiment 2 above-mentioned two in optimum response system and optimum reaction condition carry out LAMP reaction.
As shown in Figure 4, A figure is electrophoresis detection result to result, and B figure is visual inspection result, and wherein M is 100bp DNAladder, 1 positive contrast kind Duck parvovirus MDPV(AV238), 2-5 is respectively and is numbered 1-4 sample, 4 negative contrasts (water); Can find out, being numbered 1-4 sample is the MDPV positive (visual inspection is emerald green as seen, agarose gel electrophoresis expression characteristics trapezoid-shaped strips).
According to the method described above by all pattern detection, in order there to be 5 parts, to be numbered 1-5 sample be the MDPV positive to result, and all the other are negative.
Detect a kind Duck parvovirus detection according to the conventional PCR of embodiment 2 control groups respectively by being numbered each sample of 1-50, result is consistent with the qualification result of the inventive method, illustrates that method of the present invention is correct.
To be numbered each sample of 1-50 and carry out virus separation, result is consistent with the qualification result of the inventive method.
Claims (5)
1. the loop-mediated isothermal amplification (LAMP) primer group that detects kind Duck parvovirus, is made up of primer 1, primer 2, primer 3, primer 4, primer 5 and primer 6;
The nucleotide sequence of described primer 1, described primer 2, described primer 3 and described primer 4 is followed successively by respectively sequence 1, sequence 2, sequence 3 and the sequence 4 in sequence table;
The nucleotide sequence of described primer 5 and described primer 6 is followed successively by respectively sequence 5 and the sequence 6 in sequence table;
The mol ratio of described primer 1, described primer 2, described primer 3, described primer 4, described primer 5 and described primer 6 is 8:8:1:1:4:4.
2. contain the ring mediated isothermal amplification reagent of the detection kind Duck parvovirus of primer sets claimed in claim 1;
Described ring mediated isothermal amplification reagent is by primer sets claimed in claim 1, ring mediated isothermal amplification damping fluid, archaeal dna polymerase, dNTPs, magnesium sulfate, fluorexon, trimethyl-glycine, MnCl
2form with water;
Primer 1 in described primer sets and the described primer 2 final concentration in described ring mediated isothermal amplification reagent is 1.6 μ mol/L;
Primer 3 in described primer sets and described primer 4 final concentration in described ring mediated isothermal amplification reagent is 0.2 μ mol/L;
Primer 5 in described primer sets and described primer 6 final concentration in described ring mediated isothermal amplification reagent is 0.8 μ mol/L;
The final concentration of described dNTPs in described ring mediated isothermal amplification reagent is 1.4mmol/L;
The final concentration of described archaeal dna polymerase in described ring mediated isothermal amplification reagent is 320U/L;
The final concentration of described trimethyl-glycine in described ring mediated isothermal amplification reagent is 1mmol/L;
The final concentration of described magnesium sulfate in described ring mediated isothermal amplification reagent is 7mmol/L;
The yellowish green final concentration in described ring mediated isothermal amplification reagent of described calcium is 25 μ mol/L;
Described MnCl
2final concentration in described ring mediated isothermal amplification reagent is 0.5mmol/L.
3. the loop-mediated isothermal amplification kit that detects kind Duck parvovirus, comprises primer sets claimed in claim 1 or ring mediated isothermal amplification reagent claimed in claim 2.
Described in primer sets claimed in claim 1, ring mediated isothermal amplification reagent claimed in claim 2 or claim 3 loop-mediated isothermal amplification kit preparation detect and/or auxiliary detection testing sample in whether contain the application in the product of kind Duck parvovirus.
5. application according to claim 4, is characterized in that:
Described application adopts ring mediated isothermal amplification, and the condition of described ring mediated isothermal amplification is 63 DEG C of reaction 50h, 80 DEG C of effect 5min deactivation.
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Non-Patent Citations (4)
| Title |
|---|
| j. ji ,et al.Molecular detection of Muscovy duck parvovirus by loop-mediated isothermal amplification assay.《Poultry Science》.2010,第89卷(第3期),477–483. |
| j. ji,et al.Molecular detection of Muscovy duck parvovirus by loop-mediated isothermal amplification assay.《Poultry Science》.2010,第89卷(第3期),477–483. * |
| 环介导等温扩增技术检测副溶血性弧菌方法的建立与应用;薛超波 等;《海峡预防医学杂志》;20120630;第18卷(第3期);第17页倒数第二段 * |
| 薛超波 等.环介导等温扩增技术检测副溶血性弧菌方法的建立与应用.《海峡预防医学杂志》.2012,第18卷(第3期),第3-4、17页. |
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