CN112553377A - Detection kit for distinguishing Marek's disease virus vaccine strain and virulent strain by using dual fluorescence LAMP (loop-mediated isothermal amplification) and primer group of detection kit - Google Patents
Detection kit for distinguishing Marek's disease virus vaccine strain and virulent strain by using dual fluorescence LAMP (loop-mediated isothermal amplification) and primer group of detection kit Download PDFInfo
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Abstract
The invention discloses a detection primer group for distinguishing Marek's disease virus vaccine strains and virulent strains by double fluorescence LAMP, which comprises a primer 1 to a primer 8, wherein the primer 1 to the primer 8 respectively have base sequences of sequence tables SEQ.ID.No.1 to SEQ.ID.No.8, the primer 7 and the primer 8 are Taqman probes, the primers enable amplification reaction to have high specificity, can be respectively combined with differential sites for distinguishing the vaccine strains and the virulent strains by MDV meq genes, and emit fluorescence of different colors under the fluorescence of different wavelengths after the reaction, thereby realizing the quick sensitive and specific identification of the MDV vaccine strains and the virulent strains. Accordingly, the inventors also developed a corresponding kit and established a corresponding double fluorescence LAMP method. In a word, the kit has the characteristics of strong specificity, high sensitivity, rapidness, convenience and the like, is suitable for rapid detection in primary veterinary stations and farms with basic experimental instruments, and has a better application prospect.
Description
Technical Field
The invention belongs to the technical field of virus detection, and particularly relates to a detection kit for distinguishing Marek's disease virus vaccine strains and virulent strains by using double fluorescence LAMP and a primer group thereof.
Background
Marek's Disease (MD) is a tumorigenic infectious disease mainly infecting chickens and turkeys, which is caused by Marek's Disease Virus (MDV). Although MDV has only one serotype, there are significant differences in biological properties between different strains. The vaccine strain is not tumorigenic and belongs to a low virulent strain, and most of the reported separated wild strains are virulent strains and have tumorigenic characteristics. At present, the molecular markers for distinguishing MDV vaccine strains from virulent strains are mainly based on the 576-578 site of the meq gene.
Conventional detection methods require the use of expensive instruments, reagents, and the like. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technology established in 2000 by Notomi, and has the advantages of simple operation, rapid reaction, low cost, visualized result and the like, and is widely applied to the detection of some pathogenic microorganisms. In the traditional LAMP detection method, a taqman probe is innovatively added, and the taqman probe with specificity can be hybridized with an amplification product during reaction, and is hydrolyzed along with the amplification process to emit fluorescence.
According to the investigation, no related report for establishing a double-fluorescence LAMP (loop-mediated isothermal amplification) visual detection kit and method for distinguishing Marek's disease virus vaccine strains from virulent strains is found at home and abroad.
Disclosure of Invention
The invention aims to solve the technical problem of providing a detection kit for distinguishing Marek's disease virus vaccine strains and virulent strains by double fluorescence LAMP, which has strong specificity, high sensitivity, rapidness, simplicity and convenience, and a primer group thereof.
In order to solve the technical problems, the invention adopts the following technical scheme:
the detection primer group for distinguishing Marek's disease virus vaccine strains and virulent strains by the duplex fluorescence LAMP comprises a primer 1 to a primer 8, and the primer 1 to the primer 8 respectively have base sequences of sequence tables SEQ.ID.No.1 to SEQ.ID.No. 8.
The primer 7 and the primer 8 are taqman probes, the 5 'end and the 3' end of the primer 7 are respectively marked with a 5-FAM fluorescent group and a quenching group BHQ-1, and the 5 'end and the 3' end of the primer 8 are respectively marked with a CY5 fluorescent group and a quenching group BHQ-3.
The molar ratio of primer 1 to primer 8 was 8:8:1:1:4:4:1: 1.
The application of the detection primer group in loop-mediated isothermal amplification.
The conditions of the loop-mediated isothermal amplification are reaction at 68 ℃ for 60min and inactivation at 80 ℃ for 5 min.
The detection kit for distinguishing Marek's disease virus vaccine strains and virulent strains by the double fluorescent LAMP comprises primers 1 to 8 which respectively have base sequences of sequence tables SEQ.ID No.1 to SEQ.ID No. 8.
The primer 7 and the primer 8 are taqman probes, the 5 'end and the 3' end of the primer 7 are respectively marked with a 5-MEQAM fluorescent group and a quenching group BHQ-1, and the 5 'end and the 3' end of the primer 8 are respectively marked with a CY5 fluorescent group and a quenching group BHQ-3.
The detection kit further comprises the following reagents: loop mediated isothermyAmplification buffer, Bst DNA polymerase, dNTPs, magnesium sulfate, calcein, betaine and MnCl2。
The final concentrations of the primers 1 to 8 in the loop-mediated isothermal amplification reaction system were 1.6. mu. mol/L, 0.2. mu. mol/L, 0.8. mu. mol/L, 0.2. mu. mol/L and 0.2. mu. mol/L, respectively.
Aiming at the problems existing in the detection of MDV vaccine strains and virulent strains at present, the inventor designs a double fluorescence LAMP detection primer group for distinguishing Marek's disease virus vaccine strains and virulent strains according to the conserved sequence of MDV aiming at 6 specific regions, wherein the primer group comprises a primer 1 to a primer 8 which have base sequences of sequence tables SEQ ID No.1 to SEQ ID No.8 respectively, the primer 7 and the primer 8 are FENZI which enable the amplification reaction to have high specificity and can be hybridized with the site for distinguishing the strong and weak viruses of the MDV meq gene respectively, and the fluorescence with different colors is emitted under the fluorescence with different wavelengths after the reaction (the primer 7 is hybridized with the specific sequence of the strong virus strain and is hydrolyzed again in the extension process to emit green fluorescence, and the primer 8 is hybridized with the specific sequence of the weak virus strain and is hydrolyzed again in the extension process to emit red fluorescence), so that the MDV vaccine strain and the strong virus strain can be identified quickly, sensitively and specifically.
Accordingly, by optimizing the reaction system and conditions, the inventors also developed a corresponding kit and established a corresponding dual fluorescence LAMP method. The method can be completed only by reacting in a water bath kettle at 68 ℃ for 60 minutes, MDV can be specifically detected, strong and weak strains are distinguished through fluorescence, after reaction, green fluorescence is emitted and is the strong strain, red fluorescence is emitted and is the weak vaccine strain, and if the strong strain and the vaccine strain exist in a sample at the same time, yellow fluorescence is emitted. Experiments prove that the detection sensitivity of the invention is very high, and each reaction system can detect 100 copies of MDV DNA samples.
The invention can amplify all MDV meq genes only by using 1 set of primers, only hybridizes with sequences of a low-virulent vaccine strain and a high-virulent strain respectively through a taqman probe aiming at a meq gene 576-578 site specific sequence for distinguishing the high-virulent strain and the low-virulent strain, and the hybridized probe hydrolyzes to release a free fluorescent group in the reaction process. The reaction only needs to be carried out in one reaction tube, and the result can be read according to the color of the reaction under a fluorescent nucleic acid imager. And the taqman heterozygosis method has higher specificity, is not influenced by nonspecific amplification and primer dimer amplification, and solves the problem of false positive caused by the incapability of distinguishing a low virulent vaccine strain from a high virulent strain or a primer dimer in the prior art.
In a word, the invention has the characteristics of more specificity and sensitivity than the conventional detection method, only needs one temperature-controllable water bath and one fluorescence imager, is suitable for rapid detection in basic veterinary stations and farms with basic experimental instruments, and has better application prospect.
Drawings
FIG. 1 is a diagram showing the result of the specificity of the LAMP method for detecting MDV virulent strains and avirulence, in which: a is an LAMP specificity test amplification curve observed by a nephelometer, and B is an LAMP specificity test amplification result observed by a fluorescence imaging instrument; wherein, 1 is MDV vaccine strain, 2 is MDV virulent strain, 3 is the mixture of MDV vaccine strain and virulent strain, 4 is avian leukemia virus, 5 is reticuloendotheliosis virus, 6 is infectious bursal disease virus, 7 is chicken infectious anemia virus, and 8 is negative control (water).
FIG. 2 is a graph showing the sensitivity results of the LAMP method of the present invention for detecting MDV virulent strains and avirulence, in which: a is an LAMP amplification sensitivity test amplification curve observed by a turbidimeter, and templates of MDV vaccine strains and virulent strains are added in the reaction in an equal amount of 1:1 (the concentration is the total concentration); b is the LAMP amplification sensitivity test amplification result observed by a fluorescence imager, the first row is an MDV vaccine strain, the second row is an MDV virulent strain, and the third row is the same as the MDV virulent strain in a ratio of 1:1 (the concentration is the total concentration). Wherein 1 is each 107Copies, 2 being each 106Copies, 3 being 10 each5Copies, 4 being 10 each4Copies, 5 being 10 each3Copies, 6 being 10 each2Copy, 7 for each 10copies, and 8 for negative control.
FIG. 3 is a diagram showing the results of random detection of clinically isolated MDV strains and vaccine strains by the LAMP method of the present invention, in which: 1-7 are 7 samples tested at random, 8 are negative controls.
Detailed Description
The experimental procedures used in the following examples are conventional ones unless otherwise specified, and materials, reagents and the like used therein are commercially available. Wherein:
bst DNA polymerase (full length) was purchased from New England Biolabs.
Viral DNA/RNA Kit was purchased from Beijing holotype gold organisms.
MDV vaccine strain CVI988, MDV virulent strain, avian leukosis virus, reticuloendotheliosis virus, infectious bursal disease virus, chicken infectious anemia virus and the like are all known viruses which are self-maintained and can be obtained by the public from the research institute of veterinarian in the autonomous region of the Guangxi Zhuang nationality.
Example 1 design of primers
LAMP primers were designed using the online software Primer Explorer V4 (http:// Primer Explorer. jp/e/V4-manual/index. html) based on the meq gene sequence of MDV in GenBank. Primers were synthesized by Invitrogen, guangzhou, and the specific sequences are shown in table 1.
TABLE 1 LAMP primer sequences
Example 2 application of primers in differentiating Marek's disease virus vaccine strains and virulent strains by double fluorescence LAMP
Extraction of nucleic acid
Reference to
The Viral DNA/RNA Kit DNA/RNA co-extraction Kit instruction book extracts MDV vaccine strain CVI988, MDV virulent strain, avian leukemia virus, reticuloendotheliosis virus, infectious bursal disease virus and chicken infectious anemia virus genome DNA.
Second, optimizing LAMP reaction system, reaction conditions and construction of kit
25 μ L LAMP reaction system: 1-4. mu.L dNTPs (10mmol/L, final concentration 0.4mmol/L-1.6mmol/L), 2.5. mu.L 10 XBst bumeqmeqer, 1. mu.l Bst DNA polymerase 8U (final concentration 320U/L), 4-7. mu.L Betaine (5mmol/L, final concentration 0.8mmol/L-1.4mmol/L), 2-9. mu.L MgSO 24(25mmol/L, final concentration of 2mmol/L-9mmol/L), 1 μ L primer (MDV-FIP 40 μmol/L, MDV-BIP 40 μmol/L, MDV-F35 μmol/L, MDV-B35 μmol/L, MDV-LF 20 μmol/L, MDV-LB 20 μmol/L, MDV-MDV 5 μmol/L, MDV-vvMDV 5 μmol/L); the final concentrations were MDV-FIP 1.6. mu. mol/L, MDV-BIP 1.6. mu. mol/L, MDV-F30.2. mu. mol/L, MDV-B30.2. mu. mol/L, MDV-LF 0.8. mu. mol/L, MDV-LB 0.8. mu. mol/L, MDV-MDV 0.2. mu. mol/L, MDV-vvMDV 0.2. mu. mol/L and 1. mu.L template (genomic DNA of MDV), water was added to 25. mu.L.
Reaction conditions are as follows: the temperature is 60 deg.C, 62 deg.C, 64 deg.C, 66 deg.C, 68 deg.C, and gradually increased for 60min, and the inactivation is performed at 80 deg.C for 5 min.
The following optimum reaction system and conditions were obtained by searching the above reaction system and conditions, respectively:
the optimum reaction system is as follows: 25 μ L LAMP reaction system: mu.L dNTPs (10mmol/L, final concentration of 0.4mmol/L), 2.5. mu.L 10 XBst bumeqmeqer, 1. mu.L Bst DNA polymerase 8U (final concentration of 320U/L), 5. mu.L Betaine Betaine (5mmol/L, final concentration of 1mmol/L), 3. mu.L MgSO 3. mu.L4(25mmol/L, final concentration of 3mmol/L), 1. mu.L primer (MDV-FIP 40. mu. mol/L, MDV-BIP 40. mu. mol/L, MDV-F35. mu. mol/L, MDV-B35. mu. mol/L, MDV-LF 20. mu. mol/L, MDV-LB 20. mu. mol/L, MDV-MDV 5. mu. mol/L, MDV-vvMDV 5. mu. mol/L); the final concentrations were MDV-FIP 1.6. mu. mol/L, MDV-BIP 1.6. mu. mol/L, MDV-F30.2. mu. mol/L, MDV-B30.2. mu. mol/L, MDV-LF 0.8. mu. mol/L, MDV-LB 0.8. mu. mol/L, MDV-MDV 0.2. mu. mol/L, MDV-vvMDV 0.2. mu. mol/L and 1. mu.L template (genomic DNA of MDV), water was added to 25. mu.L.
The optimal reaction conditions are as follows: reacting at 68 deg.C for 60min, and inactivating at 80 deg.C for 5 min.
In order to facilitate the rapid detection of the basic layer, the detection kit can be assembled by referring to the optimal reaction system,the kit comprises related primers and the following reagents: loop-mediated isothermal amplification buffer, Bst DNA polymerase, dNTPs, magnesium sulfate, calcein, betaine and MnCl2。
Third, specific detection
Respectively taking the MDV vaccine strain CVI988 obtained in the first step, an MDV virulent strain, an avian leukosis virus, a reticuloendotheliosis virus, an infectious bursal disease virus and a chicken infectious anemia virus DNA or cDNA as templates, and carrying out LAMP reaction according to the optimal reaction system and the optimal reaction conditions in the second step.
Judging the LAMP reaction result:
1) using a fluorescent nucleic acid imager: respectively using FAM channel and CY5 channel for imaging, if the color of the reaction product is changed to green, indicating that the sample contains MDV and is a virulent strain, if the color of the reaction product is changed to red, indicating that the sample contains MDV and is a low-virulent vaccine strain, if the color of the reaction product is changed to yellow, indicating that the sample contains MDV and is a virulent strain and a low-virulent vaccine strain, and if the color of the reaction product is not changed to yellow, indicating that the sample does not contain MDV.
The results are shown in FIG. 1, in which A is the results of turbidimetric amplification and B is the results of fluorescent nucleic acid imager observation. As can be seen, 1-3 in A had amplification curves, whereas avian leukemia virus, reticuloendotheliosis virus, infectious bursal disease virus, chicken infectious anemia virus, and water were not amplified. B, 1 shows red fluorescence, MDV vaccine strain shows positive result, 2 shows green fluorescence, MDV virulent strain shows positive result, 3 shows yellow fluorescence, MDV vaccine strain and virulent strain mixed positive result; avian leukemia virus, reticuloendotheliosis virus, infectious bursal disease virus, chicken infectious anemia virus, and water have no fluorescence and show negative results.
Fourth, sensitivity detection
The DNA of MDV was diluted 10-fold to 107copies/μL、106copies/μL、105copies/μL、104copies/μL、103copies/μL、102The primers are used as templates, and the optimal reaction system and the optimal reaction conditions are adoptedPerforming LAMP reaction.
The results are shown in FIG. 2, panel A shows the results of nephelometric amplification of MDV vaccine strain and virulent strain. It can be seen that there are amplification curves from 1 to 6 and no amplification curves from 7 to 8. The minimum detection limit of LAMP on MDV DNA is 102copies. The B picture is the detection result of a fluorescence imaging instrument, wherein the first row is the sensitivity detection result of the MDV vaccine strain, 1-6 shows red fluorescence, 7-8 shows no fluorescence, and the minimum detection amount of the MDV vaccine strain is 102copies. The second row shows the sensitivity detection result of MDV virulent strain, and it can be seen that 1-6 shows green fluorescence, 7-8 shows no fluorescence, and the minimum detection amount of MDV virulent strain is 102copies. The third row is the sensitivity detection result of the mixture of the vaccine strain and the virulent strain, and can show that 1 to 6 show yellow fluorescence, 7 to 8 show no fluorescence, and the minimum detection quantity of the MDV vaccine strain and the virulent strain is 102copies。
Fifth, random detection of MD clinical disease material
Extracting genome DNA from suspected MD pathological materials to be clinically inspected as a template, and carrying out LAMP reaction according to the optimal reaction system and the optimal reaction conditions in the two steps.
The results are shown in FIG. 3, 2 and 4 are MDV virulent strains, 1 and 6 are attenuated vaccine strains, 5 and 7 are mixture of virulent strains and attenuated vaccine strains, 3 is negative, and 8 is negative control (water).
Sequence listing
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Claims (9)
1. The detection primer group for distinguishing Marek's disease virus vaccine strains and virulent strains by the double fluorescence LAMP is characterized by comprising primers 1 to 8 which respectively have base sequences of sequence tables SEQ.ID No.1 to SEQ.ID No. 8.
2. The detection primer set according to claim 1, wherein: the primer 7 and the primer 8 are taqman probes, the 5 'end and the 3' end of the primer 7 are respectively marked with a 5-FAM fluorescent group and a quenching group BHQ-1, and the 5 'end and the 3' end of the primer 8 are respectively marked with a CY5 fluorescent group and a quenching group BHQ-3.
3. The detection primer set according to claim 1, wherein: the molar ratio of the primer 1 to the primer 8 is 8:8:1:1:4:4:1: 1.
4. The use of the detection primer set according to claim 1 in loop-mediated isothermal amplification.
5. The use according to claim 4, wherein the conditions of the loop-mediated isothermal amplification are reaction at 68 ℃ for 60min and inactivation at 80 ℃ for 5 min.
6. The detection kit for distinguishing Marek's disease virus vaccine strains and virulent strains by the double fluorescent LAMP is characterized by comprising primers 1 to 8 which respectively have base sequences of sequence tables SEQ.ID No.1 to SEQ.ID No. 8.
7. The detection kit according to claim 6, characterized in that: the primer 7 and the primer 8 are taqman probes, the 5 'end and the 3' end of the primer 7 are respectively marked with a 5-FAM fluorescent group and a quenching group BHQ-1, and the 5 'end and the 3' end of the primer 8 are respectively marked with a CY5 fluorescent group and a quenching group BHQ-3.
8.The test kit according to claim 7, characterized by further comprising the following reagents: loop-mediated isothermal amplification buffer, Bst DNA polymerase, dNTPs, magnesium sulfate, calcein, betaine and MnCl2。
9. The detection kit according to claim 8, characterized in that: the final concentrations of the primers 1 to 8 in the loop-mediated isothermal amplification reaction system are 1.6. mu. mol/L, 0.2. mu. mol/L, 0.8. mu. mol/L, 0.2. mu. mol/L and 0.2. mu. mol/L, respectively.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114317827A (en) * | 2022-01-07 | 2022-04-12 | 河南省农业科学院 | Multiplex PCR (polymerase chain reaction) primer, method and application for identifying infection and immunity of Marek's disease viruses of different serotypes |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108034767A (en) * | 2018-01-03 | 2018-05-15 | 山东农业大学 | Differentiate serum I type Marek's disease virus gene deletion of vaccine, primer, probe and the kit of Attenuation vaccine and wild poison |
| CN110578019A (en) * | 2019-10-30 | 2019-12-17 | 广西壮族自治区兽医研究所 | Detection kit for distinguishing newcastle disease virulent strains and attenuated strains by double fluorescence LAMP (loop-mediated isothermal amplification) and primer group thereof |
-
2020
- 2020-12-29 CN CN202011592447.4A patent/CN112553377A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108034767A (en) * | 2018-01-03 | 2018-05-15 | 山东农业大学 | Differentiate serum I type Marek's disease virus gene deletion of vaccine, primer, probe and the kit of Attenuation vaccine and wild poison |
| CN110578019A (en) * | 2019-10-30 | 2019-12-17 | 广西壮族自治区兽医研究所 | Detection kit for distinguishing newcastle disease virulent strains and attenuated strains by double fluorescence LAMP (loop-mediated isothermal amplification) and primer group thereof |
Non-Patent Citations (2)
| Title |
|---|
| 丁维民等: "Ⅰ型马立克氏病强弱毒株Meq基因的克隆与序列分析", 安徽农业科学 * |
| 付本懂: "人参皂苷抗马立克氏病肿瘤形成作用及其机制研究", 中国优秀博硕士学位论文全文数据库 (博士)农业科技辑 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114317827A (en) * | 2022-01-07 | 2022-04-12 | 河南省农业科学院 | Multiplex PCR (polymerase chain reaction) primer, method and application for identifying infection and immunity of Marek's disease viruses of different serotypes |
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