CN103981288B - A kind of non-diagnosis and treatment object fowl tumprigenicity virus multiple PCR detection method - Google Patents
A kind of non-diagnosis and treatment object fowl tumprigenicity virus multiple PCR detection method Download PDFInfo
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Abstract
The present invention relates to gene engineering technology field, provide a kind of non-diagnosis and treatment object multi-PCR detection method for fowl tumprigenicity virus, REV can be detected simultaneously, MDV, ALV-J and ALV-A virus, mainly through studying above-mentioned four kinds of viral characteristics, 4 pairs of Auele Specific Primers are adopted to carry out multi-PRC reaction respectively, by being optimized reaction system and condition, filter out optimum reaction conditions, establish the multiplex RT-PCR method that simultaneously can detect four kinds of cause of diseases, with and in clinical sample detect judge above-mentioned four kinds of viruses whether exist, and contrast with histopathologic slide observation, rate of accuracy reached 95%, whole detection method susceptibility is high, specificity is good, easy and simple to handle, rapidly.
Description
Technical field
The present invention relates to gene engineering technology field, provide a kind of non-diagnosis and treatment object multi-PCR detection method, reticuloendothiliosis virus, marek's disease virus, avian leukosis virus J subgroup and A subgroup can be detected simultaneously.
Background technology
Reticuloendothiliosis virus (ReticuloendotheliosisVirus, REV), marek's disease virus (Marek ' sdiseaseviruses, MDV) avian leukosis virus (AvianLeukosisVirus, ALV) all can cause neoplastic disease and the immunosuppressive disease of bird.These three kinds of fowl neoplastic diseases can cause superinfection in various degree, multiple infection and secondary infection, can weaken the immunne response ability of chicken group to various vaccine, and aggravate the clinical symptom of some diseases.The healthy aquaculture of fowl industry in serious harm.At present, effective medicine is not had clinically to these three kinds of neoplastic diseases and is used for the treatment of, do not have vaccine to be on the defensive to RE and AL yet.Countries in the world are detected mainly through antigen/antibody, eliminate positive chicken, and purification population, carries out the prophylactico-therapeutic measuress such as immunity to control this three kinds of fowl neoplastic diseases to MD.
These three kinds of frequent polyinfections of disease clinically, add the difficulty of its clinical quick diagnosis, and diagnostic method conventional at present, such as serological diagnostic method, virus isolation procedure, it is low that histopathologic slide's observation etc. all also exists susceptibility, longer deficiency consuming time, isolated viral method length consuming time and be only applicable to laboratory diagnosis; And immunological detection method such as ELISA, indirect immunofluorescence etc. all need operator to possess certain expertise, and also need to be equipped with relevant device, and be not suitable for the physical condition of current common plant; Histopathologic slide's observation precisely but complicated and time consumption, is not suitable for clinical quick diagnosis; It is high and can the advantage of the multiple cause of disease of one-time detection that multiple PCR method has susceptibility, is more applicable for the clinical quick diagnosis of present polyinfection.2002, the fowl neoplastic disease rapid differential diagnosis kit of Wei equality people research and development is exactly the triple PCR according to multiplex PCR principle design, REV, MDV, ALV tri-kinds of viruses can be detected, fast and convenient, but it does not carry out Subtypes to ALV and three kinds of viral object stripe size are very close, is not easily distinguishable.Existing multiplex PCR cannot meet the requirement distinguishing above-mentioned multiple virus.
Summary of the invention
The present inventor is for the situation of above-mentioned prior art, in conjunction with studying for a long period of time and exploring, provide a kind of non-diagnosis and treatment object multi-PCR detection method for fowl tumprigenicity virus, REV can be detected simultaneously, MDV, ALV-J and ALV-A virus, mainly through studying above-mentioned four kinds of viral characteristics, 4 pairs of Auele Specific Primers are adopted to carry out multi-PRC reaction respectively, by being optimized reaction system and condition, filter out optimum reaction conditions, establish the multiplex RT-PCR method that simultaneously can detect four kinds of cause of diseases, with and in clinical sample detect judge above-mentioned four kinds of viruses whether exist, and contrast with histopathologic slide observation, rate of accuracy reached 95%, whole detection method susceptibility is high, specificity is good, easy and simple to handle, rapidly.
The concrete technical scheme that contriver provides is as follows:
Utilize existing known REV, MDV, ALV-J and ALV-A virus characteristic and conservative gene thereof, contriver obtains 4 pairs of specificity identification primers, and its sequence is as shown in the table:
Utilize above-mentioned 4 pairs of primer pair determinands to carry out multiplexed PCR amplification reaction, final contriver preferred multiplexed PCR amplification reaction system is as follows, and every 25 μ L systems are:
Multi-PRC reaction condition is: put into PCR instrument center, and to avoid fringing effect as far as possible, editor is as follows: the first step: 95 DEG C of 15min; Second step: 95 DEG C of 30s, 53 DEG C of 1min30s, 72 DEG C of 1min30s, continuous 40 circulations; 3rd step 72 DEG C 10min; Finally be stored in 4 DEG C;
After PCR has reacted, the sepharose of configuration 1.5% carries out nucleic acid electrophoresis, can detect acquisition result, wherein can directly by observing whether occur that positive band judges positive band indication cause of disease.
The template adopted that increases obtains by the following method for contriver:
REV, ALV-J, ALV-A tri-kinds of viruses being connect poison in DF-1 normal cell, is the virus liquid of amplification by supernatant collection after 7 days, and-80 DEG C of preservations, by the DF-1 cell extraction total serum IgE cultivated, by cDNA-20 DEG C of preservation after reverse transcription; The strong poison of MDV connects poison in DEF normal cell, and the DEF cell extraction total serum IgE will cultivated after 7 days, by cDNA-20 DEG C of preservation after reverse transcription.
Four kinds of virus stains of above-mentioned employing are existing strain, are REV (CHA strain) respectively, and MDV (GA strain), ALV-J (NX-0101 strain), ALV-A (SDAU09E2 strain), is commercially strain.
Four kinds of cDNA ultraviolet spectrophotometers are measured concentration, adjusts to same concentration (150ng/ μ L) respectively, the template that after equal proportion mixing, (concentration is 150ng/ μ L) reacts as PCR.
Choose the bright band identical with object stripe size after gel electrophoresis and carry out glue recovery, then link transforms order-checking, and the Reference strains that sequencing result and GenBank include is compared: REV-CHA (NC006934), REV-USA (DQ387450), MDV-GA (AF147806), NX-0101 (DQ115805), SDAU09E2 (HM452342).Comparison result proves band for the purpose of bright band namely.
Contriver obtains the reaction conditions of the multiplex PCR that the present invention limits by following mode:
1) with grads PCR instrument, under different annealing temperature (50 DEG C-58 DEG C), polymerase chain reaction is carried out to quadruple primer and quadruple template, select optimum annealing temperature (see Fig. 2), finally select 50 ~ 53 DEG C for optimum annealing temperature;
2) the best primer concentration of multiplex PCR is determined, take primer concentration as variable, get 100 μm of ol/L, 50 μm of ol/L, 25 μm of ol/L, 10 μm of ol/L, 5 μm of ol/L respectively, other conditions are the same, carry out multi-PRC reaction (see Fig. 3), finally select 8 ~ 10 μm of ol/L to be best primer concentration;
3) select peak optimization reaction cycle index, this experiment is chosen 25,30,35,40 and 45 circulations and is optimized (see Fig. 4), finally determines that 35 ~ 40 circulations are for optimum cycle number of times;
4) the minimum template amount that multiplex PCR can detect is determined, namely the susceptibility of multiplex PCR is measured, with the template concentrations of four kinds of cDNA equal proportion mixing for starting point concentration, then 101-106 doubly dilutes, other conditions are the same, carry out multi-PRC reaction (see Fig. 5), finally determine that the minimum template amount that this multiplex PCR system can detect is 60-80pg;
5) adjustment template content carries out substance, specific reaction that is double, triple and Quadruple-PCR tests (see Fig. 6), experimental result shows, REV, MDV, ALV-J and ALV-A one, two, three, quadruple reaction occur band all with expection size be consistent, and band amplification phenomenon does not appear in the negative control normally not connecing poison cell, this illustrates that this method specificity is better.
6) at different time, three kinds of viral for REV, MDV, ALV-J, ALV-A tetra-kinds template concentrations are carried out three replica tests (see Fig. 7), detect its stability and repeatability, show that the method has good stability and repeatability.
General decision method at present for above-mentioned 4 kinds of fowl tumour viruses depends on pathological observation more, and AL Detection of antigen can adopt the test kit made according to ELISA method principle, and RE antibody test can adopt the test kit made according to ELISA method principle.Pathological observation method accuracy rate is higher, needs the professional of the experience that is rich in just can be competent at, though and ELISA method can a large amount of sample of disposable process, but it is consuming time compared with PCR method, test material also needs to extract from live body, also needs data processing after detection, limited more; Detection method processing sample is simple and efficient, without the need to data processing, does not need more expertise, and easier related personnel's operation, is therefore suitable as the detection method of above-mentioned 4 kinds of fowl tumour viruses more.
In sum, the present inventor provides a kind of non-diagnosis and treatment object multi-PCR detection method for fowl tumprigenicity virus first, REV can be detected simultaneously, MDV, ALV-J and ALV-A virus, mainly through studying above-mentioned four kinds of viral characteristics, design 4 pairs of Auele Specific Primers respectively and carry out multi-PRC reaction, by being optimized reaction system and condition, filter out optimum reaction conditions, establish the multiplex RT-PCR method that simultaneously can detect four kinds of cause of diseases, with and in clinical sample detect judge above-mentioned four kinds of viruses whether exist, and contrast with histopathologic slide observation, rate of accuracy reached 95%, whole detection method susceptibility is high, specificity is good, easy and simple to handle, rapidly.
Accompanying drawing explanation
Fig. 1 is the independent pcr amplification figure of the present invention's four kinds of viruses (REV, MDV, ALV-J and ALV-A),
In figure, M is DL1000DNA relative molecular mass standard, and swimming lane 1 ~ 4 is respectively the substance pcr amplification result of REV, MDV, ALV-J, ALV-A Auele Specific Primer corresponding thereto, swimming lane 5: multiplexed PCR amplification result;
Fig. 2 is the optimization of multiple RT-PCR annealing temperature in the present invention and determines figure,
In figure, M is DL1000DNA relative molecular mass standard, swimming lane 1 ~ 12 is the annealing temperature that multiple RT-PCR sets respectively in grads PCR instrument: 50.1 DEG C, 50.2 DEG C, 50.6 DEG C, 51.3 DEG C, 52.2 DEG C, 53.2 DEG C, 54.3 DEG C, 55.4 DEG C, 56.5 DEG C, 57.3 DEG C, 58 DEG C, 58.4 DEG C, finally determines that 50 DEG C ~ 53 DEG C for optimum annealing temperature;
Fig. 3 is the determination figure of the best primer concentration of multiple RT-PCR,
In figure, M is DL1000DNA relative molecular mass standard, swimming lane 1:100 μm ol/L, swimming lane 2:50 μm ol/L, swimming lane 3:25 μm ol/L, swimming lane 4:10 μm ol/L, swimming lane 5:5 μm ol/L, as we know from the figure, under the primer concentration of 10 μm of ol/L, four bands are limpid in sight, comparatively under other primer concentrations, result is easy to observe more, so selected 10 μm of ol/L are best primer concentration;
Fig. 4 is multiple RT-PCR cycle index optimization figure,
In figure, M is DL1000DNA relative molecular mass standard, and swimming lane 1:25 circulation, a swimming lane 2:30 circulation, a swimming lane 3:35 circulation, a swimming lane 4:40 circulation, swimming lane 5:45 circulate, and finally determine that 35 ~ 40 circulations are optimum cycle number of times;
Fig. 5 is the detection figure of the minimum template amount of multiple RT-PCR,
In figure, M is DL1000DNA relative molecular mass standard, swimming lane 1:600ng, swimming lane 2:60ng, swimming lane 3:6ng, swimming lane 4:600pg, swimming lane 5:60pg, swimming lane 6:6pg, swimming lane 7:600fg;
Fig. 6 is multiple RT-PCR specific detection figure,
In figure, M is DL1000DNA relative molecular mass standard, swimming lane 1:REV, swimming lane 2:MDV, swimming lane 3:ALV-J, swimming lane 4:ALV-A, swimming lane 5:REV+MDV, swimming lane 6:REV+ALV-J, swimming lane 7:REV+ALV-A, swimming lane 8:MDV+ALV-J, swimming lane 9:MDV+ALV-A, swimming lane 10:ALV-J+ALV-A, swimming lane 11:REV+MDV+ALV-J, swimming lane 12:REV+MDV+ALV-A, swimming lane 13:MDV+ALV-J+ALV-A, swimming lane 14:REV+ALV-J+ALV-A, swimming lane 15:REV+MDV+ALV-J+ALV-A, swimming lane 16: negative control; Experimental result shows, four kinds viral one, two, three, the band that occurs of quadruple reaction is all consistent with expection size, and the negative control normally not connecing poison cell does not occur band amplification phenomenon illustrating that this method specificity is better;
Fig. 7 is multiplex PCR repeatability and Detection of Stability figure,
In figure, M is DL1000DNA relative molecular mass standard, swimming lane 1: first time test-template amount 60ng, swimming lane 2: first time test-template amount 6ng, swimming lane 3: first time test-template amount 600pg, swimming lane 4: second time test-template amount 60ng, swimming lane 5: second time test-template amount 6ng, swimming lane 6: second time test-600pg, swimming lane 7: third time test-template amount 60ng, swimming lane 8: third time test-template amount 6ng, swimming lane 9: third time test-template amount 600pg, from result, in three repeated experiments, the reaction result of same template amount is more consistent, illustrate that present method has repeatable and stability preferably.
Embodiment
In following embodiment except specified otherwise, what adopt is state of the art;
Embodiment 1 substance PCR reacts
In order to verify the Auele Specific Primer effect that contriver adopts, first contriver utilizes the virus of these its correspondences of primer pair to carry out substance PCR to react, and concrete outcome is as follows:
(four kinds of viruses are REV (CHA strain) respectively will to connect poison, MDV (GA strain), ALV-J (NX-0101 strain), ALV-A (SDAU09E2 strain)) cell harvesting of 7 days, after always carrying RNA, reverse transcription is cDNA, and carries out substance pcr amplification;
Substance pcr amplification reaction system (25 μ L) is: each 0.5 μ L of 2 × TaqPCRMasterMix12.5 μ L, upstream and downstream primer (10 μm of ol/L), ddH2O10.5 μ L, template 1 μ L;
Reaction conditions is: denaturation 95 DEG C of 5min; 94 DEG C of sex change 30s, 53 DEG C of annealing 30s, 72 DEG C of extension 50s, 30 circulations; Product end 72 DEG C extends 10min, is stored in 4 DEG C after having reacted.
After PCR primer adds sample-loading buffer, configuration adds 1.5% sepharose of nucleic acid staining agent, carries out electrophoresis detection.
The detection of 4 kinds of cause of diseases all adopts Auele Specific Primer corresponding with it in the present invention, adopts above-mentioned substance pcr amplification reaction system and condition to increase; As shown in Figure 1, visible four kinds of cause of diseases all can specificity to detect and band is clear bright reaction result.
Embodiment 2 multi-PRC reaction
Multiplexed PCR amplification reaction system, every 25 μ L are: containing 10 × MultiHotstartBuffer2.5 μ L, SuperPuredNTP (2.5mMEach) 2 μ L of+Mg2+, MultiHotStartDNAPolymerase0.5 μ L, template amount <500ng and >60pg, ddH2O are supplemented to 25 μ L
Primer is respectively each 0.5 μ L of reticuloendothiliosis virus upstream and downstream primer, each 0.5 μ L of marek's disease virus upstream and downstream primer, each 0.5 μ L of avian leukosis virus J subgroup upstream and downstream primer, each 0.5 μ L of avian leukosis virus A subgroup upstream and downstream primer;
Described primer concentration is 10 μm of ol/L;
Wherein above-mentioned all ingredients is all purchased from Tian Gen biochemical technology company limited, and primer is synthesized by Beijing six directions Hua Da company.
Multi-PRC reaction condition is: 95 DEG C of 15min; 94 DEG C of 30s, 53 DEG C of 1min30s, 72 DEG C of 1min30s, 40 circulations; 72 DEG C of 10min; Last preservation in 4 DEG C.
Described primer sequence is:
Avian leukosis virus A subgroup upstream primer, its nucleotide sequence as shown in SEDIDNO:1,
Avian leukosis virus A subgroup downstream primer, its nucleotide sequence as shown in SEDIDNO:2,
Avian leukosis virus J subgroup upstream primer, its nucleotide sequence as shown in SEDIDNO:3,
Avian leukosis virus J subgroup downstream primer, its nucleotide sequence as shown in SEDIDNO:4,
Marek's disease virus upstream primer, its nucleotide sequence as shown in SEDIDNO:5,
Marek's disease virus downstream primer, its nucleotide sequence as shown in SEDIDNO:6,
Reticuloendothiliosis virus upstream primer, its nucleotide sequence as shown in SEDIDNO:7,
Reticuloendothiliosis virus downstream primer, its nucleotide sequence is as shown in SEDIDNO:8;
After the amplified production obtained adds sample-loading buffer, configuration contains the sepharose of 1.5% of nucleic acid staining agent, carry out electrophoresis detection, can result be obtained: REV, MDV, ALV-J and ALV-A one, two, three, quadruple reaction occur band all with expection size be consistent, and band amplification phenomenon does not appear in the negative control normally not connecing poison cell, this illustrates that this method specificity better (as shown in Figure 6).
With the template concentrations (150ng/ μ L) of four kinds of cDNA equal proportion mixing for starting point concentration, template amount is 600ng, then 101-106 doubly dilutes, other conditions are the same, carry out multi-PRC reaction (as shown in Figure 5), finally determine that the minimum template amount that this multiplex PCR system can detect is 60-80pg.
With four kinds of cDNA for template, by its gradient dilution 101 ~ 103 times, the multiplex PCR system optimized at three different times with these three kinds of gradient template amounts for template carries out repeated experiment (as shown in Figure 7), it is more consistent that experimental result shows 3 repeated reaction results, and this illustrates that this multiple PCR method is repeatable and stability is better.
Claims (2)
1. a non-diagnosis and treatment object fowl tumprigenicity virus multiple PCR detection method, it is characterized in that, the conservative gene for REV, MDV, ALV-J and ALV-A virus have employed 4 pairs of Auele Specific Primers, and its sequence is as shown in the table:
Utilize above-mentioned 4 pairs of primer pair determinands to carry out multiplexed PCR amplification reaction, its multiplexed PCR amplification reaction system is as follows, and every 25 μ L systems are:
Multi-PRC reaction condition is: put into PCR instrument center, and to avoid fringing effect as far as possible, editor is as follows: the first step: 95 DEG C of 15min; Second step: 95 DEG C of 30s, 53 DEG C of 1min30s, 72 DEG C of 1min30s, continuous 35-40 circulate; 3rd step 72 DEG C 10min; Finally be stored in 4 DEG C;
After PCR has reacted, in reaction solution, add 6 × sample-loading buffer, the sepharose that configuration adds 1.5% of nucleic acid staining agent carries out nucleic acid electrophoresis, can detect acquisition result.
2. detection method according to claim 1, is characterized in that: continuous 40 circulations in multi-PRC reaction condition.
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| CN106244729A (en) * | 2016-08-31 | 2016-12-21 | 中国农业大学 | The PCR primer of detection Reticuloendotheliosis Virus and test kit thereof |
| CN106399588B (en) * | 2016-09-19 | 2019-12-17 | 中国农业大学 | kit for detecting avian leukosis virus J subgroup |
| CN106282416B (en) * | 2016-10-08 | 2018-05-25 | 广东省实验动物监测所 | A kind of the multi-fluorescence immunoassay primer, kit and method of 5 kinds of fowl immunosupress cause of diseases of quick differentiation |
| CN108085416A (en) * | 2017-12-21 | 2018-05-29 | 湖北省农业科学院畜牧兽医研究所 | A kind of fowl neoplastic disease triple fluorescent PCR detection kit and its detection method |
| CN114317827B (en) * | 2022-01-07 | 2023-03-24 | 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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