CN102212622A - Reverse transcription-polymerase chain reaction (RT-PCR) detection method and kit for quickly diagnosing bovine parainfluenza virus 3 (BPIV-3) - Google Patents
Reverse transcription-polymerase chain reaction (RT-PCR) detection method and kit for quickly diagnosing bovine parainfluenza virus 3 (BPIV-3) Download PDFInfo
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Abstract
The invention discloses a reverse transcription-polymerase chain reaction (RT-PCR) detection method and a kit for quickly diagnosing bovine parainfluenza virus 3 (BPIV-3), and belongs to the field of biomedical detection. The invention adopts the technical scheme that the method comprises acquisition of a sample, extraction of virus RNA, design optimization of a specific primer, RT-PCR amplification, agarose gel electrophoresis and result judgment; the specific primer is designed for a specific fragment of BPIV-3 nucleoprotein (NP) genes, and the upstream and downstream sequences of the specific primer are respectively P1: 5'-GGATGTTTGGGAGTGATCTTGAGTA-3' and P2: 5'-TGTGTTGAAAAATGAAGCAAGACCT-3'; the quick RT-PCR diagnosing kit for detecting the BPIV-3 comprises a sample virus RNA extracting reagent, a reverse transcription reagent, a PCR reagent, a positive reference substance and a negative reference substance; and by verifying, the method is sensitive and specific and has application prospect.
Description
Technical field
The invention belongs to biomedical detection range, relate to microorganism molecular Biological Detection method.Be specifically related to a kind of RT-PCR detection method that is used for quick diagnosis bovine parainfluenza virus 3 types.More relate to the application of the RT-PCR test kit of quick diagnosis bovine parainfluenza virus 3 types.
Background technology
(Bovine parainfluenza virus 3, BPIV-3) infection causes bovine parainfluenza, can be divided into the calf type and become the ox type by bovine parainfluenza virus 3 types.The calf type claims calf region pneumonia again, is a kind of contagious disease of invasion and attack calf in 2 thoughtful age several months, is feature with heating, expiratory dyspnea, stream slurries, mucus or purulent rhinorrhea and cough.Be born in ox after the transportation because of this disease pilosity, this virus claims transporting hot virus again.Bovine parainfluenza virus 3 types belong to paramyxovirus section paramyxovirus and belong to the member, and the RNA viruses of cyst membrane is arranged for the negative thigh of strand.
Since nineteen fifty-nine Reisinger etc. since the Niu Tizhong of the U.S. is separated to BPIV-3, states such as France, USSR (Union of Soviet Socialist Republics), Japan, Denmark, Canada, Australia, Panama and Italy also isolate this virus in succession.Because the respiratory symptom that multiple cause of disease causes is similar to this disease, the accurate diagnostic method of this disease seems extremely important.The diagnosis research of relevant BPIV-3 report is few in the world, and relating to main method has:
(1) serodiagnosis technology comprises complement fixation test, neutralization experiment, aggegation experiment, immunodiffusion(ID), precipitation experiment, immuno-electrophoresis, immunofluorescence dyeing, double-antibody sandwich elisa and immunoelectronmicroscopy etc.In these methods, generally admitted and the having of widespread use: complement fixation test, indirect hemagglutination test.Agar diffusion experiment, neutralization experiment.But because long reaction time, material is many, and the preparatory period is long, detects to have tangible hysteresis quality, can only be qualitative can not be quantitative, and repeated relatively poor.
(2) biological experiment comprises experimentation on animals, egg inoculation and cell cultures.There are problems such as not high, the consuming time length of sensitivity in this detection method, and has anticomplementary activity in some sample, and influence detects effect.Experimentation on animals cost height, and expend a large amount of manpower and materials, economic benefit is lower.
By contrast, molecular biology method has clear superiority, and is good, highly sensitive as RT-PCR method specificity, not only can detect the viral nucleic acid in the viable cell, also can directly detect the nucleic acid fragment of deactivation; Sample can derive from nose swab, throat swab, respiratory secretions etc., and can distinguish mutually with different influenza strains, and this is that immunological method is irreplaceable.Other molecular biology methods have the fluorescence quantitative RT-PCR kit patent No. (200910062399.5), at target gene HN gene, M gene or F gene etc. are arranged.
Summary of the invention
At first at the industry demand of present cattle-raising sound development, first purpose of the present invention is to utilize the RT-PCR technology to set up a kind of quick nucleic acid detection method at bovine parainfluenza virus 3 types, and this method is sensitiveer, is easier to operation.In the hope of providing technical support for the quick diagnosis of bovine parainfluenza and epidemiology survey monitoring and prevention and control; Another object of the present invention is by system optimization reaction conditions and technology, prepares that corresponding diagnostic kit detects for clinical cause of disease and the bovine parainfluenza prevention and control provide product.
Realize concrete technical scheme of the present invention, be the RT-PCR method of described detection bovine parainfluenza virus 3 types, step comprises sample collecting, the extraction of viral RNA, and the design optimization of Auele Specific Primer, the RT-PCR amplification, agarose gel electrophoresis and result judge; It is characterized in that: described Auele Specific Primer is that its upstream and downstream sequence is respectively at the design of the specificity section of bovine parainfluenza virus 3 type nucleoprotein genes (NP):
P1：5’-GGATGTTTGGGAGTGATCTTGAGTA-3’；
P2：5’-TGTGTTGAAAAATGAAGCAAGACCT-3’；
Two-step approach is adopted in described RT-PCR amplification, adding length during reverse transcription is random primer 5 '-NNNNNNNNN-3 ' of 9bp, the specificity upstream and downstream primer P1 and the P2 that in reaction tubes, add above-mentioned bovine parainfluenza virus 3 types during the PCR reaction, RT-PCR reverse transcription reaction system is 20 μ L, comprise: 8 μ L RNA, 3 μ L DEPC handle deionized water, 1 μ L random primer, behind 65 ℃ of 5min, put on ice rapidly.Add 5 * RT, 4 μ L damping fluids, 10 μ M dNTPs, 2 μ L, 0.5 μ M RNA enzyme inhibitors, 1 μ L, 25U/ μ L M-MLV 1 μ L subsequently, the continuation reaction conditions is: 30 ℃ of 30s, and 99 ℃ of 5min, 42 ℃ of 20min, 4 ℃ of 5min obtain cDNA;
The PCR system is 20 μ L, comprising: sterilization deionized water 12.5 μ L, 10 * PCR buffer2 μ L, 5U/ μ L Taq archaeal dna polymerase 0.5 μ L, dNTPs 1 μ L, bovine parainfluenza virus 3 type upstream and downstream primer P1, each 1 μ L of P2, cDNA sample 2 μ L.The PCR response procedures is: 94 ℃ of pre-sex change 5min; 94 ℃ of 30s, 58 ℃～62 ℃ 30s, 72 ℃ of 1min circulate 30 times; 72 ℃ are extended 5min, and 16 ℃ of 10min get PCR product 6 μ L and carry out 1% agarose electrophoresis, and positive sample and positive reference substance size all occurs and be the PCR product band of 425bp, the no amplified production band of negative sample and negative control product.
The RT-PCR quick diagnosis reagent kit of detection bovine parainfluenza virus 3 types of the present invention comprises: the sample viral RNA extracts reagent, reverse transcription reagent, PCR reagent, positive reference substance and negative control product; It is characterized in that: described reverse transcription reagent is made up of 5 times of RT damping fluids, dNTPs, RNA enzyme inhibitors, M-MLV, DEPC treating water and random primer, storage temperature-20 ℃.The reverse transcription reaction system is: 8 μ L RNA, 4 μ L, 5 * RT damping fluid, 3 μ L DEPC handle deionized water, 10 μ M dNTPs (Takara), 2 μ L, 0.5 μ MRNA enzyme inhibitors (Toyoba), 1 μ L, 25U/ μ LM-MLV 1 μ L (Toyoba), 1 μ L random primer; Described PCR reagent is that storage temperature is-20 ℃ by sterilization deionized water, 10 * PCR damping fluid, Taq archaeal dna polymerase, dNTPs and cDNA template.The PCR reaction system comprises: sterilization deionized water 12.5 μ l, 10 * PCR damping fluid (Fermantas), 2 μ L, 5U/ μ LTaq archaeal dna polymerase (Fermantas) 0.5 μ l, 2.5mmol/L dNTPs (Fermantas) 1 μ L, each 1 μ L of bovine parainfluenza virus 3 type specificity upstream and downstream primer P1 and P2.
The present invention has obvious superiority compared with prior art.At first, this research of the present invention is target gene with the nucleoprotein gene (NP) of BPIV-3, the critical function of NP is the genome that is wrapped in virus, combine formation complex body (ribonucleoprotein complexes with the RNA and the polymerase of virus, RNPs), the other NP that transcribes and duplicate that is beneficial to virus has the nuclear translocation function, effect is very important for the RNA of virus enters nucleus, can enter host cell by receptor-mediated endocytosis, fusion through coating and host cell membrane, RNPs is released into cytoplasm, is transported to nucleus again.Therefore, the present invention chooses the RT-PCR main candidate of NP gene as BPIV-3, set up should virus the RT-PCR detection method, and confirm this method sensitivity, special, have application prospect.
The present invention with the nucleoprotein gene NP of BPIV-3 set up should virus the RT-PCR quick diagnosis reagent kit, because this test kit sensitivity, special, quick, can save time for the detection of cause of disease, reduce and detect cost, reduce unnecessary financial loss, be highly suitable for the laboratory quick diagnosis and the epidemiological study of bovine parainfluenza virus 3 types.
Description of drawings
Fig. 1: specific detection result's 1% agarose gel electrophoresis of the present invention detects figure
RT-PCR has detected bovine parainfluenza virus 3 types, bovine viral diarrhea virus, infectious bovine rhinotrachetis, ox syncytial virus and Pestivirus suis, Mycoplasma bovis, colon bacillus, ox pasteurellosis bacillus capsular serotype A type, Salmonella typhimurium and MDBK cell RNA among Fig. 1.Each swimming lane is followed successively by M.DL2000 ladder from left to right; 1. bovine parainfluenza virus 3 types; 2. bovine viral diarrhoea/bovine diarrhoea virus; 3. infectious bovine rhinotrachetis virus; 4. ox syncytial virus; 5. Pestivirus suis; 6. Mycoplasma bovis; 7. ox pasteurella multocida; 8. Salmonella typhimurium; 9. intestinal bacteria; 10.MDBK cell negative control; 11. blank.
Have only bovine parainfluenza virus 3 types can amplify the purpose band of expection size (425bp), show that specificity of the present invention is good.
Fig. 2: sensitivity detected result of the present invention detects figure by 1% agarose gel electrophoresis
Among Fig. 2 with bovine parainfluenza virus 3 types with reference to strain from 10
9TCID
50/ 0.1mL begins 10 times and increases progressively dilution, carries out the RT-PCR amplification.Swimming lane is followed successively by M.DL2000 Ladder from left to right; 1.10
9TCID
50/ 0.1mL; 2.10
8TCID
50/ 0.1mL; 3.10
7TCID
50/ 0.1mL; 4.10
6TCID
50/ 0.1mL; 5.10
5TCID
50/ 0.1mL; 6.10
4TCID
50/ 0.1mL; 7.10
3TCID
50/ 0.1mL; 8.10
2TCID
50/ 0.1mL; 9.10
1TCID
50/ 0.1mL; 10.10
0TCID
50/ 0.1mL; 11.10
-1TCID
50/ 0.1mL; 12.10
-2TCID
50/ 0.1mL; 13.10
-3TCID
50/ 0.1mL; 14. cell RNA negative control; 15. bovine parainfluenza virus 3 type positive controls; 16. blank.
The result shows that the template minimum detectable concentration of this method is 10
-3TCID
50/ 0.1mL.
Fig. 3: the electrophoresis result figure of positive control pipe and negative control pipe PCR product
Among the figure from left to right swimming lane be followed successively by: M.DL2000 ladder; 2. positive control; 3. negative control; 4. blank.
Fig. 4: different annealing temperature expanding effect figure in the test kit PCR reaction process
Among the figure from left to right swimming lane be followed successively by: M.DL2000 ladder; 1～10 annealing temperature is respectively 56.5 ℃, and 57.3 ℃, 58.1 ℃, 58.9 ℃, 59.7 ℃, 60.5 ℃, 61.3 ℃, 62.1 ℃, 62.9 ℃, 63.7 ℃.
Fig. 5: Auele Specific Primer optimal concentration figure in the test kit PCR reaction process
Among the figure from left to right swimming lane be followed successively by: M.DL2000 ladder; 1～5 primer concentration is 1 μ mol/L respectively, 0.8 μ mol/L, 0.6 μ mol/L, 0.5 μ mol/L, 0.4 μ mol/L.
Embodiment
Case study on implementation one:
1 sample collecting and processing
Gather nose swab or the throat swab sample of disease ox, above sample is added 1: 5 suspension of sterile saline preparation, 4000r/min, centrifugal 10min, it is standby to get supernatant.
The extraction of 2 sample RNA templates
(1) get step 1 supernatant 330 μ L and add the 1.5mL centrifuge tube, add 1mTRIZOL, abundant mixing, room temperature is placed 10min.
(2) add chloroform by 200 μ L chloroform/mL Trizol, room temperature is placed 15min (annotating: forbid the vortex oscillation device, in order to avoid the genomic dna fracture) behind the thermal agitation mixing; 4 ℃, 12, the centrifugal 15min of 000g.Draw the upper strata water, to another centrifuge tube, (annotate: do not draw intermediate interface; If extract DNA and protein simultaneously, then keep lower floor's phenol and be stored in 4 ℃ of refrigerators mutually, if only carry RNA, then abandon lower floor's phenol phase).
(3) press 0.5mL Virahol/mLTrizol and add the Virahol mixing, room temperature is placed 5-10min.4 ℃, 12, the centrifugal 10min of 000g abandons supernatant, and RNA is sunken to the pipe end.
(4) add 75% ethanol by 1mL 75% ethanol/mL Trizol, gentle vibration centrifuge tube, precipitation suspends.4 ℃, 8, the centrifugal 5min of 000g abandons supernatant as far as possible.
(5) room temperature is dried or vacuum-drying 5-10min.Annotate: the RNA sample is too not dry, otherwise is difficult to dissolving.
(6) add 40 μ L DEPC treating water, prepare reverse transcription then.
3 use RT-PCR dedicated kit of the present invention to carry out following test
(1) reverse transcription reaction
Reaction system is 20 μ L, adds successively in the PCR reaction tubes, adds 3 μ L DEPC treating water, 1 μ L reverse transcription random primer and 8 μ L RNA templates.Behind 65 ℃ of 5min, put on ice rapidly.Add 4 μ L, 5 * RT damping fluid, 2 μ L dNTPs (10 μ M), 1 μ L RNA enzyme inhibitors (0.5 μ M), 1 μ L M-MLV (25U/ μ L) more successively, 30 ℃ of 30s, 99 ℃ of 5min, 42 ℃ of 20min, 4 ℃ of 5min obtain cDNA,-20 ℃ of preservations are with the template as further PCR.
Establish a positive control and a negative control simultaneously, reaction system and reaction conditions are the same, and different is to replace sample RNA with positive reference substance in this test kit and negative control product respectively.
(2) PCR reaction
Reaction system is 20 μ L, in the reverse transcription PCR pipe, add sterilization deionized water 12.5 μ L behind the reverse transcription reaction successively,, 10 * PCR Dream buffer, 2 μ L, Dream Taq archaeal dna polymerase (5U/ μ L) 0.5 μ L, dNTPs (2.5mmol/L) 1 μ L, bovine parainfluenza virus 3 type specificity upstream and downstream primer P1 and each 1 μ L of P2.
The PCR response procedures is: 94 ℃ of pre-sex change 5min; 94 ℃ of 30s, 58 ℃～62 ℃ 30s, 72 ℃ of 1min circulate 30 times; 72 ℃ are extended 5min, 16 ℃ of 10min.
(3) agarose gel electrophoresis detects
Respectively get 6 μ LPCR products and DL2000 Marker, respectively with 2 μ L sample-loading buffer mixings after 1 application of sample in 1% sepharose, 100V electrophoresis 30～45min utilizes gel imaging system to take a picture, the analytical electrophoresis result.
(4) result judges
At first check the electrophoresis result of positive control pipe and negative control pipe PCR product, should produce a size behind the positive pipe PCR product electrophoresis is the band of 425bp, and does not have band in the negative tube.And then see the result of sample hose, if produce the band of 425bp, be judged to be bovine parainfluenza virus 3 types and infect; If without any band, then be judged to feminine gender, do not infect this virus.
Above result shows, No. 1 point sample hole contrast be positive reference substance, No. 2 corresponding negative control product in point sample hole, No. 3 is blank (deionized water).
Case study on implementation two: the reaction condition optimization of the RT-PCR test kit of bovine parainfluenza virus 3 types
The result:
Fig. 4: be followed successively by M.DL2000 ladder from left to right; 1～10 annealing temperature is respectively 56.5 ℃, and 57.3 ℃, 58.1 ℃, 58.9 ℃, 59.7 ℃, 60.5 ℃, 61.3 ℃, 62.1 ℃, 62.9 ℃, 63.7 ℃.Picture shows that the suitableeest annealing temperature of this test kit PCR is 58.1 ℃.
Fig. 5: be followed successively by M.DL2000 ladder from left to right; 1～5 primer concentration is 1 μ mol/L respectively, 0.8 μ mol/L, 0.6 μ mol/L, 0.5 μ mol/L, 0.4 μ mol/L.Picture shows that the Auele Specific Primer optimal concentration is 1 μ mol/L in this test kit PCR reaction process.
Claims (2)
1. RT-PCR method that detects bovine parainfluenza virus 3 types, step comprises sample collecting, the extraction of viral RNA, the design of Auele Specific Primer, RT-PCR amplification, agarose gel electrophoresis, the result judges, it is characterized in that: described bovine parainfluenza virus 3 type specificity amplimers, and its sequence is:
P1：5’-GGATGTTTGGGAGTGATCTTGAGTA-3’；
P2：5’-TGTGTTGAAAAATGAAGCAAGACCT-3’；
Two-step approach is adopted in described RT-PCR amplification, adding length during reverse transcription is random primer 5 '-NNNNNNNNN-3 ' of 9bp, the specificity upstream and downstream primer P1 and the P2 that in reaction tubes, add above-mentioned bovine parainfluenza virus 3 types during the PCR reaction, RT-PCR reverse transcription reaction system is 20 μ L, comprise: 8 μ L RNA, 3 μ L DEPC handle deionized water, 1 μ L random primer, behind 65 ℃ of 5min, put on ice rapidly.Add 4 μ L, 5 * RT damping fluid, 10 μ M dNTPs2 μ L, 0.5 μ M RNA enzyme inhibitors, 1 μ L, 25U/ μ L M-MLV 1 μ L subsequently, the continuation reaction conditions is: 30 ℃ of 30s, and 99 ℃ of 5min, 42 ℃ of 20min, 4 ℃ of 5min obtain cDNA;
The PCR system is 20 μ L, wherein: sterilization deionized water 12.5 μ L, 10 * PCRbuffer, 2 μ L, 5U/ μ L Taq archaeal dna polymerase 0.5 μ L, 2.5mmol/L dNTPs 1 μ L, bovine parainfluenza virus 3 type upstream and downstream primer P1, each 1 μ L of P2, cDNA sample 2 μ L; The PCR response procedures is: 94 ℃ of pre-sex change 5min; 94 ℃ of 30s, 58 ℃～62 ℃ 30s, 72 ℃ of 1min circulate 30 times; 72 ℃ are extended 5min, 16 ℃ of 10min.Get PCR product 6 μ L and carry out 1% agarose electrophoresis, positive sample and positive reference substance size all occurs and are the PCR product band of 425bp, the no amplified production band of negative sample and negative control product.
2. an application rights requires the RT-PCR quick diagnosis reagent kit of 1 bovine parainfluenza virus 3 types, comprise: the sample viral RNA extracts reagent, reverse transcription reagent, PCR reagent, positive reference substance and negative control product, it is characterized in that: described reverse transcription reagent is by 5 times of RT damping fluids, dNTPs, the RNA enzyme inhibitors, M-MLV, the DEPC treating water, form with random primer, storage temperature-20 ℃, the reverse transcription reaction system is: 8 μ L RNA, 4 μ L, 5 * RT damping fluid, 3 μ L DEPC handle deionized water, 10 μ M dNTPs, 2 μ L, 1 μ L RNA enzyme inhibitors, 25U/ μ L M-MLV 1 μ L, 0.5 μ M random primer, 1 μ L; Described PCR reagent is that storage temperature is-20 ℃ by sterilization deionized water, 10 * PCR damping fluid, Taq archaeal dna polymerase, dNTPs and cDNA template.The PCR reaction system comprises: sterilization deionized water 12.5 μ l, 10 * PCR damping fluid, 2 μ L, 5U/ μ L Taq archaeal dna polymerase 0.5 μ l, 2.5mmol/L dNTPs 1 μ L, each 1 μ L of bovine parainfluenza virus 3 type specificity upstream and downstream primer P1 and P2.
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Cited By (5)
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| CN103468828A (en) * | 2013-09-16 | 2013-12-25 | 上海卓润生物科技有限公司 | PCR (Polymerase Chain Reaction) method for simultaneously detecting four bovine infectious disease RNA (Ribose Nucleic Acid) viruses by single tube |
| CN104391112A (en) * | 2014-05-27 | 2015-03-04 | 中国农业科学院特产研究所 | Expression protein detecting bovine parainfluenza virus 3 antibody and ELISA kit |
| CN106906307A (en) * | 2017-03-10 | 2017-06-30 | 中国农业科学院特产研究所 | The type of bovine parainfluenza virus 3 virus nano PCR detection kits and preparation method thereof |
| CN107988431A (en) * | 2017-12-13 | 2018-05-04 | 广西壮族自治区兽医研究所 | A kind of RT-PCR amplimers of quick detection 3 type of bovine parainfluenza virus and its application |
| CN110564858A (en) * | 2019-10-29 | 2019-12-13 | 成都益安博生物技术有限公司 | RT-PCR kit for predicting curative effect of immunodetection point regulation type medicines and prediction method thereof |
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2011
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468828A (en) * | 2013-09-16 | 2013-12-25 | 上海卓润生物科技有限公司 | PCR (Polymerase Chain Reaction) method for simultaneously detecting four bovine infectious disease RNA (Ribose Nucleic Acid) viruses by single tube |
| CN104391112A (en) * | 2014-05-27 | 2015-03-04 | 中国农业科学院特产研究所 | Expression protein detecting bovine parainfluenza virus 3 antibody and ELISA kit |
| CN106906307A (en) * | 2017-03-10 | 2017-06-30 | 中国农业科学院特产研究所 | The type of bovine parainfluenza virus 3 virus nano PCR detection kits and preparation method thereof |
| CN107988431A (en) * | 2017-12-13 | 2018-05-04 | 广西壮族自治区兽医研究所 | A kind of RT-PCR amplimers of quick detection 3 type of bovine parainfluenza virus and its application |
| CN110564858A (en) * | 2019-10-29 | 2019-12-13 | 成都益安博生物技术有限公司 | RT-PCR kit for predicting curative effect of immunodetection point regulation type medicines and prediction method thereof |
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