CN103468828A - PCR (Polymerase Chain Reaction) method for simultaneously detecting four bovine infectious disease RNA (Ribose Nucleic Acid) viruses by single tube - Google Patents
PCR (Polymerase Chain Reaction) method for simultaneously detecting four bovine infectious disease RNA (Ribose Nucleic Acid) viruses by single tube Download PDFInfo
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Abstract
The invention discloses a PCR (Polymerase Chain Reaction) method for simultaneously detecting four bovine infectious disease RNA (Ribose Nucleic Acid) viruses by a single tube. The PCR method is used for detecting a bovine parainfluenza virus, a foot and mouth disease virus, a bovine diarrhea virus and a bovine reovirus. The PCR method is rapid in operation without one step. In addition, the invention also relates to a reagent related in the PCR method, which is used in a detection kit of the method and preparation and application of the detection kit.
Description
Technical field
The invention belongs to the pathogen detection field, specifically, relate to a kind of multiple fluorescence quantitative PCR detection method of ox common transmittable disease pathogen body, more particularly, relate to a kind of fluorescence quantifying PCR method that detects to fast and parallel four kinds of ox transmissible disease RNA viruses (bovine parainfluenza virus, foot and mouth disease virus, bovine diarrhea virus, ox reovirus) in same PCR pipe.
Background technology
Bovine parainfluenza virus, foot and mouth disease virus, bovine diarrhea virus, ox reovirus are four kinds of common ox infectious disease pathogens.
Bovine parainfluenza virus belongs to haemadsorption virus 1 (PIV3), belongs to RNA viruses, under field conditions (factors), infected cattle only, sick ox and be main contagium with malicious ox, infect by the ox Contact, also can be by respiratory tract infection.This disease can cause Niu Buyu.And the Niu Changyin airway epithelial that infects BPIV3 is impaired and cause common germ and avail oneself of the opportunity to get in, and invades the respiratory tract of ill ox, thereby causes serious pneumonia, and mortality ratio is increased greatly.Foot and mouth disease virus belongs to Picornaviridae, aphthovirus genus, and infectivity is strong.Foot and mouth disease virus toxic amount in the bubble intracutaneous of ill domestic animal and lymph liquid is the highest.Row's virus quantity is with the epipharynx at ill domestic animal, lingual surface bubble or rotten to the corn place, between the hoof toe, hoof epithelial portion blister or speck place and breast place bubble maximum.The ox catched a foot and mouth disease there will be heating, walks lamely and occurs on skin and skin mucosa the symptoms such as blister macula.Pernicious foot and mouth disease also can cause ill domestic animal heart paralysis dead rapidly.Once being subject to foot and mouth disease virus, the people infects, sudden onset after the latent period of 2-18 days, show as fever, oral cavity is xeothermic, lip, gums, tongue limit, cheek, pharyngeal flush, bubble (finger tip, palm, toe) appears, simultaneously with having a headache, feel sick, vomit or diarrhoea, and sometimes can concurrent myocarditis.Bovine diarrhea virus is flaviviridae.Be a kind of single-stranded RNA, the virus of cyst membrane arranged.Virus can excrete with secretory product and movement.The persistent infection ox can be with throughout one's life, toxin expelling, thereby is the important contagium that this disease is propagated.This disease is mainly peroral infection, and susceptible animal is eaten contaminated feed, and drinking-water and through digestive tract infection also can be owing to sucking by the ill domestic animal cough, breathe the malicious spittle of the band of discharging and infect, and also can pass through the placentogenesis vertical infection.Sick ox shows as body temperature and is elevated to suddenly more than 40 ℃, continues 2-3 days, with the oligoleukocythemia of a property crossed.Conceived cow may show as embryo's Deaths, miscarriage or congenital anomaly.The reovirus gene group is linear dsRNA, from respiratory tract and the enteron aisle of humans and animals, separates at first, can cause the symptoms such as ox diarrhoea.
Now the detection of bovine parainfluenza virus, foot and mouth disease virus, bovine diarrhea virus, ox reovirus mainly be take to culture method and immunology detection as main.It is long that culture method detects the time needed; Sensitivity is low, is subject to such environmental effects larger, is prone to false negative; Operate loaded down with trivial details, high to operator's technical requirements.Immunological method is compared culture method very much progress, but also easily is subject to the other factors impact, false positive reaction occurs, brings the possibility of erroneous judgement.
Real-Time Fluorescent Quantitative PCR Technique has advantages of sensitivity, quick, high specificity, is suitable for the quick and precisely diagnosis of pathogenic agent.The method is mainly to add fluorescent probe on conventional PCR basis, accumulates the process of the whole PCR of Real-Time Monitoring by fluorescent signal, then utilizes the typical curve generated to carry out quantitative analysis to the template of unknown concentration.The continuously variation of fluorescent signal in detection system in the PCR reaction process.The method has high specificity, and has effectively solved the problem that PCR pollutes, and level of automation is high, is suitable for a large amount of characteristics such as pattern detection.At present, the method for real-time fluorescence quantitative PCR is used widely.
Pathogen detection in livestock industry needs promptly and accurately, and easy to operate, the primary treatment great amount of samples, be beneficial to the prevention and control of epidemic situation; But existing pathogen detection level still can not meet this kind of needs.Carry out primary first-order equation and can detect several livestock common disease pathogenic agent in same PCR pipe, especially whether the existence of ox infectious disease pathogens to be, provide fast result and be applicable to the large automatic analytical instrument with the methods involving that instructs next step epidemic preventing working and carry out and product at present in the real vacancy that belongs on the market.
Summary of the invention
The problems referred to above that exist for prior art, one of purpose of the present invention is to provide the PCR method that a kind of single tube detects four kinds of ox transmissible disease RNA viruses simultaneously.In multiple fluorescence PCR detection method of the present invention, reverse transcription and gene amplification one step complete, and have higher specificity and sensitivity, easy and simple to handle, and can be applicable on large automatic detection analytical instrument.
For achieving the above object, at first the present invention has designed the degree of interfering with each other acceptable primer pair and probe in PCR in real time detects for bovine parainfluenza virus, foot and mouth disease virus, bovine diarrhea virus, these four kinds of pathogenic agent of ox reovirus.The design of primers principle is: primer should be very special: Tm is between 58-65 ℃; GC content is 30～80%; Consecutive identical base is not arranged, and especially G surpasses the situation of 4; Article two, the annealing temperature of primer should be more approaching; Avoid primer dimer and hairpin structure etc.The principle of probe design is: Tm is higher 8～10 ℃ than primer; GC content is 20～80%; G must be less than 4 continuously; Probe can not form hairpin structure or can not form dimer etc. with primer.While using hybridization probe to be tested, tending to form primer-probe dimer, is mainly because probe can be hybridized with 3 ' end of primer, based on this, can further cause dimer amplification, thereby, with the raw material of goal gene competing reaction, cause reaction efficiency to descend.Probe can be with the goal gene combination due to itself, and melting temperature(Tm) is higher than primer, so it may cause extension as primer, Given this, the reply probe is well-designed, and by its terminal phosphate to avoid extension.
For getting rid of the false negative of PCR reaction, determine that negative findings does not cause because PCR reacts unsuccessfully, also need to select a suitable endogenous reference gene (From Template) to carry out augmentation detection.In the present invention, mark is template used derives from enterobacteria phage MS2 (Genebank FJ799612.1), and designs corresponding primer and probe (being mark probe in RNA), is added in each reaction vessel, carries out complete monitoring.
The concrete technical scheme adopted of the present invention is as follows:
A kind of in single PCR reaction vessel quadruple detect the real time quantitative PCR method of target nucleic acid in nucleic acid extraction liquid, described target nucleic acid is from bovine parainfluenza virus, foot and mouth disease virus, bovine diarrhea virus and ox reovirus, step comprises:
(1) extracting respectively the nucleic acid in sample, obtain nucleic acid extraction liquid, is total RNA of virus;
(2) mixed nucleus acid extraction liquid, ThermoScript II, RNA enzyme inhibitors, archaeal dna polymerase, dNTP, target nucleic acid primer pair and target nucleic acid probes in described single PCR reaction vessel, wherein said probe mark has fluorophor and quenching group, and the fluoroscopic examination wavelength of the fluorophor of various probe marks is different, wherein
The target nucleic acid primer pair comprises:
Bovine parainfluenza virus (BPIV):
BPIV-L：GGA?TGT?TAT?TTG?ACG?CCA?ACA?A
BPIV-R：TCC?CTC?TAT?CGA?GTG?GAA?GAC?ACT
Foot and mouth disease virus (FMDV):
FMDV-L：GAACACATTCTTTACACCAGGAT
FMDV-R：CATTCTTTGCCAATCAACATCAG
Bovine diarrhea virus (BVDV):
BVDV-L：TGGTTCGACGCCTTGGTATA
BVDV-R：CCCCGCTCAGGTTAAGATGT
Ox reovirus (RE0):
REO-L：GATCCTGATGTCTTGCGTCTAA
REO-R：CAGCAGTATTCCCGTCGATAAT
Internal reference:
IC-F-L：CTCGAGAACGCAAGTTC
IC-F-R：GGTGATTTCACCTCCAGTAT
Target nucleic acid probes comprises:
Bovine parainfluenza virus (BPIV):
BPIV-P：TTG?CGC?TTG?CAC?C
Foot and mouth disease virus (FMDV):
FMDV-P：ACAACCTACCGCCGAGCCAATTC
Bovine diarrhea virus (BVDV):
BVDV-P：TGGTTCGACGCCTTGGTATA
Ox reovirus (REO):
REO-P：AATTACGTGTGTCAAGGTGACGATGGA
Interior mark:
IC-F-P：CACGACAGTGGTCGCTACATA
(3) carry out the PCR reaction, detect in real time the fluorescent signal of different wave length;
(4) the Ct value calculated according to the fluoroscopic examination result has judged whether that target nucleic acid is present in sample.
The Ct value reaches for the fluorescent signal of amplified production the amplification cycles number that the threshold value of setting experiences.Now amplification is to be logarithmic phase to increase.
Nucleic acid system in sample of the present invention adopts the magnetic bead extraction method to extract, the process of extracting the nucleic acid in sample is: (the preferred nucleic acid extraction liquid comprises guanidinium isothiocyanate to add the nucleic acid extraction liquid to sample, sodium ethylene diamine tetracetate, tween 20, sodium perchlorate, ethanol and pH damping fluid (as, Tris-HCl)), add magnetic bead after insulation, after mixing, after applying magnetic field, discard wherein liquid, then washing (preferred washed twice, more preferably wherein wash for the first time washings used and comprise sodium perchlorate and ethanol, also more preferably wherein wash for the second time washings used and comprise ethanol), and wash-out (preferably wash-out elutriant used comprise the pH damping fluid (as, Tris-HCl).
In fluorescence PCR method provided by the invention, consider, when four kinds of ox transmissible disease viral template increase simultaneously, can compete enzyme, primer and probe, amplification is exerted an influence, therefore whole reaction system and reaction conditions are optimized to research.Get four kinds of viral nucleic acid extracting solutions, with corresponding primer and the probe of introducing, carry out the fluorescent PCR coamplification above, through optimizing, in fluorescent PCR system of the present invention (in single PCR reaction vessel), the concentration of each component is:
The PCR reaction system is: the reaction system cumulative volume is 60 μ l, and wherein the concentration of each component is respectively: Tris.HCl pH8.310mM; KCl50mM; MgCl25mM; DNTP0.2mM; Taq archaeal dna polymerase 5U; M-MLV ThermoScript II 150U; RNasin20U; Various primer 15pM/ kinds; Various fluorescent probe 5pM/ kinds; Glycerine 5% (v/v); Various target nucleic acid extracting solutions.Wherein various is the Nucleotide extract concentration 1000IU/mL, and consumption is 1ul.
Various probe marks have different fluorophors, and preferably fluorophor is FAM, TAMRA, ROX, Cy5, HEX.
Wherein, preferred reverse transcription and PCR condition are:
42 ℃ 30 minutes 50 ℃ 2 minutes 94 ℃ 3 minutes
(94 ℃ 10 seconds 52 ℃ 10 seconds 65 ℃ 45 seconds) 45 circulations
After reaction finishes, fluorescent collecting FAM, TAMRA, ROX, Cy5 and HEX.
The interpretation of result condition is set: the baseline of ABI7500 luminoscope is set automatically, and fluorescence threshold is set in (dR=1000).
1) FAM layer Ct value > 45 and HEX layer Ct value<40, be judged to be BPIV RNA feminine gender;
FAM layer Ct value > 45 and HEX layer Ct value >=40, need heavily to examine;
FAM layer Ct value≤40, be judged to be the BPIV RNA positive.
2) TAMRA layer Ct value > 45 and HEX layer Ct value<40, be judged to be FMDV RNA feminine gender;
TAMRA layer Ct value > 45 and HEX layer Ct value >=40, need heavily to examine;
TAMRA layer Ct value≤45, be judged to be the FMDV RNA positive.
3) Rox layer Ct value > 45 and HEX layer Ct value<40, be judged to be BVDV RNA feminine gender;
Rox layer Ct value > 45 and HEX layer Ct value >=40, need heavily to examine;
Rox layer Ct value≤45, be judged to be the BVDV RNA positive.
4) Cy5 layer Ct value > 45 and HEX layer Ct value<40, be judged to be REO RNA feminine gender;
Cy5 layer Ct value > 45 and HEX layer Ct value >=40, need heavily to examine;
Cy5 layer Ct value≤45, be judged to be the REO RNA positive.
Two of purpose of the present invention be to provide a kind of in single PCR reaction vessel quadruple detect the detection kit of the real-time PCR method of target nucleic acid in nucleic acid extraction liquid, main component is as follows:
(1) paramagnetic particle method extracts the required reagent of nucleic acid, comprising:
The nucleic acid extraction liquid, the preferred nucleic acid extraction liquid comprise guanidinium isothiocyanate, sodium ethylene diamine tetracetate, tween 20, sodium perchlorate, ethanol and pH damping fluid (as, Tris-HCl);
Wash for the first time washings used, preferably it comprises sodium perchlorate and ethanol;
Wash for the second time washings used, preferably it comprises ethanol; With,
The elutriant that wash-out is used, preferably its comprise the pH damping fluid (as, Tris-HCl).
(2) the required reagent of reverse transcription and Fluorescence PCR
ThermoScript II, RNA enzyme inhibitors, archaeal dna polymerase, dNTP, target nucleic acid primer pair and target nucleic acid probes, wherein said probe mark has fluorophor and quenching group, and the fluoroscopic examination wavelength of the fluorophor of various probe marks is different.
In test kit, various probe marks have different fluorophors, and preferably fluorophor is FAM, TAMRA, ROX, Cy5, HEX.
Test kit target nucleic acid primer pair comprises:
Bovine parainfluenza virus:
BPIV-L：GGA?TGT?TAT?TTG?ACG?CCA?ACA?A
BPIV-R：TCC?CTC?TAT?CGA?GTG?GAA?GAC?ACT
Foot and mouth disease virus:
FMDV-L：GAACACATTCTTTACACCAGGAT
FMDV-R：CATATCTTTGCCAATCAACATCAG
Bovine diarrhea virus:
BVDV-L：TGGTTCGACGCCTTGGTATA
BVDV-R：CCCCGCTCAGGTTAAGATGT
The ox reovirus:
REO-L：GATCCTGATGTCTTGCGTCTAA
REO-R：CAGCAGTATTCCCGTCGATAAT
Internal reference:
IC-F-L：CTCGAGAACGCAAGTTC
IC-F-R：GGTGATTTCACCTCCAGTAT
Target nucleic acid probes comprises:
Bovine parainfluenza virus:
BPIV-P：TTG?CGC?TTG?CAC?C
Foot and mouth disease virus:
FMDV-P：ACAACCTACCGCCGAGCCAATTC
Bovine diarrhea virus:
BVDV-P：TGGTTCGACGCCTTGGTATA
The ox reovirus:
REO-P：AATTACGTGTGTCAAGGTGACGATGGA
Internal reference:
IC-F-P：CACGACAGTGGTCGCTACATA
In test kit, every kind of target nucleic acid probes at described reaction system cumulative volume is being the preferred 5pmol/ reaction of concentration in the single PCR reaction vessel of 60 μ l.
In this article, " sample " detected is the potential vitro samples that may contain target nucleic acid or microorganism.Method of the present invention preferably only limits to the detection to vitro samples, the direct result of detection be target nucleic acid or microorganism existence whether.
In this article, " single PCR reaction vessel " refers to that described real-time PCR method carries out technically in same container.The step that just can complete pcr amplification and detect in real time in same container, realize the detection to four kinds of viral target nucleic acids.
In the real-time fluorescence PCR reaction of this paper, the cycle number experienced during the threshold value of the interior fluorescent signal arrival of each PCR reaction tubes of Ct value representation real-time fluorescence PCR instrument institute default setting, as the index of sentence read result.In detection method of the present invention, for step (4), target nucleic acid Ct value<45, this target nucleic acid is present in sample; The Ct value > 45, be not present in sample.
Beneficial effect of the present invention is: can detect in sample whether have bovine parainfluenza virus, foot and mouth disease virus, bovine diarrhea virus, ox reovirus by single tube, four kinds of primer/probes are without crossed contamination and interference, guarantee accuracy, reliability, specificity, sensitivity and repeatability, and can use the commercially available real-time fluorescence PCR equipment of current routine to complete.
The accompanying drawing explanation
The PCR in real time amplification curve diagram of Fig. 1 embodiment of the present invention to bovine parainfluenza virus, foot and mouth disease virus, bovine diarrhea virus, ox reovirus, in figure, five kinds of curves can clearly be distinguished.
Embodiment
Below in conjunction with embodiment to the present invention do further in detail, intactly explanation.
As do not specialize part, can be according to " molecular cloning experiment guide " (third edition) (Cold Spring Harbor laboratory Press), " cell experiment guide " (Science Press that those skilled in the art were familiar with, Beijing, China, calendar year 2001), " RNA experimental technique handbook (Science Press, Beijing, China, 2004), " immunoassay technology " (Science Press, Beijing, China, 1991) etc. the listed method of laboratory manual is implemented.
Probe used, primer entrust Shanghai Sangon Biological Engineering Technology And Service Co., Ltd synthetic.
Consumptive material used and instrument are all through processing to guarantee the complete and high purity of RNA without RNase.
Bovine parainfluenza virus, foot and mouth disease virus, bovine diarrhea virus, ox reovirus pseudovirion are this laboratory and synthesize and preserve; Those of ordinary skills also can obtain by conventional means.
1, nucleic acid extraction
Utilize engineered technique means, bovine parainfluenza virus, foot and mouth disease virus, bovine diarrhea virus, ox reovirus strain are made to corresponding pseudovirion, for nucleic acid extraction.
The extraction of nucleic acid is extracted according to conventional magnetic bead extraction method, method is as follows: to 10 μ l standard sampless (pseudovirion 1*104IU/ml), add 90 μ l nucleic acid extraction liquids (formula and final concentration are: guanidinium isothiocyanate 12M, sodium ethylene diamine tetracetate (pH8.0) 10mM, tween 20 2% (W/W), sodium perchlorate 1.0M, ethanol 40% (V/V), Tris-HCl (pH8.0) 10mM), in 42 ℃ of insulation 10min, then add 10 μ l Mag
bead suspension (50mg/mL, purchased from Beijing Chinese mugwort beguine Bioisystech Co., Ltd), after vibration mixes, be enclosed within on magnet stand and apply magnetic field, discard wherein liquid, then (formula and final concentration are: sodium perchlorate 1M to add 200 μ l washings A, ethanol 30% (V/V)), discard washings A after washing, (formula and final concentration are: ethanol 70% (V/V)) to add 200 μ l washings B again, discard washings B after washing, (formula and final concentration are: Tris-HCl (pH8.0) 10mM) finally to add elutriant, in 42 ℃ of insulation 10min, sucking-off retaining liquid, be nucleic acid extraction liquid, for the total RNA of the genome of every kind of virus.
2, the Fluorescence PCR system is determined
By groping, determine the combined concentration of each component, especially probe and primer in the PCR reaction system.
2.1 primer and probe sequence
In the present invention, template DNA or the RNA sequence of the reference of the synthetic institute of all primers and probe are respectively:
The template used numbering on Genebank of various virus and interior mark is respectively:
Bovine parainfluenza virus: HQ530159.1
Foot and mouth disease virus: AF5110391
Bovine diarrhea virus: KC695815.1
Ox reovirus: AY007393.1
Internal standard gene: FJ799612.1
Above Template Information those skilled in the art all can be known by open approach according to the Genebank numbering, no longer list concrete sequence in the present invention.
The primer pair of target nucleic acid comprises:
Detect the primer pair of bovine parainfluenza virus:
BPIV-L：GGA?TGT?TAT?TTG?ACG?CCA?ACA?A
BPIV-R：TCC?CTC?TAT?CGA?GTG?GAA?GAC?ACT
Detect the primer pair of foot and mouth disease virus:
FMDV-L：GAACACATTCTTTACACCAGGAT
FMDV-R：CATATCTTTGCCAATCAACATCAG
Detect the primer pair of bovine diarrhea virus:
BVDV-L：TGGTTCGACGCCTTGGTATA
BVDV-R：CCCCGCTCAGGTTAAGATGT
Detect the primer pair of ox reovirus:
REO-L：GATCCTGATGTCTTGCGTCTAA
REO-R：CAGCAGTATTCCCGTCGATAAT
Interior label primer pair:
IC-F-L：CTCGAGAACGCAAGTTC
IC-F-R：GGTGATTTCACCTCCAGTAT
Target nucleic acid probes comprises:
The bovine parainfluenza virus probe:
BPIV-P：TTG?CGC?TTG?CAC?C
The foot and mouth disease virus probe:
FMDV-P：ACAACCTACCGCCGAGCCAATTC
The bovine diarrhea virus probe:
BVDV-P：TGGTTCGACGCCTTGGTATA
Ox reovirus probe:
REO-P：AATTACGTGTGTCAAGGTGACGATGGA
Interior mark probe:
IC-F-P：CACGACAGTGGTCGCTACATA
2.2 fluorescent mark
5 ' the end at above-mentioned probe BPIV-P, FMDV-P, BVDV-P, REO-P and IC-F-P is entrusted respectively complex sign fluorescent mark FAM, TAMRA, ROX, CY5 and HEX, and at the corresponding quenching group of 3 ' end mark.
2.3 the combined concentration of primer and probe
(1) PCR reaction system cumulative volume is 60 μ l, and wherein the concentration of each component is respectively: Tris.HCl pH8.310mM; KCl50mM; MgCl
25mM; DNTP0.2mM; Taq archaeal dna polymerase 5U; M-MLV ThermoScript II 150U; RNasin20U; Various primer 15pmol/ kinds; Various fluorescent probe 5pmol/ kinds; Glycerine 5% (v/v); Various target nucleic acid extracting solutions.Wherein various is the Nucleotide extract concentration 1000IU/mL, and consumption is 1ul.
2.4 reverse transcription and PCR condition
42 ℃ 30 minutes 50 ℃ 2 minutes 94 ℃ 3 minutes
(94 ℃ 10 seconds 52 ℃ 10 seconds 65 ℃ 45 seconds) 45 circulations
2.5 test-results
Fluorescent collecting FAM, TAMRA, ROX, Cy5 and HEX.
The interpretation of result condition is set: the baseline of ABI7500 luminoscope is set automatically, and fluorescence threshold is set in (dR=1000).
FAM layer Ct value≤40, bovine parainfluenza virus (BPIV) the RNA positive.
TAMRA layer Ct value≤45, foot and mouth disease virus (FMDV) the RNA positive.
Rox layer Ct value≤45, bovine diarrhea virus (BVDV) the RNA positive.
Cy5 layer Ct value≤45, ox reovirus (REO) the RNA positive.
Various virus and interior target pcr amplification curve are referring to accompanying drawing 1.
3. quantitative fluorescent PCR condition optimizing result
Four kinds of pseudovirions (10
4iU/ml) after being increased in the fluorescent PCR system,
When primer probe ratio is 3:1, the Ct value of BPIV, FMDV, BVDV and REO is respectively 29.18-31.78,28.25-30.86,2433-2563 and 25.84-28.57; When primer probe ratio is 2:1, the Ct value of BPIV, FMDV, BVDV and REO is respectively 31.22-33.55,32.06-33.95,25.83-26.25 and 28.98-29.67; When primer probe ratio is 4:1, the Ct value of BPIV, FMDV, BVDV and REO is respectively 32.02-34.68,29.77-31.53,24.83-26.29 and 27.98-30.31.
Embodiment 2 test kit specific detection
The total RNA of the samples such as rinderpest virus, bovine leukemia virus, bovine epizootic fever virus, bovine respiratory syncytial virus, bovine parainfluenza virus, foot and mouth disease virus, bovine diarrhea virus, ox reovirus of take is template, adopt primer and the probe of this experimental design to carry out the real-time fluorescence PCR reaction, result only has bovine parainfluenza virus, foot and mouth disease virus, bovine diarrhea virus, ox reovirus to produce fluorescent signal, and the amplification situation is referring to table 1.Proof present method has specificity for the detection of bovine parainfluenza virus, foot and mouth disease virus, bovine diarrhea virus, ox reovirus.
Table 1 single tube detects four kinds of ox RNA viruses method specificity fluorescent pcr amplification results
Result shows: the specificity of this detection kit is high, with multiple factors such as rinderpest virus, bovine leukemia virus, bovine epizootic fever virus, bovine respiratory syncytial viruses, does not intersect.
The present invention adopts the fluorescence PCR method single tube to detect four kinds of ox transmissible disease RNA viruses, and traditional immunological method of comparing, can be used on the full-automatic instrument real-time PCR, and have characteristics easy and simple to handle, highly sensitive and reproducible.Therefore, the present invention can be used to carry out the external pattern detection of rapid, high volume, has good application prospect.
Finally be necessary described herein: above embodiment is only for being described in more detail technical scheme of the present invention; can not be interpreted as limiting the scope of the invention, some nonessential improvement that those skilled in the art's foregoing according to the present invention is made and adjustment all belong to protection scope of the present invention.
Claims (10)
1. detect the real time fluorescent PCR method of four kinds of target nucleic acids in single PCR reaction tubes simultaneously, described target nucleic acid is from bovine parainfluenza virus, foot and mouth disease virus, bovine diarrhea virus, ox reovirus, carry out complete monitoring with mark in RNA, step mainly comprises simultaneously:
(1) extract the nucleic acid in sample, obtain viral total nucleic acid extracting solution;
(2) in described single PCR reaction tubes mixed nucleus acid extraction liquid, ThermoScript II, RNA enzyme inhibitors, archaeal dna polymerase, dNTP, target nucleic acid primer pair and probe, interior label primer to and probe, wherein said probe mark has fluorophor and quenching group, and the fluoroscopic examination wavelength of the fluorophor of various probe marks is different, wherein
The target nucleic acid primer pair comprises:
Bovine parainfluenza virus:
BPIV-L：GGA?TGT?TAT?TTG?ACG?CCAACAA
BPIV-R：TCC?CTC?TAT?CGA?GTG?GAA?GAC?ACT
Foot and mouth disease virus:
FMDV-L：GAACACATTCTTTACACCAGGAT
FMDV-R：CATATCTTTGCCAATCAACATCAG
Bovine diarrhea virus:
BVDV-L：TGGTTCGACGCCTTGGTATA
BVDV-R：CCCCGCTCAGGTTAAGATGT
The ox reovirus:
REO-L：GATCCTGATGTCTTGCGTCTAA
REO-R：CAGCAGTATTCCCGTCGATAAT
Interior mark:
IC-F-L：CTCGAGAACGCAAGTTC
IC-F-R：GGTGATTTCACCTCCAGTAT
Target nucleic acid probes comprises:
Bovine parainfluenza virus:
BPIV-P：TTG?CGC?TTG?CAC?C
Foot and mouth disease virus:
FMDV-P：ACAACCTACCGCCGAGCCAATTC
Bovine diarrhea virus:
BVDV-P：TGGTTCGACGCCTTGGTATA
The ox reovirus:
REO-P：AATTACGTGTGTCAAGGTGACGATGGA
Interior mark:
IC-F-P：CACGACAGTGGTCGCTACATA
(3) carry out the PCR reaction, and detect in real time the fluorescent signal of different wave length;
(4) the Ct value calculated according to the fluoroscopic examination result has judged whether that target nucleic acid is present in sample.
2. method according to claim 1, the nucleic acid system that uses the method to extract in sample adopts the magnetic bead extraction method to extract, it is characterized in that step is as follows: to sample, add the nucleic acid extraction liquid, this nucleic acid extraction liquid comprises guanidinium isothiocyanate, sodium ethylene diamine tetracetate, tween 20, sodium perchlorate, ethanol and pH damping fluid Tris-HCl, add magnetic bead after insulation, mix, after applying magnetic field, discard wherein liquid, then twice washing, the washings that comprises sodium perchlorate and ethanol is used in washing for the first time, the washings that comprises ethanol is used in washing for the second time, finally adopt and comprise pH damping fluid Tris-HCl wash-out.
3. method according to claim 1, it is characterized in that: the concentration of every kind of target nucleic acid probes in described single PCR reaction tubes is preferably 5pmol/test.
4. method according to claim 1, it is characterized in that: various probe marks have different fluorophors, and preferably fluorophor is FAM, TAMRA, ROX, Cy5, HEX.
5. method according to claim 1 is characterized in that the condition of reverse transcription and PCR is:
42 ℃ 30 minutes 50 ℃ 2 minutes 94 ℃ 3 minutes
(94 ℃ 10 seconds 52 ℃ 10 seconds 65 ℃ 45 seconds) 45 circulations,
Fluorescent collecting FAM, TAMRA, ROX, Cy5, HEX.
6. method according to claim 1 is characterized in that the method for the Ct value judgement target nucleic acid that calculates according to the fluoroscopic examination result is:
1) FAM layer Ct value > 45 and HEX layer Ct value<40, be judged to be BPIV RNA feminine gender;
FAM layer Ct value > 45 and HEX layer Ct value >=40, need heavily to examine;
FAM layer Ct value≤40, be judged to be the BPIV RNA positive.
2) TAMRA layer Ct value > 45 and HEX layer Ct value<40, be judged to be the FMDVRNA feminine gender;
TAMRA layer Ct value > 45 and HEX layer Ct value >=40, need heavily to examine;
TAMRA layer Ct value≤45, be judged to be the FMDV RNA positive.
3) Rox layer Ct value > 45 and HEX layer Ct value<40, be judged to be BVDV RNA feminine gender;
Rox layer Ct value > 45 and HEX layer Ct value >=40, need heavily to examine;
Rox layer Ct value≤45, be judged to be the BVDV RNA positive.
4) Cy5 layer Ct value > 45 and HEX layer Ct value<40, be judged to be REO RNA feminine gender;
Cy5 layer Ct value > 45 and HEX layer Ct value >=40, need heavily to examine;
Cy5 layer Ct value≤45, be judged to be the REO RNA positive.
7. for detect the detection kit of the real time fluorescent PCR method of four kinds of target nucleic acids at single PCR reaction tubes simultaneously, mainly comprise:
(1) the required reagent of magnetic bead extraction method comprises:
The nucleic acid extraction liquid comprises guanidinium isothiocyanate, sodium ethylene diamine tetracetate, tween 20, sodium perchlorate, ethanol and pH damping fluid Tris-HCl:
Wash for the first time washings used, comprise sodium perchlorate and ethanol;
Wash for the second time washings used, comprise ethanol;
The elutriant that wash-out is used, comprise pH damping fluid Tris-HCl.
(2) PCR reacts required reagent
ThermoScript II, RNA enzyme inhibitors, archaeal dna polymerase, dNTP, target nucleic acid primer pair and probe, interior label primer are to reaching probe, wherein said probe two ends are marked with fluorophor and quenching group, and the fluoroscopic examination wavelength of the fluorophor of various probe marks is different.
8. test kit according to claim 7, its special medical treatment is: various probe marks have different fluorophors, and preferably fluorophor is FAM, TAMRA, ROX, Cy5, HEX.
9. test kit target nucleic acid primer pair according to claim 7 and probe comprise:
Bovine parainfluenza virus:
BPIV-L：GGA?TGT?TAT?TTG?ACG?CCA?ACAA
BPIV-R：TCC?CTC?TAT?CGA?GTG?GAA?GAC?ACT
Foot and mouth disease virus:
FMDV-L：GAACACATTCTTTACACCAGGAT
FMDV-R：CATATCTTTGCCAATCAACATCAG
Bovine diarrhea virus:
BVDV-L：TGGTTCGACGCCTTGGTATA
BVDV-R：CCCCGCTCAGGTTAAGATGT
The ox reovirus:
RE0-L：GATCCTGATGTCTTGCGTCTAA
REO-R：CAGCAGTATTCCCGTCGATAAT
Interior mark:
IC-F-L：CTCGAGAACGCAAGTTC
IC-F-R：GGTGATTTCACCTCCAGTAT
Target nucleic acid probes comprises:
Bovine parainfluenza virus:
BPIV-P：TTG?CGC?TTG?CAC?C
Foot and mouth disease virus:
FMDV-P：ACAACCTACCGCCGAGCCAATTC
Bovine diarrhea virus:
BVDV-P：TGGTTCGACGCCTTGGTATA
The ox reovirus:
RE0-P：AATTACGTGTGTCAAGGTGACGATGGA
Interior mark:
IC-F-P：CACGACAGTGGTCGCTACATA。
10. test kit according to claim 7, its special medical treatment is: every kind of target nucleic acid probes is that concentration in 60 μ l is the 5pmol/ reaction at described single PCR reaction tubes reaction system cumulative volume.
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