CN104313186A - Dual-fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit for duck tembusu viruses and duck origin Newcastle disease viruses - Google Patents
Dual-fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit for duck tembusu viruses and duck origin Newcastle disease viruses Download PDFInfo
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Abstract
The invention discloses a dual-fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit for duck tembusu viruses and duck origin Newcastle disease viruses. The dual-fluorescence quantitative RT-PCR detection kit comprises a primer group and a probe group, wherein the primer group contains primers 1 to 4 which respectively have base sequences represented by the sequence sheets SEQ.ID.No.1 and 2 and SEQ.ID.No.4 and 5; the probe group is provided with a probe A and a probe B which respectively have base sequences represented by the sequence sheets SEQ.ID.No.3 and 6. An experiment shows that the dual-fluorescence quantitative RT-PCR detection kit has the advantages of short detection time, high detection sensitivity and high specificity and can simultaneously detect two pathogens such as the duck tembusu viruses and the duck origin Newcastle disease viruses and quantify the corresponding pathogen content, so that the detection kit can be applied to a vaccine for the duck tembusu viruses and the duck origin Newcastle disease viruses and the evaluation of the medicament treating effect, as well as the research on the aspects such as the pathogenesis; therefore, the dual-fluorescence quantitative RT-PCR detection kit has an important significance for prevention and treatment on the duck tembusu viruses and the duck origin Newcastle disease viruses.
Description
Technical field
The invention belongs to RT-PCR detection kit technical field, particularly relate to a kind of duck tembusu virus and duck source Avian pneumo-encephalitis virus bifluorescence quantitative RT-PCR detecting kit.
Background technology
Duck tembusu virus (Duck Tembusu Virus, DTMUV) is harm cause of disease.This disease is infringement laying ducks and duckling mainly, and cause laying ducks to lay eggs and sharply decline with a small amount of dead, cause laying ducks high mortality, since outburst in 2010, duck industry is supported in serious harm.Duck source Avian pneumo-encephalitis virus (Newcaslte Disease Virus, NDV), causes the egg drop reduction of laying ducks equally.After duck tembusu virus and duck source newcastle disease virus infection laying ducks, cause similar symptom and egg drop reduction, therefore in the urgent need to setting up a kind of duck tembusu virus and duck source Newcastle Disease virus detection method of quick sensitivity, these virus just can be detected in early days in infection, thus strive for the time of preciousness for the control of these diseases.At present, the traditional detection method of this two-strain has virus purification, agar diffusion test etc., but these detect and have that length consuming time, susceptibility are lower, the not easily shortcoming such as stdn, have certain limitation in actual applications.
It is quantitative that fluorescent quantitative PCR technique achieves template, and have sensitivity, special, accurately and reliably, multiple reaction can be realized and the feature such as real-time is good.In actual applications, when sample size is very large, just there is certain inferior position at cost with in the time in substance fluorescent PCR, carries out rapid detection in batches in the urgent need to a kind of high-throughput, low cost, high efficiency method.Multiple fluorescence PCR adopts multipair primer amplification to detect multiple template, overcomes the deficiency of substance fluorescent PCR.But, set up a multiple fluorescence PCR method more more complex than substance, it is higher to the requirement of reagent and primer, and without interference mutually simultaneously between the fluorophor needing guarantee different probe to mark, the quantitative real time PCR Instrument of use has corresponding multiple sense channel.At present, yet there are no application multiple fluorescence quantitative PCR technology carries out diagnosis and detection report to duck tembusu virus and duck source Avian pneumo-encephalitis virus.
Summary of the invention
The technical problem to be solved in the present invention is to provide that a kind of susceptibility is good, specificity is high, accurately and reliably, duck tembusu virus and duck source Avian pneumo-encephalitis virus bifluorescence quantitative RT-PCR detecting kit quickly and easily, to realize detecting and quantitatively duck tembusu virus and duck source Avian pneumo-encephalitis virus two kinds of pathogenic agent simultaneously.
For solving the problems of the technologies described above, the present invention is by the following technical solutions: duck tembusu virus and duck source Avian pneumo-encephalitis virus bifluorescence quantitative RT-PCR detect primer sets, comprise two pairs of Auele Specific Primers, be primer 1 and 2, primer 3 and 4 respectively, they have the base sequence of sequence table SEQ .ID.No.1 and 2, SEQ.ID.No.4 and 5 respectively.
Duck tembusu virus and duck source Avian pneumo-encephalitis virus bifluorescence quantitative RT-PCR detecting kit, comprise primer sets and probe groups; Primer sets has primer 1 to 4, and they have the base sequence of sequence table SEQ .ID.No.1 and 2, SEQ.ID.No.4 and 5 respectively; Probe groups has probe A and probe B, and they have the base sequence of sequence table SEQ .ID.No.3 and 6 respectively.
5 ' the end of probe A is marked with reporter fluorescence dyestuff ROX, and 3 ' end is marked with quencher fluorescent dye BHQ2; 5 ' the end of probe B is marked with reporter fluorescence dyestuff FAM, and 3 ' end is marked with quencher fluorescent dye BHQ1.
The mol ratio of primer 1, primer 2, probe A, primer 3, primer 4 and probe B is 1:1:1:1:1:1.
This test kit contains following reagent:
A liquid: pcr amplification damping fluid, primer 1, primer 2, probe A, primer 3, primer 4, probe B;
B liquid: DTMUV+NDV template, as positive control;
C liquid: ddH
2o, as negative control.
In A liquid, primer 1, primer 2, probe A final concentration are 0.2 μm of ol/L, and primer 3, primer 4, probe B final concentration are 0.2 μm of ol/L.
For lacking the technology effectively reliably of duck tembusu virus and duck source Avian pneumo-encephalitis virus being carried out to diagnosis and detection at present simultaneously, contriver studies and has screened the two specific probe of cover and primers, and by carrying out the proportioning of different concns, obtain the concentration combination of best primer and probe, establish the detection method of the bifluorescence quantitative RT-PCR of duck tembusu virus and duck source Avian pneumo-encephalitis virus accordingly, and prepare corresponding detection kit.Experiment proves, tool of the present invention has the following advantages:
1) detection time is short
Application the present invention can realize the object that a pipe two is examined, and reverse transcription and PCR mono-step complete, only need about 30 minutes, and reaction result can directly be observed by computer, and the RT-PCR method of routine needs to have come for 3.5 hours amplified reaction at least, then must spend 1 hour and carry out gel electrophoresis and carry out observations;
2) the high and high specificity of detection sensitivity
When duck tembusu virus and duck source Avian pneumo-encephalitis virus exist simultaneously, the present invention compares with the CT value that single virus detects its CT value detected, and result is substantially the same, does not affect the susceptibility detecting and detect to duck tembusu virus and duck source Avian pneumo-encephalitis virus.In addition, utilize the present invention also can carry out quantitatively to cause of disease content corresponding in sample, and detection sensitivity is very high, all 100 duck tembusu viruses copied and duck source Avian pneumo-encephalitis virus can be detected, higher than conventional PCR method susceptibility 10 times, thus can be used for the assessment of duck tembusu virus and duck source Newcastle Disease Virus Vaccine and curative effect of medication, and the research of the aspect such as its pathogenesis, therefore the control of the present invention to duck tembusu virus and duck source Avian pneumo-encephalitis virus is significant.
Accompanying drawing explanation
Fig. 1 is that fluorescence quantitative RT-RCR detects in susceptibility (ROX passage) result figure, the figure of duck tembusu virus: 1 ~ 9 is respectively 1 × 10
10~ 1 × 10
2copy/μ l, 10 blanks (for DEPC water).
Fig. 2 is the typical curve that fluorescence quantitative RT-RCR detects the susceptibility (ROX passage) of duck tembusu virus.
Fig. 3 is that fluorescence quantitative RT-RCR detects in susceptibility (FAM passage) result figure, the figure of duck source Avian pneumo-encephalitis virus: 1 ~ 9 is respectively 1 × 10
10~ 1 × 10
2copy/μ l, 10 blanks (for DEPC water).
Fig. 4 is the typical curve that fluorescence quantitative RT-RCR detects the susceptibility (FAM passage) of duck source Avian pneumo-encephalitis virus.
Fig. 5 is specificity (ROX passage) the result figure that fluorescence quantitative RT-RCR detects duck tembusu virus, in figure: 1 duck tembusu virus, 2 duck tembusu viruses+duck source Avian pneumo-encephalitis virus, 3 Muscovy duck parvovirus, 4 duck circovirus, 5 egg-decreasing syndrome viruses, 6 duck plague viruses, 7H9 subtype avian influenza, 8ddH
2o.
Fig. 6 is specificity (FAM passage) the result figure that fluorescence quantitative RT-RCR detects duck source Avian pneumo-encephalitis virus, in figure: wherein 1 duck source Avian pneumo-encephalitis virus, 2 duck tembusu viruses+duck source Avian pneumo-encephalitis virus, 3 Muscovy duck parvovirus, 4 duck circovirus, 5 egg-decreasing syndrome viruses, 6 duck plague viruses, 7H9 subtype avian influenza, 8ddH
2o.
Fig. 7 is that fluorescence quantitative RT-RCR detects in crowd interior repeatability (ROX passage) result figure, figure of duck tembusu virus: four repeat samples in same batch of 1-4 difference, 5-7 is negative control.
Fig. 8 is that fluorescence quantitative RT-RCR detects in crowd interior repeatability (FAM passage) result figure, figure of duck source Avian pneumo-encephalitis virus: 1-4 is respectively four repeat samples in same batch, 5 and 6 blanks.
Embodiment
The experimental technique used in following examples if no special instructions, is ordinary method; Material used, reagent etc., if no special instructions, all can obtain from commercial channels.Concrete material therefor and reagent as follows:
Lightcycler2.0 quantitative real time PCR Instrument (Roche); DNA segment recovery test kit and plasmid Mini Kit are purchased from BioDev company; PGEM-T Easy test kit is purchased from Promega company; T7 in-vitro transcription test kit is purchased from Fermengtas company; One Step PrimeScript RT-PCR Kit is purchased from the precious biotech firm in Dalian; TIANamp virus genom DNA/RNA extracts test kit purchased from Tian Gen biochemical technology company limited.
Duck tembusu virus is documented in " 4 strain Guangxi Ya Yuan tembusu viruses are separated and preliminary evaluation ", Chinese Animal Quarantine, and 2013,30 (6): 31-35, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
Egg-decreasing syndrome virus is documented in " research of egg-decreasing syndrome vegetables oil emulsion seedling ", Chinese Preventive Veterinary Medicine report, and 2000, (04): 23-27, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
Duck circovirus is documented in " investigation of some areas of Guangxi duck circovirus infection conditions ", Chinese animal and veterinary, and 2010,37 (11): 156-158, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
Goose Parvovirus is documented in " foundation of Goose Parvovirus fluorescent quantitative PCR detection method ", Shanghai animal and veterinary communication, and 2008,160 (06): 30-31, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
Avian pneumo-encephalitis virus is documented in " research of newcastle disease vegetables oil emulsion seedling ", Chinese Preventive Veterinary Medicine report, and 2000, (04): 23-27, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
Duck plague virus AV1221 strain is purchased from China Veterinery Drug Inspection Office;
H9 subtype avian influenza virus is documented in " multiplex reverse polymerase chain reaction rapid detection differentiates the foundation of H9 subtype avian influenza virus method ", China Amphixenosis journal, 2006, (09): 858-860, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region.
The Design and synthesis of embodiment 1, primer and Taqman probe
According to the conserved sequence of duck tembusu virus in GenBank and duck source Avian pneumo-encephalitis virus, adopt Primer Express 3.0 software, devise many cover probes and primer, by analyzing the dimer between its primer, selected two pairs of Auele Specific Primers and two Taqmam probes (table 1).
Table 1 is primer and TaqMan probe sequence (5 '-3 ')
The foundation that embodiment 2, fluorescence quantitative RT-RCR detect
One, the determination of fluorescence quantitative RT-PCR detecting method
1, the preparation of sample
1), the extraction of nucleic acid
Extract test kit specification sheets with reference to TIANamp virus genom DNA/RNA, extract the RNA of duck tembusu virus, Avian pneumo-encephalitis virus and H9 subtype avian influenza, extract the DNA of egg-decreasing syndrome virus, duck circovirus, Goose Parvovirus and duck plague virus.
2), the reverse transcription of RNA
Duck tembusu virus, duck source Avian pneumo-encephalitis virus and H9 subtype avian influenza reverse transcription synthesis cDNA, specific as follows: to set up following reverse transcription system, total reaction volume is 50 μ L) duck tembusu virus, Avian pneumo-encephalitis virus and H9 subtype avian influenza RNA are 36.5 μ L (about 20 μ g), 10 μ L 5 × PCR damping fluids (precious biotechnology company limited in Dalian, catalog number: 2680A), 2 μ L 10mM dNTP (four kinds of bases) (the precious biotechnology company limited in Dalian, catalog number: 4019), 1 μ L RNA inhibitor (the precious biotechnology company limited in Dalian, catalog number: 2313A), 1 μ L random primer (the precious biotechnology company limited in Dalian, catalog number: 3802), 1 μ L ThermoScript II (the precious biotechnology company limited in Dalian, catalog number: 2680A), be 20 μ L with adding to cumulative volume without Rnase water, evenly centrifugal, in 25 DEG C of 10min, 42 DEG C of 60min, 95 DEG C of 5min, 4 DEG C of end, obtain duck tembusu virus, duck source Avian pneumo-encephalitis virus and H9 subtype avian influenza cDNA.
2, the preparation of standard substance
Respectively with the cDNA of duck tembusu virus obtained above and duck source Avian pneumo-encephalitis virus for template carries out pcr amplification, reaction system is that 50 μ L are (containing 0.2mmol/L dNTP, 2.5mmol/L MgCl
2, 0.5 μm of ol/L primer (table 1), 1.25U Taq polysaccharase, 1 × PCR buffer, 5 μ L DNA profiling), reaction conditions is: 95 DEG C of denaturation 5min, 94 DEG C of 60s, 50 DEG C of 60s, 70 DEG C of 60s, 35 circulations, and 72 DEG C extend 10min.
PCR primer is through 2% agarose gel electrophoresis, reclaim object segment rear clone to pGEM-T Easy carrier, positive colony bacterium (pGEM-NDV and pGEM-TMUV) send Dalian Bao Sheng Bioisystech Co., Ltd to check order, the PCR primer size contained in pGEM-TMUV bacterium is 68bp, the 1-68 position Nucleotide with SEQ.ID.No.7 in sequence table (is the NS5 sequence of TMUV GX2013H strain, No. genbank is KJ700462,9995-10062 position Nucleotide), be illustrated as positive colony, containing TMUV virus; The PCR primer size contained in pGEM-NDV bacterium is 66bp, the 1-66 position Nucleotide with SEQ.ID.No.8 in sequence table (is the NP gene of duck-China-Guangxi19-2009 strain, No. genbank is: JX193080.1,1671-1736 position Nucleotide), be illustrated as positive colony, containing NDV virus.
Extract the plasmid in pGEM-NDV bacterium and pGEM-TMUV bacterium respectively, obtain pGEM-NDV plasmid and pGEM-TMUV plasmid.
Using pGEM-NDV and pGEM-TMUV plasmid as positive criteria product, according to document (Vaitomaa, J., Rantala A., Halinen K., Rouhiainen L., Tallberg P., Mokelke L. & Sivonen K. (2003) Quantitative Real-Time PCR for Determination of Microcystin Synthetase E Copy Numbers for Microcystis and Anabaena in Lakes.Applied and Environmental Microbiology.69:7289-7297.) calculate copy number, result is copy number is 1.8 × 10
10with 2.1 × 10
10copy/μ l.
By pGEM-NDV and pGEM-TMUV plasmid, 37 DEG C of Spe I single endonuclease digestions spend the night, make plasmid linearization, agarose gel electrophoresis and test kit reclaim plasmid DNA purification linearizing product, for in-vitro transcription, illustrate by T7 in-vitro transcription test kit and add reaction reagent 37 DEG C effect 2h, then DNase I enzyme 1 μ l is added, the DNA20min do not transcribed in 37 DEG C of digestion transcription products, 70 DEG C of deactivation DNase I enzyme 15min, with the saturated phenol of equal-volume hydrochloric acid, chloroform, use equal-volume chloroform again, get supernatant 0.5 times of volume 5M Ammonium Acetate and 2 times of volume ice alcohol settling, precipitation is washed again with 75% ice ethanol, finally use DEPC water dissolution, obtain NDV-RNA and TMUV-RNA, ultraviolet spectrophotometer surveys NDV-RNA and TMUV-RNA concentration and purity,-70 DEG C save backup, result is that NDV-RNA and TMUV-RNA concentration is respectively 1.2 × 10
10with 1.3 × 10
10copy/μ l.
The establishment of primer and concentration and probe concentration in the reaction system of 3, fluorescence quantitative RT-RCR
With NDV-RNA and TMUV-RNA (copy of the two is than being 1:1) as template, be between 0.2-0.8 μm of ol/L by the primer in table 1 and probe at final concentration, the proportioning of carrying out different concns carries out fluorescence quantitative RT-RCR, selects the optimum concn of primer and probe.
Amplified reaction cumulative volume is 20 μ l, wherein 2 × One Step RT-PCR Buffer III (the precious biotechnology company limited in Dalian, catalog number: DRR064A) 10 μ l; Takara Ex Taq HS (the precious biotechnology company limited in Dalian, catalog number: DRR064A) 0.4 μ l; PrimeScript RT Enzyme Mix II (the precious biotechnology company limited in Dalian, catalog number: DRR064A) 0.4 μ l; 2 μ l templates, final concentration is TMUV-F, TMUV-R and TMUV-probe of 0.2-0.8 μm of ol/L; Final concentration is NDV-F, NDV-R and NDV-probe of 0.2-0.8 μm of ol/L; Remainder sterilizing DEPC water is supplied, and Homogeneous phase mixing is put on Lightcycler quantitative real time PCR Instrument and carried out automatization amplified reaction.Temperature transition rate is 20 DEG C/s, at the end of the extension of each circulation, carry out fluorescent signal detection.Response procedures is: 42 DEG C of 5min; 95 DEG C of 10s; Then extend 20s carry out 40 circulations by 95 DEG C of sex change 10s, 60 DEG C of annealing; Finally terminate reaction in 40 DEG C.
Result shows, different primers and probe final concentration affect larger on test-results, duck tembusu virus upstream and downstream primer and probe final concentration are respectively 0.2 μm of ol/L, duck source Avian pneumo-encephalitis virus upstream and downstream primer and probe final concentration are respectively 0.2 μm of ol/L, can obtain less Ct value to the detection of standard substance.
Therefore, the reaction system of the fluorescence quantitative RT-RCR of optimization is as follows: amplified reaction cumulative volume is 20 μ l, wherein 2 × One Step RT-PCR Buffer III (the precious biotechnology company limited in Dalian, catalog number: DRR064A) 10 μ l; Takara Ex Taq HS (the precious biotechnology company limited in Dalian, catalog number: DRR064A) 0.4 μ l; PrimeScript RT Enzyme Mix II (the precious biotechnology company limited in Dalian, catalog number: DRR064A) 0.4 μ l; 2 μ l templates, final concentration is TMUV-F, TMUV-R and TMUV-probe of 0.2 μM; Final concentration is NDV-F, NDV-R and NDV-probe of 0.2 μM; Remainder sterilizing DEPC water is supplied, and Homogeneous phase mixing is put on Lightcycler quantitative real time PCR Instrument and carried out automatization amplified reaction.Temperature transition rate is 20 DEG C/s, at the end of the extension of each circulation, carry out fluorescent signal detection.Response procedures is: 42 DEG C of 5min; 95 DEG C of 10s; Then extend 20s carry out 50 circulations by 95 DEG C of sex change 10s, 60 DEG C of annealing; Finally terminate reaction in 40 DEG C.
FAM, at 530nm exciting light fluoresces, is used for detecting duck source Avian pneumo-encephalitis virus; ROX, at 610nm exciting light fluoresces, is used for detecting duck tembusu virus;
Two, the sensitivity test of fluorescence quantitative RT-RCR
Respectively with the NDV-RNA of 10 times of serial dilutions (duck source Avian pneumo-encephalitis virus) and TMUV-RNA (duck tembusu virus), obtain copy number and be 1 × 10
9-1 × 10
2nDV-RNA and TMUV-RNA of copy/μ l, again NDV-RNA and TMUV-RNA of various copy number mixing is carried out bifluorescence quantitative pcr amplification as template, amplification system and condition are as the reaction system of the fluorescence quantitative RT-RCR optimized in, 3 and response procedures.
Result (ROX) under 610nm exciting light as illustrated in fig. 1 and 2; Result (FAM) under 530nm exciting light as shown in Figures 3 and 4.From fluorescence curve, fluorescence curve is still had to detection 100 copy of duck tembusu virus and duck source Avian pneumo-encephalitis virus, show that the sensitivity of this detection method to duck tembusu virus and duck source Avian pneumo-encephalitis virus is 100 copies, higher than conventional PCR method susceptibility 10 times, the result of duplicate detection is consistent.Increase linear as seen from typical curve, illustrate that set up method has good amplification efficiency.
Three, the specific test of fluorescence quantitative RT-RCR and interference test
1, the specific test of fluorescence quantitative RT-RCR
Fluorescence quantitative RT-RCR is carried out according to the reaction system of the fluorescence quantitative RT-RCR optimized in above-mentioned, 3 and response procedures, duck tembusu virus RNA, duck tembusu virus RNA+ duck source newcastle disease virus RNA, Muscovy duck parvovirus RNA, duck circovirus DNA, egg-decreasing syndrome viral DNA, duck plague virus DNA, H9 subtype avian influenza RNA is respectively, with ddH unlike template
2o is as negative control.
The specificity of duck tembusu virus is detected under 610nm exciting light, result (ROX) as shown in Figure 5, visible, sample 1 and 2 has PCR primer, obtain the specificity fluorescent curve of corresponding virus, and sample 3-8 does not all have specificity fluorescent curve, the primed probe designed by confirmation has specificity, the method high specificity, with other detected object no cross reaction.
The specificity of duck source Avian pneumo-encephalitis virus is detected under 530nm exciting light, result (FAM) as shown in Figure 6, visible, sample 1 and 2 has PCR primer, obtain the specificity fluorescent curve of corresponding virus, and sample 3-8 does not all have specificity fluorescent curve, the primed probe designed by confirmation has specificity, the method high specificity, with other detected object no cross reaction.
The bifluorescence quantitative PCR that application is set up, whether have an impact to the Ct value detected when carrying out detecting to determine that two kinds of templates exist, the CT value that in Fig. 5, duck tembusu virus, duck tembusu virus RNA+ duck source newcastle disease virus RNA mixing sample (2) detect is respectively 13.21 and 13.59; In Fig. 6, the CT value that duck source newcastle disease virus RNA, duck tembusu virus RNA+ duck source newcastle disease virus RNA mixing sample detect is respectively 13.81 and 13.95.Result illustrates, there is two-strain and there is single virus in sample, and the CT value variation of detection is very little, does not affect the susceptibility detecting and detect to duck tembusu virus and duck source Avian pneumo-encephalitis virus.Therefore, primer of the present invention, probe and and the bifluorescence quantitative RT-PCR detecting method set up can be applicable to qualification unknown sample whether infected duck tembusu virus and duck source Avian pneumo-encephalitis virus, the judgement of duplex fluorescent PCR reaction result is as follows:
Reaction result is straight line, be then negative; Reaction result is S type curve, be then positive;
If the reaction result of (ROX) is S type curve under 610nm exciting light, then contain duck tembusu virus in sample to be tested; Otherwise, then duck tembusu virus is not contained in sample; If the reaction result of (FAM) is S type curve under 530nm exciting light, then contain duck source Avian pneumo-encephalitis virus in sample to be tested; Otherwise, then duck source Avian pneumo-encephalitis virus is not contained in sample;
If under (ROX) and 530nm exciting light, the reaction result of (FAM) is S type curve under 610nm exciting light, then in sample containing duck tembusu virus and duck source Avian pneumo-encephalitis virus; If under (ROX) and 530nm exciting light, the reaction result of (FAM) is not all S type curve under 610nm exciting light, then in sample all not containing duck tembusu virus and duck source Avian pneumo-encephalitis virus.
Four, replica test
Carrying out fluorescence quantitative RT-RCR according to the reaction system of the fluorescence quantitative RT-RCR optimized in above-mentioned, 3 and response procedures, is that copy number is 1 × 10 unlike template
8the positive that the duck tembusu virus RNA of copy/μ l and duck source Avian pneumo-encephalitis virus DNA mixes.Be divided into 3 samples to detect simultaneously.Batch interior repeatability of fluorescence quantitative RT-RCR is verified by the standard deviation (SD) and the variation coefficient (CV) calculating Ct value.After three days, duplicate detection is stored in the template of-20 DEG C, come the stability of validation template and fluorescence quantitative RT-RCR batch between repeatability.
Detected result is shown in shown in Fig. 7-8 and table 3, Fig. 7 for detect duck tembusu virus RNA passage under 610nm exciting light (ROX), Fig. 8 for detect duck source Avian pneumo-encephalitis virus passage under 530nm exciting light (FAM), visible, and the variation coefficient is all less than 3% (table 3).Result illustrates that this method has good accuracy and repeatability.
Repeatability between table 3 batch
The assembling of embodiment 3, detection kit
According to the result of study of embodiment 1 and 2, assembling detection kit is with easy to use.
A liquid: pcr amplification damping fluid, primer 1, primer 2, probe A, primer 3, primer 4, probe B; Wherein, PrimeScript
tMreverse Transcriptase (the precious biotechnology company limited in Dalian, catalog number: 2680A); Premix Ex Taq
tM(Probe qPCR) (the precious biotechnology company limited in Dalian, catalog number: RR390A); Primer 1, primer 2, probe A final concentration are 0.2 μm of ol/L, and primer 3, primer 4, probe B final concentration are 0.2 μm of ol/L;
B liquid: DTMUV+NDV template, as positive control;
C liquid: ddH
2o, as negative control.
The detection of embodiment 4, clinical pathological material of disease
Sample to be tested is 100 parts of pathological material of diseases (being numbered 1-100) that Guangxi province duck group collects, and extracts the RNA of duck pathological material of disease (lungs of the liver of sick duck, the spleen of sick duck or sick duck) respectively.The RNA mixing that will be numbered each sample of 1-100 respectively, as template, uses the test kit of embodiment 3, carries out fluorescence quantitative RT-RCR according to the reaction system of the fluorescence quantitative RT-RCR optimized in embodiment 2 one, 3 and response procedures.
Detected result is evaluated as follows with reference to above-mentioned judging criterion:
Detect duck tembusu virus 15 parts (positive rate is 15%), detect duck source Avian pneumo-encephalitis virus 12 parts (positive rate is 12%), other sample standard deviations do not detect duck tembusu virus and duck source Avian pneumo-encephalitis virus.
Simultaneously, by 100 parts of pathological material of diseases (being numbered 1-100) respectively according to reference literature [1] Li Qingyang, Chen Fangyan, Liu Ping, Deng. the foundation [J] of duck tembusu virus TaqMan fluorescence quantitative RT-PCR detecting method. animal medicine is in progress, 2012, 33 (7): 18-22. and reference literature [2] Chen Anli. duck source Avian pneumo-encephalitis virus isolation identification, the research [D] of phylogenetic analysis and detection method. Guangxi University's master thesis, method in 2012. carries out duck tembusu virus and duck source Avian pneumo-encephalitis virus detects, result is consistent with the present invention, illustrate that method of the present invention is correct.
The above results illustrates that the duck group of Guangxi province exists the infection of duck tembusu virus and duck source Avian pneumo-encephalitis virus, and the foundation of this bifluorescence quantitative RT-PCR method, has directive significance to the Blight control of Guangxi province duck group.
Claims (6)
1. duck tembusu virus and duck source Avian pneumo-encephalitis virus bifluorescence quantitative RT-PCR detect primer sets, it is characterized in that comprising two pairs of Auele Specific Primers, be primer 1 and 2, primer 3 and 4 respectively, they have the base sequence of sequence table SEQ .ID.No.1 and 2, SEQ.ID.No.4 and 5 respectively.
2. duck tembusu virus and a duck source Avian pneumo-encephalitis virus bifluorescence quantitative RT-PCR detecting kit, is characterized in that comprising primer sets and probe groups; Primer sets has primer 1 to 4, and they have the base sequence of sequence table SEQ .ID.No.1 and 2, SEQ.ID.No.4 and 5 respectively; Probe groups has probe A and probe B, and they have the base sequence of sequence table SEQ .ID.No.3 and 6 respectively.
3. duck tembusu virus according to claim 1 and duck source Avian pneumo-encephalitis virus bifluorescence quantitative RT-PCR detecting kit, is characterized in that: the 5 ' end of described probe A is marked with reporter fluorescence dyestuff ROX, and 3 ' end is marked with quencher fluorescent dye BHQ2; 5 ' the end of described probe B is marked with reporter fluorescence dyestuff FAM, and 3 ' end is marked with quencher fluorescent dye BHQ1.
4. duck tembusu virus according to claim 2 and duck source Avian pneumo-encephalitis virus bifluorescence quantitative RT-PCR detecting kit, is characterized in that: the mol ratio of described primer 1, primer 2, probe A, primer 3, primer 4 and probe B is 1:1:1:1:1:1.
5. duck tembusu virus according to claim 3 and duck source Avian pneumo-encephalitis virus bifluorescence quantitative RT-PCR detecting kit, is characterized in that this test kit contains following reagent:
A liquid: pcr amplification damping fluid, primer 1, primer 2, probe A, primer 3, primer 4, probe B;
B liquid: DTMUV+NDV template, as positive control;
C liquid: ddH
2o, as negative control.
6. duck tembusu virus according to claim 5 and duck source Avian pneumo-encephalitis virus bifluorescence quantitative RT-PCR detecting kit, it is characterized in that: in described A liquid, primer 1, primer 2, probe A final concentration are 0.2 μm of ol/L, primer 3, primer 4, probe B final concentration are 0.2 μm of ol/L.
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| CN114438261B (en) * | 2022-01-26 | 2024-02-13 | 黑龙江省农业科学院畜牧兽医分院 | Primer group and method for detecting goose astrovirus and goose tembusu virus |
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