CN113913553A - Fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit for gene XII Newcastle disease virus and primer pair thereof - Google Patents
Fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit for gene XII Newcastle disease virus and primer pair thereof Download PDFInfo
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Abstract
The invention discloses a primer pair for fluorescent quantitative RT-PCR detection of a gene XII Newcastle disease virus, which comprises a primer 1 and a primer 2, wherein the primer 1 and the primer 2 respectively have base sequences of a sequence table SEQ.ID.No.1 and a sequence table SEQ.ID.No. 2. Accordingly, a corresponding kit is also designed, which contains a primer 1, a primer 2 and a probe A, which have base sequences of sequence tables SEQ.ID.No.1 to SEQ.ID.No.3, respectively. The method has the characteristics of good sensitivity, high specificity, accuracy, reliability, rapidness, convenience and the like, and can realize rapid detection and quantification of the gene XII type NDV. The invention can be used for evaluating the curative effect of the gene XII type NDV vaccine and the medicament and researching the pathogenic mechanism thereof and the like, thereby having important significance for preventing and treating the gene XII type NDV.
Description
Technical Field
The invention belongs to the technical field of RT-PCR detection, and particularly relates to a fluorescent quantitative RT-PCR detection kit for a gene XII Newcastle disease virus and a primer pair thereof.
Background
High-infectivity, high-pathogenicity and high-lethality infectious diseases caused by NDV (Newcastle Disease Virus) with moderate and strong toxicity can infect more than 250 kinds of poultry and birds, thus causing great harm to the poultry industry. The NDV which is mainly pathogenic to poultry such as chicken, duck, goose and the like is mainly the class II gene type VII NDV, and the gene type VII NDV is very rare due to the regulation of vaccine in recent years. From 2010, a new genotype of the newcastle disease virus is separated in China's two kingdoms and Fujian continuousy: an NDV type XII gene is proved to be virulent by animal pathogenicity tests. Although there is currently no report of the genes NDV of type XII being pathogenic to natural infection of poultry, it is likely that under immunological pressure the genes NDV of type XII will evolve to be pathogenic to poultry. Therefore, it is urgently needed to establish a rapid and sensitive detection method for the NDV of the gene XII so as to accurately detect and identify the NDV in the early stage of infection, thereby striving for precious time for controlling epidemic diseases. The traditional detection methods of the viruses comprise virus separation, agar diffusion tests and the like, but the detection has the defects of long time consumption, low sensitivity, difficult standardization and the like, and has certain limitation in practical application.
The fluorescent quantitative PCR technology realizes the quantification of the template, and has the characteristics of sensitivity, specificity, accuracy, reliability, realization of multiple reactions, good real-time property and the like. According to the inspection, reports of detection and diagnosis of NDV of the gene XII type by using a fluorescence quantitative PCR technology are not seen yet.
Disclosure of Invention
The invention aims to provide a fluorescent quantitative RT-PCR detection kit for genes XII Newcastle disease virus and a primer pair thereof, which have the advantages of good sensitivity, high specificity, accuracy, reliability, rapidness and convenience, so as to realize rapid detection and quantification of genes XII NDV.
In order to solve the technical problems, the invention adopts the following technical scheme:
a primer pair for fluorescence quantitative RT-PCR detection of gene XII Newcastle disease virus comprises a primer 1 and a primer 2 which respectively have base sequences of sequence tables SEQ.ID.No.1 and SEQ.ID.No. 2.
The RT-PCR detection primer pair is applied to amplification of HN gene of gene XII Newcastle disease virus.
The amplification reaction program is 5min at 52 ℃; 10s at 95 ℃; then carrying out 40 cycles according to denaturation at 95 ℃ for 5s and annealing extension at 60 ℃ for 34 s; the reaction was finally terminated at 40 ℃.
A fluorescence quantitative RT-PCR detection kit for a gene XII Newcastle disease virus comprises a primer 1, a primer 2 and a probe A, which respectively have base sequences of sequence tables SEQ ID No.1 to SEQ ID No. 3.
The 5 'end of the probe A is marked with a report fluorescent dye FAM, and the 3' end is marked with a quenching fluorescent dye BHQ 1.
The molar ratio of primer 1, primer 2 and probe A is 2:2: 3.
The kit comprises the following reagents:
solution A: PCR amplification buffer solution, a primer 1, a primer 2 and a probe A;
and B, liquid B: gene XIINDV template as positive control;
and C, liquid C: ddH2O, as a negative control.
The final concentrations of primer 1, primer 2 and probe A in solution A were 0.2. mu. mol/L, 0.2. mu. mol/L and 0.3. mu. mol/L, respectively.
The RT-PCR detection kit is applied to amplification of HN gene of gene XII Newcastle disease virus.
The amplification reaction program is 5min at 52 ℃; 10s at 95 ℃; then carrying out 40 cycles according to denaturation at 95 ℃ for 5s and annealing extension at 60 ℃ for 34 s; the reaction was finally terminated at 40 ℃.
Aiming at the lack of an effective and reliable technology for detecting and diagnosing NDV of the gene XII, the inventor researches and designs a primer pair for detecting fluorescent quantitative RT-PCR of the gene XII Newcastle disease virus, which comprises a primer 1 and a primer 2, wherein the primer 1 and the primer 2 respectively have base sequences of sequence tables SEQ ID No.1 and SEQ ID No. 2. Accordingly, a corresponding kit is also designed, which contains a primer 1, a primer 2 and a probe A, which have base sequences of sequence tables SEQ.ID.No.1 to SEQ.ID.No.3, respectively. The optimal concentration combination of the primer and the probe is obtained by proportioning different concentrations, and a fluorescence quantitative RT-PCR detection method of the gene XII type NDV is established. Experiments prove that the invention has the following advantages:
1) short detection time
The application of the invention can realize one-step completion of reverse transcription and PCR, only about 60 minutes is needed, and the reaction result can be directly observed by a computer, while the conventional RT-PCR method needs at least 2 hours to complete the amplification reaction, and then 1.5 hours is spent on carrying out gel electrophoresis to observe the result;
2) high detection sensitivity and strong specificity
When NDV of type XII is present, the CT values detected by the method of the invention are compared with those detected in the presence of other viruses, and the results are substantially the same without affecting the sensitivity of detection and detection of NDV of type XII. In addition, the method can also be used for quantifying the corresponding pathogen content in the sample, the detection sensitivity is very high, 1.7 copies of NDV XII (deoxyribose nucleic acid) of the gene can be detected, the sensitivity is 100 times higher than that of a conventional PCR (polymerase chain reaction) method, and the accuracy and the reliability of the method are not influenced by the existence of other genotype NDV.
In conclusion, the invention can be used for evaluating the curative effect of the NDV vaccine or medicament of the gene XII and researching the pathogenic mechanism of the NDV vaccine or medicament, and therefore has important significance for preventing and treating the NDV of the gene XII.
Drawings
FIG. 1 is a drawing of the present inventionSensitivity (FAM channel) result graph of NDV type XII of gene by fluorescent quantitative RT-PCR, wherein: 1 to 9 are 1.7X 10, respectively81.7X 100 copies/reaction tube, 10 is a blank (DEPC water).
FIG. 2 is a standard curve of sensitivity (FAM channel) of NDV type XII in the fluorescent quantitative RT-PCR assay of the present invention.
FIG. 3 is a graph showing the results of the detection of the specificity (FAM channel) of NDV type XII of the gene by the fluorescent quantitative RT-PCR of the present invention, in which: 1 gene type XII NDV, 2 duck Tembusu virus, 3 Muscovy duck parvovirus, 4 duck circovirus, 5 egg drop syndrome virus, 6 duck plague virus, 7H9 subtype avian influenza virus, 8H5 subtype avian influenza virus, 9H7 avian influenza virus, 10 infectious bronchitis virus, 11 infectious laryngotracheitis virus, 12 avian reovirus, 13 infectious bursal disease virus, 14 avian adenovirus virus, 15 other genotype NDV mixture (including NDV genotypes I, II, III, VI, VII, IX), 16ddH2O。
FIG. 4 is a graph showing the results of the quantitative fluorescence RT-PCR assay of NDV type XII of the gene (FAM channel) in batches, in which: 1-3 three replicates of the same batch, 4 is a negative control.
Detailed Description
The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified. The specific materials and reagents used were as follows:
QuantStudio 5 real-time quantitative PCR instrument (Thermo Fisher); DNA fragment recovery kit and plasmid miniprep kit were purchased from BioDev; pGEM-T Easy kit was purchased from Promega; the T7 in vitro transcription kit is purchased from Fermengtas; one Step PrimeScript RT-PCR Kit was purchased from Dalibao Bio; the TIANAmp virus genomic DNA/RNA extraction kit was purchased from Tiangen Biochemical technology Ltd.
Duck Tembusu virus is described in 'isolation and preliminary identification of 4 Guangxi Duck source Tembusu virus', Chinese animal quarantine, 2013, 30(6):31-35, and is available to the public from Guangxi Zhuang autonomous region veterinary research institute;
egg drop syndrome virus is described in "research on egg drop syndrome vegetable oil emulsion vaccine", Chinese preventive veterinary newspaper 2000, (04):23-27, publicly available from Guangxi Zhuang autonomous region veterinary research institute;
the duck circovirus is recorded in the research on the infection condition of the duck circovirus in Guangxi part of China, Chinese animal husbandry veterinarians, 2010, 37(11): 156-;
muscovy duck parvovirus is recorded in the establishment of a fluorescence quantitative PCR detection method for gosling plague virus, Shanghai zootechnical veterinary communication, 2008, 160(06):30-31, and the public can obtain from Guangxi Zhuang autonomous region veterinary research institute;
newcastle disease viruses are described in "molecular epidemiological analysis of Newcastle disease viruses in Guangxi live bird market in 2018", Chinese poultry, 2020,42, (03):22-28, and "etiological detection and genetic evolution analysis of Newcastle disease of 4 wild birds in Guangxi in 2016-2017", Chinese animal quarantine, 2018,35, (05): 21-24,50. The public is available from the Guangxi Zhuang autonomous region veterinary research institute;
the duck plague virus AV1221 strain is purchased from the Chinese veterinary medicine inspection institute;
infectious bronchitis virus, infectious laryngotracheitis virus, avian reovirus, infectious bursal disease virus and avian adenovirus are recorded in the establishment of a method for detecting nine chicken respiratory disease pathogens GeXP, and Chinese veterinary science, 2013,10: 1040-.
The avian influenza virus is recorded in the establishment of a method for rapidly detecting and identifying H9 subtype avian influenza virus by multiple reverse transcription polymerase chain reaction, which is reported by Chinese veterinary community, 2006, (09): 858) 860, and can be obtained by the public from Guangxi Zhuang autonomous region veterinary research institute.
Example 1 design and Synthesis of primers and Taqman probes
The complete gene sequences of the different genotypes (3 genotypes 1.1.1,1.1.2 and 1.2 of class I NDV; 21 genotypes I-XXI of class II NDV, with at least 3 strain sequences downloaded per genotype) NDV were downloaded in GenBank. Alignment of the sequences using DNASTAR revealed that the differences in gene sequence between the different genotypes were mainly between the F and HN genes, and thus the method designed primer regions to address both genes. According to the sequence difference between the NDV type XII gene and other genotypes, a plurality of sets of probes and primers are designed by using Primer Express 3.0 software, and finally a pair of specific primers and a Taqmam probe are selected by analyzing dimers among the primers (Table 1). The amplification regions of the primers and probes are located in the HN gene. Through NCBI Blast comparison, the primer and the probe only coincide with the sequence of the gene XII type NDV and have base difference with other genotype NDV.
TABLE 1 primer and TaqMan probe sequences (5 '-3')
Example 2 establishment of fluorescent quantitative RT-PCR detection
Determination of fluorescent quantitative RT-PCR detection method
1. Preparation of samples
1) Extraction of nucleic acid
Referring to the TIANamp virus genome DNA/RNA extraction kit specification, RNA and DNA of genes type XII NDV, duck tembusu virus, Muscovy duck parvovirus, duck circovirus, egg drop syndrome virus, duck plague virus, H9 subtype avian influenza virus, H5 subtype avian influenza virus, H7 avian influenza virus, infectious bronchitis virus, infectious laryngotracheitis virus, avian reovirus, infectious bursal disease virus, avian adenovirus and other genotype NDV (genes I, II, III, VI, VII and IX, wherein the genes I, II and III NDV have vaccine strains and are used in China or are used in China at present, and the genes VI, VII and IX all have large areas or are dispersed in China and are main epidemic strain genotypes in China.
2) Reverse transcription of RNA
cDNA was synthesized by reverse transcription of RNA, and the following reverse transcription system (total reaction volume 20. mu.L) was established: NDV type XII cDNA was 2. mu.L (about 20. mu.g), 4. mu.L of 8mM MgCl2(Dalibao bioengineering Co., Ltd., catalog No.: DRR0019A), 2. mu.L of 10 XPCR buffer (Dalibao bioengineering Co., Ltd., catalog No.): DRR0019A), 2 μ L of 10mM dNTP (four bases), 1 μ L of RNA inhibitor (dallopore bioengineering limited, catalog No.: DRR0019A), 1 μ L of random primer (dalibobao bioengineering limited, catalog No.: DRR0019A), 1 μ L AMV reverse transcriptase (dalibobao bioengineering limited, catalog No.: DRR0019A), adding RNase-free water to the total volume of 20 mu L, centrifuging uniformly, and finishing at 25 ℃ for 10min, 42 ℃ for 60min, 95 ℃ for 5min and 4 ℃ to obtain gene XII type NDVcDNA.
2. Preparation of standards
PCR amplification was carried out using the cDNA of the gene type XII NDV obtained above as a template, and the reaction system was 50. mu.L (containing 0.2mmol/L dNTP and 2.5mmol/L MgCl)20.5. mu. mol/L primer (Table 1), 1.25U Taq polymerase, 1 XPCR buffer, 5. mu.L DNA template) under the following reaction conditions: pre-denaturation at 95 ℃ for 5min, at 94 ℃ for 60s, at 50 ℃ for 60s, at 70 ℃ for 60s, for 35 cycles, and extension at 72 ℃ for 10 min.
The PCR product is subjected to 2% agarose gel electrophoresis, a target fragment is recovered and then cloned to a pGEM-T Easy vector, a positive clone bacterium (pGEM-NDV XII) is sent to Dalian Baobaosheng biotechnology limited company for sequencing, the PCR product contained in the pGEM-EDSV bacterium has the size of 172bp, has the 1 st to 172 th nucleotides (HN sequence of gene XII type NDV Francolin/China/GX01/2017 strain, the genbank number is MZ306226, the 7202 and 7373 th nucleotides) of SEQ ID No.4 in a sequence table, and is indicated to be a positive clone containing gene XII type NDV.
And extracting the plasmid of pGEM-NDV XII to obtain the plasmid pGEM-NDV XII.
Plasmid pGEM-NDV XII was used as a positive standard according to the literature (Vaitoma, J., Rantala A., Halinen K., Rouhiaine L., Tallberg P., Mokelke L).&(2003) Quantitative Real-Time PCR for Determination of Microcystin Synthesis E Copy Numbers for Microcystis and Anabaena in lakes, applied and Environmental microbiology.69:7289-7297.) the number of copies was calculated as 2.2X 109copies/μl。
Carrying out single digestion on the plasmid pGEM-NDV XII of the gene by SalI at 37 ℃ overnight to linearize the plasmid, carrying out agarose gel electrophoresis and recovering and purifying a plasmid DNA linearized product by using a kit forIn vitro transcription, adding a reaction reagent according to the instruction of a T7 in vitro transcription kit for 2 hours at 37 ℃, adding 1 mu l of DNase I enzyme, digesting untranscribed DNA in a transcription product at 37 ℃ for 20 minutes, inactivating the DNase I enzyme at 70 ℃ for 15 minutes, extracting with isovolumetric hydrochloric acid saturated phenol and chloroform, extracting with isovolumetric chloroform, taking supernatant, precipitating with 0.5 times of 5M ammonium acetate and 2 times of glacial ethanol, washing the precipitate with 75% of glacial ethanol, dissolving with DEPC water to obtain DHV-RNA, and storing at-70 ℃ for later use by using an ultraviolet spectrophotometer to measure the concentration and purity of the DHV-RNA, wherein the result is that the concentration of pGEM-NDV XII-RNA is 1.7 multiplied by 10 and the pGEM-NDV XII-RNA is 1.7 multiplied by 10 for later use9Copies/. mu.l.
3. Establishment of primer and probe concentration in reaction system of fluorescent quantitative RT-PCR
pGEM-NDV XII-RNA is used as a template, the primers and the probes in the table 1 are subjected to fluorescent quantitative RT-PCR at different concentration ratios between 0.2 and 0.8 mu mol/L, and the optimal concentrations of the primers and the probes are selected.
The amplification reaction was performed in a total volume of 20. mu.l, 2 Xone Step PrimeScriptTMIII RT-qPCR Mix (Dalianbao bioengineering Co., Ltd., product catalog number: RR600A) 10. mu.l; 1 mul of template, NDV XII-F, NDV XII-R and NDV XII-probe with final concentration of 0.2-0.8 mul/L; and (3) complementing 0.4 mu l of ROX Reference Dye II with sterilized DEPC water, uniformly mixing, and carrying out automatic amplification reaction on a QuantStudio 5 real-time quantitative PCR instrument. The temperature conversion rate was 20 ℃/s and the fluorescence signal detection was performed at the end of the extension of each cycle. The reaction procedure is as follows: 5min at 52 ℃; 10s at 95 ℃; then carrying out 40 cycles according to denaturation at 95 ℃ for 5s and annealing extension at 60 ℃ for 34 s; the reaction was finally terminated at 40 ℃.
The result shows that the final concentrations of different primers and probes have larger influence on the test result, the final concentrations of NDV XII-F, NDV XII-R are respectively 0.2 mu mol/L, the final concentrations of NDV XII-probe are respectively 0.3 mu mol/L, and the detection on the standard substance can obtain a smaller Ct value.
Therefore, the optimized reaction system of the fluorescent quantitative RT-PCR is as follows: the amplification reaction was performed in a total volume of 20. mu.l, 2 Xone Step PrimeScriptTMIII RT-qPCR Mix (Dalianbao bioengineering Co., Ltd., product catalog number: RR600A) 10. mu.l; 1 μ l template, final concentration 0.2 μ MNDV XII-F, NDV XII-R, NDV XII-probe with a final concentration of 0.3. mu. mol/L; ROX Reference Dye II 0.4. mu.l; and supplementing the rest with sterilized DEPC water, uniformly mixing, and performing automatic amplification reaction on the mixture on a QuantStaudio 5 real-time quantitative PCR instrument. The temperature conversion rate was 20 ℃/s and the fluorescence signal detection was performed at the end of the extension of each cycle. The reaction procedure is as follows: 5min at 52 ℃; 10s at 95 ℃; then carrying out 40 cycles according to denaturation at 95 ℃ for 5s and annealing extension at 60 ℃ for 34 s; the reaction was finally terminated at 40 ℃.
FAM fluoresces under excitation light of 530nm and is used to detect gene type XII NDV.
Second, sensitivity test of fluorescent quantitative RT-PCR
pGEM-NDV XII-RNA (Gene XII type NDV) each diluted 10-fold in series gave copy numbers of 1.7X 108-1.7×100And copying/mu l of pGEM-NDV XII-RNA plasmid, and performing fluorescent quantitative RT-PCR amplification by using pGEM-NDV XII-RNA with various copy numbers as a template, wherein the amplification system and conditions are as the optimized fluorescent quantitative RT-PCR reaction system and reaction program in step one and 3.
The results (FAM) under 530nm excitation light are shown in FIG. 1. As can be seen from the fluorescence curve, the fluorescence curve still remains for 1.7 copies of NDV of the gene XII, which indicates that the sensitivity of the detection method to NDV of the gene XII is 1.7 copies, 10 times higher than that of the conventional PCR method, and the repeated detection results are consistent. Amplification is linear as seen from the standard curve, indicating that the established method has good amplification efficiency.
Third, specificity test of fluorescent quantitative RT-PCR
Performing fluorescent quantitative RT-PCR according to the optimized fluorescent quantitative RT-PCR reaction system and reaction program in the first step and the second step, except that the templates are respectively 1 gene type XII NDV, 2 duck tembusu virus, 3 Muscovy duck parvovirus, 4 duck circovirus, 5 egg drop syndrome virus, 6 duck plague virus, 7H9 subtype avian influenza virus, 8H5 subtype avian influenza virus, 9H7 avian influenza virus, 10 infectious bronchitis virus, 11 infectious laryngotracheitis virus, 12 avian reovirus, 13 infectious bursal disease virus, 14 avian adenovirus, 15 other genotype NDV mixture, 16ddH2O。
The specificity of the gene XII type NDV is detected under 530nm exciting light, and the result (FAM) is shown in figure 3, so that the specific fluorescence curve of the corresponding virus is obtained by using the sample 1CR product, but no specific fluorescence curve exists in the samples 2-16, and the designed primer probe is proved to have specificity, the method has strong specificity, no cross reaction with other detection objects exists, particularly no amplification reaction exists on other gene type NDV, and the influence of other gene type NDV on the application of the invention can be eliminated.
Fourth, repeatability test
Performing fluorescent quantitative RT-PCR according to the optimized fluorescent quantitative RT-PCR reaction system and reaction program in the step one and 3, wherein the copy number of the template is 1.7 multiplied by 107Copies/. mu.l of a positive sample of type XII NDV gene. The samples were divided into 3 specimens and tested simultaneously. The in-batch reproducibility of the fluorescent quantitative RT-PCR was verified by calculating the Standard Deviation (SD) and Coefficient of Variation (CV) of the Ct values. The template stored at-80 ℃ was examined repeatedly three days later to verify the stability of the template and the batch-to-batch reproducibility of the fluorescent quantitative RT-PCR.
The results are shown in FIGS. 4 and 3, and FIG. 4 shows that the NDV channel type XII gene was detected under 530nm excitation light (FAM), and the coefficients of variation were all less than 3% (Table 3). The results show that the method has good accuracy and repeatability.
TABLE 3 repeatability between batches
Example 3 Assembly of assay kit
Based on the results of the studies of examples 1 and 2, the test kit was assembled for convenient use.
Solution A: PCR amplification buffer solution, a primer 1, a primer 2 and a probe A; wherein, 2 xOne Step PrimeScriptTMIII RT-qPCR Mix (Dalianbao bioengineering Co., Ltd., catalog No.: RR 600A); the final concentrations of the primer 1 and the primer 2 are both 0.2 mu mol/L, and the final concentration of the probe A is both 0.3 mu mol/L;
and B, liquid B: gene type XII NDV template as positive control;
and C, liquid C:ddH2o, as a negative control.
Example 4 detection of clinical Agents
The sample to be detected is 100 parts (numbered as 1-100) of poultry larynx/cloaca cotton swab collected in live poultry market in Guangxi area, and total RNA and DNA of the cotton swab are respectively extracted. The total DNA and RNA of each sample, numbered 1 to 100, were used as templates, and the quantitative fluorescence RT-PCR was performed using the kit of example 3 according to the reaction system and reaction procedure of the quantitative fluorescence RT-PCR optimized in examples 2 and 3. 2 portions of NDV type XII gene were detected (positive rate: 2%) by evaluation according to the above criteria.
Meanwhile, 100 parts of disease material (numbered 1-100) are respectively subjected to gene XII type NDV detection according to the method in the reference document [1] Liuhualei, Luyan, Wangjing, and the like [ J ] distribution and characteristics of new gene XII type Newcastle disease virus in China [ 9 ] Chinese animal quarantine, 2017,34(9):1-3 and 18 ], and the result is consistent with the method, so that the method is correct.
The result indicates that the infection of the gene XII type NDV exists in Guangxi regions, and the establishment of the fluorescent quantitative RT-PCR method has guiding significance on the control of epidemic diseases of the gene XII type NDV.
Sequence listing
<110> Guangxi Zhuang nationality autonomous region veterinary research institute
<120> gene XII Newcastle disease virus fluorescent quantitative RT-PCR detection kit and primer pair thereof
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<213> Artificial Sequence (Artificial Sequence)
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taggaggtgg gtctttcatt aacgaccgtg tatggttccc agtttatgga gg 172
Claims (10)
1. A primer pair for fluorescence quantitative RT-PCR detection of gene XII Newcastle disease virus is characterized by comprising a primer 1 and a primer 2 which respectively have base sequences of sequence tables SEQ.ID.No.1 and SEQ.ID.No. 2.
2. Use of the RT-PCR detection primer pair of claim 1 in amplification of the HN gene of XII Newcastle disease virus.
3. Use according to claim 2, characterized in that: the amplification reaction program is 5min at 52 ℃; 10s at 95 ℃; then carrying out 40 cycles according to denaturation at 95 ℃ for 5s and annealing extension at 60 ℃ for 34 s; the reaction was finally terminated at 40 ℃.
4. A fluorescence quantitative RT-PCR detection kit for a gene XII Newcastle disease virus is characterized by comprising a primer 1, a primer 2 and a probe A, wherein the primer 1, the primer 2 and the probe A respectively have base sequences of sequence tables SEQ.ID.No.1 to SEQ.ID.No. 3.
5. The RT-PCR detection kit according to claim 4, characterized in that: the 5 'end of the probe A is marked with a report fluorescent dye FAM, and the 3' end is marked with a quenching fluorescent dye BHQ 1.
6. The RT-PCR detection kit according to claim 4, characterized in that: the molar ratio of the primer 1 to the primer 2 to the probe A is 2:2: 3.
7. The RT-PCR detection kit according to claim 4, characterized in that the kit comprises the following reagents:
solution A: PCR amplification buffer solution, a primer 1, a primer 2 and a probe A;
and B, liquid B: gene XIINDV template as positive control;
and C, liquid C: ddH2O, as a negative control.
8. The RT-PCR detection kit according to claim 7, characterized in that: the final concentrations of the primer 1, the primer 2 and the probe A in the solution A are respectively 0.2 mu mol/L, 0.2 mu mol/L and 0.3 mu mol/L.
9. Use of the RT-PCR detection kit of claim 4 to amplify the HN gene of the gene XII Newcastle disease virus.
10. Use according to claim 9, characterized in that: the amplification reaction program is 5min at 52 ℃; 10s at 95 ℃; then carrying out 40 cycles according to denaturation at 95 ℃ for 5s and annealing extension at 60 ℃ for 34 s; the reaction was finally terminated at 40 ℃.
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