CN102618668A - Duplex PCR (polymerase chain reaction) detection kit for duck Newcastle disease virus and duck circovirus - Google Patents
Duplex PCR (polymerase chain reaction) detection kit for duck Newcastle disease virus and duck circovirus Download PDFInfo
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Abstract
The invention discloses a duplex PCR (polymerase chain reaction) detection kit for duck Newcastle disease virus and duck circovirus, and particularly relates to a primer group for detecting Newcastle disease virus and duck circovirus, which comprises a first primer, a second primer, a third primer and a fourth primer. Nucleotide sequences of the first primer, the second primer, the third primer and the fourth primer are respectively a first sequence, a second sequence, a third sequence and a fourth sequence in a sequence list. Testing shows that the two pairs of primers are designed to establish a duplex PCR detection method for Newcastle disease virus and duck circovirus, and the duplex PCR technique capable of detecting two pathogens, namely the Newcastle disease virus and the duck circovirus has the advantages of simplicity in operation, high sensitivity, high specificity, high repeatability and the like.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of duck NDV and duck circovirus two-fold PCR detection kit.
Background technology
Duck new city eqpidemic disease is that a type of being caused by duck source NDV (Duck Newcastle Diseases duck NDV) is acute, the transmissible disease of height contact and lethality.Discover that in early days duck has extremely strong resistibility to pathogenic APMV-1, only show as healthy band poison, even strong poison infects also not pathogenic.But discovered in recent years causes duck source NDV popular naturally of highly pathogenic and death property, already endangers seriously supporting duck.The generation of this disease and popular is reported on ground such as Hebei, Foochow, Zhejiang, Shanxi, Shandong, Henan successively, and above-mentioned research shows that the infection of duck source NDV is in rising trend, will already produce significant damage to the foster duck of China.
(Duck circo virus is that PCV-II section (Circoviridae) PCV-II belongs to a member of (Circovirus) DuCV) to duck circovirus, reports in Germany first.In recent years, there are ground such as Hungary, the U.S., TaiWan, China and China Shandong, Liaoning, Fujian, Guangdong and Guangxi to find have duck circovirus to cause duck to infect or the report of morbidity in succession.It is that dysplasia, weight loss and feather are in disorder etc. that DuCV causes the main clinical manifestation of duck morbidity, and pathological tissue shows as the fabricius bursa and downright bad lymphopenia and histiocytosis occur.The Lymphoid tissue of knitting hyperplasia phenomenon infection animal atrophy gradually causes lower immune function or immunoreation to suppress and then the probability that improved two-fold or multiple infection is the characteristic that infects of PCV-II and the unusual complicacy of the polyinfection situation of other pathogenic bacteria or virus.Chai Tongjie shows that to the cherry valley duck crowd duck circovirus in Shandong and the investigation of polyinfection duck circovirus and duck I type hepatitis, riemerella anatipestifer and colibacillary polyinfection rate are higher.Bend white and pure grade some areas, Guangxi duck circovirus infection conditions is investigated, the result shows existence and cause of disease polyinfection phenomenons such as Riemerlla anatipestifer, NDV, bird flu virus and DHV.
The aquatic bird PCV-II can not be grown on cell and fowl embryo, and should virus how to occur with the subclinical infection form, so can't diagnose with traditional isolated viral, animal test method.Initial diagnosis PCV-II mainly leans on pathological tissue and electron microscopic observation, but these two kinds of methods all need the technical ability and the susceptibility of specialty not high.
Polyinfection has brought many difficulties for the duck medical diagnosis on disease, can not only make diagnosis through clinical manifestation, need be by means of molecular diagnostic techniques.These methods such as separation, agar diffusion test and EUSA of virus are consuming time, are unfavorable for the control of virus.Advantages such as that PCR method has is easy and simple to handle, susceptibility is high, high specificity and good reproducibility have become the important method that animal pathogenic detects.Particularly multiplex PCR has the advantages that to detect simultaneously, to differentiate that the several diseases substance is outstanding, in the differential diagnosis of clinical multiple cause of disease polyinfection, has special advantages and very high practical value.But in a PCR system,, need prevent the non-specific binding between primer and the template, avoid the appearance of non-characteristic band, also will avoid the generation of primer dimer as far as possible owing to have multiple template and primer.Simultaneously, the segmental size of purpose has suitable gradient identical as far as possible with each primer annealing condition, and to guarantee 2 kinds of amplified production amount relative equilibriums, condition is groped very important.
So far do not see as yet to have and use the report of double PCR NDV and duck circovirus detection and diagnosis.
Summary of the invention
An object of the present invention is to provide a kind of primer sets that detects NDV and duck circovirus.
Primer sets provided by the invention is made up of primer 1, primer 2, primer 3 and primer 4;
The nucleotide sequence of said primer 1, said primer 2, said primer 3 and said primer 4 is followed successively by sequence 1, sequence 2, sequence 3 and sequence 4 in the sequence table respectively.
In the above-mentioned primer sets, the mol ratio of said primer 1, said primer 2, said primer 3 and said primer 4 is 0.1-0.5: 0.1-0.5: 1: 1;
In the above-mentioned primer sets, the mol ratio of said primer 1, said primer 2, said primer 3 and said primer 4 was specially 0.2: 0.2: 1: 1.
Another object of the present invention provides a kind of PCR reagent that detects NDV and duck circovirus.
PCR reagent provided by the invention is made up of above-mentioned primer sets, PCR damping fluid and water;
Above-mentioned PCR damping fluid is 2 * Taq PCR Mix (available from sky root .KT201).
The 3rd purpose of the present invention provides a kind of PCR test kit that detects NDV and duck circovirus.
PCR test kit provided by the invention comprises above-mentioned primer sets or above-mentioned PCR reagent.
The application whether above-mentioned primer sets or above-mentioned PCR reagent or mentioned reagent box contain in NDV and the duck circovirus product in preparation detection and/or auxiliary detection testing sample also is the scope that the present invention protects.
Whether contain NDV and duck circovirus in above-mentioned detection and/or the auxiliary detection testing sample for said testing sample being carried out double pcr amplification with above-mentioned primer sets or above-mentioned PCR reagent or mentioned reagent box.
In the above-mentioned application, the annealing temperature of said double pcr amplification is 50 ℃-65 ℃, and the annealing temperature of said double pcr amplification is specially 55 ℃.
The 4th purpose of the present invention provides a kind of primer that detects NDV to A.
Primer provided by the invention is made up of said primer in the above-mentioned primer sets 1 and said primer 2 A;
The 5th purpose of the present invention provides a kind of primer that detects duck circovirus to B.
Primer provided by the invention is made up of said primer in the above-mentioned primer sets 3 and said primer 4 B.
Above-mentioned primer also is the scope that the present invention protects to the application whether A contains in the NDV product in preparation detection and/or auxiliary detection testing sample;
Above-mentioned primer also is the scope that the present invention protects to the application whether B contains in the duck circovirus product in preparation detection and/or auxiliary detection testing sample.
Above-mentioned NDV can also can be non-duck source for the duck source.
Experiment of the present invention proves; This research and design 2 pairs of primers; Set up the detection method of the double PCR of NDV and duck circovirus; Can detect and differentiate the double round pcr of NDV and two kinds of pathogenic agent of duck circovirus simultaneously, have easy and simple to handle, advantage such as susceptibility is high, high specificity and good reproducibility.Therefore; The detection method of the duck circovirus that this research is set up and the double PCR of duck NDV can be used for the polyinfection of the duck source NDV that hypoimmunity that the duck circovirus infection causes causes; Both can time-saving cost, can reduce pollution again.
Description of drawings
Fig. 1 is double PCR temperature optimization test
Fig. 2 is the optimum result of double PCR
Fig. 3 is double PCR specificity test
Fig. 4 is the susceptibility that double PCR method detects DuCV and DuNDV
Fig. 5 is that double PCR part clinical sample detects
Fig. 6 is that conventional PCR detects DuCV virus in the clinical sample
Fig. 7 is that conventional PCR detects NDV virus in the clinical sample
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
Virus, reagent used among the following embodiment are specific as follows:
Duck plague virus AV1221 is available from China Veterinery Drug Inspection Office;
NDV is duck NDV, NDV-F48E9, NDV-Lasota; Wherein the duck NDV is documented in " research of duck source newcastle disease oil emulsion inactivated vaccine preparation "; Shandong Agricultural University's Master's thesis, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region; NDV-F48E9 and NDV-Lasota are all available from China Veterinery Drug Inspection Office;
Duck I Hepatitis virus AV2111 strain (being designated hereinafter simply as DHV I) is available from China Veterinery Drug Inspection Office;
Kind duck parvovirus (MDPV) is documented in " separation and the evaluation of Guangxi kind duck parvovirus ", the Guangxi animal and veterinary, and 2002,18 (6): 5-7, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
Duck circovirus is documented in " investigation of some areas, 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;
Gosling plague virus is documented in " foundation of gosling plague virus 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;
The H9 subtype avian influenza virus is documented in " foundation that multiple reverse transcriptional PCR rapid detection is differentiated 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 fowl pasteurella multocida is documented in " research that using polymerase chain reaction detects the fowl pasteurella multocida ", Chinese Preventive Veterinary Medicine newspaper, and 1999, (06) public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
Colon bacillus 0157: H7 is documented in " Guangxi livestock and poultry colon bacillus 0157: H7 epidemiology survey ", Chinese Amphixenosis's journal, and 2011,27 (12): 1151-1155, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
Riemerella anatipestifer is documented in " foundation and the applied research of riemerella anatipestifer real-time fluorescence quantitative PCR method for quick ", biotechnology, and 2010,37 (10): 87-91, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
Reagent: 2 * Taq PCR Mix is available from sky, Beijing root biotech company; TIANamp blood/cell/tissue gene DNA extracts test kit available from sky root company; RNA extracts reagent TRIzol LS Reagent available from Invitrogen company.
The V1/rep gene order of F gene of NDV strain and duck circovirus among the GenBank utilizes Lasergene software to carry out the multisequencing comparison, uses online software Primer Premier 5.0 design primers in conserved regions.Primer is synthetic by Shanghai Invitrogen company.Primer sequence is seen table 1.
Table 1 is double PCR primer sequence
One, the extraction of nucleic acid
Extract test kit with reference to TIANamp blood/cell/tissue gene DNA and tell a story, extract the DNA of duck circovirus, kind duck parvovirus, duck plague virus AV1221, gosling plague virus, riemerella anatipestifer, intestinal bacteria, fowl pasteurella multocida;
With reference to the RNA of TRIzol LS Reagent working instructions difference extracting duck NDV, NDV NDV-F48E9, NDV NDV-Lasota, DHV I, H9 subtype avian influenza virus, reverse transcription becomes cDNA respectively, and-70 ℃ of preservations are subsequent use.
Two, the foundation of double pcr amplification system
1, reaction system optimization
Consumptions such as duck NDV primer, duck circovirus primer, template are optimized; Repeatedly confirm the optimum response consumption after the revision test; Final definite double PCR reaction system is 25 μ L:2 * Taq PCR Mix (available from sky root .KT201), 12.5 μ L, (primer concentration is 50 μ mol/mL to each 0.1ul of DuNDV upstream and downstream primer; Final concentration in reaction system is 0.2 μ mol/mL), (primer concentration is 50 μ mol/mL to DuCV upstream and downstream primer;, the final concentration in reaction system is 1 μ mol/mL) and each 0.5 μ L, each 2 μ L of template, add water to 25 μ L.
2, reaction condition optimization
According to above-mentioned reaction system, template is the equal-volume mixture of DNA of cDNA and the duck circovirus of duck NDV, and annealing temperature is increased progressively by 50 ℃-65 ℃ successively, repeatedly confirms optimum annealing temperature after the revision test.
Reaction annealing temperature optimum result is as shown in Figure 1, wherein, and M:100bp DNA ladder; 1:50.0 ℃; 2:50.4 ℃; 3:51.5 ℃; 4:53.0 ℃; 5:54.8 ℃; 6:56.6 ℃; 7:58.4 ℃; 8:60.2 ℃; 9:62.0 ℃; 10:63.5 ℃; 11:64.6 ℃; 12:65.0 ℃, can find out that optimum annealing temperature is 55 ℃.
Confirm The optimum reaction conditions: 94 ℃ of 5min; 94 ℃ of 1min; 55 ℃ of 1min; 72 ℃ of 1min, 35 circulations; 72 ℃ of 10min, according to above-mentioned reaction system, template is the equal-volume mixture, the DNA of duck circovirus, the cDNA of duck NDV of DNA of cDNA and the duck circovirus of duck NDV, reacts.
The result sees shown in Figure 2, wherein, and M:100bp DNA ladder; 1: the equal-volume mixture of the cDNA of duck NDV and the DNA of duck circovirus; 2: the DNA of duck circovirus; 3: the cDNA of duck NDV, can find out that this condition can amplify the purpose fragment, its expanding effect is good.
Three, the specificity of double PCR test
PCR reaction system and optimized reaction conditions according to above-mentioned two optimizations are carried out double pcr amplification, and different is that template is distinguished as follows:
The DNA of the DNA of the cDNA of the DNA (equal-volume mixing) of the cDNA+ duck circovirus DNA (equal-volume mixing) of the DNA of the cDNA+ duck circovirus of duck NDV (equal-volume mixing), NDV-F48E9, the cDNA+ duck circovirus of NDV-Lasota, the DNA of kind duck parvovirus, DHV I, gosling plague virus, the DNA of duck plague virus AV1221, the cDNA of H9 subtype influenza virus, the DNA of riemerella anatipestifer, colibacillary DNA, fowl pasteurella multocida.The result is as shown in Figure 3, wherein, and M:100bp DNA ladder; 1: the DNA of the cDNA+ duck circovirus of duck NDV (equal-volume mixing); The DNA of the cDNA+ duck circovirus of 2:NDV-F48E9 (equal-volume mixing); The DNA of the cDNA+ duck circovirus of 3:NDV-Lasota (equal-volume mixing); 4: the DNA of kind duck parvovirus; The cDNA of 5:DHV I; 6: the DNA of gosling plague virus; 7: the DNA of duck plague virus AV1221; The cDNA of 8:H9 subtype influenza virus; 9: the DNA of riemerella anatipestifer; 10: colibacillary DNA; 11: the DNA of fowl pasteurella multocida; 12: negative control (water), can find out that 1-3 has the purpose fragment of 493bp and 218bp, all the other all do not have the purpose fragment.
Explain that primer of the present invention and method have high specific.
Therefore, above-mentioned primer and method can be applicable to the identified unknown sample and whether infect NDV (can be the duck source or for the duck source) and duck circovirus:
If obtain the fragment of 493bp, then contain NDV in the sample, otherwise then do not have;
If obtain the fragment of 218bp, then contain duck circovirus in the sample, otherwise then do not have;
If obtain the fragment of 493bp and 218bp, then contain NDV and duck circovirus in the sample, otherwise then do not have.
Four, the sensitivity test of double PCR
Record duck NDV cDNA concentration and duck circovirus DNA concentration is respectively 40ng/ul and 20ng/ul with DU 800 ultraviolet spectrophotometers; The two is carried out respectively equal-volume mixes as template behind 10 times of gradient dilutions; PCR reaction system and optimized reaction conditions according to above-mentioned two optimizations are carried out double pcr amplification
The result is as shown in Figure 4, M:100bp DNA ladder; 1:40ng duck NDV cDNA and 20ng duck circovirus DNA; 2:4ng duck NDV cDNA and 2ng duck circovirus DNA; 3:400pg duck NDV cDNA and 200pg duck circovirus DNA; 4:40pg duck NDV cDNA and 20pg duck circovirus DNA; 5:4pg duck NDV cDNA and 2pg duck circovirus DNA; 6:400rg duck NDV cDNA and 200fg duck circovirus DNA; 7:40fg duck NDV cDNA and 20fg duck circovirus DNA; 8:4fg duck NDV cDNA and 2fg duck circovirus DNA; Can find out that 1-7 all has the purpose fragment to expand, the double PCR method of therefore setting up is 40fg to the nucleic acid minimum detectability of duck NDV; Nucleic acid minimum detectability to duck circovirus is 20fg.
38 parts of (being numbered 1-38) duck pathological material of diseases (adopting liver) of Yulin, Nanning, Haikou censorship; Extract duck pathological material of disease DNA and RNA respectively; And the RNA reverse transcription obtained cDNA, and the DNA and the cDNA of each sample mixed (volume ratio is 1: 1), obtain being numbered the mixing sample of 1-38.
Respectively with the above-mentioned mixing sample that is numbered 1-38 as template, carry out double pcr amplification according to PCR reaction system and optimized reaction conditions that embodiment 2 two optimizes.
If obtain the fragment of 493bp, then contain NDV in the sample, otherwise then do not have;
If obtain the fragment of 218bp, then contain duck circovirus in the sample, otherwise then do not have;
If obtain the fragment of 493bp and 218bp, then contain NDV and duck circovirus in the sample, otherwise then do not have.
Amplification is as shown in Figure 5, and M is 100bp DNA ladder; The positive contrast of Y (duck NDV cDNA and duck circovirus DNA equal-volume mix); 39 negative contrasts (water); 1-38 is respectively the duck pathological material of disease that is numbered 1-38; Can find out that 5,13,22,24,32 have only 218bp purpose fragment, all the other all do not have other purpose fragments, explain in the duck pathological material of disease that is numbered 1-38 and have only duck circovirus, and the infection rate that contains duck circovirus is 13.1% (5/38).
Conventional method detects the result of the duck pathological material of disease of 1-38:
With the duck pathological material of disease DNA that is numbered 1-38 as template, with the special primer upper reaches of DuCV virus: 5 '-TATATTATTACCGGCGC (C/T) TGTA-3 '; Downstream: 5 '-TCAGGAATCCCTG (A/C) AGGTGA-3 ', (be shown in article " Development of a polymerase chain reaction procedure for detection and differentiation of duck and goose circovirus "; Amplified fragments is long to be 228bp.Amplification is as shown in Figure 6, and M is 100bp DNA ladder; D is the duck circovirus positive control; 39 negative contrasts (water); 1-38 is respectively the duck pathological material of disease that is numbered 1-38; Can find out that 5,13,22,24,32 have 228bp purpose fragment, all the other all do not have the purpose fragment, and explaining in the duck pathological material of disease that is numbered 1-38 has duck circovirus, and the infection rate that contains duck circovirus is 13.1% (5/38).
With the duck pathological material of disease cDNA that is numbered 1-38 as template, with the special primer P1:5 '-AGGCCTCTTGCRGCTGC-3 ' of NDV virus; P2:5 '-TTGTTCCCTAYACCTAAC-3 '; (being shown in article " multiple RT2PCR rapid detection is differentiated the foundation of NDV virulent strain and attenuated vaccine strain method "); The general fragment of NDV virulent strain and attenuated vaccine strain 671bp (this fragment is the Nucleotide of 103nt-774nt position of the F gene of NDV).Amplification is as shown in Figure 7, and M is 100bp DNA ladder; N is the newcastle disease positive control; 1-38 is respectively the duck pathological material of disease that is numbered 1-38; Can find out that 1-38 does not all have the purpose fragment, explain in the duck pathological material of disease that is numbered 1-38 and all do not have NDV.
Claims (9)
1. detect the primer sets of NDV and duck circovirus, form by primer 1, primer 2, primer 3 and primer 4;
The nucleotide sequence of said primer 1, said primer 2, said primer 3 and said primer 4 is followed successively by sequence 1, sequence 2, sequence 3 and sequence 4 in the sequence table respectively.
2. primer sets according to claim 1 is characterized in that:
The mol ratio of said primer 1, said primer 2, said primer 3 and said primer 4 is 0.1-0.5: 0.1-0.5: 1: 1;
The mol ratio of said primer 1, said primer 2, said primer 3 and said primer 4 was specially 0.2: 0.2: 1: 1.
3. detect the PCR reagent of NDV and duck circovirus, form by claim 1 or 2 described primer sets, PCR damping fluid and water;
Primer 1 in the said primer sets all is specially 0.1 μ mol/mL-0.5 μ mol/mL with the final concentration of primer 2 in said PCR reagent; Said primer 1 all further is specially 0.2 μ mol/mL with the final concentration of said primer 2 in said PCR reagent;
Primer 3 in the said primer sets all is specially 0.8 μ mol/mL-1.2 μ mol/mL with the final concentration of primer 4 in said PCR reagent; Said primer 3 all further is specially 1 μ mol/mL with the final concentration of said primer 4 in said PCR reagent.
4. detect the PCR test kit of NDV and duck circovirus, comprise claim 1 or 2 described primer sets or the said PCR reagent of claim 3.
5. whether claim 1 or 2 described primer sets or the said PCR reagent of claim 3 or the said test kit of claim 4 contain the application in NDV and the duck circovirus product in preparation detection and/or auxiliary detection testing sample.
6. according to the said application of claim 5, it is characterized in that: whether contain NDV and duck circovirus in said detection and/or the auxiliary detection testing sample for said testing sample being carried out double pcr amplification with claim 1 or 2 described primer sets or the said PCR reagent of claim 3 or the said test kit of claim 4.
7. according to claim 5 or 6 said application, it is characterized in that:
The annealing temperature of said double pcr amplification is 50 ℃-65 ℃, and the annealing temperature of said double pcr amplification is specially 55 ℃.
8. a primer that detects NDV is made up of said primer in claim 1 or the 2 described primer sets 1 and said primer 2 A;
Or a kind of primer that detects duck circovirus is made up of said primer in claim 1 or the 2 described primer sets 3 and said primer 4 B.
9. whether the described primer of claim 8 contains the application in the NDV product to A in preparation detection and/or auxiliary detection testing sample;
Or whether the described primer of claim 8 contains the application in the duck circovirus product to B in preparation detection and/or auxiliary detection testing sample.
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| CN103725801A (en) * | 2014-01-16 | 2014-04-16 | 广西壮族自治区兽医研究所 | Amphimorphic RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit of duck flavivirus and duck circovirus |
| CN104894285A (en) * | 2015-06-29 | 2015-09-09 | 天津市农业生物技术研究中心 | Method for detecting genes Cf-9 and I-2 according to dual-PCR (polymerase chain reaction) technology |
| CN105886666A (en) * | 2016-05-13 | 2016-08-24 | 佛山科学技术学院 | Duck circovirus real-time fluorescent quantitative PCR (polymerase chain reaction) detection method |
| CN107201400A (en) * | 2017-05-17 | 2017-09-26 | 中国农业科学院上海兽医研究所 | The five weight PCR detection methods and detection kit of avian escherichia coli, Salmonella gallinarum, Salmonella Pullorm etc. |
| CN109182607A (en) * | 2018-10-19 | 2019-01-11 | 天津市畜牧兽医研究所 | A kind of Dual-PCR Kit and application for detecting avian reovirus and turkey reovirus |
| CN112575122A (en) * | 2020-12-29 | 2021-03-30 | 商丘美兰生物工程有限公司 | Dual PCR primer group for rapidly detecting duck type 2 adenovirus and duck circovirus as well as detection method and application thereof |
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| CN103320539B (en) * | 2013-07-11 | 2015-11-18 | 广西壮族自治区兽医研究所 | The duplex RT-PCR test kit of duck tembusu virus and Avian pneumo-encephalitis virus |
| CN103725801A (en) * | 2014-01-16 | 2014-04-16 | 广西壮族自治区兽医研究所 | Amphimorphic RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit of duck flavivirus and duck circovirus |
| CN103725801B (en) * | 2014-01-16 | 2016-03-09 | 广西壮族自治区兽医研究所 | Duck flavivirus and duck circovirus duplex RT-PCR detection kit |
| CN104894285A (en) * | 2015-06-29 | 2015-09-09 | 天津市农业生物技术研究中心 | Method for detecting genes Cf-9 and I-2 according to dual-PCR (polymerase chain reaction) technology |
| CN105886666A (en) * | 2016-05-13 | 2016-08-24 | 佛山科学技术学院 | Duck circovirus real-time fluorescent quantitative PCR (polymerase chain reaction) detection method |
| CN107201400A (en) * | 2017-05-17 | 2017-09-26 | 中国农业科学院上海兽医研究所 | The five weight PCR detection methods and detection kit of avian escherichia coli, Salmonella gallinarum, Salmonella Pullorm etc. |
| CN109182607A (en) * | 2018-10-19 | 2019-01-11 | 天津市畜牧兽医研究所 | A kind of Dual-PCR Kit and application for detecting avian reovirus and turkey reovirus |
| CN112575122A (en) * | 2020-12-29 | 2021-03-30 | 商丘美兰生物工程有限公司 | Dual PCR primer group for rapidly detecting duck type 2 adenovirus and duck circovirus as well as detection method and application thereof |
| CN112575122B (en) * | 2020-12-29 | 2024-04-09 | 商丘美兰生物工程有限公司 | Dual PCR primer set for rapidly detecting duck type 2 adenovirus and duck circovirus, and detection method and application thereof |
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