CN108998575B - Establishment of double PCR detection method for chicken parvovirus and chicken newcastle disease virus - Google Patents
Establishment of double PCR detection method for chicken parvovirus and chicken newcastle disease virus Download PDFInfo
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Abstract
The invention discloses establishment of a double PCR detection method for chicken parvovirus and newcastle disease virus. The invention provides a set of primers for identifying chicken parvovirus and chicken newcastle disease virus, which consists of the following primers 1 to 4: the primer 1 is a single-stranded DNA molecule shown in a sequence 1 of a sequence table; the primer 2 is a single-stranded DNA molecule shown in a sequence 2 of a sequence table; the primer 3 is a single-stranded DNA molecule shown in a sequence 3 of a sequence table; the primer 4 is a single-stranded DNA molecule shown in a sequence 4 of a sequence table. The ChPV and NDV double PCR method established by the test can be used for quickly detecting clinical mixed infection of ChPV and NDV, is suitable for large-scale detection, can save cost and time, can reduce pollution, and has high practical value.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to establishment of a double PCR detection method for chicken parvovirus and newcastle disease virus.
Background
Chicken parvovirus (ChPV) is one of the important pathogens causing intestinal diseases of chickens, and can cause acute or chronic intestinal diseases, short-stature syndrome and malnutrition syndrome of chickens characterized by diarrhea, mental depression, thermoregulatory disturbance, growth retardation, increased feed consumption and the like. The chicken flock is ubiquitous, and mainly affects chicks, wherein the infection rate of commercial broilers is high, and the number of laying hens or breeding hens is low. Since 2010, countries in North America, Poland, Brazil, Hungary, Croda and Korea have successively outbreaked the disease, causing great economic losses to the poultry industry. Newcastle Disease (ND) is a multi-pathogenic type of acute, highly contagious disease caused by Newcastle Disease Virus (NDV). When poultry are infected with NDV, the disease is acute and serious, symptoms such as high fever, mucosal hemorrhage, dyspnea and diarrhea are often caused, and the mortality is high. NDV consists of an unfragmented single-stranded negative-strand RNA comprising NP, P, M, F, HN, and L, 6 structural proteins in total. At present, ND is a main disease influencing the development of poultry industry, which is classified as a type of animal epidemic disease in China and is also a frequently encountered disease and frequently encountered disease in large-scale chicken farms in China. ChPV is a new infectious disease virus in poultry industry and is popular worldwide, and diseases caused by ChPV and NDV bring great economic loss to poultry industry in China, so that the establishment of a diagnostic method for rapidly detecting ChPV and NDV is necessary. Zsak and the like establish a rapid specific PCR detection method of ChPV according to the conserved sequence of ChPV NS1 gene; carratal, et al established TaqMan probe fluorescent quantitative PCR for detection of ChPV; finkler et al established a SYBR Green-based fluorescent quantitative PCR method according to the conserved sequence of ChPV NS1 gene; fengxin et al established a semi-nested PCR method for detection of ChPV. One-step RT-PCR for identifying the strong and weak toxicity of NDV is established in plaintext dragon and the like; real-time RT-PCR for detecting NDV is established by Tung Zhou.
Disclosure of Invention
An object of the present invention is to provide a set of primers for identifying chicken parvovirus and chicken newcastle disease virus.
The invention provides a set of primers for identifying chicken parvovirus and chicken newcastle disease virus, which consists of the following primers 1 to 4:
the primer 1 is (a1) or (a 2):
(a1) a single-stranded DNA molecule shown in sequence 1 of the sequence table;
(a2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and have the same functions as the sequence 1;
the primer 2 is (a3) or (a 4):
(a3) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
(a4) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and have the same functions as the sequence 2;
the primer 3 is (a5) or (a 6):
(a5) a single-stranded DNA molecule shown in sequence 3 of the sequence table;
(a6) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and have the same functions as the sequence 3;
the primer 4 is (a7) or (a 8):
(a7) a single-stranded DNA molecule shown in a sequence 4 of the sequence table;
(a8) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 4 and has the same function as the sequence 4.
In the above set of primers, the molar ratio of the primer 1, the primer 2, the primer 3, and the primer 4 is 1:1:1: 1.
The primers in the primer set are packaged independently.
Another objective of the invention is to provide PCR reagents for identifying chicken parvovirus and chicken Newcastle disease virus.
The PCR reagent provided by the invention comprises the primer set,
the concentrations of the primer 1, the primer 2, the primer 3 and the primer 4 in the PCR reagent were all 0.4 pmol/. mu.L.
A kit comprising the primer set or the PCR reagent set is also within the scope of the present invention.
The application of the above primer set or the above PCR reagent or the above kit in any one of the following (c1) to (c6) is also within the scope of the present invention:
(c1) identifying chicken parvovirus and/or chicken newcastle disease virus;
(c2) preparing a product for identifying the chicken parvovirus and/or the chicken newcastle disease virus;
(c3) detecting whether the pathogenic microorganism to be detected is chicken parvovirus and/or chicken newcastle disease virus;
(c4) preparing a product for detecting whether pathogenic microorganisms to be detected are chicken parvovirus and/or chicken newcastle disease virus;
(c5) detecting whether a sample to be detected contains chicken parvovirus and/or chicken newcastle disease virus;
(c6) preparing a product for detecting whether the sample to be detected contains the chicken parvovirus and/or the chicken Newcastle disease virus.
The 3 rd object of the present invention is to provide a method for preparing the above kit.
The preparation method of the kit provided by the invention comprises the step of packaging each primer separately.
The 4 th purpose of the invention is to provide a method for identifying whether a sample to be detected contains chicken parvovirus and/or chicken newcastle disease virus.
The method provided by the invention comprises the following steps: extracting nucleic acid of a sample to be detected as a template, carrying out double PCR amplification by using the complete set of primers, detecting a PCR amplification product, and judging as follows:
if the PCR amplification product meets the following conditions A and B: the condition A is that the PCR amplification product contains a fragment with the size of 650-700bp, the condition B is that the PCR amplification product contains a fragment with the size of 450-500bp, the sample to be detected contains or is candidate to contain the chicken parvovirus and the newcastle disease virus, and if the condition A and the condition B are not met, the sample to be detected does not contain or is candidate to contain the chicken parvovirus and the newcastle disease virus;
or, if the PCR amplification product meets the condition A, the sample to be detected contains or is candidate to contain the chicken parvovirus; if the condition A is not met, the sample to be detected does not contain or is candidate to contain chicken parvovirus;
or, if the PCR amplification product meets the condition B, the sample to be detected contains or is candidate to contain the Newcastle disease virus; and if the condition B is not met, the sample to be detected does not contain or candidate does not contain the Newcastle disease virus.
The 5 th purpose of the invention is to provide a method for detecting whether pathogenic microorganisms are chicken parvovirus and/or chicken newcastle disease virus.
The method provided by the invention comprises the following steps: extracting nucleic acid of pathogenic microorganism to be detected as a template, performing double PCR amplification by using the complete set of primers, detecting PCR amplification products, and judging as follows:
if the PCR amplification product meets the following condition A: the fragment with the size of 650-plus 700bp is contained, and the pathogenic microorganism to be detected is or is selected as chicken parvovirus; if the condition A is not met, the pathogenic microorganism to be detected is not or is not a candidate chicken parvovirus;
or, if the PCR amplification product meets the following condition B: if the fragment contains 450-and 500bp fragments, the sample to be detected is or is selected as the Newcastle disease virus; if the condition B is not met, the pathogenic microorganism to be detected is not the newcastle disease virus or the candidate pathogenic microorganism is not the newcastle disease virus.
In the above-mentioned method, the first step of the method,
the 650-plus 700bp fragment is a 687bp fragment, and the specific nucleotide sequence is sequence 5 or a sequence with homology of more than 90 percent;
or, the 450-500bp fragment is a 453bp fragment, and the specific nucleotide sequence is sequence 6 or a sequence with homology of more than 90 percent;
or, the template of the double PCR amplification is DNA or cDNA obtained by reverse transcription of RNA;
and/or the annealing temperature of the double PCR amplification is 58.5 ℃.
In the method, the sample is a chicken larynx swab or a cloaca swab;
or the pathogenic microorganism is a virus.
The double PCR technology established in the test can be used for quickly identifying and diagnosing ChPV and NDV common poultry infectious disease pathogens, compared with the conventional detection technology, the multiple PCR is improved on the basis of the common PCR, a plurality of templates are simultaneously amplified by utilizing a plurality of pairs of primers, the defects of the common PCR are overcome, the detection time is greatly shortened, the detection efficiency is improved, a large number of resources such as manpower and material resources are saved, and the prevention, control and extinguishment of the type of epidemic diseases are facilitated. Although the number of primers may not be limited, the primers do interact with each other, and the more the primers are, the more the interaction between the primers is complicated, which affects the amplification effect of the primers. Therefore, the conditions should be optimized individually in the experiment to avoid as much as possible the occurrence of non-specific amplification due to non-specific binding between the primer pair and the template. In addition, the amplified target fragment needs to have a proper annealing gradient, so that the annealing conditions of each primer pair are kept as consistent as possible to ensure the relative balance of the amounts of the amplified products.
The experiment designs primers aiming at conserved sequences of ChPV NS1 gene and NDV F gene, can simultaneously amplify 2 sections of specific fragments of ChPV and NDV, the lengths are 687 and 453bp respectively, the target fragments have obvious length difference (more than 100bp), the identification is convenient in the same system, and the amplification efficiency of each pair of primers is high and the specificity is strong. The method established by the test can detect ChPV and NDV nucleic acid in the sample, and therefore, the method can be used for monitoring the chicken flock infected with ChPV and NDVToxic conditions and clinical epidemiological monitoring of ChPV and NDV. The quality of nucleic acid is another important factor that directly affects the sensitivity of PCR. In identifying the sensitivity of duplex PCR, the lowest amount of nucleic acid detected by duplex PCR was compared with that of single PCR, which was reduced by 1X 101～1×102The kit has the advantages of high sensitivity (the minimum content of the ChPV nucleic acid and the NDV nucleic acid can be detected to be 6.4pg and 2.5pg respectively), and capability of completely meeting the requirements of epidemic disease detection. Feces (cotton swabs) are often used as samples in clinical tests, but since the feces contain various PCR reaction inhibiting factors, the use of a commercial nucleic acid extraction kit is suggested to obtain nucleic acid with good quality. In addition, in order to reduce the false negative of the sample, the sample should be processed as soon as possible after sampling, and the sample is transported and stored under low temperature condition.
According to the research, 50 morbid materials from Guangxi district are detected by using the established ChPV and NDV duplex PCR method, the clinical morbid materials are respectively detected and compared by adopting one-step amplification and the ChPV and NDV duplex PCR method, the coincidence rate of the results is 100%, and the detection result shows that mixed infection of ChPV and NDV exists in chicken flocks in Guangxi district, and the fact that mixed infection of ChPV and NDV should be prevented in chicken flock breeding is prompted. The method can simultaneously detect two pathogens in the same sample, meets the clinical requirement on synchronous diagnosis of various diseases, and has important clinical application value for differential diagnosis and mixed infection detection of the two diseases. The ChPV and NDV double PCR method established by the test can be used for quickly detecting clinical mixed infection of ChPV and NDV, is suitable for large-scale detection, can save cost and time, can reduce pollution, and has high practical value.
Drawings
FIG. 1 shows the optimization of ChPV and NDV duplex PCR primer volumes.
FIG. 2 is a ChPV and NDV double temperature gradient PCR.
FIG. 3 shows the results of the ChPV and NDV duplex PCR specificity assay.
FIG. 4 shows the results of ChPV and NDV duplex PCR susceptibility assays.
FIG. 5 shows the results of ChPV and NDV duplex PCR assays on a portion of clinical samples.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The viruses and reagents used in the following examples are as follows:
chicken parvovirus (chicken parovirus, abbreviated ChPV): are described in the following references: day J M, Zsak L.determination and analysis of the full-length chip partial genes [ J ] Virology,2010,399(1): 59-64.; the public is available from the Guangxi Zhuang autonomous region veterinary research institute.
Newcastle disease virus NDV lassota strain, newcastle disease virus NDV F48E9 strain, NDV i strain: are described in the following references: establishment of triple RT-PCR detection method for Newcastle disease virus, H9 subtype avian influenza virus and avian pneumovirus [ J ] China veterinary magazine, 2017(2): 6-9%; the public is available from the Guangxi Zhuang autonomous region veterinary research institute.
Marek's virus (MDV): are described in the following references: the establishment of LAMP rapid visual detection method for chicken infectious anemia virus disease [ J ] livestock ecology newspaper, 2011, 32(6): 57-60%; the public is available from the Guangxi Zhuang autonomous region veterinary research institute.
Infectious bronchitis virus (IBV Mass 41): are described in the following references: establishment of triple RT-PCR detection method for Newcastle disease virus, H9 subtype avian influenza virus and avian pneumovirus [ J ] China veterinary magazine, 2017(2): 6-9%; the public is available from the Guangxi Zhuang autonomous region veterinary research institute.
Infectious laryngotracheitis virus (AILTV Beijing): are described in the following references: establishment of triple RT-PCR detection method for Newcastle disease virus, H9 subtype avian influenza virus and avian pneumovirus [ J ] China veterinary magazine, 2017(2): 6-9%; the public is available from the Guangxi Zhuang autonomous region veterinary research institute.
Avian pneumovirus (APV MN-10) is described in the following references: establishment of triple RT-PCR detection method for Newcastle disease virus, H9 subtype avian influenza virus and avian pneumovirus [ J ] China veterinary magazine, 2017(2): 6-9%; the public is available from the Guangxi Zhuang autonomous region veterinary research institute.
Avian escherichia coli (e.coli O2) is described in the following references: establishment of triple RT-PCR detection method for Newcastle disease virus, H9 subtype avian influenza virus and avian pneumovirus [ J ] China veterinary magazine, 2017(2): 6-9%; the public is available from the Guangxi Zhuang autonomous region veterinary research institute.
Avian reovirus strain S1133 is described in the following references: establishment of triple RT-PCR detection method for Newcastle disease virus, H9 subtype avian influenza virus and avian pneumovirus [ J ] China veterinary magazine, 2017(2): 6-9%; the public is available from the Guangxi Zhuang autonomous region veterinary research institute.
The DNA/RNA co-extraction kit is purchased from Beijing all-purpose gold biotechnology limited; the MiniBEST Bacteria Genomic DNA extraction kit is purchased from TaKaRa company; the gel recovery kit was purchased from Axygen Biotechnology (Hangzhou) Inc.; 100bp ladder Marker, reverse transcription kit, pMD18-T vector kit, 2 XTAQA PCR Mix were purchased from Takara Bio engineering (Dalian) Co., Ltd.
Example 1 establishment of primers and method for double PCR detection of chicken parvovirus and newcastle disease virus
Design synthesis of primer special for double PCR detection of chicken parvovirus and newcastle disease virus
Referring to conserved sequences of ChPV NS1 gene and NDV F gene in GenBank, Primer design software DNASTAR and Primer 5.0 is applied, and finally 2 pairs of specific primers are designed through comparison and verification of NCBI BLAST. The primers were synthesized by Beijing Liuhe Huada Gene science and technology Co., Ltd (see Table 1).
Table 1 shows ChPV and NDV primer sequences
Second, establishment of double PCR detection method for chicken parvovirus and newcastle disease virus
1. DNA/RNA extraction and RNA reverse transcription
Extracting chicken parvovirus (ChPV) DNA according to the instruction of a DNA/RNA co-extraction kit; simultaneously, RNA of the NDV LaSota strain is extracted, the RNA is reversely transcribed into cDNA according to a reverse transcription instruction and is directly used for the test, and then the DNA is stored at the temperature of minus 30 ℃ for standby.
2. Optimization of ChPV and NDV double PCR reaction conditions
The total volume of the double PCR reaction system is 25.0 mu L, and the double PCR reaction system comprises 0.2-1.6 mu L of each of ChPV upstream and downstream primers and NDV upstream and downstream primers (the concentration of each primer is diluted to 10 pmol/mu L, and the concentration of each primer in the system is 0.4 pmol/mu L); 2 XPCR Mix (TaKaRa Ex Taq Mix, RR902A) 12.5. mu.L; DNA of ChPV and NDV 1.0. mu.L each for 2.0. mu.L of mixed template, and ddH2Make up to 25.0. mu.L of O.
The reaction procedure is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 deg.C for 1min, annealing at 8 temperature gradients (50.0-64.0 deg.C), and allowing reaction at 72 deg.C for 1min for 35 cycles; finally, the reaction is ended after 10min of extension at 72 ℃.
The optimization content comprises the concentration and volume of each primer of the duplex PCR; the annealing temperature is optimized.
And verifying PCR products obtained under the conditions of the concentration, the volume and the annealing temperature of each primer by using 1.2% agarose gel electrophoresis, and photographing to store results.
Detecting PCR amplification products, wherein the judgment standard is as follows:
if the PCR amplification product meets the following conditions A and B: the condition A is that the PCR amplification product contains a 650-plus 700bp fragment (specifically 687bp, sequence 5), the condition B is that the PCR amplification product contains a 450-plus 500bp fragment (specifically 453bp, sequence 6), the sample to be detected contains or is candidate to contain the chicken parvovirus and the newcastle disease virus, and if the condition A and the condition B are not met, the sample to be detected does not contain or is candidate to not contain the chicken parvovirus and the newcastle disease virus;
if the PCR amplification product meets the condition A, the sample to be detected contains or is candidate to contain the chicken parvovirus; if the condition A is not met, the sample to be detected does not contain or is candidate to contain chicken parvovirus;
if the PCR amplification product meets the condition B, the sample to be detected contains or is candidate to contain the Newcastle disease virus; and if the condition B is not met, the sample to be detected does not contain or candidate does not contain the Newcastle disease virus.
The optimization results of the ChPV and NDV duplex PCR primer volumes are shown in FIG. 1, M: 100bp DNA Ladder Marker; 1-8: 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 μ L ChPV primer; 1-8: 1.6, 1.4, 1.2, 1.0, 0.8, 0.6, 0.4, 0.2 μ L NDV primer; n: negative control; the optimal primer concentration combination was determined as: the upper and lower primers of ChPV, NDV and the upper and lower primers are 1.0. mu.L each.
The ChPV and NDV duplex PCR annealing temperature optimization results are shown in FIG. 2, M: 100bp DNA Ladder Marker; 1: 50.0 ℃; 2: 51.0 ℃; 3: 52.7 ℃; 4: 55.3 ℃; 5: 58.5 ℃; 6: 61.2 ℃; 7: 62.9 ℃; 8: 64.0 ℃; n: negative control; it can be seen that the optimum annealing temperature is 58.5 ℃.
Therefore, the optimal dual PCR reaction system is: 2 XPCR Mix 12.5. mu.L, ChPV upstream and downstream primers 1. mu.L each (final concentration of each primer in the system: 0.4 pmol/. mu.L), NDV upstream and downstream primers 1.0. mu.L each (final concentration of each primer in the system: 0.4 pmol/. mu.L), mixed template ChPV and NDV DNA 1.0. mu.L each, ddH2Make up to 25.0. mu.L of O.
The optimal reaction procedure is: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 deg.C for 1min, annealing at 58.5 deg.C for 1min, and acting at 72 deg.C for 1min for 35 cycles; finally, the reaction is ended after 10min of extension at 72 ℃.
Preparation of triple, ChPV and NDV double PCR kit
Independently packaging the ChPV upstream and downstream primers and the NDV upstream and downstream primers to obtain a ChPV and NDV double PCR kit;
or packaging the double PCR reaction system separately to obtain the ChPV and NDV double PCR kit.
Example 2 ChPV and NDV Duplex PCR identification
One, ChPV and NDV duplex PCR specificity assay
DNA or RNA of ChPV, NDV, ARV, MDV, APV, IBV, AILTV and e.coli was extracted, and double PCR reaction was performed using cDNA reverse transcribed from the DNA or RNA as a template, wherein the reaction system of the double PCR reaction was the optimal double PCR reaction system of example 1, and the reaction procedure was the optimal reaction procedure of example 1, and was judged according to the criteria of example 1.
The results are shown in FIG. 3, where M: 100bp DNA Ladder Marker; n: negative control; 1: NDV; 2: ChPV; 3: ChPV + NDV F48E 9; 4: ChPV + NDV lassota; 5: ChPV + NDV I; 6: avian reovirus; 7: marek's virus; 8: avian pneumovirus; 9: infectious bronchitis virus of chicken; 10: infectious laryngotracheitis virus of chicken; 11: avian escherichia coli; as can be seen, 3: ChPV + NDV F48E 9; 4: ChPV + NDV lassota; 5: ChPV + NDV I can amplify specific bands of ChPV 687bp 239bp and NDV 405 bp; no specific bands appeared for APV, MDV, ARV, IBV, AILTV and e.
Duplex PCR susceptibility assays for ChPV and NDV
The nucleic acid of ChPV and NDV F48E9 was extracted and the concentration of the nucleic acid was determined by Beckman UV-800 UV spectrophotometer to be 63.6 ng/. mu.L and 25.3 ng/. mu.L for ChPV and NDV F48E9, respectively.
The double PCR reaction was performed using 10-fold gradient diluted ChPV and NDV F48E9 nucleic acids as templates, wherein the reaction system of the double PCR reaction was the optimal double PCR reaction system of example 1, and the optimal reaction procedure of example 1 was determined according to the criteria of example 1. A negative control was also set.
The results are shown in FIG. 4, where M: 100bp DNA Ladder Marker; 1: 636pg ChPV +253pg NDV; 2: 64pg ChPV +25pg NDV; 3: 6.4pg ChPV +2.5pg NDV; 4: 636fg ChPV +253fg NDV; 5: 64fg ChPV +25fg NDV; 6: 6.4fg ChPV +2.5fg NDV; 7: 0.64fg ChPV +0.25 fg NDV; 8: 0.064fg ChPV +0.025fg NDV; n: negative control; it can be seen that the method of the invention detects both 64fg ChPV and 25fg NDV with high sensitivity at the lowest energy.
Triple, ChPV and NDV duplex PCR clinical sample detection
50 parts of samples (chicken larynx and cloaca double-cotton swab) collected from 10 months-2017 months in 2014 are randomly extracted, and the nucleic acid is extracted by a DNA/RNA co-extraction kit.
The extracted nucleic acid was used as a template to perform a double PCR reaction, wherein the reaction system of the double PCR reaction was the optimal double PCR reaction system of example 1, and the reaction procedure was the optimal reaction procedure of example 1. The sample was tested in duplicate and judged according to the criteria of example 1 above.
The results are shown in FIG. 5, where M: 100bp DNA Ladder Marker; p: a positive control; n: negative control; 3-23: clinical pathological material; as can be seen, there are 9 ChPV positive samples and 1 NDV positive sample, which includes 1 ChPV and NDV mixed infection, and no ChPV and NDV double infection is observed.
Nucleic acid extracted from 50 samples is used as a template to carry out one-step PCR reaction, the one-step PCR reaction is the same system, the annealing temperature is the same, and only one pair of primers is added respectively; clinical pathological materials are detected and compared by adopting a one-step amplification and ChPV and NDV double PCR method, and the coincidence rate of the results is 100%, which shows that the method of the invention has accurate detection.
Sequence listing
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Claims (6)
1. The complete set of primers for identifying the chicken parvovirus and the chicken newcastle disease virus consists of the following primers 1 to 4:
the primer 1 is a single-stranded DNA molecule shown in a sequence 1 of a sequence table;
the primer 2 is a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
the primer 3 is a single-stranded DNA molecule shown in a sequence 3 of a sequence table;
the primer 4 is a single-stranded DNA molecule shown in a sequence 4 of a sequence table.
2. The set of primers according to claim 1, wherein: the molar ratio of the primer 1 to the primer 2 to the primer 3 to the primer 4 is 1:1:1: 1.
3. PCR reagents for identifying chicken parvovirus and chicken Newcastle disease virus comprising the set of primers of claim 1 or 2,
the concentrations of the primer 1, the primer 2, the primer 3 and the primer 4 in the PCR reagent were all 0.4 pmol/. mu.L.
4. A kit comprising the primer set of claim 1 or 2 or the PCR reagent of claim 3.
5. Use of the primer set of claim 1 or 2 or the PCR reagent of claim 3 or the kit of claim 4 in any one of (c1) to (c3) as follows:
(c1) preparing a product for identifying the chicken parvovirus and/or the chicken newcastle disease virus;
(c2) preparing a product for detecting whether pathogenic microorganisms to be detected are chicken parvovirus and/or chicken newcastle disease virus;
(c3) preparing a product for detecting whether the sample to be detected contains the chicken parvovirus and/or the chicken Newcastle disease virus.
6. A method for preparing the kit according to claim 4, comprising the step of packaging each primer individually.
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| CN110643743B (en) * | 2019-10-30 | 2023-05-05 | 广西壮族自治区兽医研究所 | Double LAMP (loop-mediated isothermal amplification) detection kit for chicken infectious anemia and chicken parvovirus |
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| CN103805719A (en) * | 2014-02-20 | 2014-05-21 | 广西壮族自治区兽医研究所 | Tripe RT-PCR (Reverse Transcription-Polymerase Chain Reaction) kit for Newcastle disease virus, H9 subtype avian influenza virus and avian pneumovirus and application of kit |
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| CN105567877A (en) * | 2016-03-09 | 2016-05-11 | 广西壮族自治区兽医研究所 | Primer combination for identifying ChPV (Chicken Parvovirus) and ARV (Avian Reoviruses) and application thereof |
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