CN108866245A - The foundation of chicken parvovirus, avian influenza virus and newcastle disease virus triple PCR detection method - Google Patents
The foundation of chicken parvovirus, avian influenza virus and newcastle disease virus triple PCR detection method Download PDFInfo
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Abstract
The invention discloses the foundation of chicken parvovirus, avian influenza virus and newcastle disease virus triple PCR detection method.The present invention provides primer sets, are made of following primer 1 to primer 6：The primer 1 is single strand dna shown in the sequence 1 of sequence table；The primer 2 is single strand dna shown in the sequence 2 of sequence table；The primer 3 is single strand dna shown in the sequence 3 of sequence table；The primer 4 is single strand dna shown in the sequence 4 of sequence table；The primer 5 is single strand dna shown in the sequence 5 of sequence table；The primer 6 is single strand dna shown in the sequence 6 of sequence table.ChPV, AIV and NDV triple PCR method that this test is established can be used for the quick detection of the mixed infection of clinical ChPV, AIV and NDV, be suitable for mass detection, not only cost-saved and save the time, but also can be reduced pollution, with very high practical value.
Description
Technical field
The invention belongs to field of biotechnology, are related to a breeder parvovirus, avian influenza virus and newcastle disease virus three
The foundation of weight PCR detection method.
Background technique
Bird flu (Avian influenza, AI) is the abbreviation of Bird Flu, is that one kind is drawn by influenza A
The birds infectious disease risen, is usually expressed as sudden onset and non-evident sympton；The course of disease slightly length then has apparent nervous system, digestion
The symptoms such as road, respiratory tract and genital tract, and may be reduced with feed intake, egg production decline, and other concurrent diseases of easy infection
Disease causes serious economic loss.Avian influenza virus (Avian influenza virus, AIV) is by 8 individual segments groups
At sub-thread strand RNA, include HA, NA, M, PB1, PB2, NP, PA, NS.
Newcastle disease (Newcastle disease, ND) be by newcastle disease virus (Newcastle disease virus,
NDV a kind of acute, the highly contagious disease of more sick types caused by).When birds infect NDV, morbidity is anxious, state of an illness weight, often
There are the symptoms such as high fever, mucosa hemorrhage, expiratory dyspnea and diarrhea, and causes the higher death rate.NDV is by non-segmented negative
Sub-thread strand RNA composition includes NP, P, M, F, HN and L, totally 6 kinds of structural proteins.Currently, AI and ND are to influence aviculture development
Principal disease, China has been classified as the commonly-occurring disease and frequently-occurring disease of a kind of animal epidemic and China's scale chicken house.AI with
ND is caused by virus infection, and winter and spring is high-incidence, and tool is highly infectious, because the two is similar in symptom, therefore infects the chicken of ND
Group is easily mistakenly considered AI by raiser, and it is improper to delay chicken group's state of an illness to cause to select because for the treatment of measures.
Chicken parvovirus (Chincken parvovirus, ChPV) be cause chicken intestinal class disease important pathogen body it
One, chicken group can be caused to occur characterized by diarrhea, mental depression, thermoregulatory dysfunctions, growth retardation, increase food consumption etc.
Acute or chronic enteron aisle disease, undernutrition syndrome, depauperation and runting syndrome (runting and
stunting syndrome,RSS).ChPV chicken group in it is generally existing, mainly encroach on chick, wherein commercial broiler infection rate compared with
Height, laying hen or breeder take second place.Since two thousand and ten, North America, Poland, Hungary, Brazil, Croatia, the country such as Korea S is in succession
The disease has been broken out, has caused biggish economic loss to poultry husbandry.
When two kinds and a variety of virus mixed infections, it is difficult to make accurate judgement in clinical diagnosis, conventional method (such as
Antigen isolation and identification and serological test etc.) it is time-consuming and laborious, and be not easy to carry out antidiastole.
Summary of the invention
It is an object of the present invention to provide identification chicken parvovirus, avian influenza virus and the complete of newcastle disease virus to draw
Object.
Primer set provided by the invention is made of following primer 1 to primer 6：
The primer 1 is following (a1) or (a2)：
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) sequence 1 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 1
The DNA molecular of identical function；
The primer 2 is following (a3) or (a4)：
(a3) single strand dna shown in the sequence 2 of sequence table；
(a4) sequence 2 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 2
The DNA molecular of identical function；
The primer 3 is following (a5) or (a6)：
(a5) single strand dna shown in the sequence 3 of sequence table；
(a6) sequence 3 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 3
The DNA molecular of identical function；
The primer 4 is following (a7) or (a8)：
(a7) single strand dna shown in the sequence 4 of sequence table；
(a8) sequence 4 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 4
The DNA molecular of identical function；
The primer 5 is following (b1) or (b2)：
(b1) single strand dna shown in the sequence 5 of sequence table；
(b2) sequence 5 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 5
The DNA molecular of identical function；
The primer 6 is following (b3) or (b4)：
(b3) single strand dna shown in the sequence 6 of sequence table；
(b4) sequence 6 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 6
The DNA molecular of identical function.
It is the primer 1, the primer 2, the primer 3, the primer 4, the primer 5, described in above-mentioned primer set
The molar ratio of primer 6 is 1:1:2:2:1:1.
Another object of the present invention is to provide the PCR examination of identification chicken parvovirus, avian influenza virus and newcastle disease virus
Agent.
PCR reagent provided by the invention, including above-mentioned primer set,
The primer 1, the concentration of the primer 2, the primer 5, the primer 6 in the PCR reagent are
0.2pmol/μL；
The concentration of the primer 3 and the primer 4 in the PCR reagent is 0.4pmol/ μ L.
Kit containing above-mentioned primer set or above-mentioned PCR reagent is also the scope of protection of the invention.
Above-mentioned primer set or above-mentioned PCR reagent or mentioned reagent box are in following (c1) any into (c6)
Application be also the scope of protection of the invention：
(c1) identify chicken parvovirus, avian influenza virus and/or newcastle disease virus；
(c2) preparation is for identifying chicken parvovirus, avian influenza virus and/or the product of newcastle disease virus；
(c3) detect whether pathogenic microorganism to be measured is chicken parvovirus, avian influenza virus and/or newcastle disease virus；
(c4) preparation is for detecting whether pathogenic microorganism to be measured is chicken parvovirus, avian influenza virus and/or chicken new city
The product of epidemic disease poison；
(c5) it detects in sample to be tested and whether contains chicken parvovirus, avian influenza virus and/or newcastle disease virus；
(c6) preparation is for detecting in sample to be tested whether contain chicken parvovirus, avian influenza virus and/or newcastle disease
The product of virus.
3rd purpose of the invention is to provide the preparation method of the kit.
Preparation method provided by the invention includes the steps that individually packing each primer.
4th purpose of the invention be to provide a kind of identification sample to be tested whether contain chicken parvovirus, avian influenza virus and/
Or the method for newcastle disease virus.
Method provided by the invention, includes the following steps：The nucleic acid of sample to be tested is extracted as template, with above-mentioned complete
Primer carries out triple PCR amplification, detects pcr amplification product, makes the following judgment：
If the eligible A of the pcr amplification product, condition B and condition C：The condition A is that the pcr amplification product contains
Having the segment of 650-700bp size, the condition B is the segment, described that the pcr amplification product contains 450-500bp size
Condition C is the segment that the pcr amplification product contains 200-250bp size, then the sample to be tested contains or candidate contains chicken
Parvovirus, avian influenza virus and newcastle disease virus, it is described to test sample if not meeting the condition A, condition B and condition C
Originally it does not contain or candidate without containing chicken parvovirus, avian influenza virus and newcastle disease virus；
If the pcr amplification product meets the condition A and the condition B, contains in sample to be tested or candidate is contained
Chicken parvovirus and newcastle disease virus；If not meeting the condition A and the condition B, the sample to be tested is not contained or is waited
Choosing does not contain chicken parvovirus and newcastle disease virus；
If the pcr amplification product meets the condition A and the condition C, contains in sample to be tested or candidate is contained
Chicken parvovirus and avian influenza virus；If not meeting the condition A and the condition C, the sample to be tested is not contained or is waited
Choosing does not contain chicken parvovirus and avian influenza virus；
If the pcr amplification product meets the condition B and the condition C, contains in sample to be tested or candidate is contained
Newcastle disease virus and avian influenza virus；If not meeting the B and the condition C, the sample to be tested do not contain or it is candidate not
Contain newcastle disease virus and avian influenza virus；
If the pcr amplification product meets the condition A, contains in sample to be tested or candidate contains chicken parvovirus；
If not meeting the condition A, the sample to be tested is not contained or candidate is without containing chicken parvovirus；
If the pcr amplification product meets the condition B, contains in sample to be tested or candidate contains newcastle disease virus；
If not meeting the condition B, the sample to be tested is not contained or candidate is without containing newcastle disease virus；
If the pcr amplification product meets the condition C, contains in sample to be tested or candidate contains avian influenza virus；
If not meeting the condition C, the sample to be tested is not contained or candidate is without containing avian influenza virus.
4th purpose of the invention be to provide a kind of detection pathogenic microorganism whether be chicken parvovirus, avian influenza virus and/
Or the method for newcastle disease virus.
Method provided by the invention, includes the following steps：The nucleic acid of pathogenic microorganism to be measured is extracted as template, use is above-mentioned
Primer set carry out triple PCR amplification, detect pcr amplification product, make the following judgment：
If the pcr amplification product meets the condition A, pathogenic microorganism to be measured is or candidate is chicken parvovirus；
If not meeting the condition A, the pathogenic microorganism to be measured is not or candidate is not chicken parvovirus；
If the pcr amplification product meets the condition B, pathogenic microorganism to be measured is or candidate is newcastle disease virus；
If not meeting the condition B, the pathogenic microorganism to be measured is not or candidate is not newcastle disease virus；
If the pcr amplification product meets condition C as described below, pathogenic microorganism to be measured is or candidate is avian flu
Poison；If not meeting the condition C, the pathogenic microorganism to be measured is not or candidate is not avian influenza virus.
In the above method, the segment of the 650-700bp size is the segment of 687bp size, and nucleotides sequence is classified as sequence
Column 7 or with its homology be greater than 90% sequence；
The segment of the 450-500bp size is the segment of 453bp size, and nucleotides sequence is classified as sequence 8 or same with it
Source property is greater than 90% sequence；
The segment of the 200-250bp size is the segment of 239bp size, and nucleotides sequence is classified as sequence 9 or same with it
Source property is greater than 90% sequence；
Or, the template of the triple PCR amplification is DNA or obtains cDNA by RNA reverse transcription；
And/or the annealing temperature of the triple PCR amplification is 58.5 DEG C.
In the above method, the sample is chicken larynx swab or cloacal swab；
Or the pathogenic microorganism is virus.
The triple PCR technology that the present invention establishes can carry out tri- kinds of poultry common transmittable disease cause of diseases of ChPV, AIV and NDV
Quick antidiastole, compared with conventional detection technique, multiplex PCR is improved on the basis of regular-PCR, using more
Multiple template is expanded simultaneously to primer, the deficiency of regular-PCR is compensated for, highly shortened detection time, improves detection effect
Rate saves the resources such as a large amount of manpower and material resources, is conducive to the prevention of such epidemic disease, controls and put out, still, multiplex PCR
There are different primer pairs and template in same system, may make in amplified reaction between different templates, between primer pair and
There are Reverse transcriptases between primer pair and template.Although the quantity of primer can be without restriction, deposited really between primer
It is interacting, primer is more, and the interaction between primer is more complicated, influences the expanding effect of primer.Therefore, it is answered in test
This optimizes various conditions respectively, avoids the non-spy generated by the non-specific binding between primer pair and template as far as possible
Property amplification.In addition, the target fragment of amplification needs suitable annealing gradient, so that each primer pair annealing conditions are kept as far as possible
Unanimously, to guarantee the relative equilibrium of its amplified production amount.
Conserved sequence design primer of the present invention for ChPV NS1 gene, AIV M gene and NDV F gene, Neng Goutong
When amplify 3 sections of specific fragments of ChPV, AIV and NDV, length is respectively 687,239 and 453bp, is had between target fragment
There is apparent difference in length (> 100bp), convenient for identifying in same system, and the amplification efficiency of every pair of primers is high, special
Property is strong.The method that this test is established is able to detect that ChPV, AIV and NDV nucleic acid in sample, therefore, can be used for monitoring chicken group's sense
Toxin expelling situation and the clinically monitoring of ChPV, AIV and NDV epidemiology after contaminating ChPV, AIV and NDV.In identification triple PCR
Sensibility when, the nucleic acid detection limit of triple PCR and substance PCR is compared, accordingly reduces 1 × 101~1 × 102
A order of magnitude, still with very high sensibility (minimum content that can detecte ChPV, AIV and NDV nucleic acid is respectively 6.4,
2.0,2.5pg), it can satisfy the requirement of epidemic disease detection completely.Often with excrement (cotton swab) for sample in clinical detection, but
Be due in excrement containing there are many suppression PCR response factor, it is therefore proposed that the nucleic acid extraction kit of commodity in use is to obtain
The nucleic acid of good quality.In addition, should be handled as early as possible sample after sampling to reduce the false negative of sample, and by sample
It is placed under cryogenic conditions and transports, saves.This method can detect three kinds of pathogen simultaneously in same a sample, meet clinic
On requirement to the synchronous diagnosis of a variety of diseases, antidiastoles and mixed infection detection for these three diseases have important faces
Bed application value.ChPV, AIV and NDV triple PCR method that this test is established can be used for clinical ChPV, AIV and NDV
The quick detection of mixed infection is suitable for mass detection, not only cost-saved and save the time, but also can be reduced pollution, with very
High practical value.
Detailed description of the invention
Fig. 1 is the optimization of ChPV, AIV and NDV triple PCR primer volume.
Fig. 2 is the triple temperature gradient PCR of ChPV, AIV and NDV.
Fig. 3 is ChPV, AIV and NDV triple PCR specific test result.
Fig. 4 is ChPV, AIV and NDV triple PCR sensitivity tests result.
Fig. 5 is ChPV, AIV and NDV triple PCR testing result of partial clinical sample.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Virus as used in the following examples is as follows with reagent：
Chicken parvovirus (chicken parvovirus, be abbreviated as ChPV) is documented in following bibliography：Day J M,
Zsak L.Determination and analysis of the full-length chicken parvovirus
genome.[J].Virology,2010,399(1):59-64.；The public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region
?.
LaSota plants of newcastle disease virus NDV, F48E9 plants of newcastle disease virus NDV, I plant of NDV be documented in it is following with reference to text
It offers：Xie Zhiqin, Xie Zhixun, Deng Xianwen wait newcastle disease virus, H9 subtype avian influenza virus and the triple RT- of avian pneumovirus
Foundation [J] the China Veterinary Journal of PCR detection method, 2017 (2):6-9.；The public can grind from Guangxi Zhuang Autonomous Region animal doctor
Study carefully and is obtained.
Marek's disease poison (MDV) is documented in following bibliography：Deng Xianwen, Xie Zhixun, Xie Zhiqin wait avian infectious poor
Foundation [J] ecology of domestic animals report of blood virosis LAMP quick visualization detection method, 2011,32 (6):57-60.；The public can
To be obtained from Veterinary Institute of Guangxi Zhuang Autonomous Region.
Infectious bronchitis virus (IBV Mass 41) is documented in following bibliography：Xie Zhiqin, Xie Zhixun, Deng Xian
Text waits in foundation [J] of newcastle disease virus, H9 subtype avian influenza virus and the triple RT-PCR detection methods of avian pneumovirus
State's Veterinary Journal, 2017 (2):6-9.；The public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region.
Infectious laryngotracheitis virus (AILTV Beijing) is documented in following bibliography：Xie Zhiqin, Xie Zhixun, Deng
Aobvious text, waits foundation [J] of newcastle disease virus, H9 subtype avian influenza virus and the triple RT-PCR detection methods of avian pneumovirus
Chinese Veterinary Journal, 2017 (2):6-9.；The public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region.
Avian pneumovirus (APV MN-10) is documented in following bibliography：Xie Zhiqin, Xie Zhixun, Deng Xianwen wait chicken new
Foundation [J] the China animal doctor of city epidemic disease poison, H9 subtype avian influenza virus and the triple RT-PCR detection methods of avian pneumovirus is miscellaneous
Will, 2017 (2):6-9.；The public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region.
Avian escherichia coli (E.coli O2) is documented in following bibliography：Xie Zhiqin, Xie Zhixun, Deng Xianwen wait chicken new
Foundation [J] the China animal doctor of city epidemic disease poison, H9 subtype avian influenza virus and the triple RT-PCR detection methods of avian pneumovirus is miscellaneous
Will, 2017 (2):6-9.；The public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region.
S1133 plants of Avianreovirus are documented in following bibliography：Xie Zhiqin, Xie Zhixun, Deng Xianwen wait chicken new city
Foundation [J] the China Veterinary Journal of epidemic disease poison, H9 subtype avian influenza virus and the triple RT-PCR detection methods of avian pneumovirus,
2017(2):6-9.；The public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region.
Viral DNA/RNA is total to extraction reagent kit purchased from Beijing Quanshijin Biotechnology Co., Ltd；MiniBEST
Bacteria Genomic DNA extraction agent box is purchased from Dalian TaKaRa company；Plastic recovery kit is purchased from Axygen biology skill
Art (Hangzhou) Co., Ltd；100bp ladder Marker, reverse transcription reagent box, pMD18-T support agent box, 2 × Taq
PCR Mix is purchased from precious bioengineering (Dalian) Co., Ltd.
Embodiment 1, chicken parvovirus, avian influenza virus and newcastle disease virus triple PCR detection primer special and method
Foundation
One, the design synthesis of chicken parvovirus, avian influenza virus and newcastle disease virus triple PCR detection primer special
Referring to the conserved sequence of ChPV NS1 gene, NDV F gene and AIV M gene in GenBank, using design of primers
Software DNASTAR and Primer 5.0 finally passes through NCBI BLAST comparison, designs three pairs of specific primers.Primer
It synthesizes in Beijing Liuhe Huada Genomics Technology Co., Ltd (being shown in Table 1).
Table 1 is ChPV, AIV and NDV primer sequence
Two, the foundation of chicken parvovirus, avian influenza virus and newcastle disease virus triple PCR detection method
1, DNA/RNA extracting and RNA reverse transcription
The DNA that extraction reagent kit specification extracts ChPV is total to according to Viral DNA/RNA；The RNA of AIV, NDV are extracted simultaneously,
RNA reverse transcription is directly used in test, later saves backup DNA in -30 DEG C at cDNA referring to reverse transcription specification.According to
MiniBEST Bacteria Genomic DNA extraction agent box specification extracts E.coli O2DNA, -30 DEG C of preservations are standby
With.
2, the optimization of ChPV, AIV and NDV triple PCR reaction condition
Triple PCR reaction system total volume is 25.0 μ L, comprising on ChPV upstream and downstream primer, AIV upstream and downstream primer and NDV
Each 0.5-1 μ L of downstream primer (each primer concentration is diluted to 10pmol/ μ L, concentration 0.4pmol/ μ L of the primer in system)；2×
PCR Mix(TaKaRa Ex Taq Mix,RR902A)12.5μL；Each 1.0 μ L of the DNA of ChPV, AIV and NDV totally 3.0 μ L's is mixed
Shuttering finally uses ddH2O complements to 25.0 μ L.
Response procedures are：95 DEG C of initial denaturation 5min；8 temperature gradients are arranged in 94 DEG C of denaturation 1min, annealing temperature altogether
(50.0-64.0 DEG C), 72 DEG C of effect 1min carry out 35 circulations altogether；Last 72 DEG C of extensions 10min, reaction was completed.
Optimizing content is to each primer concentration of triple PCR, volume；Annealing temperature is optimized.
By 1.2% agarose gel electrophoresis of the PCR product obtained under each primer concentration, volume, annealing temperature condition
It is verified, takes pictures and save result.
Pcr amplification product is detected, judgment criteria is as follows：
If the eligible A of the pcr amplification product, condition B and condition C：The condition A is to contain 650-700bp size
Segment (specially the segment of 687bp size, nucleotides sequence are classified as sequence 7), the condition B be 450-500bp size piece
Section (specially the segment of 453bp size, nucleotides sequence are classified as sequence 8), the segment (tool that the condition C is 200-250bp size
Body is the segment of 239bp size, and nucleotides sequence is classified as sequence 9), then the sample to be tested contains or candidate contains the tiny disease of chicken
Poison, avian influenza virus and newcastle disease virus, if not meeting the condition A, condition B and condition C, the sample to be tested is free of
Have or candidate without containing chicken parvovirus, avian influenza virus and newcastle disease virus；
If the pcr amplification product meets the condition A and the condition B, contains in sample to be tested or candidate is contained
Chicken parvovirus and newcastle disease virus；If not meeting the condition A and the condition B, the sample to be tested is not contained or is waited
Choosing does not contain chicken parvovirus and newcastle disease virus；
If the pcr amplification product meets the condition A and the condition C, contains in sample to be tested or candidate is contained
Chicken parvovirus and avian influenza virus；If not meeting the condition A and the condition C, the sample to be tested is not contained or is waited
Choosing does not contain chicken parvovirus and avian influenza virus；
If the pcr amplification product meets the condition B and the condition C, contains in sample to be tested or candidate is contained
Newcastle disease virus and avian influenza virus；If not meeting the B and the condition C, the sample to be tested do not contain or it is candidate not
Contain newcastle disease virus and avian influenza virus；
If the pcr amplification product meets the condition A, contains in sample to be tested or candidate contains chicken parvovirus；
If not meeting the condition A, the sample to be tested is not contained or candidate is without containing chicken parvovirus；
If the pcr amplification product meets the condition B, contains in sample to be tested or candidate contains newcastle disease virus；
If not meeting the condition B, the sample to be tested is not contained or candidate is without containing newcastle disease virus；
If the pcr amplification product meets the condition C, contains in sample to be tested or candidate contains avian influenza virus；
If not meeting the condition C, the sample to be tested is not contained or candidate is without containing avian influenza virus.
The optimum results of ChPV, AIV and NDV triple PCR primer volume are as shown in Figure 1, M：100bp DNA Ladder
Marker；1-5：0.5, each ChPV upstream and downstream primer of 0.5,0.5,1.0,1.0 μ L；1-5：0.5,1.0,0.5,0.5,1.0 μ L are each
AIV upstream and downstream primer；1-5：0.5, each NDV upstream and downstream primer N of 0.5,1.0,0.5,1.0 μ L：Negative control；Best primer concentration
Combination is determined as：The upstream and downstream each 0.5 μ L, NDV of the upstream and downstream ChPV and AIV primer each 1.0 μ L of primer.
ChPV, AIV and NDV triple PCR annealing temperature optimum results are as 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 Negative control；As can be seen that optimal annealing temperature is 58.5 DEG C.
Therefore, optimal triple PCR reaction system is：2 × PCR Mix, 12.5 μ L, ChPV upstream and downstream primer and AIV
Each 0.5 μ L of upstream and downstream primer (final concentration 0.2pmol/ μ L of each primer in system), each 1.0 μ L of the upstream and downstream NDV primer
(final concentration 0.4pmol/ μ L of each primer in system), the DNA of hybrid template ChPV, AIV and NDV each 1.0 μ L, ddH2O
Complement to 25.0 μ L.
Optimal response procedures are：95 DEG C of initial denaturation 5min；95 DEG C of denaturation 1min, 58.5 DEG C of annealing 1min, 72 DEG C act on
1min carries out 35 circulations altogether；Last 72 DEG C of extensions 10min, reaction was completed.
Three, the preparation of ChPV, AIV and NDV triple PCR kit
The upstream and downstream ChPV primer, the upstream and downstream AIV primer and the upstream and downstream NDV primer are individually packed, ChPV, AIV are obtained
With NDV triple PCR kit；
Or individually pack triple PCR reaction system, obtain ChPV, AIV and NDV triple PCR kit.
Embodiment 2, the identification of ChPV, AIV and NDV triple PCR
One, ChPV, AIV and NDV triple PCR specific test
Extract MDV, ARV S1133, AEV Van, APV MN-10, the IBV Mass of ChPV, AIV and NDV and control
41, AILTV Beijing and E.coli O2DNA or RNA, using the cDNA that DNA RNA reverse transcription obtains as template into
The reaction of row triple PCR, wherein the reaction system of triple PCR reaction is the optimal triple PCR reaction system of embodiment 1, reaction
Program is the optimal response procedures of embodiment 1, and judgment criteria is the same as embodiment 1.
As a result as shown in figure 3, M：100bp DNA Ladder Marker；1：AIV；2：NDV；3：ChPV；4：ChPV+AIV
H9+NDV(laSota)；5：ChPV+ H5 hypotype AIV+NDV (laSota)；6：ChPV+ H9 hypotype AIV+NDV (F48E9)；
7：Avian pneumovirus；8：Marek's disease poison；9：Avian encephalomyclitis virus；10：Avian infectious bronchitis virus；11：Chicken is infected
Property laryngotracheitis virus；12：Avianreovirus；13：Avian escherichia coli；It can be seen that 4：ChPV+AIV H9+NDV
(laSota)；5：ChPV+ H5 hypotype AIV+NDV (laSota)；6：ChPV+ H9 hypotype AIV+NDV (F48E9) can be expanded
The specific band of ChPV 687bp, AIV 239bp and NDV 405bp out；And MDV, ARV, AEV, APV, IBV, AILTV and
E.coli does not occur specific band.
Two, ChPV, AIV and NDV triple PCR sensitivity tests
The nucleic acid for extracting ChPV, AIV and NDV carries out nucleic acid concentration through Beckman UV-800 ultraviolet specrophotometer
Measurement, the nucleic acid concentration of ChPV, AIV and NDV are respectively 63.6ng/ μ L, 20.1ng/ μ L and 25.3ng/ μ L.
Triple PCR reaction is carried out using ChPV, AIV and NDV nucleic acid after 10 times of gradient dilutions as template respectively, wherein three
The reaction system of weight PCR reaction is the optimal triple PCR reaction system of embodiment 1, and response procedures are the optimal of embodiment 1
Response procedures, judgment criteria is the same as embodiment 1.Negative control is set simultaneously.
As a result as shown in figure 4, M：100bp DNA Ladder Marker；1：63.6ng ChPV+25.3ng NDV +
20.1ng AIV；2：6.4ng ChPV+2.5ng NDV+2.0ng AIV；3：636pg ChPV +253pg NDV +201pg
AIV；4：63.6pg ChPV+25.3pg NDV +20.1pg AIV；5：6.4pg ChPV +2.5pg NDV +2.0pg AIV；
6：636fg ChPV +253fg NDV +201fg AIV；7：63.6fg ChPV +25.3fg NDV +20.1fg AIV；8：
6.4fg ChPV +2.5fg NDV+2.0fg AIV；N：Negative control Negative control；As can be seen that of the invention
Method most low energy is detected simultaneously by 6.4pg ChPV, 2.5pg NDV and 2.0pg AIV, high sensitivity.
Three, ChPV, AIV and NDV triple PCR clinical sample detect
50 are randomly selected from the sample (the double cotton swabs of chicken larynx, cloaca) that in October, -2017 in October, 2014 acquires
Part, DNA/RNA is total to extraction reagent kit and extracts nucleic acid.
Using the nucleic acid of extraction as template, triple PCR reaction is carried out, wherein the reaction system of triple PCR reaction is to implement
The optimal triple PCR reaction system of example 1, response procedures are the optimal response procedures of embodiment 1.Sample repeats detection two
It is secondary.
As a result as shown in figure 5, M：100bp DNA Ladder Marker；P：Positive control；1-23：Clinical pathological material of disease；It can be with
Find out there is 9 parts of ChPV positive samples, 2 parts of AIV positive samples, 1 part of NDV positive sample is mixed including 2 parts of ChPV with AIV
Infection and 1 part of ChPV and NDV mixed infection, have no ChPV, AIV and NDV triple infection.
Using the nucleic acid of 50 parts of sample extractions as template, one-step method PCR reaction is carried out, one-step method, that is, identical system is moved back
Fiery temperature only adds the amplification method of pair of primers respectively；Using one step amplification and ChPV, AIV and NDV triple PCR method pair
Clinical pathological material of disease has carried out detection comparison respectively, and as a result the coincidence rate of the two is 100%, shows that method detection of the invention is accurate.
Sequence table
<110>Veterinary Institute of Guangxi Zhuang Autonomous Region
<120>The foundation of chicken parvovirus, avian influenza virus and newcastle disease virus triple PCR detection method
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
aaaaggatga gaagggaact 20
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence
<400> 2
gcacagtctg atggctaag 19
<210> 3
<211> 19
<212> DNA
<213>Artificial sequence
<400> 3
tggaggcata caacaggac 19
<210> 4
<211> 19
<212> DNA
<213>Artificial sequence
<400> 4
gagttaaggc aggggaagt 19
<210> 5
<211> 19
<212> DNA
<213>Artificial sequence
<400> 5
cctaaatggg aatggagac 19
<210> 6
<211> 19
<212> DNA
<213>Artificial sequence
<400> 6
tgtgggaccg atgttgtgc 19
<210> 7
<211> 687
<212> DNA
<213>Artificial sequence
<400> 7
aaaaggatga gaagggaact ttgcaacaaa gctgcagaaa tgttgcacat gttttgtcct 60
gatttcccca aaagtcaaat ctggggatcg ctggtgaata tccaaaacgc tatgctttcg 120
ctgaataggg gatactcacc gaggattggg aaaccaatcc cccaaccagt aaatccgtac 180
tcatttctaa aatactacat gttcaattct gctaaaatga gctttctgag aggggctact 240
ccaaaattag ctgaaaaatt accaattggt acaagatatg ctagatttga agggtctcct 300
gacctggacg agggtcccga aattccatcg caggaattaa ctccagggga cactctacca 360
gtgtgctatg taagtactga aactgattgg gggaatggtg ggggggaaac cgtaatcaaa 420
acaagtataa tcgaaaaatt atgcatggaa gcattaagat tatgcaggga aaattatatt 480
tttaccatga aagcgtttaa actgaaattc ccagacaaat tcatgcaatt cagctcacga 540
aatcaggggt tagtcaagtt agacgaaaca ataaacctat attgcgagac cattatccac 600
gatcacaacg cgtgggaaat agctaaatcg atacatggag atgttgacac atctgatctg 660
gatgataact tagccatcag actgtgc 687
<210> 8
<211> 453
<212> DNA
<213>Artificial sequence
<400> 8
tggaggcata caacaggaca ctgactactt tgctcacccc ccttggtgat tctatccgca 60
ggatacaaga gtctgcgact acgtccggag gaaggaggca gagacgcttt ataggtgcca 120
ttatcggcag tgtagctctt ggggttgcca cagatgccca gataacagca gcctcagctc 180
tgatacaagc caaccagaat gctgccaaca tcctccggct taaagagagc attgctgcaa 240
ctaatgaagc tgtacatgaa gtcactgacg gattatcgca actagcagtg gcagttggga 300
agatgcagca gtttgttaat gaccagttta ataacacagc tcaggaattg gactgtataa 360
aaattacaca gcaggttggt gtagaactca acctgtacct aactgaattg actacagtat 420
tcgggccaca aatcacttcc cctgccttaa ctc 453
<210> 9
<211> 239
<212> DNA
<213>Artificial sequence
<400> 9
cctaaatggg aatggagacc caaacaacat ggacaaggca gttaaattgt acaagaaact 60
gaagagagaa atgacatttc atggagcaaa ggaagttgca ctcagttact caactggtgc 120
gcttgccagc tgcatgggtc tcatatacaa caggatgggg acagtgaccg cagaaggggc 180
tcttggacta gtatgtgcca cttgtgagca gattgctgac gcacaacatc ggtcccaca 239
Claims (10)
1. the primer set of chicken parvovirus, avian influenza virus and newcastle disease virus is identified, by following primer 1 to primer 6
Composition：
The primer 1 is following (a1) or (a2)：
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) have by sequence 1 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 1 identical
The DNA molecular of function；
The primer 2 is following (a3) or (a4)：
(a3) single strand dna shown in the sequence 2 of sequence table；
(a4) have by sequence 2 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 2 identical
The DNA molecular of function；
The primer 3 is following (a5) or (a6)：
(a5) single strand dna shown in the sequence 3 of sequence table；
(a6) have by sequence 3 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 3 identical
The DNA molecular of function；
The primer 4 is following (a7) or (a8)：
(a7) single strand dna shown in the sequence 4 of sequence table；
(a8) have by sequence 4 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 4 identical
The DNA molecular of function；
The primer 5 is following (b1) or (b2)：
(b1) single strand dna shown in the sequence 5 of sequence table；
(b2) have by sequence 5 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 5 identical
The DNA molecular of function；
The primer 6 is following (b3) or (b4)：
(b3) single strand dna shown in the sequence 6 of sequence table；
(b4) have by sequence 6 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 6 identical
The DNA molecular of function.
2. primer set according to claim 1, it is characterised in that：The primer 1, the primer 2, the primer 3, institute
State primer 4, the primer 5, the primer 6 molar ratio be 1:1:2:2:1:1.
3. identifying the PCR reagent of chicken parvovirus, avian influenza virus and newcastle disease virus, including of any of claims 1 or 2
Primer set,
The primer 1, the concentration of the primer 2, the primer 5, the primer 6 in the PCR reagent are 0.2pmol/ μ
L；
The concentration of the primer 3 and the primer 4 in the PCR reagent is 0.4pmol/ μ.
4. the kit containing primer set of any of claims 1 or 2 or PCR reagent as claimed in claim 3.
5. kit described in primer set of any of claims 1 or 2 or PCR reagent as claimed in claim 3 or claim 4
Application in following (c1) any into (c6)：
(c1) identify chicken parvovirus, avian influenza virus and/or newcastle disease virus；
(c2) preparation is for identifying chicken parvovirus, avian influenza virus and/or the product of newcastle disease virus；
(c3) detect whether pathogenic microorganism to be measured is chicken parvovirus, avian influenza virus and/or newcastle disease virus；
(c4) preparation is for detecting whether pathogenic microorganism to be measured is chicken parvovirus, avian influenza virus and/or Newcastle disease
The product of poison；
(c5) it detects in sample to be tested and whether contains chicken parvovirus, avian influenza virus and/or newcastle disease virus；
(c6) preparation is for detecting in sample to be tested whether contain chicken parvovirus, avian influenza virus and/or newcastle disease virus
Product.
6. the preparation method of kit described in claim 4 includes the steps that individually packing each primer.
7. a kind of identification sample to be tested whether the method containing chicken parvovirus, avian influenza virus and/or newcastle disease virus, packet
Include following steps：The nucleic acid of sample to be tested is extracted as template, carries out triple PCR with primer set of any of claims 1 or 2
Amplification detects pcr amplification product, makes the following judgment：
If the eligible A of the pcr amplification product, condition B and condition C：The condition A is the piece containing 650-700bp size
Section, the segment that the condition B is 450-500bp size, the segment that the condition C is 200-250bp size, then it is described to test sample
Originally contain or candidate containing chicken parvovirus, avian influenza virus and newcastle disease virus, if do not meet the condition A, condition B and
Condition C, then the sample to be tested does not contain or candidate is without containing chicken parvovirus, avian influenza virus and newcastle disease virus；
If the pcr amplification product meets the condition A and the condition B, contain in sample to be tested or candidate thin containing chicken
Small virus and newcastle disease virus；If not meeting the condition A and the condition B, the sample to be tested do not contain or it is candidate not
Contain chicken parvovirus and newcastle disease virus；
If the pcr amplification product meets the condition A and the condition C, contain in sample to be tested or candidate thin containing chicken
Small virus and avian influenza virus；If not meeting the condition A and the condition C, the sample to be tested do not contain or it is candidate not
Contain chicken parvovirus and avian influenza virus；
If the pcr amplification product meets the condition B and the condition C, contains in sample to be tested or candidate contains new city
Epidemic disease poison and avian influenza virus；If not meeting the B and the condition C, the sample to be tested is not contained or candidate does not contain
Newcastle disease virus and avian influenza virus；
If the pcr amplification product meets the condition A, contains in sample to be tested or candidate contains chicken parvovirus；If no
Meet the condition A, then the sample to be tested does not contain or candidate is without containing chicken parvovirus；
If the pcr amplification product meets the condition B, contains in sample to be tested or candidate contains newcastle disease virus；If no
Meet the condition B, then the sample to be tested does not contain or candidate is without containing newcastle disease virus；
If the pcr amplification product meets the condition C, contains in sample to be tested or candidate contains avian influenza virus；If no
Meet the condition C, then the sample to be tested does not contain or candidate is without containing avian influenza virus.
8. it is a kind of detection pathogenic microorganism whether be chicken parvovirus, avian influenza virus and/or newcastle disease virus method, packet
Include following steps：The nucleic acid of pathogenic microorganism to be measured is extracted as template, is carried out with primer set of any of claims 1 or 2
Triple PCR amplification, detects pcr amplification product, makes the following judgment：
If the eligible A of pcr amplification product：The pcr amplification product contains the segment of 650-700bp size, then to be measured
Pathogenic microorganism is or candidate is chicken parvovirus；If not meeting the condition A, the pathogenic microorganism to be measured is not or waits
Choosing is not chicken parvovirus；
If the pcr amplification product meets following condition B：The pcr amplification product contains the segment of 450-500bp size, then
Pathogenic microorganism to be measured is or candidate is newcastle disease virus；If not meeting the condition B, the pathogenic microorganism to be measured is not
Or candidate is not newcastle disease virus；
If the pcr amplification product meets following condition C：The pcr amplification product contains the segment of 200-250bp size, then
Pathogenic microorganism to be measured is or candidate is avian influenza virus；If not meeting the condition C, the pathogenic microorganism to be measured is not
Or candidate is not avian influenza virus.
9. method according to claim 7 or 8, it is characterised in that：
The segment of the 650-700bp size is the segment of 687bp size；
The segment of the 450-500bp size is the segment of 453bp size；
The segment of the 200-250bp size is the segment of 239bp size；
Or, the template of the triple PCR amplification is DNA or obtains cDNA by RNA reverse transcription；
And/or the annealing temperature of the triple PCR amplification is 58.5 DEG C.
10. according to any method of claim 7-9, it is characterised in that：The sample is chicken larynx swab or cloaca
Swab；
Or the pathogenic microorganism is virus.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110777219A (en) * | 2019-10-31 | 2020-02-11 | 广西壮族自治区兽医研究所 | Triple PCR detection primer group, kit and method for chicken parvovirus and H9 subtype avian influenza virus |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103773899A (en) * | 2014-01-26 | 2014-05-07 | 广西壮族自治区兽医研究所 | GeXP (Gene Expression Profiler) detection kit for differentiating 11 kinds of duck viral diseases |
| CN106636473A (en) * | 2017-01-20 | 2017-05-10 | 广西壮族自治区兽医研究所 | Complete reagent and method for detecting H5 subtype avian influenza virus and chicken parvovirus |
| CN106636472A (en) * | 2017-01-20 | 2017-05-10 | 广西壮族自治区兽医研究所 | Reagent set and method for detecting avian influenza viruses and chicken parvoviruses |
-
2018
- 2018-08-31 CN CN201811008894.3A patent/CN108866245B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103773899A (en) * | 2014-01-26 | 2014-05-07 | 广西壮族自治区兽医研究所 | GeXP (Gene Expression Profiler) detection kit for differentiating 11 kinds of duck viral diseases |
| CN106636473A (en) * | 2017-01-20 | 2017-05-10 | 广西壮族自治区兽医研究所 | Complete reagent and method for detecting H5 subtype avian influenza virus and chicken parvovirus |
| CN106636472A (en) * | 2017-01-20 | 2017-05-10 | 广西壮族自治区兽医研究所 | Reagent set and method for detecting avian influenza viruses and chicken parvoviruses |
Non-Patent Citations (3)
| Title |
|---|
| ZONGYAN CHEN等: "Rapid diagnosis of goose viral infections by multiplex PCR", 《J VIROL METHODS》 * |
| 刘晓东主编: "《兽医传染病学实验指导》", 30 November 2017, 中国农业大学出版社 * |
| 张艳芳等: "鸭新城疫病毒与番鸭细小病毒二重PCR方法的建立", 《中国畜牧兽医》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110777219A (en) * | 2019-10-31 | 2020-02-11 | 广西壮族自治区兽医研究所 | Triple PCR detection primer group, kit and method for chicken parvovirus and H9 subtype avian influenza virus |
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