CN103981286B - Differentiate GeXP rapid detection kit and the primer sets thereof of 8 kinds of virus diseases of pigs - Google Patents
Differentiate GeXP rapid detection kit and the primer sets thereof of 8 kinds of virus diseases of pigs Download PDFInfo
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Abstract
The invention discloses a kind of GeXP rapid detection kit differentiating 8 kinds of virus diseases of pigs and primer sets thereof, inventor devises nine pairs of specific primers and a pair universal primer based on GeXP system research, establish the GeXP method for quick simultaneously differentiating 8 kinds of virus diseases of pigs accordingly, and be prepared for corresponding detection kit.The present invention has advantages such as the best, highly sensitive, convenience and high-efficiency, and the application present invention can detect simultaneously and differentiate 8 kinds of pathogen, and compared with the qualification result of the normal experiment method such as virus purification and blood clotting Inhibition test, coincidence rate reaches 100%.The present invention efficiently solves the amplification preferences during multiplex PCR, and sick for Main Pig common transmittable and pathogen detection provides detection means easy, high-throughout, more meets the needs of reality, has a extensive future.
Description
Technical field
The invention belongs to virus diseases of pigs detection technique field, the GeXP particularly relating to 8 kinds of virus diseases of pigs of a kind of discriminating is quick
Detection kit and primer sets thereof.
Background technology
H1, H3 hypotype swine influenza virus, PRV, CSFV, pig breeding and distress syndrome virus, pig second
Encephalovirus, pig parvoviral, porcine circovirus 2 type are 8 kinds of main infection diseases of serious harm pig.Along with pig aquaculture
Development, the incidence of disease of pig transmissible virosis is also gradually rising, it has also become limit a big key factor of pig industry development.Mirror
Do not diagnose the conventional method of these pig transmissible diseases, mainly include Antigen isolation and identification and serological test etc., but these sides
Method is often limited by clinical pathological material of disease freshness, pollution level or the course of disease, operates the most loaded down with trivial details, time-consuming, to multiple mixed infection
Antidiastole increasingly difficult.In recent years, along with the development of molecular biology, round pcr has been widely used in pig infectious disease
Detection and establish multiplex PCR detection technique, can detect several cause of disease simultaneously, but need several primer sets is combined the most competing
Expanding with striving, can produce and interfere, increase pair of primers more between primer, sensitiveness is the lowest.Meanwhile, PCR primer need to be led to
Crossing agarose electrophoresis just observable result, this electrophoresis is difficult to differentiate between the band within 50bp-100bp, accomplishes 2-4 weight the most only
PCR, it is difficult to reach high-throughout detection.Multiple fluorescence PCR typically only detects 3 weights, owing to probe to mark different luminous ripple
Long fluorophor, as marked too many fluorophor, mutually can produce interference, therefore, also be only able to detect 2-4 weight.Due to
Multi-PRC reaction system exist simultaneously several to primer so that the probability forming complicated primer dimer is greatly increased, simultaneously
The genes of interest limited amount (how at 2-4 gene) of detection so that multiplex PCR does not also reach high flux and quickly detects and analyze
Purpose.
GeXP system (Gene Expression Profiler Genetic Analysis System) is the U.S.
The platform for studying multi-gene expression quantitative analysis of Beckman Coulter company research and development, is made up of two parts: be used for setting
Count the GeXP eXpression Profiler software of primer and be used for the GenomeLabTM GeXP Genetic of interpretation of result
Analysis System HPCE, the latter can clearly isolate the adjacent amplified fragments of difference more than 7bp.GeXP is many
Weight PCR amplification uses fluorescence labeling universal primer and specific chimeric primer, and (i.e. gene-specific primer 5 ' end connection universal draws
Thing sequence) combine initiation multiple system amplification.At the beginning of PCR reaction, first tied with primary template by reverse specific chimeric primer
Conjunction carries out reverse transcription reaction, then the second chain by forward specific chimeric primer synthesis cDNA, hereafter, forward and reverse being fitted together to draws
The specific sequence of thing starts PCR reaction with cDNA for template, amplifies the complementary series of universal primer respectively；Again by reactant
Prevailing fluorescence labeling universal primer in system, is complementary to sequence and combines, cause following amplification, universal primer and reaction
With fluorescently-labeled base sequence complementary in system, PCR primer is through GeXP capillary electrophoresis separation, containing fluorescently-labeled
PCR primer detects through GeXP detection window, moves with standard molecule fragment (DNA Size Standard, DSS) according to detection fragment
Shift time calculates the length of amplified fragments, and fluorescence signal intensity represents the amplification content of this isolated fragment.GeXP system can be
In same system, up to 40 genes of interest are effectively analyzed.
Utilize GeXP multiple gene expression genetic analysis systems, set up a kind of detection side that can simultaneously differentiate multiple pathogens
Method and detection kit, crucial and difficult point is design specific primer, and by multi-primers efficient combination, utilizes universal primer handle
The amplification of multi-primers is converted into the amplification of 1 pair of universal primer, thus reaches the purpose of high flux detection.At present, not yet report
Based on reagent or the kit that can detect the multiple infectious disease pathogen of virus diseases of pigs while GeXP system.
Summary of the invention
The technical problem to be solved in the present invention is to provide that a species specificity is good, highly sensitive, discriminating 8 boar of convenience and high-efficiency
The GeXP rapid detection kit of virosis and primer sets thereof.
For solve above-mentioned technical problem, the present invention by the following technical solutions:
Differentiate the GeXP quick detection primer group of 8 kinds of virus diseases of pigs, including nine pairs of specific primers, be primer pair respectively
SIV-M-1 and SIV-M-2, primer to SIV-H1-1 and SIV-H1-2, primer to SIV-H3-1 and SIV-H3-2, primer to PRV-
1 and PRV-2, primer to CSFV-1 and CSFV-2, primer to PRRSV-1 and PRRSV-2, primer to JEV-1 and JEV-2, primer
To PPV-1 and PPV-2, primer to PCV-1 and PCV-2, they are respectively provided with sequence table SEQ .ID.No.1 to SEQ.ID.No.18
Base sequence.
The GeXP quick detection primer group of above-mentioned 8 kinds of virus diseases of pigs of discriminating, also includes a pair universal primer, be respectively upper,
Downstream universal primer Cy5-Tag-F and Tag-R, they are respectively provided with the alkali of sequence table SEQ .ID.No.19 to SEQ.ID.No.20
Basic sequence.
Differentiate the GeXP rapid detection kit of 8 kinds of virus diseases of pigs, including reverse transcription RT reactant liquor, PCR reactant liquor, DEPC
Water, ultra-pure water, containing nine pairs of specific primers and a pair universal primer in PCR reactant liquor, be primer respectively to SIV-M-1 and
SIV-M-2, primer to SIV-H1-1 and SIV-H1-2, primer to SIV-H3-1 and SIV-H3-2, primer to PRV-1 and PRV-2,
Primer to CSFV-1 and CSFV-2, primer to PRRSV-1 and PRRSV-2, primer to JEV-1 and JEV-2, primer to PPV-1 and
PPV-2, primer are to PCV-1 and PCV-2 and upstream and downstream universal primer Cy5-Tag-F and Tag-R, and they are respectively provided with sequence
The base sequence of table SEQ.ID.No.1 to SEQ.ID.No.20.
The 9 pairs of specific primers to SIV-M-1 and SIV-M-2, primer to SIV-H1-1 and SIV-H1-2, primer to SIV-
H3-1 and SIV-H3-2, primer to PRV-1 and PRV-2, primer to CSFV-1 and CSFV-2, primer to PRRSV-1 and PRRSV-
2, primer is corresponding in PCR reaction system to PCV-1 with PCV-2 to PPV-1 with PPV-2, primer to JEV-1 with JEV-2, primer
Molar concentration is respectively 50nmol/L, 50nmol/L, 100nmol/L, 50nmol/L, 50nmol/L, 50nmol/L, 50nmol/
L, 50nmol/L, 50nmol/L；Universal primer molar concentration in PCR reaction system is 500nmol/L.
Reverse transcription RT reactant liquor often pipe 9 μ L, containing 5 × Reverse Transcriptase Buffer5 μ L, 50pmol
Random Primer (9mer) 1 μ L, 10mM dNTP Mixture2 μ L, 40U Ribonuclease Inhibitor0.5 μ L and
5U/μL AMV Reverse Transcriptase0.5μL；PCR reactant liquor often pipe 10.7 μ L, containing 10 × PCR
Buffer2.5 μ L, 25mM MgCl22.5 μ L, 2.5mM dNTP Mixture2 μ L, 9 couples of specific primer mixture 1.25 μ L,
10 μMs/L upstream and downstream universal primer mixture 1.25 μ L, JumpStart Taq DNA Polymerase1.2 μ L；DEPC water and
The each 1mL of ultra-pure water.
The cause of disease of 8 kinds of virus diseases of pigs is H1 hypotype swine influenza virus, H3 hypotype swine influenza virus, porcine pseudorabies respectively
Poison, CSFV, the breeding of american type pig and distress syndrome virus, pig encephalitis B virus, pig parvoviral, porcine circovirus 2 type.
Above-mentioned nine pairs of specific primers to SIV-M-1 and SIV-M-2, primer to SIV-H1-1 and SIV-H1-2, primer to SIV-
H3-1 and SIV-H3-2, primer to PRV-1 and PRV-2, primer to CSFV-1 and CSFV-2, primer to PRRSV-1 and PRRSV-
2, PCV-1 and PCV-2 is respectively used to identify or auxiliary by primer by JEV-1 and JEV-2, primer by PPV-1 and PPV-2, primer
Identify H1 hypotype swine influenza virus, H3 hypotype swine influenza virus, PRV, CSFV, the breeding of american type pig and barrier
Hinder syndrome virus, pig encephalitis B virus, pig parvoviral, porcine circovirus 2 type.
For lacking at present effective robust techniques Prevention of Common Occurrence Porcine Disease viral disease being detected and diagnosing simultaneously, inventor based on
GeXP system research devises nine pairs of specific primers and a pair universal primer, establishes 8 kinds of virus diseases of pigs of discriminating simultaneously accordingly
GeXP method for quick, and be prepared for corresponding detection kit.The present invention has the best, highly sensitive, convenient
The advantage such as efficiently, the application present invention can detect simultaneously and differentiate 8 kinds of pathogen, to H1 hypotype swine influenza virus, H3 hypotype pig stream
Influenza Virus, Pseudorabies virus, CSFV, the breeding of american type pig and distress syndrome virus, pig encephalitis B virus, the tiny disease of pig
Poison, the high specificity of porcine circovirus 2 type, sensitivity respectively 10 copy/μ l, 10 copy/μ l, 10 copy/μ l, 1000 copy
Shellfish/μ l, 100 copy/μ l, 10 copy/μ l, 100 copy/μ l, 100 copy/μ l, 100 copy/μ l, with virus purification and blood clotting
The qualification result of the normal experiment methods such as Inhibition test is compared, and coincidence rate reaches 100%.The present invention efficiently solves multiplex PCR mistake
Amplification preferences in journey, sick for Main Pig common transmittable and pathogen detection provides simplicity, high-throughout detection hand
Section, more meets the needs of reality, has a extensive future.
Accompanying drawing explanation
Fig. 1 is the capillary electrophoresis analysis figure of the GeXP nine weight PCR of H1, H3 hypotype swine influenza virus cDNA hybrid template.
Fig. 2 is the cDNA of cDNA, H3N2 hypotype swine influenza virus of H1N2 hypotype swine influenza virus, CSFV
CDNA, the cDNA of the breeding of american type pig and distress syndrome virus and the GeXP nine of DNA hybrid template of porcine circovirus 2 type
The capillary electrophoresis analysis figure of weight PCR.
Fig. 3 is the capillary electrophoresis analysis figure of the GeXP nine weight PCR of 8 boar communicable disease cause of diseases.
In Fig. 1-3, abscissa is the base number of pcr amplification product, and ordinate is fluorescence signal value.
Detailed description of the invention
Swine influenza virus RNA (H3N2, H1N2), the breeding of american type pig and distress syndrome used in following example
RNA is given by Guangxi University；Swine influenza virus (H1N1), pig parvoviral (PPV) and porcine circovirus 2 type (PCV2) are by extensively
Western Zhuang autonomous region veterinary institute separates and preserves；PRV vaccine, pig encephalitis B virus vaccine herd reality in being purchased from
Industry Co., Ltd；CSFV is purchased from Guangxi beautiful protozoa Science and Technology Co., Ltd..
Embodiment 1, the design of specific primer pair
With GenBank announce primer sequence as reference, from NCBI download known array use DNASTAR software carry out
Sequence alignment, chooses for each target gene high conservative and special genetic fragment, primer premier5.0 software sets
Count 9 weight specific primers, and at 5 ' ends interpolation GeXP universal primer (table 1) of primer.
Table 1 primer information
In table 1, annexing base code R=A/G, D=A/G/T, Y=C/T, what underscore represented is that upstream and downstream are general draws
Thing sequence；Fluorescent dye Cy5 mark upstream universal primer label, downstream primer does not marks.According to actually detected pathogen
Strain difference and GeXP system (such as GenomeLabTM GeXP Genetic Analysis System HPCE)
Error, the actual amplified production length using above-mentioned primer to obtain A I and GeXP universal primer to detection can be anticipated
Fluctuate on the length of amplified production 3bp up and down.
A I is respectively used to detect whether containing H1 hypotype swine influenza virus, H3 hypotype swine flu disease by the primer in table 1
Poison, PRV, CSFV, american type pig breeding with distress syndrome virus, pig encephalitis B virus, pig parvoviral,
Porcine circovirus 2 type.
Embodiment 2, the specific detection of PCR primer pair
One, the preparation of template
1, the extraction of viral RNA and the acquisition of cDNA
1) extraction of viral RNA
(being purchased from Beijing Quanshijin Biotechnology Co., Ltd, catalog number (Cat.No.) is to use DNA/RNA to extract kit
ER201), according to kit specification, extract swine influenza virus (H1N1, H1N2, H3N2), pig encephalitis B virus (JEV), U.S. respectively
Continent type pig breeding and disorders syndrome virus (PRRSV), the RNA of CSFV (CSFV)
2) acquisition of cDNA
By step 1) RNA sample that obtains carries out reverse transcription according to following reaction system and reaction condition respectively, obtains
cDNA；Using DEPC water as the comparison of total serum IgE.
Reaction system (25 μ L): 5 × Reverse Transcriptase Buffer5 μ L, 50pmol Random
Primer(9mer)1μL、dNTP Mixture(10mM)2μL,40U Ribonuclease Inhibitor0.5μL,5U/μL
AMV Reverse Transcriptase0.5 μ L, template ribonucleic acid 1 μ g, DEPC water complements to 25 μ L.Reverse transcription temperature 42 DEG C
1.5h, is placed in-20 DEG C of preservations.
2, the extraction of genomic DNA
(being purchased from Beijing Quanshijin Biotechnology Co., Ltd, catalog number (Cat.No.) is to use DNA/RNA to extract kit
ER201), according to kit specification, extract PRV (PRV), pig parvoviral (PPV) and pig circular ring virus respectively
The DNA of 2 types (PCV2).
3, nucleic acid content is measured
Measure its OD value with nucleic acid-protein analyzer BioPhotometer, draw its concentration and Reinheitszahl, and control with this
Nucleic acid quality.
Two, substance RT-PCR method checking primer is used
1, substance specific primer (SP-Primer) is diluted to working concentration 1 μm ol/L, Cy5-Tag-F and Tag-R dilutes
To working concentration 10 μm ol/L.25 μ L systems are prepared according to the specification of Sigma JumpStartTaqDNA Polymerase,
Including 10 × PCR buffer2.5 μ L, 25mM MgCl22.5μL、2.5mM dNTP Mixture2μL、 SP-Primer F/R
(1 μm ol/L) respectively takes 1.25 μ l (PCR system final concentration 50nmol/L), Cy5-Tag-F and Tag-R (10 μm ol/L) respectively takes 1.25
μ l (PCR system final concentration 500nmol/L), JumpStart Taq DNA Polymerase1.2 μ L, cDNA or DNA2 μ L, adds
Aqua sterilisa is to 25 μ L.Each cDNA/DNA arranges 3 repetitions.
Response procedures: 95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 10 circulations；95 DEG C of 30s, 63 DEG C of 30s, 72 DEG C of 30s, 10
Individual circulation；95 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 30s, 20 circulations；4 DEG C of terminations.
Capillary Electrophoresis: use each PCR primer of GenomeLab GeXP genetic analysis systems simultaneously to carry out capillary electricity
Swimming detection, operating procedure is as follows: be sample-loading buffer (Beckman Coulter Inc. of the U.S., catalog number with formamide
608082), DNA size standard Kit-400Base Pairs (Beckman Coulter Inc. of the U.S., catalog number
608098) with sample-loading buffer 1:(80-160 by volume) thoroughly mix, in sample panel, every hole adds what 39 μ L mixed
Liquid, carries out 10-100 times by PCR primer and dilutes, and takes the product after dilution 1 μ L and adds to sample panel, and piping and druming mixing, finally often
Hole instills a dropstone wax oil and closes, in order to avoid formamide oxidation and sample evaporate.On buffer solution plate, every hole adds the buffering of 2/3
Liquid, carries out Capillary Electrophoresis.The condition of Capillary Electrophoresis is as follows: capillary heats up: temperature 50 C；Sex change: 90 DEG C, 120s；Note
Enter sample: 2.0KV, 30s；Separate: 6.0KV, 35min.GenomeLab GeXP genetic analysis systems is utilized to determine various special
The actually detected size of primer amplification fragment.
2, the primer specific detection to A
1) PCR amplification
Swine influenza virus (H1N1, H3N2, H1N2), the breeding of american type pig and barrier A obtained in step one 1 with primer
Hinder 2 PRVs obtained, pig in syndrome virus, pig encephalitis B virus, the cDNA sample of CSFV and step one
Parvovirus, porcine circovirus 2 type DNA sample carry out PCR amplification, reaction system and response procedures and electrophoresis respectively according to step
In two, the step of 1 is carried out.
2) result: the cDNA sample of swine influenza virus strain (H1N1, H1N2, H3N2) is the most amplifiable obtains 181 183bp
Amplified production, through order-checking confirm, the sequence of this amplified production is the primer target sequence to A；Non-swine influenza virus and feminine gender are right
According to without any amplified production.Result shows, primer to A specific very well, can the swine influenza virus of special 3 kinds of hypotypes of detection
(H1N1, H3N2, H1N1 hypotype).
3, the primer specific detection to B
1) PCR amplification and Capillary Electrophoresis
Swine influenza virus (H1N1, H3N2, H1N2), the breeding of american type pig and barrier B obtained in step one 1 with primer
Hinder 2 PRVs obtained, pig in syndrome virus, pig encephalitis B virus, the cDNA sample of CSFV and step one
Parvovirus, porcine circovirus 2 type DNA sample carry out PCR amplification, reaction system and response procedures and electrophoresis respectively according to step
In rapid two, the step of 1 is carried out.
2) result: the most amplifiable amplification obtaining 119-121bp of cDNA sample of swine influenza virus strain (H1N1, H1N2)
Product, confirms through order-checking, the sequence of this amplified production is the primer target sequence to A；The swine influenza virus (H3N2) of non-H1 hypotype
And negative control is without any amplified production.Result shows, primer to B specific very well, can special detection H1N1 and H1N2
The swine influenza virus of hypotype.
4, the primer specific detection to C
1) PCR amplification and Capillary Electrophoresis
Swine influenza virus (H1N1, H3N2, H1N2), the breeding of american type pig and barrier C obtained in step one 1 with primer
Hinder 2 PRVs obtained, pig in syndrome virus, pig encephalitis B virus, the cDNA sample of CSFV and step one
Parvovirus, porcine circovirus 2 type DNA sample carry out PCR amplification, reaction system and response procedures and electrophoresis respectively according to step
In two, the step of 1 is carried out.
2) result: the amplifiable amplification obtaining 322 325bp of the cDNA sample of H3N2 hypotype swine influenza virus strain is produced
Thing, confirms through order-checking, the sequence of this amplified production is the primer target sequence to C；Non-H3N2 hypotype swine influenza virus and feminine gender are right
According to without any amplified production.Result shows, primer to C specific very well, can special detection H3N2 hypotype swine influenza virus.
5, the primer specific detection to D
1) PCR amplification
With primer, in the step one 2 PRV DNA sample obtained are expanded by D, simultaneously sick to swine flu
Poison (H1N1, H3N2, H1N2), the breeding of american type pig and disorders syndrome virus, pig encephalitis B virus, the cDNA sample of CSFV
And 2 pig parvovirals obtained, porcine circovirus 2 type DNA sample carry out PCR amplification respectively in step one, reaction system and
Response procedures and electrophoresis are carried out according to the step of 1 in step 2.
2) result: the amplifiable amplified production obtaining 225-227bp of the DNA sample of PRV virus stain, warp
Order-checking confirms, the sequence of this amplified production is the primer target sequence to D；Non-PRV virus and negative control are without any
Amplified production.Result shows, primer to D specific very well, can special detection PRV.
6, the primer specific detection to E
1) PCR amplification
With primer, the cDNA sample of in step one 1 CSFV obtained is expanded, the most respectively to swine flu by E
Virus (H1N1, H3N2, H1N2), the breeding of american type pig and disorders syndrome virus, the cDNA sample of pig encephalitis B virus and step
In rapid one, 2 PRVs obtained, pig parvoviral, porcine circovirus 2 type DNA sample carry out PCR amplification respectively, reaction
System and response procedures and electrophoresis are carried out according to the step of 1 in step 2
2) result: the amplifiable amplified production obtaining 159 161bp of cDNA sample of CSFV, confirms through order-checking, should
The sequence of amplified production is the primer target sequence to E；Non-HCV viruses and negative control are without any amplified production.Result shows,
Primer to E specific very well, can special detection CSFV.
7, the primer specific detection to F
1) PCR amplification
The american type pig breeding obtained F in step one 1 with primer is carried out with the cDNA sample of disorders syndrome virus
Amplification, the most respectively to swine influenza virus (H1N1, H3N2, H1N2), pig encephalitis B virus, the cDNA sample of CSFV and step
In rapid one, 2 PRVs obtained, pig parvoviral, porcine circovirus 2 type DNA sample carry out PCR amplification respectively, reaction
System and response procedures and electrophoresis are carried out according to the step of 1 in step 2.
2) result: the amplifiable expansion obtaining 260 263bp of cDNA sample that the breeding of american type pig is viral with disorders syndrome
Volume increase thing, confirms through order-checking, the sequence of this amplified production is the primer target sequence to F；The breeding of non-american type pig is comprehensive with obstacle
Syndrome virus and negative control are without any amplified production.Result shows, primer to F specific very well, can specifically detect U.S.
Continent type pig breeding and disorders syndrome virus.
8, the primer specific detection to G
1) PCR amplification
With primer, the cDNA sample of in the step one 1 pig encephalitis B virus obtained is expanded by G, simultaneously sick to swine flu
Poison (H1N1, H3N2, H1N2), the breeding of american type pig and disorders syndrome virus, the cDNA sample of CSFV and step one
In 2 PRVs obtained, pig parvoviral, porcine circovirus 2 type DNA sample carry out PCR amplification, reaction system respectively
Carry out according to the step of 1 in step 2 with response procedures and electrophoresis.
2) result: the amplifiable amplified production obtaining 301 303bp of cDNA sample of pig encephalitis B virus, confirms through order-checking,
The sequence of this amplified production is the primer target sequence to G；Non-pig encephalitis B virus and negative control are without any amplified production.Result table
Bright, primer to G specific very well, can special detection pig encephalitis B virus.
9, the primer specific detection to H
1) PCR amplification
With primer, H is carried out PCR amplification, the most respectively amplification step to the DNA sample of the pig parvoviral of 2 in step one
1 swine influenza virus (H1N1, H3N2, H1N2) obtained, pig encephalitis B virus, the breeding of american type pig and disorders syndrome disease in one
In poison, the cDNA sample of CSFV and step one, 2 PRVs obtained, porcine circovirus 2 type DNA sample are respectively
Carry out PCR amplification, reaction system and response procedures and electrophoresis to carry out according to the step of 1 in step 2.
2) result: the amplifiable amplified production obtaining 137 139bp of the DNA sample of pig parvoviral, confirms through order-checking,
The sequence of this amplified production is the primer target sequence to H；Non-pig parvoviral and negative control are without any amplified production.Result table
Bright, primer to H specific very well, can special detection pig parvoviral.
10, the primer specific detection to I
1) PCR amplification
With primer, I is carried out PCR amplification respectively to the DNA sample of the porcine circovirus 2 type of 2 in step one, distinguish simultaneously
In amplification step one, 1 swine influenza virus (H1N1, H1N2, H3N2) obtained, pig encephalitis B virus, the breeding of american type pig are combined with obstacle
Close 2 PRVs obtained, pig parvoviral DNA sample in syndrome virus, the cDNA sample of CSFV and step one
Carry out PCR amplification, reaction system and response procedures and electrophoresis respectively to carry out according to the step of 1 in step 2.2) result: pig annulus
The amplifiable amplified production obtaining 273 275bp of DNA sample of virus 2 types, confirms through order-checking, the sequence of this amplified production is
The primer target sequence to I；Non-porcine circovirus 2 type and negative control are without any amplified production.Result shows, the primer spy to I
The opposite sex very well, can special detection porcine circovirus 2 type.
Three, GeXP multiple reaction system specificity verification
1) primer is mixed into multiple mix primer (Mix-Primer) working solution to A, B, C, D, E, F, G, H and I, makes
In PCR system, SP-Primer final concentration is 50nmol/L, except SIV-H3-1 and SIV-H3-2 primer is to final concentration of
100nmol/L (the i.e. 9 pairs specific primers to SIV-M-1 and SIV-M-2, primer to SIV-H1-1 and SIV-H1-2, primer pair
SIV-H3-1 and SIV-H3-2, primer to PRV-1 and PRV-2, primer to CSFV-1 and CSFV-2, primer to PRRSV-1 and
PRRSV-2, primer to JEV-1 and JEV-2, primer to PPV-1 and PPV-2, primer to PCV-1 and PCV-2 in PCR system right
Answer final concentration be 50nmol/L, 50nmol/L, 100nmol/L, 50nmol/L, 50nmol/L, 50nmol/L respectively,
50nmol/L, 50nmol/L, 50nmol/L), to J upstream and downstream universal primer, primer is mixed into working concentration is 10 μm ol/L,
Final concentration of 500nmol/L in PCR system, remaining composition is identical with what primer was verified.With many strains known viruse nucleic acid
Single positive sample as template, carries out the reaction of many primer PCRs, instead as the mixing cDNA of template or 8 kinds of cause of diseases and DNA sample
Answer program and electrophoresis identical with what primer was verified.
2) result: (H1N1, H1N2, H3N2 hypotype swine flu is sick to 8 kinds of pathogen respectively to after A-I mixing for 9 pairs of primers
Poison, CSFV, pig encephalitis B virus, the breeding of american type pig and distress syndrome syndrome virus, PRV, pig are tiny
Virus, porcine circovirus 2 type) single cDNA or mixing cDNA and DNA of 8 kinds of cause of diseases detect.Result shows many primers
Single mode version has detected 121bp and 183bp, 181bp and 325bp, 227bp, 160bp, 260bp, 303bp, 137bp, 274bp respectively
Amplified production, negative control is without any amplified production；Many primers multi-template result shows that the gene of 8 kinds of swine disease viruses can be same
Time detection, demonstrate the primer specificity in Multiple detection detection architecture.
The above results shows: primer A I is mixed after on each primer on specific without impact, can be used for examining specifically
Go out swine influenza virus (H1N1, H1N2 and H3N2), CSFV, pig encephalitis B virus, the breeding of american type pig and distress syndrome to combine
Simulator sickness virus, PRV, pig parvoviral, every kind of pathogen of porcine circovirus 2 type.
Embodiment 3, the sensitivity technique of PCR primer pair
One, the preparation monoclonal plasmid standard containing target gene
Respectively with the swine influenza virus (H1N1, H1N2, H3N2) of step one acquisition, PRV, pig in embodiment 2
Pestivirus, the breeding of american type pig and distress syndrome virus, pig encephalitis B virus, pig parvoviral, the cDNA of porcine circovirus 2 type
Or DNA sample is template, by the swine influenza virus M gene of PCR amplification acquisition, H1 hypotype swine influenza virus HA gene, H3 hypotype
Swine influenza virus HA gene, PRV GD gene, E 2 gene of Classical Swine Fever, the breeding of american type pig and distress syndrome disease
Poison NSP2 gene, pig encephalitis B virus M GFP, pig parvoviral VP2 gene, porcine circovirus 2 type ORF1 gene, respectively with
Carrier pMD18-T vector connects (purchased from Dalian treasured biotech firm) and connects, it is thus achieved that 9 recombinant plasmid PA, PB, PC, PD, PE,
Through order-checking, PF, PG, PH, PI, confirm that these 9 recombinant plasmids are that plasmid pMD18-T vector inserts a kind of said gene respectively
Recombinant plasmid, and respectively can be as the above-mentioned 9 pairs of primers target gene to A I.
Ultraviolet specrophotometer is utilized to measure the concentration respectively containing target gene recombinant plasmid, and according to the molecular weight of recombinant plasmid
Corresponding copy number is calculated with concentration.Each recombinant plasmid of high concentration is diluted to target gene concentration respectively is 103、102、101Copy
The single plasmid dilution of shellfish/μ L.Each recombinant plasmid is mixed, is formed in the concentration of each plasmid in mixed solution and is 106Copy
The mixed liquor of shellfish/μ L, then be 10 by 10 doubling dilutions5、104、103、102The mixing plasmid dilution of copy/μ L.
Two, the 9 pairs of primers sensitivity technique to mixing
1. with primer, A I being mixed into primer, the different single plasmid dilutions of variable concentrations are template, according to enforcement
In example 2, the method for step 2 carries out PCR amplification and electrophoresis detection；As a result, detection swine influenza virus (H1N1, H1N2, H3N2), H1
Hypotype swine influenza virus, H3 hypotype swine influenza virus, PRV, CSFV, american type pig breed comprehensive with obstacle
Levy virus, pig encephalitis B virus, pig parvoviral, the sensitivity of porcine circovirus 2 type are 10 copies/μ L.
2. with primer, A I being mixed into primer, ten kinds of plasmid dilutions of the mixing of variable concentrations are template, according to enforcement
In example 2, the method for step 2 carries out PCR amplification and electrophoresis detection；As a result, swine influenza virus (H1N1, H1N2, H3N2) is detected
Sensitivity is 10 copies/μ L, and the sensitivity of detection H1 hypotype swine influenza virus is 10 copies/μ L, and detection H3 subtype avian influenza is sick
The sensitivity of poison is 10 copies/μ L, and the sensitivity of detection PRV is 1000 copies/μ L, the spirit of detection CSFV
Sensitivity is 100 copies/μ L, and detection american type pig breeding is 10 copies/μ L with the sensitivity of distress syndrome virus, detects pig second
The sensitivity of encephalovirus is 100 copies/μ L, and the sensitivity of detection pig parvoviral is 100 copies/μ L, detects 2 porcine circovirus
The sensitivity of type is 100 copies/μ L.
Embodiment 4, the assembling of detection kit
According to the result of study of embodiment 1 to 3, assemble detection kit to facilitate use.
Differentiate 8 kinds of virus diseases of pigs GeXP rapid detection kit, including reverse transcription RT reactant liquor, PCR reactant liquor,
DEPC water, ultra-pure water, containing nine pairs of specific primers and a pair universal primer in PCR reactant liquor, be that primer is to SIV-M-1 respectively
With SIV-M-2, primer to SIV-H1-1 and SIV-H1-2, primer to SIV-H3-1 and SIV-H3-2, primer to PRV-1 and PRV-
2, primer to CSFV-1 and CSFV-2, primer to PRRSV-1 and PRRSV-2, primer to JEV-1 and JEV-2, primer to PPV-1
With PPV-2, primer to PCV-1 and PCV-2 and upstream and downstream universal primer Cy5-Tag-F and Tag-R, they are respectively provided with sequence
The base sequence of list SEQ.ID.No.1 to SEQ.ID.No.20.
Reverse transcription RT reactant liquor often pipe 9 μ L, containing 5 × Reverse Transcriptase Buffer5 μ L, 50pmol
Random Primer (9mer) 1 μ L, 10mM dNTP Mixture2 μ L, 40U Ribonuclease Inhibitor0.5 μ L and
5U/μL AMV Reverse Transcriptase0.5μL；PCR reactant liquor often pipe 10.7 μ L, containing 10 × PCR
Buffer2.5 μ L, 25mM MgCl22.5 μ L, 2.5mM dNTP Mixture2 μ L, 9 couples of specific primer mixture 1.25 μ L,
10 μMs/L upstream and downstream universal primer mixture 1.25 μ L, JumpStart Taq DNA Polymerase1.2 μ L；DEPC water and
The each 1mL of ultra-pure water.
The 9 pairs of specific primers to SIV-M-1 and SIV-M-2, primer to SIV-H1-1 and SIV-H1-2, primer to SIV-
H3-1 and SIV-H3-2, primer to PRV-1 and PRV-2, primer to CSFV-1 and CSFV-2, primer to PRRSV-1 and PRRSV-
2, primer is corresponding in PCR reaction system to PCV-1 with PCV-2 to PPV-1 with PPV-2, primer to JEV-1 with JEV-2, primer
Molar concentration is respectively 50nmol/L, 50nmol/L, 100nmol/L, 50nmol/L, 50nmol/L, 50nmol/L, 50nmol/
L, 50nmol/L, 50nmol/L；Universal primer molar concentration in PCR reaction system is 500nmol/L.
Embodiment 5, the GeXP rapid detection kit simulated experiment to clinical infection of 8 kinds of virus diseases of pigs of application
1, simulation clinical infection
CDNA or the DNA mixing of several pathogen is randomly choosed from the cDNA or DNA sample of above-mentioned 8 kinds of pathogen,
Point following two groups of tests:
The cDNA mixing of cDNA and the H3 hypotype swine influenza virus of the 1st group: H1 hypotype swine influenza virus (H1N1, H1N2) is made
For template.
The cDNA of cDNA, H3N2 hypotype swine influenza virus of the 2nd group: H1N2 hypotype swine influenza virus, CSFV
CDNA that cDNA, the breeding of american type pig are viral with distress syndrome and the DNA of porcine circovirus 2 type mix as template；
By in embodiment 2 step 3 method detection, the 1st group can detect 121.01bp and 182.66,182.66bp and
324.50 3 purpose peaks (Fig. 1), show the template containing H1, H3 hypotype swine influenza virus in sample, are consistent with actual；2nd
Group can detect 119.56bp and 180.98bp, six mesh of 160.07bp, 260.53p, 273.81bp, 180.98bp and 322.55bp
Peak, without other miscellaneous peaks, show in sample the cDNA pig of cDNA, H3 hypotype swine influenza virus containing H1 hypotype swine influenza virus
The cDNA of pestivirus, the cDNA of the breeding of american type pig and distress syndrome virus and the DNA (Fig. 2) of porcine circovirus 2 type, with
Actual it is consistent.
2,8 kinds of pathogen mixing
9 purpose peaks (as shown in Figure 3) can be detected, without other miscellaneous peaks, show to detect in sample containing H1 hypotype swine flu
Virus, H3 hypotype swine influenza virus, PRV, CSFV, the breeding of american type pig and distress syndrome virus, pig second
The cDNA of the whole pathogen of encephalovirus, pig parvoviral, porcine circovirus 2 type or DNA profiling, be consistent with actual.
Claims (4)
1. differentiate the GeXP quick detection primer group of 8 kinds of virus diseases of pigs, it is characterised in that include nine pairs of specific primers, be respectively
Primer to SIV-M-1 and SIV-M-2, primer to SIV-H1-1 and SIV-H1-2, primer to SIV-H3-1 and SIV-H3-2, primer
To PRV-1 and PRV-2, primer to CSFV-1 and CSFV-2, primer to PRRSV-1 and PRRSV-2, primer to JEV-1 and JEV-
2, primer to PPV-1 and PPV-2, primer to PCV-1 and PCV-2, they be respectively sequence table SEQ .ID.No.1 extremely
The base sequence of SEQ.ID.No.18.
The GeXP quick detection primer group of 8 kinds of virus diseases of pigs of discriminating the most according to claim 1, it is characterised in that also include
A pair universal primer, is upstream and downstream universal primer Cy5-Tag-F and Tag-R respectively, and they are sequence table respectively
The base sequence of SEQ.ID.No.19 to SEQ.ID.No.20.
3. differentiate the GeXP rapid detection kit of 8 kinds of virus diseases of pigs, including reverse transcription RT reactant liquor, PCR reactant liquor, DEPC
Water, ultra-pure water, it is characterised in that: containing nine pairs of specific primers and a pair universal primer in described PCR reactant liquor, draw respectively
Thing to SIV-M-1 and SIV-M-2, primer to SIV-H1-1 and SIV-H1-2, primer to SIV-H3-1 and SIV-H3-2, primer pair
PRV-1 and PRV-2, primer to CSFV-1 and CSFV-2, primer to PRRSV-1 and PRRSV-2, primer to JEV-1 and JEV-2,
Primer to PPV-1 and PPV-2, primer to PCV-1 and PCV-2 and upstream and downstream universal primer Cy5-Tag-F and Tag-R, they
It is the base sequence of sequence table SEQ .ID.No.1 to SEQ.ID.No.20 respectively；Described nine couples of specific primer SIV-M-1 and
SIV-M-2, primer to SIV-H1-1 and SIV-H1-2, primer to SIV-H3-1 and SIV-H3-2, primer to PRV-1 and PRV-2,
Primer to CSFV-1 and CSFV-2, primer to PRRSV-1 and PRRSV-2, primer to JEV-1 and JEV-2, primer to PPV-1 and
PPV-2, primer are respectively 50nmol/L, 50nmol/L to the molar concentration that PCV-1 and PCV-2 is corresponding in PCR reaction system,
100nmol/L, 50nmol/L, 50nmol/L, 50nmol/L, 50nmol/L, 50nmol/L, 50nmol/L；Described universal primer
Molar concentration in PCR reaction system is 500nmol/L；Described reverse transcription RT reactant liquor often pipe 9 μ L, containing 5 × Reverse
Transcriptase Buffer 5μL、50pmol Random Primer 1μL、10mM dNTP Mixture 2μL、40U
Ribonuclease Inhibitor 0.5 μ L and 5U/ μ L AMV Reverse Transcriptase 0.5 μ L；Described PCR is anti-
Answer liquid often pipe 10.7 μ L, containing 10 × PCR buffer 2.5 μ L, 25mM MgCl22.5 μ L, 2.5mM dNTP Mixture 2 μ
L, 9 couples of specific primer mixture 1.25 μ L, 10 μMs/L upstream and downstream universal primer mixture 1.25 μ L, JumpStart Taq
DNA Polymerase 1.2μL；Described DEPC water and each 1mL of ultra-pure water.
The GeXP rapid detection kit of 8 kinds of virus diseases of pigs of discriminating the most according to claim 3, it is characterised in that: described 8
The cause of disease planting virus diseases of pigs is H1 hypotype swine influenza virus, H3 hypotype swine influenza virus, PRV, hog cholera respectively
Poison, the breeding of american type pig and distress syndrome virus, pig encephalitis B virus, pig parvoviral, porcine circovirus 2 type.
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| CN104293978A (en) * | 2014-09-29 | 2015-01-21 | 四川农业大学 | Gene chip and kit for detecting swine fever viruses (SFVs) and/or porcine reproductive and respiratory syndrome viruses (PRRSVs) |
| CN105200049B (en) * | 2015-10-21 | 2018-11-09 | 广西壮族自治区兽医研究所 | Differentiate the GeXP rapid detection kit of H5 subtype avian influenza virus and its four kinds of difference NA hypotypes simultaneously |
| CN105316431B (en) * | 2015-11-27 | 2018-09-25 | 广西壮族自治区兽医研究所 | Differentiate the quick detection primer groups of GeXP, kit and its application of three kinds of H5 subtype avian influenza virus combination of infection people simultaneously |
| CN106987639B (en) * | 2017-04-27 | 2020-07-03 | 郑州大学第一附属医院 | Multiple PCR primer group and kit for detecting micro-deletion of AZFa and AZFb of Y chromosome and application |
| CN109207635B (en) * | 2018-09-19 | 2021-10-22 | 浙江农林大学 | MLPA detection kit for detecting pathogenic nucleic acid of porcine viral reproductive disorder syndrome |
| CN110735005B (en) * | 2019-11-26 | 2023-01-10 | 广西大学 | SIV and PRRSV multiple RT-PCR rapid detection kit and primer |
| CN112760413A (en) * | 2021-03-22 | 2021-05-07 | 苏州大学 | Application of public primer-mediated multiple quantitative PCR detection technology in transgenic soybean detection |
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