CN105779650A - Triple fluorescent quantitative PCR primer, probe and kit for identifying pseudorabies virus strains - Google Patents
Triple fluorescent quantitative PCR primer, probe and kit for identifying pseudorabies virus strains Download PDFInfo
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Abstract
The invention discloses a triple fluorescent quantitative PCR primer, probe and kit for identifying pseudorabies virus classical strains, variant strains and Bartha-K61 vaccine strains. The sequences of the primer are represented by SEQ ID No. 8-9 and SEQ ID No. 14-15, and the sequences of the probe are represented by SEQ ID No. 1, 4 and 6. According to the kit, the pseudorabies virus classical strains, the variant strains and the Bartha-K61 vaccine strains can be accurately identified based on TaqMan probes marked with different fluorescence signals, and the kit has good sensitivity and specificity and can be applied to the discriminative detection of pseudorabies virus strains.
Description
Technical field
The present invention relates to for differentiating the triple glimmering of pseudorabies virus classics strain, variant and Bartha-K61 vaccine strain
The primer of Fluorescent Quantitative PCR and probe, further relate to a kind of discriminating pseudorabies virus classics strain, variant and Bartha-K61 vaccine
The triple fluorescent quantitative PCR detection kit of strain, belongs to the detection and identification field of pseudorabies virus difference strain.
Background technology
Pseudorabies (Pseudorabies, PR) is sick also known as Ao Yecijishi, is by pseudorabies virus
(Pseudorabies virus, PRV) infects the domestic animal and the acute infectious disease of wild animal caused, and is to cause world's pig industry
One of cause of disease of heavy economic losses.Pig is reservoir and the major source of infection of this disease, and clinical symptoms is with heating, nervous system
It is principal character with respiratory disorder.Nursery-age pig and children pig are most sensitive to this virus, and after infection, body weight and appetite decline,
Mortality rate is 50-100%.PRV is also referred to as porcine herpesvirus 1, belongs to herpetoviridae.PRV is double-stranded DNA virus, and length is about
For 143kb, codified 70-100 kind albumen (Mettenleiter TC.2000.Aujeszky ' s disease
(Pseudorabies)virus:the virus and molecular pathogenesis–state of the art.Vet
Res 31:99–115.)。
From the end of the year 2011, the new epidemic situation of pseudorabies is broken out on the pig farm in Chinese multiple provinces successively, though immunity mistake
Bartha-K61 attenuated vaccine, still cannot effectively resist new epidemic situation.Some study display, and this time epidemic situation is to be made a variation by PRV
Strain causes.Compared with PRV classics strain, there is regular insertion and disappearance in the multiple gene of variant, such as gE, gI and gB gene.
These variants define a new branch (An TQ, et al., 2013.Pseudorabies virus on evolving
variant in Bartha-K61-vaccinated pigs,China,2012.Emerg Infect Dis 19:1749–
1755；Luo Y,et al.,2014.Pathogenicity and genomic characterization of a
pseudorabies virus variant isolated from Bartha-K61-vaccinated swine
population in China.Vet Microbiol 174:107–115.).At present, Bartha-K61 vaccine strain can not be right
PRV variant provides sufficiently protection, thus China's pig industry is constituted a serious threat by PRV variant.
Classical PRV detection method is virus purification and immunofluorescence analysis (Pensaert MB, Kluge
JP.1989.Pseudorabies virus(Aujeszky’s disease),p 39–65.In:Pensaert MB(ed),
Virus Infections of Porcines.Elsevier,Amsterdam；Ducatelle R,Coussement W,
Hoorens J.1982.Immunoperoxidase study of Aujeszky’s disease virus in pigs.Res
Vet Sci 32:294 302.), but both approaches is long for experimental period, and experiment consumes big.At present, multiple PRV inspection has been set up
Survey method, such as polymerase chain amplified reaction (PRV), loop-mediated isothermal amplification (Loop-mediated isothermal
Amplification, LAMP), electrochemical reaction based on gold colloidal, immune chromatography test paper, elisa
And quantitative fluorescent PCR (Zanella EL, et al., 2012.Evaluation of a real-time (ELISA)
polymerase chain reaction assay for Pseudorabies virus surveillance
purposes.J Vet Diagn Invest 24:739–745.).But, these methods can not differentiate distinguish PRV classics strain and
Variant.Can be used for differentiating to distinguish different strain although virus purification combines DNA sequencing, but this method wastes time and energy.
Therefore, set up a kind of can quickly difference and detect the new of PRV classics strain, variant and Bartha-K61 vaccine strain
Triple fluorescent quantitative PCR detection method, distinguishes detection for PRV strain and the most serious PR epidemic situation of reply will have weight
The meaning wanted and using value.
Summary of the invention
The technical problem to be solved be to provide a kind of for differentiate pseudorabies virus classics strain, variant and
The primer of the triple fluorescent quantitative PCR of Bartha-K61 vaccine strain and probe；
Another technical problem to be solved by this invention is to provide a kind of for differentiating pseudorabies virus classics strain, change
The triple fluorescent quantitative PCR detection kit of different strain and Bartha-K61 vaccine strain.
For solving above-mentioned technical problem, the technical solution used in the present invention is:
The present invention is by analyzing the different strain gene order of pseudorabies virus (PRV), at Bartha-K61 vaccine strain
The design many groups PRV classics strain of (Bartha-K61 vaccine strain is gE/gI gene-deleted strain) gene delection position and variant common primers
To and probe, in Bartha-K61 vaccine strain sequence design many groups primer to and probe.Concrete, the present invention is used for differentiating puppet
The primer of the triple fluorescent quantitative PCR of rabies virus classics strain, variant and Bartha-K61 vaccine strain includes: SEQ ID
The primer of the nucleotide sequence composition shown in No.8 and SEQ ID No.9 is to 1, shown in SEQ ID No.10 and SEQ ID No.11
The drawing of nucleotide sequence composition to 2, shown in SEQ ID No.12 and SEQ ID No.13 of the primer of nucleotide sequence composition
Thing to any pair (above-mentioned 3 pairs of primers be classical strain and variant common primers to) in 3, and SEQ ID No.14 and
The primer of the nucleotide sequence composition shown in SEQ ID No.15 to 4 (for detection Bartha-K61 vaccine strain primer to).
Described probe includes: appointing in nucleotide sequence shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3
Meaning is a kind of (above-mentioned 3 kinds of probes are used for detecting pseudorabies virus classics strain), core shown in SEQ ID No.4 or SEQ ID No.5
Any one (above-mentioned 2 kinds of probes are used for detecting pseudorabies virus variant) in nucleotide sequence, and SEQ ID No.6 or
Any one (above-mentioned 2 kinds of probes are used for detecting Bartha-K61 vaccine strain) in nucleotide sequence shown in SEQ ID No.7.Its
In, nucleotides sequence is classified as 5 ' ends of the probe shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3 and is marked with respectively
FAM fluorescent reporter group, 3 ' ends are marked with BHQ1 fluorescent quenching group respectively；Nucleotides sequence is classified as SEQ ID No.4 or SEQ
5 ' ends of the probe shown in ID No.5 are marked with HEX fluorescent reporter group respectively, and 3 ' ends are marked with BHQ1 fluorescent quenching base respectively
Group；Nucleotides sequence is classified as 5 ' ends of the probe shown in SEQ ID No.6 or SEQ ID No.7 and is marked with Cy5 fluorescence report respectively
Group, 3 ' ends are marked with BHQ3 fluorescent quenching group respectively.
The various combination of above-mentioned primer and probe is optimized by the present invention, and result shows, applies SEQ ID No.1, SEQ
The probe of nucleotide sequence shown in ID No.4 and SEQ ID No.6, and by core shown in SEQ ID No.8 and SEQ ID No.9
The primer of nucleotide sequence composition to 1, shown in SEQ ID No.14 and SEQ ID No.15 the primer of nucleotide sequence composition to 4 groups
Pseudorabies virus classics strain, variant and three kinds of strains of Bartha-K61 vaccine strain can be detected also by the primer sets become simultaneously
Being distinguished, CT value is minimum simultaneously, and detection limit is minimum, and sensitivity is the highest；And the combination of other primer and probe can not be examined simultaneously
Survey and distinguish classical strain, variant and Bartha-K61 vaccine strain.
Visible by primer of the present invention and the design of probe and optimization, due to the genome sequence of the different strains of same virus
The most close, the amino acid structure of especially PRV classics strain and variant is essentially identical, and major gene is the most individual other amino acid whose
Inserting and disappearance, homology is high, differentiates the more difficult design of probe of the different strain of PRV tri-kinds and more than three kinds the most simultaneously.This
Invention tactic Bartha-K61 vaccine strain gene delection position design two probes (SEQ ID No.1, SEQ ID
Shown in No.4), distinguish classical strain and variant；Link position at Bartha-K61 deleted areas designs the 3rd probe simultaneously
(shown in SEQ ID No.6), is used for detecting vaccine strain.The present invention utilizes above-mentioned three kinds of probes, establishes a kind of triple fluorescent fixed
Amount PCR method is in order to differentiate detection PRV classics strain, variant and Bartha-K61 vaccine strain.
(FAM (special to classical strain), HEX are (special to variant for TaqMan probe based on three kinds of labelling difference fluorescence signals
Different) and Cy5 (special to vaccine strain)), triple fluorescent quantitative PCR method of the present invention can distinguish simultaneously PRV classics strain (SC strain),
Variant (TJ strain) and Bartha-K61 vaccine strain.The method high specificity, is not detected by CSFV, PCV2, PPV and PRRSV
Fluorescence signal.The detection of SC strain, TJ strain and Bartha-K61 is limited and is respectively by triple fluorescent quantitative PCR method of the present invention
0.5TCID50、0.2TCID50And 0.05TCID50.Replica test in group and between group shows, the coefficient of variation of the method is less than
3%.
Triple fluorescent quantitative PCR method of the present invention is applied accurately PRV difference strain to be differentiated, identification result
Identical with sequencing result.Meanwhile, mix two-by-two or the situation of three kinds of mixing at PRV SC strain, TJ strain and Bartha-K61 vaccine strain
Under, the method can also differentiate the strain type in virus mixture accurately.Triple fluorescent quantitative PCR method of the present invention is with sick
Poison separation method is respectively 100%, 99.5% and 100% to the detection coincidence rate of classical strain, variant and vaccine strain.It addition,
In the various organ of pig body, the virus load of different PRV equally detects by the method.
Pseudorabies virus classics strain of the present invention includes but not limited to: pseudorabies virus SC strain or Min-A strain；Institute
State variant to include but not limited to: pseudorabies virus TJ strain, HLJMDJ2013 strain, HeBLP2014 strain or BJKJZ2015 strain.
Primer of the present invention and probe can be applied to preparation differentiate pseudorabies virus classics strain, variant and
The reagent of Bartha-K61 vaccine strain or material.
The present invention further discloses a kind of discriminating pseudorabies virus classics strain, variant and Bartha-K61 vaccine strain
Triple fluorescent quantitative PCR detection kit, including primer, probe, Premix Ex Taq Probe qPCR and distilled water；
The primer that wherein said primer is made up of the nucleotide sequence shown in SEQ ID No.8 and SEQ ID No.9 to 1, SEQ ID
The primer of the nucleotide sequence composition shown in No.14 and SEQ ID No.15 is to 4 compositions；Described probe be SEQ ID No.1,
Nucleotide sequence shown in SEQ ID No.4 and SEQ ID No.6.Wherein, nucleotides sequence is classified as the spy shown in SEQ ID No.1
5 ' ends of pin are marked with FAM fluorescent reporter group, and 3 ' ends are marked with BHQ1 fluorescent quenching group；Nucleotides sequence is classified as SEQ ID
5 ' ends of the probe shown in No.4 are marked with HEX fluorescent reporter group, and 3 ' ends are marked with BHQ1 fluorescent quenching group；Nucleotides sequence
5 ' the ends being classified as the probe shown in SEQ ID No.6 are marked with Cy5 fluorescent reporter group, and 3 ' ends are marked with BHQ3 fluorescent quenching base
Group.
Triple fluorescent quantitative PCR detection kit of the present invention also includes dimethyl sulfoxide (DMSO).Wherein, described
The consumption of dimethyl sulfoxide be by primer, probe, Premix Ex Taq Probe qPCR, distilled water, testing gene group DNA and
0%-10%, the preferably 2.5%-10% of the triple fluorescent quantitative PCR reaction system cumulative volume of dimethyl sulfoxide composition, optimum
Elect 5% as.
Existing document report, adding DMSO in common PCR reaction system can increase the susceptiveness of reaction.But fluorescence
Quantitative PCR itself is the detection method that a kind of susceptiveness is higher, and the addition of DMSO simultaneously may affect the reading of fluorescence signal
Take.Pseudorabies virus double-stranded DNA combines more tight, and when 95 DEG C of degeneration, double-strand can not fully open, and primer and probe are not
Can engage with double-stranded DNA and expand, reduce the identification to reacting of probe and primer and distinguishing ability.The present invention is at triple fluorescent
Quantitative PCR reaction system adds DMSO, enhances the distinguishing ability of probe, significantly improve the sensitivity of the method；Work as DMSO
Addition when being 5% (v/v), CT value is minimum, and sensitivity is the highest, the rate of accuracy reached of differentiation to 100%.
Technical solution of the present invention compared with prior art, has the advantages that
Tactic of the present invention design two probes at Bartha-K61 vaccine strain gene delection position, distinguish classical strain and
Variant, simultaneously at the link position of Bartha-K61 deleted areas, designs the 3rd probe, is used for detecting vaccine strain.This
Three kinds of probes designed by bright utilization, establish a kind of discriminating pseudorabies virus classics strain, variant and Bartha-K61 epidemic disease
The triple fluorescent quantitative PCR method of Seedling strain.It addition, the present invention adds DMSO in triple fluorescent quantitative PCR reaction system, significantly
Improve the sensitivity of the method.TaqMan probe based on three kinds of labelling difference fluorescence signals, triple fluorescent quantitative PCR of the present invention
Method can differentiate PRV classics strain, variant and Bartha-K61 vaccine strain, has good Sensitivity and Specificity, and
The shortest, low cost, accuracy rate is high, can apply to the difference detection of PRV strain and the PR epidemic situation that reply is the most serious.
Accompanying drawing explanation
Fig. 1 is the selection of triple fluorescent quantitative PCR primer and probe；A-F is respectively the amplification curve of scheme 1-scheme 6；
Fig. 2 is to expand PRV classics strain and the specific primer of variant and probe；Wherein, dashed box is PRV classics strain and
The primer of variant and probe design attitude；Shi Kuang mesogen strain is PRV variant, and other strains are classical strain；Identical sequence is used
(.) represents；
Fig. 3 is specific primer and the probe design attitude of amplification Bartha-K61 vaccine strain；
Fig. 4 is the selection of DMSO concentration；
Fig. 5 is the amplification curve of triple fluorescent quantitative PCR；Wherein, A is SC strain amplification curve；B is TJ strain amplification curve；C
For Bartha-K61 amplification curve；
Fig. 6 is the standard curve of triple fluorescent quantitative PCR；With the logarithm of standard concentration as abscissa, CT value is sat for vertical
Mark；Wherein, A is SC strain standard curve；B is TJ strain standard curve；C is Bartha-K61 standard curve；
Fig. 7 is the specificity of triple fluorescent quantitative PCR.
Detailed description of the invention
Below in conjunction with specific embodiment further describe the present invention, advantages of the present invention and feature will be with describe and
Apparent.It should be understood that described embodiment is only exemplary, the scope of the present invention is not constituted any restriction.This area
Skilled artisans appreciated that, lower without departing from the spirit and scope of the present invention can to the details of technical solution of the present invention and
Form is modified or replaces, but these amendments or replacement each fall within protection scope of the present invention.
Embodiment 1 differentiates detection PRV classics strain, variant and the triple fluorescent quantitative PCR side of Bartha-K61 vaccine strain
The foundation of method
1, material and method
1.1 it is viral
Swine fever virus (Classical swine fever virus, CSFV) Strain Shimen, pig parvoviral (Porcine
Parvovirus, PPV) Z strain, porcine circovirus 2 type (Porcine circovirus type2, PCV2) JXL strain, pig breeding with
Breath syndrome virus (Porcine reproductive and porcine respiratory syndrome virus,
PRRSV) HuN4 strain, PRV SC strain, TJ strain, HLJMDJ2013 strain, HeBLP2014 strain, BJKJZ2015 strain (variant) and
Bartha-K61 (vaccine strain) is preserved (wherein pseudorabies virus TJ strain, HLJMDJ2013 strain, HeBLP2014 by the present inventor
Strain and BJKJZ2015 strain, separated by the present inventor, identify, preserved, and its partial sequence is uploaded to GenBank simultaneously, and sequence logs in
Number be respectively KJ789182, KU670431, KU670430 and KU937306)；Min-A strain (classical strain) is purchased from China's veterinary medicament
Supervision institute.
1.2 viral genome are extracted
Tissue homogenate and the viral DNA cultivated in cell, after-80 DEG C of multigelations three times, use DNA extraction kit
(Omega, USA) extracts.
1.3 primers and the selection of probe
NCBI website Blast analyzes PRV difference strain gene order, at Bartha-K61 vaccine strain gene delection position
Design many groups PRV classics strain and variant common primers to and probe, Bartha-K61 vaccine strain sequence designs many groups and draws
Thing to and probe (table 1, table 2).Meanwhile, design 6 kinds of assembled schemes (table 3), probe and primer are optimized.Experiment condition is such as
Under: each 0.5 μ L of probe (10 μMs)；The each 1 μ L of primer (10 μMs)；Classical strain, variant and each 1.5 μ of Bartha-K61 genomic DNA
L；Premix Ex Taq Probe qPCR (TaKaRa, Japan) 10 μ L；Often pipe adds water to 20 μ L.Triple fluorescent quantitative PCR is anti-
Program is answered to be provided that initial denaturation temperature, 95 DEG C, 5min；Denaturation temperature 95 DEG C, 35s, annealing and elongating temperature 60 DEG C,
35s, 40 circulations；After each loop ends, detect fluorescence signal.By detection fluorescence signal, experimental program is selected.
CT value is defined as the period that amplification curve reaches to be experienced during threshold value.By select primer and probe application to subsequent experimental
In.
Table 1 triple fluorescent quantitative PCR probe implementation sequence
Table 2 triple fluorescent quantitative PCR primer implementation sequence
The primer of table 3 triple fluorescent quantitative PCR and probe combinations scheme
Note: a plus sige (+) represent that probe or primer are added in experimental program.
2, the selection result of triple fluorescent quantitative PCR primer and probe
The selection result of primer and probe shows, scheme 1～4 can not detect simultaneously and distinguish classical strain, variant and
Bartha-K61 vaccine strain, and three kinds of strains can be detected simultaneously and be distinguished by application scheme 5, CT value is minimum simultaneously, detection
Limit minimum, sensitivity the highest (Fig. 1, table 4).Therefore, the present invention is by the probe in scheme 5 and primer (FAM-PRV-Cla (SEQ
Shown in ID No.1), HEX-PRV-Var (shown in SEQ ID No.4), Cy5-PRV-Bar (shown in SEQ ID No.6), PRV-F1
(shown in SEQ ID No.8), PRV-R1 (shown in SEQ ID No.9), PRV-F2 (shown in SEQ ID No.14) and PRV-R2
(shown in SEQ ID No.15)) in subsequent experimental (Fig. 2, Fig. 3).
Table 4 uses the Detection results of the triple fluorescent quantitative PCR of various combination scheme to compare
The optimization of embodiment 2 triple fluorescent quantitative PCR method condition
The present invention is on the basis of the triple fluorescent quantitative PCR method that embodiment 1 is set up, further to triple fluorescent quantitative
PCR condition is optimized.
1, experimental technique
Triple fluorescent quantitative PCR experiment application Mx3005p instrument (Stratagene, USA) is carried out, primer and probe dense
Degree is optimized by fluorescence signal (Δ Rn) numerical value.Experiment condition is as follows: PCR reaction cumulative volume is 20 μ L, FAM-PRV-Cla
(shown in SEQ ID No.1), HEX-PRV-Var (shown in SEQ ID No.4) and Cy5-PRV-Bar (shown in SEQ ID No.6)
(10 μMs) each 0.5 μ L；PRV-F1 (shown in SEQ ID No.8), PRV-R1 (shown in SEQ ID No.9), PRV-F2 (SEQ ID
Shown in No.14) and PRV-R2 (shown in SEQ ID No.15) (10 μMs) each 1 μ L；Genomic DNA 2 μ L；Premix Ex Taq
Probe qPCR (TaKaRa, Japan) 10 μ L；Meanwhile, in PCR reaction system, it is separately added into 0%, 2.5%, 5% and 10%
(v/v) dimethyl sulfoxide (DMSO), to screen the optimum concentration of DMSO.Triple fluorescent quantitative PCR response procedures has been provided that
Beginning denaturation temperature, 95 DEG C, 5min；Denaturation temperature 95 DEG C, 35s, annealing and elongating temperature 60 DEG C, 35s, 45 circulations；Each follow
After ring terminates, detect fluorescence signal.The CT value of sample exceedes limit value, needs again to check.Will optimize after experiment condition and
Program is arranged to be applied to subsequent experimental.
2, experimental result
The result of DMSO condition optimizing shows, the addition of DMSO can significantly improve the sensitivity of the method.With being not added with
The matched group of DMSO is compared, and when the addition of DMSO is 5% (v/v), CT value is minimum, sensitivity the highest (Fig. 4, table 5).
The optimization of table 5 DMSO concentration
Specificity, sensitivity and the replica test of embodiment 3 triple fluorescent quantitative PCR method
Experiment condition and the program of the triple fluorescent quantitative PCR after Application Example 2 optimization are arranged.
1, experimental technique
1.1 standard curves are set up
With primer PRV-F1/PRV-R1, PRV classics strain and variant are expanded respectively, use primer PRV-F2/PRV-
Bartha-K61 vaccine strain is expanded by R2, is cloned into respectively by amplified production in pUC-19T carrier, builds pUC-19T-
SC, pUC-19T-TJ and pUC-19T-Bartha recombiant plasmid, as the examination criteria product of triple fluorescent quantitative PCR.Three weights
Group plasmid carries out 10 times of gradient dilutions, respectively from 5 × 108To 5 copy/μ L ,-20 DEG C store for future use.
Standard substance are detected by the triple fluorescent quantitative PCR detection method after Application Example 2 optimizes.
1.2 specificitys, sensitivity and replica test
Infect PK-15 cell respectively with PRV SC strain, TJ strain and Bartha-K61 vaccine strain, extract viral genome, should
Triple fluorescent quantitative PCR after optimizing by embodiment 2 detects.CSFV, PCV2, PPV and PRRSV are tested simultaneously,
The specificity of detection the method.Subsequently, after PRV SC strain, TJ strain and Bartha-K61 vaccine strain infect PK-15 cell respectively, enter
Ten times of gradient dilutions of row, the sensitivity of detection the method.PRV SC strain, TJ strain and Bartha-K61 vaccine strain are carried out three not
With titre repeat test, including organize between interior repetition and group repeat, detection the method repeatability.
PRV difference strain is differentiated by 1.3 triple fluorescent quantitative PCR method
PRV difference strain is differentiated by triple fluorescent quantitative PCR method, checks order strain simultaneously.SC strain, TJ
Strain and Bartha-K61 vaccine strain mix or three kinds of mixing two-by-two, and the method that application is set up is mixed situation to virus and enters
Row detection.
1.4 virus purification experimental verification triple fluorescent quantitative PCR method
234 parts are detected by triple fluorescent quantitative PCR method from the sample in different provinces.All samples is all by virus
Separating experiment and immunofluorescence experiment are rechecked, and positive are carried out DNA sequencing simultaneously.
2, experimental result
2.1 triple fluorescent quantitative PCR standard curves are set up
Standard substance are detected (Fig. 5 A-C) by the triple fluorescent quantitative PCR detection method that the application present invention sets up.Standard
The logarithm of product concentration is abscissa, and CT value is vertical coordinate, sets up triple fluorescent quantitative PCR standard curve.Result shows, SC strain and
The standard curve of TJ strain is 5 × 108To 5 × 101In the range of individual copy, linear, the standard curve of Bartha-K61 strain
5 × 107To 5 copies, linear (Fig. 6 A-C).The linear of SC strain, TJ strain and Bartha-K61 strain returns
Equation is returned to be respectively y=-3.436 × log (x)+45.84, (R2=0.995)；Y=-3.434 × log (x)+43.93 (R2=
0.997)；Y=-3.054 × log (x)+40.7, (R2=0.995).Detection range based on the method, SC strain, TJ strain and
The limit value of Bartha-K61 strain is respectively 39.0,38.1 and 38.5.
Specificity, sensitivity and the repeatability of 2.2 triple fluorescent quantitative PCR method
Glimmering by detection FAM (special to classical strain), HEX (special to variant) and Cy5 (special to vaccine strain) three kinds
Optical signal, the method can distinguish SC strain, TJ strain and Bartha-K61 strain simultaneously.The present invention set up detection method to CSFV,
PCV2, PPV and PRRSV test, and are not detected by fluorescence signal, show the method high specificity (Fig. 7).Triple fluorescent quantitative
The detection of SC strain, TJ strain and Bartha-K61 is limited and is respectively 0.5TCID by PCR method50、0.2TCID50And 0.05TCID50.Group
In and group between replica test show, the coefficient of variation of the method be less than 3%.
2.3 PRV difference strain is differentiated by triple fluorescent quantitative PCR method
PRV difference strain is detected by application triple fluorescent quantitative PCR method, and result shows, the method can be accurate
Different strains are differentiated, identification result is identical with sequencing result.Meanwhile, at PRV SC strain, TJ strain and Bartha-K61
Vaccine strain mixes two-by-two or in the case of three kinds of mixing, the method can also differentiate the strain type in virus mixture accurately
(table 6).
Table 6 triple fluorescent quantitative PCR differentiates PRV strain
Note: a plus sige (+) represent that this Air conduct measurement result is for the positive；
B minus sign (-) represent that this Air conduct measurement result is for feminine gender.
2.4 verify triple fluorescent quantitative PCR method with virus purification
234 parts of samples are detected by application triple fluorescent quantitative PCR method, and result shows, has 8 parts of samples to contain PRV
Classical strain；34 parts of samples contain variant；6 parts of samples contain Bartha-K61 strain.Virus purification result shows, has 8 parts of samples
Containing PRV classics strain；35 parts of samples contain variant；6 parts of samples contain Bartha-K61 strain.Two kinds of methods are to classical strain, change
The detection coincidence rate of different strain and vaccine strain is respectively 100%, 99.5% and 100% (table 7).The DNA sequencing result of positive
Showing, triple fluorescent quantitative PCR method can accurately differentiate PRV difference strain.
Table 7 triple fluorescent quantitative PCR and the accordance of virus purification
Claims (10)
1. for differentiating the triple fluorescent quantitative PCR's of pseudorabies virus classics strain, variant and Bartha-K61 vaccine strain
Primer and probe, it is characterised in that described primer includes: nucleotide sequence shown in SEQ ID No.8 and SEQ ID No.9 forms
Primer to 1, shown in SEQ ID No.10 and SEQ ID No.11 the primer of nucleotide sequence composition to 2, SEQ ID No.12
With the primer of the composition of nucleotide sequence shown in SEQ ID No.13 to any pair in 3, and SEQ ID No.14 and SEQ
The primer of the composition of nucleotide sequence shown in ID No.15 is to 4；
Described probe includes: any one in nucleotide sequence shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3
Kind, any one in nucleotide sequence shown in SEQ ID No.4 or SEQ ID No.5, and SEQ ID No.6 or SEQ ID
Any one in nucleotide sequence shown in No.7.
2. according to the primer described in claim 1 and probe, it is characterised in that described primer includes: SEQ ID No.8 and SEQ
The primer of the composition of nucleotide sequence shown in ID No.9 is to 1, and nucleotides sequence shown in SEQ ID No.14 and SEQ ID No.15
The primer of row composition is to 4；
Described probe includes: nucleotide sequence shown in SEQ ID No.1, SEQ ID No.4 and SEQ ID No.6.
3. according to the primer described in claim 1 and probe, it is characterised in that: nucleotides sequence is classified as SEQ ID No.1, SEQ ID
5 ' ends of the probe shown in No.2 or SEQ ID No.3 are marked with FAM fluorescent reporter group respectively, and 3 ' ends are marked with BHQ1 respectively
Fluorescent quenching group；
Nucleotides sequence is classified as 5 ' ends of the probe shown in SEQ ID No.4 or SEQ ID No.5 and is marked with HEX fluorescence report respectively
Group, 3 ' ends are marked with BHQ1 fluorescent quenching group respectively；
Nucleotides sequence is classified as 5 ' ends of the probe shown in SEQ ID No.6 or SEQ ID No.7 and is marked with Cy5 fluorescence report respectively
Group, 3 ' ends are marked with BHQ3 fluorescent quenching group respectively.
4. according to the primer described in claim 1 and probe, it is characterised in that described pseudorabies virus classics strain includes: pseudo-
Rabies virus SC strain or Min-A strain；Described variant includes: pseudorabies virus TJ strain, HLJMDJ2013 strain,
HeBLP2014 strain or BJKJZ2015 strain.
5. primer described in claim 1 or 2 and probe preparation differentiate pseudorabies virus classics strain, variant and
Application in the reagent of Bartha-K61 vaccine strain or material.
6. according to the application described in claim 5, it is characterised in that: described pseudorabies virus classics strain includes: pseudorabies
Virus SC strain or Min-A strain；Described variant includes: pseudorabies virus TJ strain, HLJMDJ2013 strain, HeBLP2014 strain or
BJKJZ2015 strain.
7. the triple fluorescent quantitative PCR inspection differentiating pseudorabies virus classics strain, variant and Bartha-K61 vaccine strain
Test agent box, including: primer, probe, Premix Ex Taq Probe qPCR and distilled water；It is characterized in that, described primer
It is the primer described in claim 1 or 2 and probe with probe.
8. according to the triple fluorescent quantitative PCR detection kit described in claim 7, it is characterised in that also include that dimethyl is sub-
Sulfone.
9. according to the triple fluorescent quantitative PCR detection kit described in claim 8, it is characterised in that described dimethyl sulfoxide
Consumption be by primer, probe, Premix Ex Taq Probe qPCR, distilled water, testing gene group DNA and dimethyl sulfoxide
The 0%-10%, preferably 2.5%-10% of the triple fluorescent quantitative PCR reaction system cumulative volume of composition.
10. according to the triple fluorescent quantitative PCR detection kit described in claim 9, it is characterised in that described dimethyl sulfoxide
Consumption be by primer, probe, Premix Ex Taq Probe qPCR, distilled water, testing gene group DNA and dimethyl sulfoxide
The 5% of the triple fluorescent quantitative PCR reaction system cumulative volume of composition.
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| CN106636460A (en) * | 2016-10-21 | 2017-05-10 | 四川出入境检验检疫局检验检疫技术中心 | Nucleic acid combination for detecting pseudorabies virus and application of nucleic acid combination, and kit and method for detecting pseudorabies virus |
| CN108842003A (en) * | 2018-07-18 | 2018-11-20 | 中国农业科学院特产研究所 | For identifying the Taqman fluorescence probe quantitative PCR detection kit and application of canine distemper virus street strain and vaccine strain |
| WO2019090971A1 (en) * | 2017-11-09 | 2019-05-16 | 先正达参股股份有限公司 | Improvement in copy number analysis based on real-time fluorescence quantitative pcr by using dimethyl sulfoxide |
| CN111154917A (en) * | 2020-02-22 | 2020-05-15 | 南京农业大学 | PCR primer and method for identifying porcine pseudorabies virus variant strain |
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| WO2002052043A1 (en) * | 2000-12-26 | 2002-07-04 | Takara Bio Inc. | Method of detecting pathogenic microorganism |
| CN105018645A (en) * | 2015-07-29 | 2015-11-04 | 福建省农业科学院畜牧兽医研究所 | Real-time fluorescence PCR-HRM primers for detecting classical pseudorabies virus (PRV) and variable PRV |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106636460A (en) * | 2016-10-21 | 2017-05-10 | 四川出入境检验检疫局检验检疫技术中心 | Nucleic acid combination for detecting pseudorabies virus and application of nucleic acid combination, and kit and method for detecting pseudorabies virus |
| WO2019090971A1 (en) * | 2017-11-09 | 2019-05-16 | 先正达参股股份有限公司 | Improvement in copy number analysis based on real-time fluorescence quantitative pcr by using dimethyl sulfoxide |
| CN108842003A (en) * | 2018-07-18 | 2018-11-20 | 中国农业科学院特产研究所 | For identifying the Taqman fluorescence probe quantitative PCR detection kit and application of canine distemper virus street strain and vaccine strain |
| CN111154917A (en) * | 2020-02-22 | 2020-05-15 | 南京农业大学 | PCR primer and method for identifying porcine pseudorabies virus variant strain |
| CN111154917B (en) * | 2020-02-22 | 2022-12-27 | 南京农业大学 | PCR primer and method for identifying porcine pseudorabies virus variant strain |
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