CN107460194A - The dual droplet digital pcr absolute quantitation detection kit of Pseudorabies virus gB, gE - Google Patents
The dual droplet digital pcr absolute quantitation detection kit of Pseudorabies virus gB, gE Download PDFInfo
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Abstract
The invention discloses a kind of Pseudorabies virus droplet digital pcr absolute quantitation detection kit.Primer combination of probe provided by the invention is made up of gB F1R1P1 and gE F1R1P1；Primer gB F1 are shown in sequence 1；Primer gB R1 are shown in sequence 2；Probe is shown in sequence 3；Primer gE F1 are shown in sequence 4；Primer gE R1 are shown in sequence 5；Probe gE P1 are as shown in sequence 6.The present invention also protects the application of the primer combination of probe：Identify whether virus to be measured is porcine pseudorabies virus street strain or porcine pseudorabies virus vaccine strain；Whether detection sample to be tested contains porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine strain；Detect the content of porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine strain in sample to be tested；Prevention and control of the present invention for porcine pseudorabies virus have great application value, contribute to from Sources controlling epidemic situation, the effectively sick large-scale outbreak of prevention schweineseuche.
Description
Technical field
The invention belongs to field of virus detection, and in particular to a kind of dual droplet digital pcr of Pseudorabies virus gB, gE is absolute
Immue quantitative detection reagent box.
Background technology
Porcine pseudorabies virus (porcine pseudorabies virus, PRV) belongs to herpetoviridae, herpesviral
Category, particle is spherical in shape, and ripe virion diameter is about 150-180nm, and virion internal package icosahedral core
Capsid, its diameter are about 100-110nm, and genome is located inside nucleocapsid, are disease outside nucleocapsid in curling winding shape arrangement
Malicious cyst membrane, is lipid bilayer Rotating fields, and cyst membrane surface is that 10nm or so fibre is dashed forward.PRV can cause the infection of a variety of domestic animals,
Wherein pig is most important reservoir and the infection sources, and the virus can cause porcine pseudorabies (PR) in pig body, is a kind of high
Spend communicable disease.Breaking out for porcine pseudorabies can bring huge economic loss to pig industry, mainly including in-pig
High abortion ratio, production the breeding difficulty such as stillborn foetus or mummy tire, the respiratory disease of Adult Pig, piglet nervous system disease
Shape, tremble, ataxia, diarrhoea etc..The how resistance to mistake of Adult Pig and the death rate is up to 100% in newborn piglet, the adult of resistance to mistake
Continue band poison and toxin expelling in pig body, cause the multiple of this disease.
Traditional PRV detection methods mainly include separation and identification, the immunoperoxidase monolayer assay of virus
(IPMA), indirect immunofluorescence assay (IFA) and indirect enzyme-linked immunosorbent assay (indirect ELISA) etc..Current the most frequently used core
Sour detection method has PCR and Realtime PCR.Traditional PRV detection methods, which exist, needs to use high cost instruments equipment, tries
The deficiencies of longer the time required to testing.Although method has specific stronger, sensitivity to PCR and Realtime PCR as compared with the past
The features such as high, reproducible, and automaticity is high, but the above method can only realize the detection of quantitative and semi-quantitative, nothing
Method carries out accurate quantification to viral nucleic acid, and certain limitation is still suffered from sensitivity and Sensitivity Specificity.
The concept for digitizing PCR (Digital PCR, dPCR) was just used simultaneously early in 1999 by Bert Vogelstein
Pertinent literature is delivered, its original intention is to be able to substantial amounts of normal from clinical sample (such as urine, lymph, blood plasma, excrement)
Micro mutant cell is detected in body cell, but because the consumptive material that can be used for dilute sample at that time is 384 orifice plates, therefore also
The core concept of digital pcr can not very well be embodied --- " infinite dilution " (terminal dilution).Bio-Rad is public
A sample can be divided into the droplet of 20,000 nanoliter level by the droplet technology of the QX200 system cores of department, substantially will
A traditional quantitative PCR test becomes 20,000 test, substantially increases sensitivity and the precision of nucleotide sequence detection,
It is the perfection deduction to " infinite dilution " this concept, its Method And Principle can be described as droplet type digitlization PCR (Droplet
Digital PCR, ddPCR).QX200ddPCR systems include two instruments：Drop generator and droplet analyzer, and its it is related
Consumptive material.Each sample is divided into 20,000 uniform nanoliter level droplet by drop generator, wherein each droplet or without treating
Nucleic acid target molecule is examined, or contains one to several nucleic acid target molecules to be checked.The PCR reaction independent as one of each droplet
Device.Subsequent droplet is transferred in 96 hole PCR plates, carries out terminal PCR amplifications.Using droplet analyzer (droplet reader) by
Individual that each droplet is detected, the droplet interpretation for having fluorescence signal is 1, and the droplet interpretation without fluorescence signal is 0, final root
According to Poisson distribution principle and the ratio of positive droplet, analysis software can calculate the concentration or copy number for providing target molecule to be checked.
Compared with traditional quantitative PCR, the accuracy of digital pcr and sensitivity are more preferably.Using droplet type digital pcr technology, people is studied
Member can detect rare mutation, accurate measure copy number variation, and carry out absolute quantitation to gene expression.By QX200 systems
High sensitivity, researcher nowadays can detect concentration as little as 1/1,000,000 target molecule.
The content of the invention
It is an object of the invention to provide a kind of Pseudorabies virus droplet digital pcr absolute quantitation detection kit.
The invention provides a kind of primer combination of probe, by primed probe group gB-F1R1P1 and primed probe group gE-
F1R1P1 is formed；
The primed probe group gB-F1R1P1 is made up of primer gB-F1, primer gB-R1 and probe gB-P1；
The primer gB-F1 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 of one or several nucleotides and/or missing and/or addition and with sequence 1
The DNA molecular of identical function；
The primer gB-R1 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 of one or several nucleotides and/or missing and/or addition and with sequence 2
The DNA molecular of identical function；
The probe gB-P1, an end have fluorescent reporter group, and another end has fluorescent quenching group, core
Nucleotide sequence is following (a5) or (a6)：
(a5) as shown in the sequence 3 of sequence table；
(a6) as shown in by sequence 3 by the substitution of one or several nucleotides and/or missing and/or addition；
The primed probe group gE-F1R1P1 is made up of primer gE-F1, primer gE-R1 and probe gE-P1；
The primer gE-F1 is following (a1) or (a2)：
(a1) single strand dna shown in the sequence 4 of sequence table；
(a2) sequence 4 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 4
The DNA molecular of identical function；
The primer gE-R1 is following (a3) or (a4)：
(a3) single strand dna shown in the sequence 5 of sequence table；
(a4) sequence 5 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 5
The DNA molecular of identical function；
The probe gE-P1, an end have fluorescent reporter group, and another end has fluorescent quenching group, core
Nucleotide sequence is following (a5) or (a6)：
(a5) as shown in the sequence 6 of sequence table；
(a6) as shown in by sequence 6 by the substitution of one or several nucleotides and/or missing and/or addition；
The probe gB-P1 and probe gE-P1 has the fluorescent reporter group of different colours.
Specifically, 5 ' the end mark fluorophor FAM of the probe gB-P1,3 ' end mark fluorescent quenching groups
BHQ1。
Specifically, 5 ' the end mark fluorophor HEX of the probe gE-P1,3 ' end mark fluorescent quenching groups
BHQ1。
The purposes of the primer combination of probe is any one in following (b1) to (b6)：
(b1) identify whether virus to be measured is porcine pseudorabies virus street strain or porcine pseudorabies virus vaccine strain；
(b2) prepare for identifying whether virus to be measured is porcine pseudorabies virus street strain or porcine pseudorabies virus epidemic disease
The kit of seedling strain；
(b3) whether detection sample to be tested contains porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine
Strain；
(b4) prepare for whether detecting sample to be tested containing porcine pseudorabies virus street strain and/or porcine pseudorabies disease
The kit of toxic vaccine strain；
(b5) containing for porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine strain in sample to be tested is detected
Amount；
(b6) prepare for detecting porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine in sample to be tested
The kit of strain content.
The present invention also protects the application of the primer combination of probe, for any one in following (b1) to (b6)：
(b1) identify whether virus to be measured is porcine pseudorabies virus street strain or porcine pseudorabies virus vaccine strain；
(b2) prepare for identifying whether virus to be measured is porcine pseudorabies virus street strain or porcine pseudorabies virus epidemic disease
The kit of seedling strain；
(b3) whether detection sample to be tested contains porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine
Strain；
(b4) prepare for whether detecting sample to be tested containing porcine pseudorabies virus street strain and/or porcine pseudorabies disease
The kit of toxic vaccine strain；
(b5) containing for porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine strain in sample to be tested is detected
Amount；
(b6) prepare for detecting porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine in sample to be tested
The kit of strain content.
The present invention also protects a kind of kit, including the primer combination of probe；The function of the kit is as follows
Any one in (c1) to (c3)：(c1) identify whether virus to be measured is porcine pseudorabies virus street strain or porcine pseudorabies
Virus vaccine strain；(c2) whether detection sample to be tested contains porcine pseudorabies virus street strain and/or porcine pseudorabies virus epidemic disease
Miao Zhu；(c3) content of porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine strain in sample to be tested is detected.
The kit also includes recording method I and/or method II and/or method III carrier.
A kind of detection of the present invention also protection virus to be measured whether be whether be that porcine pseudorabies virus street strain or pig puppet are mad
The method (method I) of dog disease virus vaccine strain, comprises the following steps：Using viral genomic DNA to be measured as template, using described
Primer combination of probe carries out digital pcr；If testing result of the virus based on the corresponding fluorophors of probe gB-P1 to be measured is sun
Property, virus to be measured be or candidate is porcine pseudorabies virus, if virus to be measured is based on the corresponding fluorophors of probe gB-P1
Testing result is that virus negative, to be measured is or candidate is non-porcine pseudorabies virus；If or candidate be porcine pseudorabies disease
The viral testing result based on the corresponding fluorophors of probe gE-P1 to be measured of poison is that virus positive, to be measured is or candidate is pig
Pseudorabies virus street strain, if or candidate for porcine pseudorabies virus it is to be measured virus based on probe gE-P1 it is corresponding
The testing result of fluorophor is that virus negative, to be measured is or candidate is porcine pseudorabies virus vaccine strain.
Whether a kind of detection sample to be tested of the present invention also protection contains porcine pseudorabies virus street strain or porcine pseudorabies
The method (method II) of virus vaccine strain, comprises the following steps：Using the STb gene of sample to be tested as template, visited using the primer
Pin combination carries out digital pcr；If testing result of the sample to be tested based on the corresponding fluorophors of probe gB-P1 is the positive, treated
Test sample originally contains porcine pseudorabies virus, if testing result of the sample to be tested based on the corresponding fluorophors of probe gB-P1 is
Negative, sample to be tested does not contain porcine pseudorabies virus；If the sample to be tested containing porcine pseudorabies virus is based on probe gE-
The testing result of the corresponding fluorophors of P1 contains porcine pseudorabies virus street strain for positive, sample to be tested, if containing pig
Testing result of the sample to be tested of pseudorabies virus based on the corresponding fluorophors of probe gE-P1 contains for negative, sample to be tested
There is porcine pseudorabies virus vaccine strain.
Porcine pseudorabies virus street strain and/or porcine pseudorabies virus in a kind of detection sample to be tested of the present invention also protection
The method (method III) of the content of vaccine strain, comprises the following steps：Using the STb gene of sample to be tested as template, using the primer
Probe combinations carry out digital pcr；Porcine pseudorabies in sample to be tested are obtained according to the quantity of porcine pseudorabies virus positive droplet
The content of virus, porcine pseudorabies virus positive droplet are that the testing result based on the corresponding fluorophors of probe gB-P1 is sun
The droplet of property；According to the quantity of porcine pseudorabies virus street strain obtain in sample to be tested porcine pseudorabies virus street strain and/
Or the content of porcine pseudorabies virus vaccine strain, porcine pseudorabies virus street strain positive droplet are corresponding based on probe gE-P1
The testing result of fluorophor be positive droplet.
In methods described I or method II or method III, in the reaction system of the digital pcr, primer gB-F1 concentration is
0.9 μM, primer gB-R1 concentration is 0.9 μM, and probe gB-P1 concentration is 0.2 μM, and primer gE-F1 concentration is 0.9 μM, is drawn
Thing gE-R1 concentration is 0.9 μM, and probe gE-P1 concentration is 0.2 μM.
In methods described I or method II or method III, the reaction system of the digital pcr can be by 2 × ddPCR
Supermix for Probes, primer gB-F1, primer gB-R1, probe gB-P1, primer gE-F1, primer gE-R1, probe gE-
P1 and template composition.
In methods described I or method II or method III, the reaction system of the digital pcr can be by 2 × ddPCR
Supermix for Probes, primer gB-F1, primer gB-R1, probe gB-P1, primer gE-F1, primer gE-R1, probe gE-
P1, template and RNase Free dH2O is formed.
In methods described I or method II or method III, the response procedures of the digital pcr can be：95 DEG C of pre-degenerations
10min；94 DEG C of denaturation 30sec, 55 DEG C of annealing 60sec, totally 40 circulations；98℃10min.
In methods described I or method II or method III, in the response procedures of the digital pcr, warming and cooling rate is arranged to
2.5℃/sec。
The present invention also protects a kind of premixed liquid, wherein containing primer gB-F1, primer gB-R1, probe gB-P1, primer gE-
F1, primer gE-R1 and probe gE-P1；In the premixed liquid, primer gB-F1 concentration is 1 μM, and primer gB-R1 concentration is 1 μ
M, probe gB-P1 concentration are 2/9 μM, and primer gE-F1 concentration is 1 μM, and primer gE-R1 concentration is 1 μM, probe gE-P1
Concentration be 2/9 μM.The function of the premixed liquid is any one in following (c1) to (c3)：(c1) identify that virus to be measured is
No is porcine pseudorabies virus street strain or porcine pseudorabies virus vaccine strain；(c2) whether mad containing pig puppet sample to be tested is detected
Dog disease field virus and/or porcine pseudorabies virus vaccine strain；(c3) the wild poison of porcine pseudorabies virus in sample to be tested is detected
Strain and/or the content of porcine pseudorabies virus vaccine strain.
The present invention also protects a kind of kit, including the premixing and droplet that oil occurs.The kit also includes the moon
Property control and positive control；The negative control is RNase Free dH2O；The positive control is containing porcine pseudorabies disease
The solution of the genomic DNA of malicious street strain.The function of the kit is any one in following (c1) to (c3)：(c1) reflect
Whether fixed virus to be measured is porcine pseudorabies virus street strain or porcine pseudorabies virus vaccine strain；(c2) detection sample to be tested is
It is no containing porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine strain；(c3) it is mad to detect pig puppet in sample to be tested
The content of dog disease field virus and/or porcine pseudorabies virus vaccine strain.
The kit also includes recording method IV and/or the carrier of method V and/or method VI.
Method IV be it is a kind of by digital pcr detect virus to be measured whether be whether be porcine pseudorabies virus street strain or
The method of porcine pseudorabies virus vaccine strain, comprises the following steps：Using viral genomic DNA to be measured or its dilution as mould
Plate solution, oil occurs with 70 μ L droplets after premixed liquid described in 2 μ L template solutions and 18 μ L is mixed and is generated with droplet is placed in instrument
Droplet is formed, then enters performing PCR amplification, is then detected in droplet analyzer；If virus to be measured is based on probe gB-P1
The testing result of corresponding fluorophor is that virus positive, to be measured is or candidate is porcine pseudorabies virus, if virus to be measured
Testing result based on the corresponding fluorophors of probe gB-P1 is that virus negative, to be measured is or candidate is non-porcine pseudorabies disease
Poison；If or candidate for porcine pseudorabies virus the viral detection knot based on the corresponding fluorophors of probe gE-P1 to be measured
Fruit is that virus positive, to be measured is or candidate is porcine pseudorabies virus street strain, if or candidate be porcine pseudorabies virus
The viral testing result based on the corresponding fluorophors of probe gE-P1 to be measured be that virus negative, to be measured is or candidate is pig puppet
Rabies virus vaccine strain.
Whether method V detects sample to be tested containing porcine pseudorabies virus street strain or pig puppet to be a kind of by digital pcr
The method of rabies virus vaccine strain, comprises the following steps：Using the STb gene of sample to be tested or its dilution as template solution,
Will described in 2 μ L template solutions and 18 μ L premixed liquid mix after with 70 μ L droplets occur oil be placed in droplet generation instrument in formed it is micro-
Drop, then enter performing PCR amplification, then detected in droplet analyzer；It is corresponding that if sample to be tested is based on probe gB-P1
The testing result of fluorophor contains porcine pseudorabies virus for positive, sample to be tested, if sample to be tested is based on probe gB-P1
The testing result of corresponding fluorophor does not contain porcine pseudorabies virus for negative, sample to be tested；If contain pseudorabies
Testing result of the sample to be tested based on the corresponding fluorophors of probe gE-P1 of virus contains pig puppet for positive, sample to be tested
Hydrophobin street strain, if the sample to be tested containing porcine pseudorabies virus is based on the corresponding fluorophors of probe gE-P1
Testing result contain porcine pseudorabies virus vaccine strain for negative, sample to be tested.
Method VI is mad for porcine pseudorabies virus street strain in a kind of detection sample to be tested by digital pcr and/or pig puppet
The method of the content of dog disease virus vaccine strain, comprises the following steps：The STb gene of sample to be tested or its dilution is molten as template
Liquid, same be placed in droplet generation instrument of oil occurs with 70 μ L droplets after premixed liquid described in 2 μ L template solutions and 18 μ L is mixed and is formed
Droplet, then enter performing PCR amplification, then detected in droplet analyzer；According to porcine pseudorabies virus positive droplet
Quantity obtains the content of porcine pseudorabies virus in sample to be tested, and porcine pseudorabies virus positive droplet is based on probe gB-P1
The testing result of corresponding fluorophor is positive droplet；Obtain treating test sample according to the quantity of porcine pseudorabies virus street strain
The content of porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine strain in this, the wild poison of porcine pseudorabies virus
The positive droplet of strain is positive droplet for the testing result based on the corresponding fluorophors of probe gE-P1.
In methods described IV or method V or method VI, the response procedures of the digital pcr can be：95 DEG C of pre-degenerations
10min；94 DEG C of denaturation 30sec, 55 DEG C of annealing 60sec, totally 40 circulations；98℃10min.
In methods described I or method II or method III, in the response procedures of the digital pcr, warming and cooling rate is arranged to
2.5℃/sec。
The present invention also protects the application of the premixed liquid or the kit, is any one in following (c1) to (c3)
Kind：(c1) identify whether virus to be measured is porcine pseudorabies virus street strain or porcine pseudorabies virus vaccine strain；(c2) detect
Whether sample to be tested contains porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine strain；(c3) test sample is treated in detection
The content of porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine strain in this.
Concretely droplet type digitizes PCR to digital pcr described in any of the above.
The concretely droplet digital pcr absolute quantitation detection kit of kit described in any of the above.
Digital pcr described in any of the above specifically uses QX200Droplet Digital PCR systems.
Sample to be tested described in any of the above can be porcine tissue sample to be measured, such as pig lung tissue sample.
Virus to be measured described in any of the above can be tiny for porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus, pig
Virus or 2 type pig circular ring virus or CSFV.
Porcine pseudorabies virus described in any of the above can be porcine pseudorabies virus street strain or porcine pseudorabies virus epidemic disease
Miao Zhu.
Porcine reproductive and respiratory syndrome virus described in any of the above can be porcine reproductive and respiratory syndrome virus american type or
Porcine reproductive and respiratory syndrome virus Europe class.
Porcine pseudorabies virus street strain described in any of the above can be Qihe547 strains.
Porcine pseudorabies virus vaccine strain described in any of the above can be Bartha-K61 strains.
Porcine reproductive and respiratory syndrome virus american type described in any of the above can be VR-2332 strains.
Porcine reproductive and respiratory syndrome virus Europe class described in any of the above can be LV strains.
Pig parvoviral described in any of the above can be NADL-2 strains.
2 type pig circular ring virus described in any of the above can be 08TJ strains.
CSFV described in any of the above can be C-strain strains.
The present inventor's design is screened the primed probe group for identifying porcine pseudorabies virus and is used for
The primed probe group of porcine pseudorabies virus street strain is identified, has further groped the concentration of primer and probe, have found most
The working concentration of suitable primer and probe, to improve ddPCR amplification efficiency.
The invention provides with high sensitivity, high specific, high accuracy, pinpoint accuracy, accurate quantification can be achieved
Droplet type digitizes PCR kit, the inspection for porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine strain
Survey, can direct quantitative, without standard curve, easy to operate, result accurately and reliably, be particularly suitable for Site Detection.The present invention for
The prevention and control of porcine pseudorabies virus have great application value, contribute to from Sources controlling epidemic situation, effectively prevent schweineseuche disease
Large-scale outbreak.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, it is conventional method unless otherwise specified.Test material used in following embodiments, it is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, it is respectively provided with and repeats to test three times, as a result make even
Average.
Digital pcr in embodiment uses Bio-Rad Laboratories, Inc. QX200Droplet
Digital PCR systems (including two instruments：QX200 droplets generate instrument and QX200 droplets analyzer).Used in embodiment
Heat-sealing instrument is PX1TMPCR seals instrument, and it is DG8 that card, which occurs, for droplet used in embodimentTMCard (8 × 3), embodiment occur for droplet
In used base to occur card base, droplet used occurs oil and oily (being used for probe) occurs for droplet in embodiment, is
Bio-Rad Laboratories, Inc. product.2 × ddPCR Supermix for Probes, it is Bio-Rad
Laboratories, Inc. product.
Porcine reproductive and respiratory syndrome virus american type strain used is in embodimentVR-2332TM,
Serial No. U87392 in GENBANK.Porcine reproductive and respiratory syndrome virus Europe class strain used is LV in embodiment
(Lelystad virus), the Serial No. M96262 in GENBANK.Pig parvoviral strain used is in embodiment
NADL-2,VR-742TM, the Serial No. KF913351 in GENBANK.2 type pig circular ring virus used in embodiment
Strain is 08TJ, the Serial No. HQ395021 in GENBANK.CSFV strain used is C- in embodiment
Strain, the sequence number Z46258 in GENBANK.
Porcine pseudorabies virus (porcine pseudorabies virus, PRV), also known as porcine herpesvirus 1 (Suid
herpesvirus 1).Porcine pseudorabies virus vaccine strain used is Bartha-K61 strains in embodiment.In embodiment
Porcine pseudorabies virus street strain used is Qihe547 strains.
The design and screening of embodiment 1, primer and probe
First, for the design and screening of the primer and probe for detecting porcine pseudorabies virus
By a large amount of retrievals, analysis, comparison and preliminary experiment, design and preliminary screening obtains four primers and two
Probe, nucleotide sequence are as follows：
GB-F1 (sequence 1 of sequence table)：5’-GTAGTACACGTACYCGCTCCCCA-3’；
GB-R1 (sequence 2 of sequence table)：5’-TCGCBGAGCTGGCCCTCGATCA-3’；
GB-P1 (sequence 3 of sequence table)：5’-TCGCGCGAGATGARGAGCTCGT；
gB-F2：5’-AGCTGTAGTCSTCGTAGTACACGT-3’；
gB-R2：5’-GAGCTGGCCVTCGATCACGCCCG-3’；
gB-P2：5’-AGGTCGCKCGAGATGAGGAGCT；
In above nucleotide sequence, Y represents C or T, B represent G, T or C, R represents A or G, S represent G or C, V represent G, A or
C, K represent G or T.
GB-F1 and gB-F2 is sense primer, and gB-R1 and gB-R2 are anti-sense primer, and gB-P1 and gB-P2 are to visit
Pin.GB-P1 5 ' end mark fluorophor FAM, 3 ' end mark fluorescent quenching group BHQ1.GB-P2 5 ' end marks
Fluorophor FAM, 3 ' end mark fluorescent quenching group BHQ1.Fluorophor FAM shows blue-fluorescence.
2nd, for the screening for the primed probe group for detecting porcine pseudorabies virus
Four primers and two probes progress random combines that step 1 is screened to obtain, form eight primed probe groups：
The primed probe group gB-F1R1P1 being made up of gB-F1, gB-R1 and gB-P1, the primer being made up of gB-F1, gB-R1 and gB-P2
Probe groups gB-F1R1P2, the primed probe group gB-F1R2P1 being made up of gB-F1, gB-R2 and gB-P1, by gB-F1, gB-R2 and
Primed probe group gB-F1R2P2, the primed probe group gB-F2R1P1 being made up of gB-F2, gB-R1 and gB-P1 of gB-P2 compositions,
The primed probe group gB-F2R1P2 being made up of gB-F2, gB-R1 and gB-P2, the primer being made up of gB-F2, gB-R2 and gB-P1
Probe groups gB-F2R2P1, the primed probe group gB-F2R2P2 being made up of gB-F2, gB-R2 and gB-P2.
1st, porcine pseudorabies virus vaccine strain is taken, extracts genomic DNA.
2nd, using the genomic DNA that step 1 obtains as template, each primed probe group is respectively adopted and carries out real time fluorescent quantitative
PCR。
QPCR reaction system (20 μ L)：Probe qPCR Master Mix, 10 μM of upstreams are drawn
The μ L of thing solution 1.2,10 μM of μ L of anti-sense primer solution 1.2,10 μM of μ L of probe solution 0.6, the μ L of template 2, surplus is water.
QPCR response procedures：95℃2min；95 DEG C of 15s, 60 DEG C of 1min, 40 circulations.
Real-time fluorescence quantitative PCR is carried out using primed probe group gB-F1R1P1, △ Rn peak values are 1000000.Using in addition
Seven primed probe groups carry out real-time fluorescence quantitative PCR, and △ Rn peak values are 300000-500000.Using primed probe group gB-
The signal value of F1R1P1 amplification curve is most strong.
3rd, for differentiating that the primer and probe of porcine pseudorabies virus vaccine strain and porcine pseudorabies virus street strain is set
Meter and screening
By a large amount of retrievals, analysis, comparison and preliminary experiment, design and preliminary screening obtains four primers and two
Probe, nucleotide sequence are as follows：
GE-F1 (sequence 4 of sequence table)：5’-TCCACTCBCAGCTCTTCTCGCC-3’；
GE-R1 (sequence 5 of sequence table)：5’-TCGCGCGAGAACTTTAMCGCCA-3’；
GE-P1 (sequence 6 of sequence table)：5’-CGCCCGKGGACACGTTCGA-3’；
gE-F2：5’-ACGCGCTCGKCTTCCACTCGCA-3’；
gE-R2：5’-ACATGBGCGACTCGCGCGAGA-3’；
gE-P2：5’-AGCTCTTCTCGCHCGGGGACA-3’；
In above nucleotide sequence, B represents G, T or C, and M represents A or C, K represent G or T, H represent A, T or C.
GE-F1 and gE-F2 is sense primer, and gE-R1 and gE-R2 are anti-sense primer, and gE-P1 and gE-P2 are to visit
Pin.GE-P1 5 ' end mark fluorophor HEX, 3 ' end mark fluorescent quenching group BHQ1.GE-P2 5 ' end marks
Fluorophor HEX, 3 ' end mark fluorescent quenching group BHQ1.Fluorophor HEX shows green fluorescence.
4th, for the sieve for the primed probe group for differentiating porcine pseudorabies virus vaccine strain and porcine pseudorabies virus street strain
Choosing
Four primers and two probes progress random combines that step 3 is screened to obtain, form eight primed probe groups：
The primed probe group gE-F1R1P1 being made up of gE-F1, gE-R1 and gE-P1, the primer being made up of gE-F1, gE-R1 and gE-P2
Probe groups gE-F1R1P2, the primed probe group gE-F1R2P1 being made up of gE-F1, gE-R2 and gE-P1, by gE-F1, gE-R2 and
Primed probe group gE-F1R2P2, the primed probe group gE-F2R1P1 being made up of gE-F2, gE-R1 and gE-P1 of gE-P2 compositions,
The primed probe group gE-F2R1P2 being made up of gE-F2, gE-R1 and gE-P2, the primer being made up of gE-F2, gE-R2 and gE-P1
Probe groups gE-F2R2P1, the primed probe group gE-F2R2P2 being made up of gE-F2, gE-R2 and gE-P2.
1st, porcine pseudorabies virus street strain is taken, extracts genomic DNA.
2nd, using the genomic DNA that step 1 obtains as template, each primed probe group is respectively adopted and carries out real time fluorescent quantitative
PCR。
QPCR reaction system (20 μ L)：Probe qPCR Master Mix, 10 μM of upstreams are drawn
The μ L of thing solution 1.2,10 μM of μ L of anti-sense primer solution 1.2,10 μM of μ L of probe solution 0.6, the μ L of template 2, surplus is water.
QPCR response procedures：95℃2min；95 DEG C of 15s, 60 DEG C of 1min, 40 circulations.
Real-time fluorescence quantitative PCR is carried out using primed probe group gE-F1R1P1, △ Rn peak values are 1000000.Using in addition
Seven primed probe groups carry out real-time fluorescence quantitative PCR, and △ Rn peak values are 300000-500000.Using primed probe group gE-
F1R1P1 carries out real-time fluorescence quantitative PCR, and the signal value of amplification curve is most strong.
The optimization of correlated response parameter in embodiment 2, digital pcr
First, the optimization of annealing temperature
1st, porcine pseudorabies virus street strain is taken, extracts genomic DNA.
2nd, using the genomic DNA that step 1 obtains as template, using primed probe group gB-F1R1P1 and primed probe group gE-
The primer combination of F1R1P1 compositions carries out digital pcr.
(1) it is formulated as follows system (20 μ L)：10 μ L 2 × ddPCR Supermix for Probes, 1 μ L gB-F1,1 μ L
GB-R1,0.2 μ L gB-P1,1 μ L gE-F1,1 μ L gE-R1,0.2 μ L gE-P1,2 μ L templates, 3.6 μ L RNase Free
dH2O.In system, gB-F1 concentration is 0.5 μM, and gB-R1 concentration is 0.5 μM, and gB-P1 concentration is 0.1 μM, gE-F1's
Concentration is 0.5 μM, and gE-R1 concentration is 0.5 μM, and gE-P1 concentration is 0.1 μM.
(2) droplet is taken to occur to fix due on base, every hole adds 20 μ L steps (1) and prepared in 8 holes of a middle row
System, add 70 μ L droplets per hole in 8 holes of bottom row and oil occur, then put the base for being fixed with droplet card
Be placed in droplet generation instrument and form droplet (droplet is created on droplet and occurred in 8 holes of one row of card top).
(3) 96 orifice plates (being named as 96 orifice plates I) are taken, after completing step (2), occur the 8 of one row of card top from droplet
In individual hole, each hole takes 40 μ L, is added in a manner of one-to-one in 8 holes of 96 orifice plates I, and sealer is carried out with heat-sealing instrument.Take
One 96 orifice plate (being named as 96 orifice plates II), after completing step (2), from 8 holes that one row of card top occurs for droplet, Mei Gekong
40 μ L are taken, are added in a manner of one-to-one in 8 holes of 96 orifice plates II, sealer is carried out with heat-sealing instrument.Take 96 orifice plates (life
Entitled 96 orifice plate III), after completing step (2), from 8 holes that one row of card top occurs for droplet, each hole takes 40 μ L, with one by one
Corresponding mode is added in 8 holes of 96 orifice plates III, and sealer is carried out with heat-sealing instrument.96 orifice plates are taken (to be named as 96 orifice plates
IV) after, completing step (2), from 8 holes that one row of card top occurs for droplet, each hole takes 40 μ L, in a manner of one-to-one
Add in 8 holes of 96 orifice plates IV, sealer is carried out with heat-sealing instrument.96 orifice plates (being named as 96 orifice plates V) are taken, complete step
(2) after, from 8 holes that one row of card top occurs for droplet, each hole takes 40 μ L, and 96 orifice plates V are added in a manner of one-to-one
8 holes in, with heat-sealing instrument carry out sealer.96 orifice plates (being named as 96 orifice plates VI) are taken, after completing step (2), from droplet
In 8 holes that one row of card top occurs, each hole takes 40 μ L, is added in a manner of one-to-one in 8 holes of 96 orifice plates VI, uses
Seal instrument and carry out sealer.
(4) each 96 orifice plate for completing step (3) is placed in different PCR instruments, enters performing PCR amplification.
PCR amplification programs：95 DEG C of pre-degeneration 10min；94 DEG C of denaturation 30sec, annealing 60sec, totally 40 circulations；98℃
10min.The warming and cooling rate of use is arranged to 2.5 DEG C/sec.
PCR instrument where 96 orifice plate, I to 96 orifice plate VI, sets gradually following annealing temperature：52.0℃、54.0℃、55.0
℃、56.0℃、58.0℃、60.0℃。
(5) after completing step (4), 96 orifice plates is taken, are detected in droplet analyzer, the droplet for showing blue-fluorescence is
The positive droplet (cannot distinguish between vaccine strain and street strain) of porcine pseudorabies virus, the droplet for showing green fluorescence is pseudorabies
The positive droplet (being not porcine pseudorabies virus vaccine strain) of sick field virus.
Using 52.0 DEG C of annealing temperature, in every microlitre of template, the detected value of porcine pseudorabies virus is 1280copies,
The detected value of porcine pseudorabies virus street strain is 1180copies.Using 54.0 DEG C of annealing temperature, in every microlitre of template, pig
The detected value of pseudorabies virus is 1320copies, and the detected value of porcine pseudorabies virus street strain is 1240copies.Adopt
With 55.0 DEG C of annealing temperature, in every microlitre of template, the detected value of porcine pseudorabies virus is 1680copies, porcine pseudorabies
The detected value of field virus is 1560copies.Using 56.0 DEG C of annealing temperature, in every microlitre of template, porcine pseudorabies disease
The detected value of poison is 1590copies, and the detected value of porcine pseudorabies virus street strain is 1420copies.Using 58.0 DEG C
Annealing temperature, in every microlitre of template, the detected value of porcine pseudorabies virus is 1550copies, porcine pseudorabies virus street strain
Detected value be 1440copies.Using 60.0 DEG C of annealing temperature, in every microlitre of template, the detected value of porcine pseudorabies virus
For 1490copies, the detected value of porcine pseudorabies virus street strain is 1380copies.
It is closest with actual value using 55.0 DEG C of annealing temperature, detected value.
As a result show, annealing temperature can select 55-58 DEG C, and optimal annealing temperature is 55 DEG C.
2nd, the optimization of primer, concentration and probe concentration
1st, porcine pseudorabies virus street strain is taken, extracts genomic DNA.
2nd, using the genomic DNA that step 1 obtains as template, using primed probe group gB-F1R1P1 and primed probe group gE-
The primer combination of F1R1P1 compositions carries out digital pcr.
(1) different systems are prepared, are specifically shown in Table 1 (numeral in form is the addition volume of each component, and unit is μ L).
Sense primer concentration, anti-sense primer concentration and concentration and probe concentration for preparing system are 10 μM.
Table 1
(2) droplet is taken to occur to fix due on base, every hole adds 20 μ L steps (1) and prepared in 8 holes of a middle row
System, add 70 μ L droplets per hole in 8 holes of bottom row and oil occur, then put the base for being fixed with droplet card
Be placed in droplet generation instrument and form droplet (droplet is created on droplet and occurred in 8 holes of one row of card top).
(3) 96 orifice plates are taken, after completing step (2), from 8 holes that one row of card top occurs for droplet, each hole takes 40 μ L,
Added in a manner of one-to-one in 8 holes of 96 orifice plates, sealer is carried out with heat-sealing instrument.
(4) each 96 orifice plate for completing step (3) is placed in different PCR instruments, enters performing PCR amplification.
PCR amplification programs：95 DEG C of pre-degeneration 10min；94 DEG C of denaturation 30sec, 55 DEG C of annealing 60sec, totally 40 circulations；98
℃10min.The warming and cooling rate of use is arranged to 2.5 DEG C/sec.
(5) after completing step (4), 96 orifice plates is taken, are detected in droplet analyzer, the droplet for showing blue-fluorescence is
The positive droplet (cannot distinguish between vaccine strain and street strain) of porcine pseudorabies virus, the droplet for showing green fluorescence is pseudorabies
The positive droplet (being not porcine pseudorabies virus vaccine strain) of sick field virus.
Using system 1, in every microlitre of template, the detected value of porcine pseudorabies virus is 1060copies, porcine pseudorabies
The detected value of field virus is 1100copies.Using system 2, in every microlitre of template, the detected value of porcine pseudorabies virus
For 1130copies, the detected value of porcine pseudorabies virus street strain is 1120copies.Using system 3, in every microlitre of template,
The detected value of porcine pseudorabies virus is 1220copies, and the detected value of porcine pseudorabies virus street strain is 1180copies.
Using system 4, in every microlitre of template, the detected value of porcine pseudorabies virus is 1400copies, the wild poison of porcine pseudorabies virus
The detected value of strain is 1390copies.
Using system 4, detected value is closest with actual value.
As a result show, in reaction system, optimal primed probe concentration is：gB-F1 0.9μM、gB-R1 0.9μM、gB-
0.9 μM of 0.2 μM of P1, gE-F1,0.9 μM of gE-R1,0.2 μM of gE-P1.
The preparation of embodiment 3, kit
First, the preparation of each reagent
Solution A is one-step method ddPCR sonde method premixed liquids.The composition of every 900 μ L solution As is as follows：500μL 2×ddPCR
Supermix for Probes, 90 μ L gB-F1 solution (upper gB-F1 concentration is 10 μM in gB-F1 solution), 90 μ L gB-R1
Solution (gB-R1 concentration is 10 μM in gB-R1 solution), (gB-P1 concentration is 10 μ to 20 μ L gB-P1 solution in gB-P1 solution
M), 90 μ L gE-F1 solution (upper gE-F1 concentration is 10 μM in gE-F1 solution), 90 μ L gE-R1 solution are (in gE-R1 solution
GE-R1 concentration is 10 μM), 20 μ LgE-P1 solution (gE-P concentration is 10 μM in gE-P1 solution).
Solution B is that oil occurs for droplet.
Solution C is positive control.The preparation method of solution C：The genomic DNA of porcine pseudorabies virus street strain is extracted,
Using Tris-EDTA buffer solutions (pH8.0,0.01M) dilute, make its concentration for 10000 copy numbers/microlitre.
Solution D is negative control.Solution D is RNase Free dH2O。
2nd, the assembling of droplet digital pcr absolute quantitation detection kit
Kit forms are as follows：Solution A, solution B, solution C, solution D, are independently packed.
3rd, the application method of kit
Sample to be tested is to be measured viral or porcine tissue to be measured, porcine tissue concretely pig lung.
1st, the DNA of sample to be tested is extracted, using DNA or its dilution as template solution.When sample to be tested is to be measured viral,
Extract genomic DNA.When sample to be tested is porcine tissue to be measured, STb gene is extracted.
2nd, 18 μ L solution As are taken, add 2 μ L template solutions.Template solution is replaced as at negative control by the use of isometric solution D
Reason.Template solution is replaced as positive control processing by the use of isometric solution C.
3rd, take droplet to occur to fix due on base, the body that 20 μ L steps 2 are prepared is added per hole in 8 holes of a middle row
It is to add 70 μ L solution Bs in 8 holes of bottom row per hole, the base for being fixed with droplet card is then positioned over droplet
Droplet is formed in generation instrument (droplet is created on droplet and occurred in 8 holes of one row of card top).
4th, 96 orifice plates are taken, after completing step 3, from 8 holes that one row of card top occurs for droplet, each hole takes 40 μ L, with
One-to-one mode is added in 8 holes of 96 orifice plates, and sealer is carried out with heat-sealing instrument.
5th, 96 orifice plates for completing step 4 are placed in PCR instrument, enter performing PCR amplification.
PCR amplification programs：95 DEG C of pre-degeneration 10min；94 DEG C of denaturation 30sec, 55 DEG C of annealing 60sec, totally 40 circulations；98
℃10min.The warming and cooling rate of use is arranged to 2.5 DEG C/sec.
6th, after completing step 5,96 orifice plates is taken, are detected in droplet analyzer, the droplet for showing blue-fluorescence is pig
The positive droplet (cannot distinguish between vaccine strain and street strain) of pseudorabies virus, the droplet for showing green fluorescence is porcine pseudorabies
The positive droplet (being not porcine pseudorabies virus vaccine strain) of field virus.
Meet following four condition stub credible result simultaneously：1. in every microlitre of positive control, porcine pseudorabies virus
Detected value is 10000 ± 100 copy numbers；2. in every microlitre of positive control, the detected value of porcine pseudorabies virus street strain is
10000 ± 100 copy numbers；3. in every microlitre of negative control, the detected value of porcine pseudorabies virus is 0；4. every microlitre of feminine gender is right
According in, the detected value of porcine pseudorabies virus street strain is 0.If the micro- of the porcine pseudorabies virus positive is detected in template
Drop, illustrates to contain porcine pseudorabies virus in sample to be tested, can obtain copying for porcine pseudorabies virus according to positive number of droplets
Shellfish number；If not detecting the positive droplet of porcine pseudorabies virus in template, illustrate not contain pig puppet in sample to be tested mad
Dog disease virus.If detecting the positive droplet of porcine pseudorabies virus street strain in template, illustrate to contain pig in sample to be tested
Pseudorabies virus street strain, the copy number of porcine pseudorabies virus street strain can be obtained according to positive number of droplets；If mould
The positive droplet of porcine pseudorabies virus street strain is not detected in plate, illustrates not containing porcine pseudorabies disease in sample to be tested
Malicious street strain.
Embodiment 4, specific assay
Sample to be tested is respectively：Porcine pseudorabies virus vaccine strain, porcine reproductive and respiratory syndrome virus american type strain,
Porcine reproductive and respiratory syndrome virus Europe class strain, the strain of 3D4/21 pig pneumonocytes, pig parvoviral, 2 type pig circular ring virus, pig
Pestivirus.
The kit prepared using embodiment 3, is detected according to the application method of kit.
After porcine pseudorabies virus vaccine strain is detected, the positive droplet of porcine pseudorabies virus can be detected, not
Detect the positive droplet of porcine pseudorabies virus street strain.After other each samples to be tested are detected, pig puppet is not detected by
The positive droplet of hydrophobin, it is not detected by the positive droplet of porcine pseudorabies virus street strain.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, some improvements and modifications can also be made, these improvements and modifications
Also it should be regarded as protection scope of the present invention.
SEQUENCE LISTING
<110>China Animal Disease Control And Prevention Center
<120>The dual droplet digital pcr absolute quantitation detection kit of Pseudorabies virus gB, gE
<130> GNCYX171439
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (14)
<223> y =c or t
<400> 1
gtagtacacg tacycgctcc cca 23
<210> 2
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (5)
<223> b =g t or c
<400> 2
tcgcbgagct ggccctcgat ca 22
<210> 3
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (14)
<223> r =a or g
<400> 3
tcgcgcgaga tgargagctc gt 22
<210> 4
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (8)
<223> b =g t or c
<400> 4
tccactcbca gctcttctcg cc 22
<210> 5
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (17)
<223> m =a or c
<400> 5
tcgcgcgaga actttamcgc ca 22
<210> 6
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (7)
<223> k =g or t
<400> 6
cgcccgkgga cacgttcga 19
Claims (10)
1. a kind of primer combination of probe, it is made up of primed probe group gB-F1R1P1 and primed probe group gE-F1R1P1；
The primed probe group gB-F1R1P1 is made up of primer gB-F1, primer gB-R1 and probe gB-P1；
The primer gB-F1 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 of one or several nucleotides and/or missing and/or addition and with sequence 1 identical
The DNA molecular of function；
The primer gB-R1 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 of one or several nucleotides and/or missing and/or addition and with sequence 2 identical
The DNA molecular of function；
The probe gB-P1, an end have fluorescent reporter group, and another end has fluorescent quenching group, nucleotides
Sequence is following (a5) or (a6)：
(a5) as shown in the sequence 3 of sequence table；
(a6) as shown in by sequence 3 by the substitution of one or several nucleotides and/or missing and/or addition；
The primed probe group gE-F1R1P1 is made up of primer gE-F1, primer gE-R1 and probe gE-P1；
The primer gE-F1 is following (a1) or (a2)：
(a1) single strand dna shown in the sequence 4 of sequence table；
(a2) have by sequence 4 by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 4 identical
The DNA molecular of function；
The primer gE-R1 is following (a3) or (a4)：
(a3) single strand dna shown in the sequence 5 of sequence table；
(a4) have by sequence 5 by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 5 identical
The DNA molecular of function；
The probe gE-P1, an end have fluorescent reporter group, and another end has fluorescent quenching group, nucleotides
Sequence is following (a5) or (a6)：
(a5) as shown in the sequence 6 of sequence table；
(a6) as shown in by sequence 6 by the substitution of one or several nucleotides and/or missing and/or addition；
The probe gB-P1 and probe gE-P1 has the fluorescent reporter group of different colours.
2. the application of primer combination of probe described in claim 1, for any one in following (b1) to (b6)：
(b1) identify whether virus to be measured is porcine pseudorabies virus street strain or porcine pseudorabies virus vaccine strain；
(b2) prepare for identifying whether virus to be measured is porcine pseudorabies virus street strain or porcine pseudorabies virus vaccine strain
Kit；
(b3) whether detection sample to be tested contains porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine strain；
(b4) prepare for detecting whether sample to be tested contains porcine pseudorabies virus street strain and/or porcine pseudorabies virus epidemic disease
The kit of seedling strain；
(b5) content of porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine strain in sample to be tested is detected；
(b6) prepare and contain for detecting porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine strain in sample to be tested
The kit of amount.
3. a kind of kit, including primer combination of probe described in claim 1；The function of the kit be following (c1) extremely
(c3) any one in：
(c1) identify whether virus to be measured is porcine pseudorabies virus street strain or porcine pseudorabies virus vaccine strain；
(c2) whether detection sample to be tested contains porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine strain；
(c3) content of porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine strain in sample to be tested is detected.
4. one kind detection it is to be measured virus whether be whether be porcine pseudorabies virus street strain or porcine pseudorabies virus vaccine strain
Method, comprise the following steps：Using viral genomic DNA to be measured as template, using primer combination of probe described in claim 1
Carry out digital pcr；If testing result of the virus based on the corresponding fluorophors of probe gB-P1 to be measured is virus positive, to be measured
For or candidate be porcine pseudorabies virus, if it is to be measured virus the testing result based on the corresponding fluorophors of probe gB-P1 be
Virus negative, to be measured is or candidate is non-porcine pseudorabies virus；If or candidate be porcine pseudorabies virus disease to be measured
The malicious testing result based on the corresponding fluorophors of probe gE-P1 is that virus positive, to be measured is or candidate is porcine pseudorabies disease
Malicious street strain, if or candidate for porcine pseudorabies virus it is to be measured virus based on the corresponding fluorophors of probe gE-P1
Testing result is that virus negative, to be measured is or candidate is porcine pseudorabies virus vaccine strain.
5. it is a kind of detection sample to be tested whether the side containing porcine pseudorabies virus street strain or porcine pseudorabies virus vaccine strain
Method, comprise the following steps：Using the STb gene of sample to be tested as template, numeral is carried out using primer combination of probe described in claim 1
PCR；If testing result of the sample to be tested based on the corresponding fluorophors of probe gB-P1 contains pig puppet for positive, sample to be tested
Hydrophobin, if testing result of the sample to be tested based on the corresponding fluorophors of probe gB-P1 for negative, sample to be tested not
Contain porcine pseudorabies virus；If the sample to be tested containing porcine pseudorabies virus is based on the corresponding fluorescent bases of probe gE-P1
The testing result of group contains porcine pseudorabies virus street strain for positive, sample to be tested, if containing porcine pseudorabies virus
Testing result of the sample to be tested based on the corresponding fluorophors of probe gE-P1 contains porcine pseudorabies disease for negative, sample to be tested
Toxic vaccine strain.
6. a kind of detect the content of porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine strain in sample to be tested
Method, comprise the following steps：Using the STb gene of sample to be tested as template, line number is entered using primer combination of probe described in claim 1
Word PCR；The content of porcine pseudorabies virus in sample to be tested is obtained according to the quantity of porcine pseudorabies virus positive droplet, pig is pseudo-
Hydrophobin positive droplet is that the testing result based on the corresponding fluorophors of probe gB-P1 is positive droplet；According to pig
The quantity of pseudorabies virus street strain obtains porcine pseudorabies virus street strain and/or porcine pseudorabies virus in sample to be tested
The content of vaccine strain, porcine pseudorabies virus street strain positive droplet are the detection based on the corresponding fluorophors of probe gE-P1
As a result it is positive droplet.
7. a kind of be used for by digital pcr detection porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine strain
Premixed liquid, wherein containing the primer gB-F1 described in claim 1, primer gB-R1, probe gB-P1, primer gE-F1, primer
GE-R1 and probe gE-P1；In the premixed liquid, primer gB-F1 concentration is 1 μM, and primer gB-R1 concentration is 1 μM, probe
GB-P1 concentration is 2/9 μM, and primer gE-F1 concentration is 1 μM, and primer gE-R1 concentration is 1 μM, probe gE-P1 concentration
For 2/9 μM.
8. a kind of be used for by digital pcr detection porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine strain
Oil occurs for kit, including premixing described in claim 7 and droplet.
9. kit as claimed in claim 8, it is characterised in that：The kit also includes negative control and positive control；
The negative control is RNase Free dH2O；The positive control is the genome containing porcine pseudorabies virus street strain
DNA solution.
10. the application of kit described in kit described in premixed liquid described in claim 7 or claim 8 or claim 9, is
Any one in (c1) to (c3) as follows：
(c1) identify whether virus to be measured is porcine pseudorabies virus street strain or porcine pseudorabies virus vaccine strain；
(c2) whether detection sample to be tested contains porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine strain；
(c3) content of porcine pseudorabies virus street strain and/or porcine pseudorabies virus vaccine strain in sample to be tested is detected.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710845509.XA CN107460194A (en) | 2017-09-19 | 2017-09-19 | The dual droplet digital pcr absolute quantitation detection kit of Pseudorabies virus gB, gE |
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| CN201710845509.XA CN107460194A (en) | 2017-09-19 | 2017-09-19 | The dual droplet digital pcr absolute quantitation detection kit of Pseudorabies virus gB, gE |
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| CN107460194A true CN107460194A (en) | 2017-12-12 |
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| CN201710845509.XA Pending CN107460194A (en) | 2017-09-19 | 2017-09-19 | The dual droplet digital pcr absolute quantitation detection kit of Pseudorabies virus gB, gE |
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| CN (1) | CN107460194A (en) |
Citations (4)
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|---|---|---|---|---|
| CN103509877A (en) * | 2012-06-18 | 2014-01-15 | 武汉中博生物股份有限公司 | Fluorescence quantitative PCR kit used for detecting PRV, and application thereof |
| CN104561374A (en) * | 2014-12-18 | 2015-04-29 | 河南省动物疫病预防控制中心 | Detection reagent and method for identifying porcine pseudorabies virus vaccine strain and wild strain |
| CN106048094A (en) * | 2016-07-19 | 2016-10-26 | 金宇保灵生物药品有限公司 | Wild porcine pseudorabies strain and gene-deleted strain dual real-time fluorescence quantification PCR detection kit, primers and probe |
| 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 |
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2017
- 2017-09-19 CN CN201710845509.XA patent/CN107460194A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103509877A (en) * | 2012-06-18 | 2014-01-15 | 武汉中博生物股份有限公司 | Fluorescence quantitative PCR kit used for detecting PRV, and application thereof |
| CN104561374A (en) * | 2014-12-18 | 2015-04-29 | 河南省动物疫病预防控制中心 | Detection reagent and method for identifying porcine pseudorabies virus vaccine strain and wild strain |
| CN106048094A (en) * | 2016-07-19 | 2016-10-26 | 金宇保灵生物药品有限公司 | Wild porcine pseudorabies strain and gene-deleted strain dual real-time fluorescence quantification PCR detection kit, primers and probe |
| 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 |
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| MA ET AL: "Development of real-time polymerase chain reaction assays for rapid detection and differentiation of wild-type pseudorabies and gene-deleted vaccine viruses", 《J VET DIAGN INVEST》 * |
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Application publication date: 20171212 |