CN106636460A - Nucleic acid combination for detecting pseudorabies virus and application of nucleic acid combination, and kit and method for detecting pseudorabies virus - Google Patents
Nucleic acid combination for detecting pseudorabies virus and application of nucleic acid combination, and kit and method for detecting pseudorabies virus Download PDFInfo
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Abstract
The invention provides a nucleic acid combination for detecting pseudorabies virus and application of the nucleic acid combination, and a kit and a method for detecting the pseudorabies virus, and relates to the technical field of pseudorabies virus detection. The nucleic acid combination for detecting the pseudorabies virus comprises a first primer pair designed according to a conserved sequence of a gB gene of the pseudorabies virus, and a first probe, wherein the first primer pair comprises two primers, and the base sequences of the two primers are respectively as shown in SEQ ID No. 1 and SEQ ID No. 2; the base sequence of the first probe is as shown in SEQ ID No. 3. After the nucleic acid combination is adopted, rabies virus can be rapidly and conveniently detected on a digital polymerase chain reaction (PCR) platform, samples with low rabies virus content can be detected, and missed detection is avoided; the nucleic acid combination has the characteristics of being high in sensitivity, high in specificity, high in detection rate, and the like.
Description
Technical field
The present invention relates to pseudorabies virus detection technique field, in particular to a kind of detection pseudorabies virus
Nucleic acid combinations and its application, kit and method.
Background technology
At present, pseudorabies virus (pseudorabies, PRV) category herpetoviridae (Herpesvirdae) a- blister sores
Malicious subfamily (Alpherpesvirinae), is a kind of with farrowing sow miscarriage, stillborn foetus, weak tire, mummy tire, and newborn piglet is sent out
The infectious disease that heat, nervous symptoms, paralysis, exhaustion death are characterized, its death rate is up to 100%.Pseudoabies is harm pig
One of infectious disease of industry most serious, to pig industry huge economic loss is caused, and the disease is widely present in China and causes huge
Big loss.
At present the detection method in terms of nucleic acid is mainly normal PCR, quantitative fluorescent PCR and nucleic acid hybridization technique etc..It is fixed
Amount or semiquantitative PCR method have had the research report of many, often also show that good effect.However, for puppet
The detection of nucleic acids of hydrophobin, is largely dependent upon the collection position of sample.Sample is such as gathered just in tonsillotome, three
The high positions of viral level such as fork neuromere, schneiderian membrane can then cause detection smooth, and recall rate is high.But, in actual detection
In, all it is that tester samples in person when not all, some positions such as gasserian ganglion is also to be difficult precisely collection, if inspection
The sample for using the viral levels such as blood few during survey, it is possible to missing inspection occurs.
The content of the invention
The first object of the present invention is to provide a kind of Nucleic acid combinations of detection pseudorabies virus, and the Nucleic acid combinations include
The primer and probe of detection pseudorabies virus, using digital pcr technology for detection the pseudorabies virus of low content in sample is gone out,
Avoid missing inspection.
The second object of the present invention is the Nucleic acid combinations for providing above-mentioned detection pseudorabies virus in detection pseudoabies
Application in virus, improves the sensitivity and specificity of detection, it is to avoid missing inspection.
The third object of the present invention is to provide a kind of kit of detection pseudorabies virus, and the reagent includes above-mentioned core
Acid combination, the kit can be used to detect the sample of low pseudorabies virus content, it is to avoid missing inspection, and it has, and sensitivity is good, spy
Different in nature strong, detection speed is fast, it is easy to operate the features such as.
The fourth object of the present invention is to provide a kind of method of detection pseudorabies virus, and the detection method is with above-mentioned
Nucleic acid combinations, with reference to digital round pcr, can detect have as detection basis to the sample of pseudorabies virus content
Sensitivity is high, high specificity feature.
What embodiments of the invention were realized in：
A kind of Nucleic acid combinations of detection pseudorabies virus, it includes the first primer pair and corresponding with the first primer pair
First probe, the first primer pair includes base sequence primer respectively as shown in SEQ ID NO.1-2, the base sequence of the first probe
Row as shown in SEQ ID NO.3, the first probe 5 ' end fluorescent reporter groups be HEX or VIC, the first probe 3 ' hold quench
The group that goes out is BHQ or TAMRA.
Application of the Nucleic acid combinations of detection pseudorabies virus described above in detection pseudorabies virus.
A kind of kit of detection pseudorabies virus, it includes the Nucleic acid combinations of above-mentioned detection pseudorabies virus.
A kind of method of detection pseudorabies virus, it includes：
The first primer pair and first probe corresponding with the first primer pair are added toward PCR system；
PCR system is formed into droplet, and carries out droplet digital pcr；
Wherein, the first primer pair includes base sequence respectively as shown in SEQ ID NO.1-2, the base sequence of the first probe
As shown in SEQ ID NO.3, the first probe 5 ' end fluorescent reporter groups be HEX or VIC, the first probe 3 ' end be quenched
Group is BHQ or TAMRA.
A kind of Nucleic acid combinations of detection pseudorabies virus of the embodiment of the present invention and its application, kit and method
Beneficial effect is：The Nucleic acid combinations of the detection pseudorabies virus of the present invention are sick according to pseudoabies with based on data PCR platform
The conserved sequence of the gB genes of poison is designed, and the Nucleic acid combinations include base sequence respectively as shown in SEQ ID NO.1-2
First probe of first primer pair and base sequence of primer as shown in SEQ ID NO.1-2, so, the Nucleic acid combinations can counted
By the detection to gB genes on word PCR platforms, the detection to pseudorabies virus is realized, avoided Lou during detection
Inspection, improves recall rate, the characteristics of it has sensitivity high, high specificity.
Description of the drawings
In order to be illustrated more clearly that the technical scheme of the embodiment of the present invention, below will be attached to what is used needed for embodiment
Figure is briefly described, it will be appreciated that the following drawings illustrate only certain embodiments of the present invention, thus be not construed as it is right
The restriction of scope, for those of ordinary skill in the art, on the premise of not paying creative work, can be with according to this
A little accompanying drawings obtain other related accompanying drawings.
Fig. 1 for the embodiment of the present invention 4 standard plasmid by detecting the calibration curve that obtains after 10 times of gradient dilutions；
Fig. 2 for the embodiment of the present invention 4 standard plasmid by detecting the calibration curve that obtains after 2 times of gradient dilutions；
Fig. 3 is the specific detection result of the test of the embodiment of the present invention 5；
Fig. 4 is the fluorescence signal testing result of the experimental group and control group of the embodiment of the present invention 7 in FAM passages；
Fig. 5 is the fluorescence signal testing result of the experimental group and control group of the embodiment of the present invention 7 in HEX passages.
Specific embodiment
To make purpose, technical scheme and the advantage of the embodiment of the present invention clearer, below will be in the embodiment of the present invention
Technical scheme be clearly and completely described.Unreceipted actual conditions person, builds according to normal condition or manufacturer in embodiment
The condition of view is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can pass through the conventional product that commercially available purchase is obtained
Product.
The Nucleic acid combinations and its application, kit and method of a kind of detection pseudorabies virus of the invention are entered below
Row is illustrated.
Droplet digital pcr (Droplet digital PCR, ddPCR) be it is developed in recent years it is quick, accurately, can
Realize the PCR method of DNA absolute quantitations.Its principle is by being distributed in certain amount certain density DNA molecular is diluted to
Droplet in, make DNA molecular number in most of droplet be 1 or 0, then by PCR amplifications and the accumulative reading of fluorescence signal
Take positive droplet number, calculate further according to Poisson distribution (existing digital pcr instrument be substantially all automatic corresponding software can from
It is dynamic to calculate DNA molecular number) DNA molecular number in sample.The method need not prepare calibration curve without the need for reference gene, micro
Good Detection results are shown in the detection of nucleic acid samples.
Based on this, inventor has been designed out by the conserved sequence of the gB genes to pseudorabies virus and can be used for
The Nucleic acid combinations of the detection pseudorabies virus of ddPCR platforms, it includes：First primer pair and corresponding with the first primer pair
First probe, the first primer pair includes base sequence primer respectively as shown in SEQ ID NO.1-2, the base sequence of the first probe
Row as shown in SEQ ID NO.3, the first probe 5 ' end fluorescent reporter groups be HEX or VIC, the first probe 3 ' hold quench
The group that goes out is BHQ or TAMRA.
Specifically, the first primer pair includes upstream primer gB-F and downstream primer gB-R, the base sequence of upstream primer gB-F
As shown in SEQ ID NO.1, the base sequence of downstream primer gB-R is as shown in SEQ ID NO.2 for row.
By above-mentioned Nucleic acid combinations, by the detection of gB genes, the detection to pseudorabies virus is realized, it has higher
Sensitivity and specificity, pseudorabies virus can be detected from low pseudorabies virus content sample, be effectively prevented from pass
The detection leakage phenomenon that system detection method occurs.
Additionally, gE gene delections are all had according to the gene-deleted vaccine of great majority commercialization pseudorabies virus, some
Polygene deletion vaccine is also situations such as TK or gI genes are lacked on the basis of gE gene delections again.The present inventor is also directed to puppet
The gE genes of hydrophobin have devised corresponding primer and probe.To while accurate quick detection is realized to cause of disease
Can be street strain to the pseudorabies virus that detected, or the vaccine strain of gE disappearances is effectively distinguished, and makes testing result
It is more reliable accurate.
Preferably, the Nucleic acid combinations of the detection pseudorabies virus that the present invention is provided may also include：Second primer pair and with
The second corresponding probe of second primer pair.Second primer pair includes base sequence respectively as shown in SEQ ID NO.4-5, second
The base sequence of probe is as shown in SEQ ID NO.6.The fluorescent reporter group at 5 ' ends of the second probe is FAM, the second probe
The quenching group at 3 ' ends is BHQ or TAMRA.It should be noted that in this case, the fluorescent reporter gene of the first probe
Classification can be selected according to actual conditions, as long as ensureing that the classification of the fluorescent reporter gene of its first probe and the second probe is different
.The quenching group of both the first probe and the second probe then can be identical, it is also possible to differs.
Wherein, the second primer pair includes upstream primer gE-F and downstream primer gE-R.The base sequence of upstream primer gE-F
As shown in SEQ ID NO.4, the base sequence of downstream primer gE-R is as shown in SEQ ID NO.5.
, to gE genetic tests, it coordinates with the first primer pair and the first probe, forms two for second primer pair and the second probe
Weight ddPCR, non-specific amplification is not only avoided in the detection to pseudorabies virus simultaneously, and pseudorabies virus is capable of achieving again is
The differentiation of the vaccine strain of street strain or gE disappearances, also just says and detects that gB genes and gE genes have fluorescence signal for open country
Strain, detects that gB genes have fluorescence signal, gE genes not to have the fluorescence signal vaccine strain that then gE is lacked, and so can ensure that inspection
Survey result more accurately more credible.
Present invention also offers the Nucleic acid combinations of any one above-mentioned detection pseudorabies virus are in detection pseudoabies
Application in virus.It not only can be applied the Nucleic acid combinations of the detection pseudorabies virus in ddPCR platforms, can also be led to
Cross the modes such as quantitative fluorescent PCR, regular-PCR to realize the detection to detecting pseudorabies virus, as long as using present invention offer
Nucleic acid combinations be used for detect that pseudorabies virus belongs to protection scope of the present invention.
The kit of the detection pseudorabies virus that the present invention is provided includes any of the above-described kind of Nucleic acid combinations.
The kit can be used for whether containing pseudorabies virus in detection sample on ddPCR platforms, and while to inspection
The cause of disease for going out makes a distinction, i.e., the pseudorabies virus to being detected is the vaccine strain of street strain or gE gene delections, together
The characteristics of sample has good sensitivity, high specificity.
Preferably, the kit of the detection pseudorabies virus that the present invention is provided is additionally may included in and carries out matching somebody with somebody during ddPCR
The composition such as one or more in PCR reaction buffers, dNTP, Taq archaeal dna polymerase of set.
Additionally, the method for detecting pseudorabies virus that the present invention is provided is comprised the following steps：
(1) PCR reaction systems are prepared
Above-mentioned the first primer pair and first probe corresponding with the first primer pair are added toward PCR system, to carry out
PCR, realizes the high sensitivity detection to pseudorabies virus in sample.Wherein, PCR system includes PCR reaction mixtures and treats
The DNA profiling of test sample sheet.PCR reaction mixtures can be bought by market, also can voluntarily prepare, and PCR reaction mixtures are mainly wrapped
Include：The compositions such as PCR reaction buffers, dNTP, Taq archaeal dna polymerase.
Preferably, above-mentioned the second primer pair and corresponding with the second primer pair second can also be added in the PCR system
Probe.To carry out double PCR, the high sensitivity of pseudorabies virus detects that simultaneously it is right further to realize in realizing to sample
The classification of the pseudorabies virus for being detected detected, differentiation detect cause of disease be pseudorabies virus be street strain or gE
The vaccine strain of gene delection, so that result is more accurately and reliably.
(2) droplet is generated
PCR system is formed into droplet, and carries out droplet digital pcr.
Preferably, generating oil using droplet carries out droplet process formation droplet to PCR system, and droplet generates oil and PCR bodies
The volume ratio of system is 6.8～7.2:2.Preferably, droplet generation oil is with the volume ratio of PCR system:7:2..
Certainly, the present invention in other examples, it would however also be possible to employ by PCR reaction systems be distributed to ddPCR reaction core
Mode on piece forms droplet, however it is not limited to generated by the way of oil parcel using droplet.
Preferably, the PCR reaction conditions for carrying out the droplet digital pcr are：95 DEG C of denaturations 10min；95 DEG C of denaturation
30s, 50-60 DEG C of annealing 1min, 40 circulations；98 DEG C of 10min, 4 DEG C stop reaction.
It is highly preferred that the PCR reaction conditions for carrying out the droplet digital pcr are：95 DEG C of denaturations 10min；95 DEG C of changes
Property 30s, 58.3 DEG C annealing 1min, 40 circulation；98 DEG C of 10min, 4 DEG C stop reaction.
(3) detect
Corresponding fluorescence signal is detected on digital pcr instrument, testing result is obtained.
To sum up, the Nucleic acid combinations and its application, kit and method tool of the detection pseudorabies virus that the present invention is provided
There is very high sensitivity and relatively strong specificity, effectively the sample of low viral level can be detected, recall rate is high, while
The classification of detection cause of disease is distinguished, testing result more accurately and reliably, overcomes the defect of existing detection method, be pseudoabies disease
The detection of poison provides more scientific basis and support.
The feature and performance of the present invention are described in further detail with reference to embodiments.
Embodiment 1
Present embodiments provide detection pseudorabies virus Nucleic acid combinations, it include the first primer pair and with the first primer
To the first corresponding probe, and the second primer pair and second probe corresponding with the second primer pair.First primer pair and
Whether the first probe is designed according to the conserved sequence of the gB genes of pseudorabies virus, can be used to detect sample containing pseudo- mad dog
Virus.Second primer pair and the second probe are designed according to the conserved sequence of the gE genes of pseudorabies virus, are drawn by second
The addition of thing pair and the second probe, can cause while whether detection sample is containing pseudorabies virus, to further discriminate between institute
The pseudorabies virus for detecting is street strain (gB positive, gE positive), or the commercial vaccine strains that lack of gE (gB is positive
Property, gE are negative).
It should be noted that in other examples, it is wild poison detected pseudorabies virus is needed not distinguish between
In the case of strain or gE disappearance commercial vaccine strains.The Nucleic acid combinations of the detection pseudorabies virus of the present invention can be wrapped only
Include the first primer pair and the first probe, you can realize the Detection results to pseudorabies virus.
Specifically, the upstream primer gB-F of the first primer pair is：
5 '-TGAAGCGGTTCGTGATGG-3 ' (its base sequence is as shown in SEQ ID NO.1), under the first primer pair
Swimming primer gB-R is：
5 '-CCCCGCACAAGTTCAAGG-3 ' (its base sequence is as shown in SEQ ID NO.2).First probe gB-
Probe is：
HEX-5 '-CGCGTACGTGCTCCCGGACC-3 '-BHQ (its base sequence is as shown in SEQ ID NO.3).
Specifically, the upstream primer gE-F of the second primer pair is：5 '-CTTCCACTCGCAGCTCTTCTC-3 ' (its base
Sequence is as shown in SEQ ID NO.4), the downstream primer gE-R of the second primer pair is：5’-GTRAAGTTCTCGCGCGAGT-3’
(its base sequence is as shown in SEQ ID NO.5).Second probe gE-probe is：FAM-5’-TTCGACCTGATGCCGC-3’-
BHQ (its base sequence is as shown in SEQ ID NO.6).
To sum up, whether the Nucleic acid combinations that the present embodiment is provided can be by using double digital pcr technology, detecting in sample
Containing gB and gE gene elements, improve detection limit, prevent missing inspection, realize to pseudorabies virus quick detection, while by many
Weight PCR, while detection, it is street strain or gE disappearance commercial vaccine strains that cause of disease is examined in differentiation.
Embodiment 2
The kit of detection pseudorabies virus is present embodiments provided, the kit includes the nucleic acid that embodiment 1 is provided
Combination.The kit can be used for whether containing pseudorabies virus in detection sample on ddPCR platforms, and while to detection
Cause of disease makes a distinction, i.e. the pseudorabies virus to being detected is the vaccine strain of street strain or gE gene delections, and this reality
The kit for applying example offer has good sensitivity and specificity, and the virus quantity contained in sample can be avoided few and caused
Missing inspection situation occurs.
Certainly, in other examples, the kit of the detection pseudorabies virus that the present invention is provided can also include
The composition such as one or more in PCR reaction buffers, dNTP, Taq archaeal dna polymerase supporting when ddPCR is carried out.
Embodiment 3
The method for present embodiments providing detection pseudorabies virus, it is specific as follows.
1. ddPCR reaction systems are prepared：The μ L of 2 × ddPCR master mix 10, each 0.9 μ L of primer (900nM) (gB-F,
The each 0.9 μ L of gB-R, gE-F, gE-R), each 0.5 μ L of probe (250nM) (each 0.5 μ L of gB-probe, gE-probe), testing sample
The μ L of DNA profiling 2, DEPC process water complement to 20 μ L.
The base sequence of gB-F, gB-R, gE-F, gE-R, gB-probe, gE-probe is with embodiment 1.
2. droplet is generated：Oil parcel ddPCR reaction system modes are generated using droplet and form droplet, droplet generate oil with
The volume ratio 7 of ddPCR reaction systems:2, the quantity of droplet is 1 × 104It is individual.In the present embodiment, on ddPCR instruments
(QX100 droplet type digital pcr instrument, Bio Rad Laboratories) generates oil parcel ddPCR reaction systems and forms droplet with 75 μ L droplets.
3. reacted by following ddPCR reaction conditions：95 DEG C of denaturations 10min；95 DEG C of denaturation 30s, 58.3 DEG C of annealing
1min, 40 circulations；98 DEG C of 10min, 4 DEG C stop reaction.
4. detect, under FAM passages and VIC passages distinguish FAM and VIC fluorescence signals, FAM Air conduct measurement gE genes,
HEX Air conduct measurement gB genes.According to fluorescence signal value, the pseudorabies virus of testing sample is detected.If FAM passages and HEX
Passage has fluorescence signal, then show to contain pseudorabies virus street strain in testing sample；If HEX passages have fluorescence signal,
FAM passages do not have fluorescence signal, then show in testing sample containing the pseudorabies virus vaccine of gE disappearances.
Embodiment 4
The sensitivity test of the Nucleic acid combinations of detection pseudorabies virus is provided embodiment 1.
Specification Curve of Increasing is tested with sensitivity
Expanded as template with the standard plasmid of gradient dilution, using estimate standard plasmid concentration as abscissa,
The plasmid concentration for getting is detected as ordinate using ddPCR, calibration curve is drawn and with R2Evaluate linear relationship.Draw and meeting
Linear relationship, and on the premise of FAM and HEX passages can detect positive findings, it is minimum dense that the method can be detected out
Degree, as the sensitivity judge index of above-mentioned Nucleic acid combinations.Comprise the following steps that.
1. plasmid template is prepared：To be connected to the standard plasmid (pMD19-T-gE) of gE genes and the standard of gB genes
Plasmid (pMD19-T-gB) carries out isoconcentration equal-volume mixing, prepares the plasmid template of 10 times and 2 times gradient dilutions.Wherein, 10
The concentration of times gradient dilution is respectively：1×104、1×103、1×102、1×101And 0copies/ μ L；2 times of gradient dilutions
Concentration is respectively：76、38、19、9.5、4.75copies/μL.
2. ddPCR reaction systems are prepared：The μ L of 2 × ddPCR master mix 10 (mix include PCR reaction buffers,
DNTP, Taq archaeal dna polymerase, precious bioengineering (Dalian) Co., Ltd), primer (900nM) each 0.9 μ L (gB-F, gB-R, gE-
The each 0.9 μ L of F, gE-R), each 0.5 μ L of probe (250nM) (each 0.5 μ L of gB-probe, gE-probe), plasmid template 2 μ L, DEPC
Process water and complement to 10 μ L.By above-mentioned ddPCR reaction systems, the ddPCR reaction systems of each gradient dilution concentration are made.By upper
State the ddPCR reaction systems that ddPCR reaction systems make each gradient concentration.
3. droplet is generated：Using digital pcr instrument (QX100 droplet type digital pcr instrument, Bio Rad Laboratories), with 75 μ L
Droplet generate oil correspondence 25 μ L ddPCR reaction systems generate 10000 droplets, generate droplet after sealer go forward side by side performing PCR expansion
Increase.DdPCR conditions：95 DEG C of denaturations 10min；95 DEG C of denaturation 30s, 58.3 DEG C of annealing 1min, carry out 40 circulations；98℃
10min, 4 DEG C of preservations.
4. detect：After reaction terminates, existed with digital pcr instrument (QX100 droplet type digital pcr instrument, Bio Rad Laboratories)
FAM passages and VIC passages detect respectively FAM the and VIC fluorescence signals corresponding to the ddPCR reaction systems of each gradient concentration, FAM
Air conduct measurement gE genes, HEX Air conduct measurement gB genes.According to fluorescence signal value, digital pcr instrument is counted automatically by carrying software
Corresponding standard plasmid copy number (i.e. detection gained standard plasmid copy number) is calculated, with the ddPCR reactants of each gradient concentration
Expected plasmids copy number is as abscissa, with detection gained plasmid copy number in the ddPCR reaction systems of each gradient concentration in system
As ordinate, respectively draw out 10 times and 2 times gradient dilutions plasmid template calibration curve, as a result such as Fig. 1～Fig. 2 and Biao
Shown in 1.
As shown in Figure 1 (in figure), under 10 times of diluted concentrations, the R of the calibration curve of gB genes is detected2For 0.998, detection
The R of the calibration curve of gE genes2For 999；As shown in Figure 2, under 2 times of diluted concentrations, the R of the calibration curve of gB genes is detected2For
0.9966, detect the R of the calibration curve of gE genes2For 9968.It can be seen that the Nucleic acid combinations of present invention offer and inspection
Survey method is regardless of (1 × 10 in the case of high concentration or low concentration4～4.75copies/ μ L) good linear pass can be kept
System.And as shown in Table 2, minimum detectable concentration is 4.75copies/ μ L, further demonstrate that present invention offer Nucleic acid combinations,
The sensitivity of kit and detection method is preferable.
The standard plasmid concentration that table 1. is detected under 2 times of gradient dilution concentration
Embodiment 5
The specific test of the Nucleic acid combinations of the detection pseudorabies virus that embodiment 1 is provided.
With hog cholera vaccine, blue ear vaccine, pseudorabies virus vaccine Bartha K61, Wei Kuang dogs street strain, Vaccinum Encephalitidis Epidemicae,
It is tiny vaccine, the type vaccine of annulus 2 and health pig tissue DNA detect the specificity of above-mentioned Nucleic acid combinations as by sample product.
Comprise the following steps that.
1. the genomic DNA by sample product is extracted：Above-mentioned receiving is extracted by the operating instruction of viral DNA/RNA extracts kits
The genomic DNA of sample product, and respectively the μ g/ μ L of same mass concentration 100 are diluted to by the DNA of sample product by what is extracted, as entering
The DNA profiling of row ddPCR.
2. ddPCR reaction systems are prepared：The μ L of 2 × ddPCR master mix 10, each 0.9 μ L of primer (900nM) (gB-F,
The each 0.9 μ L of gB-R, gE-F, gE-R), each 0.5 μ L of probe (250nM) (each 0.5 μ L of gB-probe, gE-probe), the μ of DNA profiling 2
L, DEPC process water and complement to 20 μ L.By above-mentioned ddPCR reaction systems, make respectively by the ddPCR reaction systems of sample product.Together
When using the ultra-pure water that sterilizes as template, make the ddPCR reaction systems of blank.
3. droplet is generated：Upper machine (QX100 droplet type digital pcr instrument, Bio Rad Laboratories), generate oil with the droplet of 75 μ L
Correspondence 25 μ L ddPCR reaction systems generate 10000 droplets, generate droplet after sealer go forward side by side performing PCR amplification.DdPCR conditions：
95 DEG C of denaturations 10min；95 DEG C of denaturation 30s, 58.3 DEG C of annealing 1min, carry out 40 circulations；98 DEG C of 10min, 4 DEG C of preservations.
4. detect：After reaction terminates, FAM passages and VIC passages detect respectively respectively by the FAM corresponding to sample product and
VIC fluorescence signals, as a result as shown in Figure 3.
As shown in Figure 3 (in figure：A represents the fluorescence signal testing result in FAM passages, and B represents glimmering in HEX passages
Optical signal detecting, A11 is blank, and B11 is hog cholera vaccine, and C11 is blue ear vaccine, and D11 is pseudo- rabies vaccine Bartha
K61 (the pseudorabies virus vaccine of gE disappearances), E11 Shi Weikuang dogs street strain (pseudoabies street strain), G11 Vaccinum Encephalitidis Epidemicaes,
H11 is tiny vaccine, and A12 is the type vaccine of annulus 2, and H12 is healthy porcine tissue), in FAM and HEX sense channels, blank
And hog cholera vaccine, blue ear vaccine, Vaccinum Encephalitidis Epidemicae, tiny vaccine, the type vaccine of annulus 2 and the no fluorescence letter of healthy porcine tissue
Number；Pseudo- rabies vaccine Bartha K61 (D11) only have fluorescence signal under FAM passages, but believe without fluorescence in VIC passages
Number, illustrate that puppet rabies vaccine Bartha K61 are missing from the pseudorabies virus vaccine of gE genes, it is consistent with actual result；And it is pseudo-
Kuang Quan street strains (E11) detect fluorescence signal in FAM passages and VIC passages, illustrate that Wei Kuang dogs street strain is pseudo- mad dog
Sick street strain, it is consistent with actual result；It is indicated above that the Nucleic acid combinations for detecting pseudorabies virus, examination that the present invention is provided
The specificity of agent box and detection method preferably, is not result in non-specific amplification, and while can efficiently differentiate pseudo- mad dog
The vaccine strain that wild poison is lacked with gE,
Embodiment 6
The method of the detection pseudorabies virus provided embodiment 3 carries out repeatability detection test.
Prepare plasmid template：To be connected to the standard plasmid (pMD19-T-gE) of gE genes and the standard matter of gB genes
Grain (pMD19-T-gB) carries out isoconcentration equal-volume mixing, and gradient dilution goes out 3 gradient concentrations, respectively：7.6×104、7.6
×103And 7.6 × 102Copies/ μ L, each gradient concentration detects each gradient concentration by the detection method of embodiment 3
Under plasmid copy number, as a result represented with mean value, in comparative group and between-group variation coefficient (CV), evaluate repeatability, each ladder
Degree concentration is repeated 3 times.Replica test result is as shown in table 2.
The replica test result of table 2.
Note：Sample number 1,2,3 represents respectively 7.6 × 10 in table4、7.6×103And 7.6 × 102copies/μL
As shown in Table 2, coefficient of variation mean value is 0.66% in organizing, and between-group variation coefficient is 0.74%, is below 3%,
The method for being indicated above the detection pseudorabies virus of present invention offer has preferably repeatability, further shows that the present invention
Nucleic acid combinations, the kit of offer equally has preferably repeatability.
Embodiment 7
The checking test of the annealing temperature of the method for the detection pseudorabies virus that embodiment 3 is provided.Step specific as follows
It is rapid as follows.
1. ddPCR reaction systems are prepared：The μ L of 2 × ddPCR master mix 10, each 0.9 μ L of primer (900nM) (gB-F,
The each 0.9 μ L of gB-R, gE-F, gE-R), each 0.5 μ L of probe (250nM) (each 0.5 μ L of gB-probe, gE-probe), testing sample
The μ L of DNA profiling 2, DEPC process water complement to 20 μ L.
The base sequence of gB-F, gB-R, gE-F, gE-R, gB-probe, gE-probe is with embodiment 1.
2. droplet is generated：Oil parcel ddPCR reaction system modes are generated using droplet and form droplet, droplet generate oil with
The volume ratio 7 of ddPCR reaction systems:2, the quantity of droplet is 1 × 104.In the present embodiment, on ddPCR instruments
(QX100 droplet type digital pcr instrument, Bio Rad Laboratories) generates oil parcel ddPCR reaction systems and forms droplet with 75 μ L droplets.
3. with 95 DEG C of denaturations 10min；95 DEG C of denaturation 30s, 58.3 DEG C of annealing 1min, 40 circulations；98 DEG C of 10min, 4 DEG C
Stop the ddPCR reaction conditions of reaction as test group.50 DEG C are respectively with annealing temperature, 50.7 DEG C, 52 DEG C, 53.9 DEG C,
56.3 DEG C, 59.4 DEG C, 60 DEG C of group (totally 7 contrast groups) as a comparison, the setting of other ddPCR reaction conditions of contrast groups is with trying
Test group.
4. detect, detect FAM and VIC fluorescence signals, FAM Air conduct measurement gE bases respectively in FAM passages and VIC passages
Cause, HEX Air conduct measurement gB genes.Testing result is as shown in Figure 4 and Figure 5.
Understood (in figure by Fig. 4 and Fig. 5：Abscissa represents droplet number, and ordinate represents amplitude, and A12 is 50 DEG C, and B12 is
50.7 DEG C, C12 is 52 DEG C, and D12 is 53.9 DEG C, and E12 is 56.3 DEG C, and F12 is 58.3 DEG C, and G12 is 59.4 DEG C, and H12 is 60 DEG C),
In FAM passages, the Positive fluorescence signal of test group (F12) and the difference of background signal are maximum, with obvious reaction effect,
Show to carry out ddPCR using 58.3 DEG C as degenerate temperature, with preferably reaction result.
Embodiment 8
In the present embodiment, the method for the detection pseudorabies virus for providing by embodiment 3 is to 23 parts of tissue samples and 21
Part blood sample carries out pseudorabies virus detection.Testing result is as shown in table 2.
The testing result of the tissue sample of table 2. and blood sample
As shown in Table 2, in 23 parts of tissue samples, the dual-gene positive findingses of gE and gB are 18 parts, gE feminine gender gB positive findingses
It is dual-gene negative for 4 parts for 1 part, it is 100% with the uniformity of known testing result.21 parts of blood samples are fixed with fluorescence in advance
It is that 47.6%, ddPCR testing result total positiveses rate is 81% with the monogenic total positives rates of gB that amount PCR testing results are dual-gene,
Prove that the present invention is tied using specific primer (the first primer pair and the second primer pair) and probe (the first probe and the second probe)
The testing result that conjunction ddPCR technologies carry out the detection method to pseudorabies virus is higher with the accordance of actual result, and enters
One step proves that Nucleic acid combinations, kit and the method for the detection pseudorabies virus that the present invention is provided has very high sensitivity
Relatively strong specificity, effectively can detect to the sample of low viral level, while distinguishing the classification of detection cause of disease, be
The detection of pseudorabies virus provides more scientific basis and support.
The preferred embodiments of the present invention are these are only, the present invention is not limited to, for those skilled in the art
For member, the present invention can have various modifications and variations.All any modifications within the spirit and principles in the present invention, made,
Equivalent, improvement etc., should be included within the scope of the present invention.
Claims (10)
1. a kind of Nucleic acid combinations of detection pseudorabies virus, it is characterised in that it includes the first primer pair and with described first
The first corresponding probe of primer pair, first primer pair includes base sequence respectively such as SEQ ID NO.1 and SEQ ID
Primer shown in NO.2, the base sequence of first probe as shown in SEQ ID NO.3, first probe 5 ' end it is glimmering
Light reporter group is HEX or VIC, and the quenching group at 3 ' ends of first probe is BHQ or TAMRA.
2. it is according to claim 1 detection pseudorabies virus Nucleic acid combinations, it is characterised in that the Nucleic acid combinations are also
Including：Second primer pair and second probe corresponding with second primer pair, second primer pair includes base sequence
Primer as shown in SEQ ID NO.4-5 respectively, the base sequence of second probe as shown in SEQ ID NO.6, described
The fluorescent reporter group at 5 ' ends of two probes is FAM, and the quenching group at 3 ' ends of second probe is BHQ or TAMRA.
3. application of the Nucleic acid combinations of the detection pseudorabies virus described in claim 1 or 2 in detection pseudorabies virus.
4. a kind of kit of detection pseudorabies virus, it is characterised in that it includes that detection described in claim 1 or 2 is pseudo-
The Nucleic acid combinations of hydrophobin.
5. it is according to claim 4 detection pseudorabies virus kit, it is characterised in that the kit also includes
One or more in PCR reaction buffers, dNTP, Taq archaeal dna polymerase.
6. it is a kind of detection pseudorabies virus method, it is characterised in that it includes：
The first primer pair and first probe corresponding with first primer pair, the PCR system bag are added toward PCR system
Include PCR reaction mixtures and sample to be tested；
The PCR system is formed into droplet, and carries out droplet digital pcr；
Wherein, first primer pair includes base sequence primer respectively as shown in SEQ ID NO.1-2, first probe
Base sequence as shown in SEQ ID NO.3, the fluorescent reporter groups at 5 ' ends of first probe are HEX or VIC, described the
The quenching group at 3 ' ends of one probe is BHQ or TAMRA.
7. it is according to claim 6 detection pseudorabies virus method, it is characterised in that also add in the PCR system
Enter the second primer pair and second probe corresponding with second primer pair, second primer pair is distinguished including base sequence
As shown in SEQ ID NO.4-5, the base sequence of second probe as shown in SEQ ID NO.6, the 5 ' of second probe
The fluorescent reporter group at end is FAM, and the quenching group at 3 ' ends of second probe is BHQ or TAMRA.
8. it is according to claim 6 detection pseudorabies virus method, it is characterised in that using droplet generate oil to institute
State PCR system and carry out droplet process and form the droplet, the droplet generate the oily volume ratio with the PCR system be 7.8～
7.2:2。
9. it is according to claim 6 detection pseudorabies virus method, it is characterised in that the quantity of the droplet be 1
～2 × 104It is individual.
10. the method for detection pseudorabies virus according to claim 6, it is characterised in that carry out droplet numeral
The reaction condition of PCR is：95 DEG C of denaturations 10min；95 DEG C of denaturation 30s, 50～60 DEG C of annealing 1min, 40 circulations；98℃
10min, 4 DEG C stop reaction.
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