CN103740859B - Primer pair, probe and kit for specific detection of porcine lymphotropic herpesviruses III - Google Patents
Primer pair, probe and kit for specific detection of porcine lymphotropic herpesviruses III Download PDFInfo
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Abstract
The invention provides primers and a probe for real-time fluorescent quantitive PCR (Polymerase Chain Reaction) detection of porcine lymphotropic herpesviruses III through gene sequence analysis and comparison of genes of porcine lymphotropic herpesviruses III. The nucleotide sequences of the primers and probe are shown as follows: SEQIDNO.1-3. The invention further provides a method of detecting the porcine lymphotropic herpesviruses III and a detection kit. The detection kit and a detection reagent provided by the invention have the advantages of accuracy in detection, high sensitivity, strong specificity and simplicity and rapidness, and have a good detection capacity.
Description
Technical field
The present invention relates to Measurement for Biotechnique, being specifically related to a kind of PCR primer pair, probe and test kit for detecting III type pig lymphotrophic herpes virus.
Background technology
III type pig lymphotrophic herpes virus (Porcine Lymphotropic herpesviruses 3, PLHV3) belongs to herpetoviridae gamma herpes viruses and belongs to.Can a kind of transmissible disease of infected pigs, may by this viral communication to people by the cell of heteroplastic transplantation pig, tissue and organ.Pig has three hypotypes addicted to lymph blister sore, is respectively PLHV-1, PLHV-2 and PLHV-3.PLHV-1 and PLHV-2 is found in 1999 by people such as EhLers, and PHLV-3 is found in 2003 years by ChmieLewicz.Still know very few to the epidemiology of this virus at present.As tropic virus, lymphoid member, organize often PLHV sequence fragment can be detected at the peripheral blood cells of blood, lymphoglandula, spleen and tonsilla etc.Pig is donor the most promising in the animal replacement source of human organ transplantation, and existing pig source organ successful implantation enters the precedent of human body.But pig is also the carrier of each viroid, in pig body, found the multiple virus that can infect people, but these viruses are not fallen ill in pig body, to be not easy be familiar with by the mankind, only have by constantly testing and verifying, just can guarantee the security of pig source organ.Confirm that III type pig lymphotrophic herpes virus can be transmitted to people by the cell of transplanting pig, tissue and organ, the human body interior generation illness accepting to transplant may have been caused.How to carry out the detection of pig lymphotrophic herpes virus before transplantation to for the pig Organ and tissue transplanted, thus guarantee the security of transplant organ and tissue from source, be one and there is the clinical medicine of reality and the problem of economic research value.
Summary of the invention
In view of this, the present invention disclose a kind of energy accurately, the primer pair of rapid detection III type pig lymphotrophic herpes virus.
Another object of the present invention be to provide a kind of and above-mentioned primer pair with the use of fluorescent probe.
3rd object of the present invention is to provide the detection reagent of detection III type pig lymphotrophic herpes virus, test kit and reaction system thereof.
First object of the present invention is achieved through the following technical solutions: a kind of PCR primer pair for detecting III type pig lymphotrophic herpes virus, the nucleotide sequence of its upstream primer is as shown in SEQ ID NO.1, and the nucleotide sequence of downstream primer is as shown in SEQ ID NO.2.
Second object of the present invention is achieved through the following technical solutions: a kind of and above-mentioned primer pair with the use of fluorescent probe, its sequence is for PLHV3 D P1 is as shown in EQ ID NO.3; 5 ' end of described probe is marked with fluorescent reporter group, and 3 ' end is marked with fluorescent quenching group.
Described fluorescent reporter group is any one in FAM, VIC; Described fluorescent quenching group is any one in TAMRA, Eclipse.
In the present invention, described probe is TaqMan probe.
The present invention is by obtaining all Homologous gene sequences with III type pig lymphotrophic herpes virus type strain (GeneBankNo.AY170316) in ncbi database, and adopt MegAlign software screening method to go out one section of highly conserved sequence (as shown in SEQ ID NO.3) in PLHV3 DNA polymerase gene (GeneBankNo.AAO12315.1), and using as template, with primer Express3.0 software, design primer and TaqMan probe.
Of the present invention detection PLHV3 primer pair and unlike routine gene primer design, as long as based on the genome sequence of PLHV3, primer-design software well known in the art is adopted to design primer, and by logical analysis, reasoning and limited test, the detection primer of high specificity just can be obtained.Because the genome sequence of PLHV3 is not too cautious, only has the amino acid sequence homology of 66% with PLHV1 and PLHV2.Though there is the encoding gene of the archaeal dna polymerase (DPOL) between report PLHV1 and PLHV2 comparatively conservative, between the two, still there is the difference of 50 base pairs.Therefore compared with said two devices, more there is the difference of 245 base pairs in the DPOL encoding gene of PLHV-3, and the primer of design energy specific detection PLHV-3 is very difficult, and this is also prior art insurmountable difficulty always, and the present invention passes through
by contrasting (I type to the DPOL gene of type III Virus Standard strain and the Homologous gene sequences of I type and II typegeneBankNo.AY177148 and AF478169,
iI typegeneBankNo.AF191043, AY170314 and AY170317
), filter out conserved sequence---the sequence PLHV3 of 17 base pairs of one section of high conservative in PLHV3 DPOL encoding gene existed in the such as PLHV3 shown in SEQ ID NO.4
dPOL gene conservative fragments, and then design the Auele Specific Primer of a pair energy specific detection PLHV3.
The design of primers more complicated of PLHV3, and to primer amplification
gorequirement higher.So design of primers of the present invention not have general primer to design so simple, excellent primer be designed, need time-consuming screening is carried out on the basis assessed at various bioinformatics software.Due to designed primer have height selection specificity, when carrying out the detection to PLHV3, require primer for target sequence site should be quite conservative, such set up reaction system just has general suitability.Also can design some degenerate primers in research to strengthen the popularity of application, but annex and spend high primer and can cause negative impact to amplification.Contriver finds under study for action, and in the present invention, the length of target sequence should control at about 210bp, is just conducive to the carrying out of amplified reaction, and long meeting is reacted to the formation of start material and later cycles and brought difficulty, does not sometimes even obviously increase in 1 h.Some are had to the aim sequence of the features such as GC content is high, variability is strong, conserved regions is discrete, well-adapted primer of having no idea to design, makes the application of round pcr be restricted.
By continuous assay optimization, grope in reaction system and reaction conditions two respectively, to obtaining best reagent concentration and temperature of reaction.Reaction system aspect: for Mg
2+the working concentration of concentration, primer working concentration and probe carries out repetition test, carries out doubling dilution respectively to related reagent, obtains its best working concentration in system.Such as Mg
2+for stable base pairing, the reactive behavior maintaining polysaccharase all plays an important role, it is similar to LAMP to the influence mode of PCR, and concentration is too high or too low all makes reaction carry out or in time lengthening simultaneous reactions process, the precipitation of magnesium pyrophosphate precipitation makes Mg in reaction system
2+concentration constantly decline, therefore grope Mg2+ optimum concn highly significant.Through test of many times screening, Mg in PCR reaction system of the present invention
2+the preferred 1.0-1.8mM of concentration, the best is 1.5mM.Reaction conditions aspect, with reference to temperature of reaction and the time of methods involving, repeatedly adjusts, determines suitable reaction conditions.And in experimentation, repeatability, susceptibility, specificity are groped and verified.
The present invention provides a kind of above-mentioned primer pair and the application of fluorescent probe in the detection kit or diagnostic kit of preparation III type pig lymphotrophic herpes virus simultaneously.
Concrete, above-mentioned application is that 25mM magnesium ion 1.2 μ L(generally uses MgCL by pure water 13.8 μ L, 10 × PCR reaction buffer 2 μ L
2), 25mM dNTPs mixing solutions (comprising dATPs, dTTPs, dGTPs, dCTPs of equal proportion) 0.5 μ L, 10 μMs of PLHV3 D F1 0.5 μ L, 10 μMs of PLHV3 D R1 0.5 μ L, 20 μMs of Taqman fluorescent probe 0.3 μ L, the Taq enzyme 0.2 μ L of 5 u/μ L, testing sample DNA profiling 1 μ L forms the detection reaction system of 20 μ L.
Concrete, in above-mentioned application, test kit working conditions is 95 DEG C, 2min; 95 DEG C, 10s; 60 DEG C, 40s, does 40 circulations altogether, then stops.
3rd object of the present invention is by technical scheme realization once: a kind of diagnostic reagent of III type pig lymphotrophic herpes virus, it contains above-mentioned primer and fluorescent probe.
The present invention also provides a kind of detection kit, its above-mentioned primer and fluorescent probe of III type pig lymphotrophic herpes virus.
Further, the specific configuration of its 20 μ L reaction system of described detection kit is pure water 13.8 μ L, 10 × PCR reaction buffer 2 μ L, 25mM magnesium ion 1.2 μ L, 25mM dNTPs mixing solutions 0.5 μ L, 10 μMs of PLHV3 D F1 0.5 μ L, 10 μMs of PLHV3 D R1 0.5 μ L, 20 μMs of PLHV3 D P1 0.3 μ L, the Taq enzyme 0.2 μ L of 5 u/μ L, testing sample DNA profiling 1 μ L.
Concrete, its working conditions is 95 DEG C, 2min; 95 DEG C, 10s; 60 DEG C, 40s, does 40 circulations altogether, then stops.
In the present invention, when probe is complete time, the 5 ' fluorescent energy launched of end reporter gene to be gone out group absorptions by 3 ' end quenching, and instrument can't detect fluorescent signal, along with the carrying out of real-time fluorescence quantitative PCR, Taq enzyme runs into the probe be combined with template in chain extension process, probe will cut off by its 5 ' → 3 ' exonuclease activity, reporter group dissociates probe, free reporter group is away from quenching group, the energy excited can not be absorbed, therefore the fluorescent signal sent is detected by instrument, each circulation receives image data, therefore often through a PCR circulation, fluorescent signal is along with the process being extended with a sync index growth of object fragment, reaction terminates rear according to amplification curve result of determination, thus to realize the detection of III type pig lymphotrophic herpes virus and quantitatively.
Above-mentioned primer pair can be utilized in addition to carry out pcr amplification to sample, then carry out electrophoresis to reaction product, if electrophoresis occurs band at 170bp place, then detected result is positive.
The present invention more provides a kind of and adopts described test kit to detect the method supplying transplanting porcine tissue or organ, comprises the steps: that a. extracts its STb gene in porcine tissue to be transplanted or organ; B. adopting test kit according to claim 5, is that template carries out real-time fluorescence quantitative PCR reaction with STb gene, image data after each loop ends, and reaction terminates rear according to amplification curve and typical curve result of determination.
PLHV3 does not express in the body of live hog, but after migrating to human body, then easily falls ill.Treat PLHV3 detected result in the porcine tissue of transplanting or organ when adopting the present invention, for time positive, should stop to adopt this infected tissue or organ to carry out organ transfer operation.
The present invention is directed to conservative region design specific probe and amplimer in the gene of PLHV3.Room condition optimizing by experiment, establishes easy, detection system and the detection kit of highly sensitive, high specificity that builds based on this system accurately and rapidly for PLHV3.Compared with prior art, the main tool of the present invention has the following advantages:
1. high specific: adopt PCR reaction system provided by the invention to organize STb gene to be template to the live hog infecting PLHV3, commonly DNA virus in live hog body is present in for contrast with other, changed by real-time fluorescence quantitative PCR fluorescence intensity, result shows, the DNA extracted from illing tissue is template, detected by real-time fluorescence quantitative PCR and can be observed obvious fluorescence intensity change and good positive amplification result, and the DNA profiling fluorescence intensity of other kind Viral extraction does not change.
2. highly sensitive: amplification template can reach 8.2 copies, 1 to 3 order of magnitude higher than Standard PCR method, accurately can detect the PLHV3 in sample, sample of protecting from infection is used to human body therapy.
3. the reaction times is shorter, testing efficiency is high: the cycle of each circulation in the present invention is 40S, lower than the reaction time of general PCR, thus promotes the raising of testing efficiency.
4. applied range: PLHV3 Auele Specific Primer provided by the present invention and probe, can the detection of complete paired samples under existing equipment; The goal gene that Auele Specific Primer provided by the present invention increases simultaneously also can adopt common gel electrophoresis to detect, and these all make this technology have more wide application prospect small research institute, veterinary station and even common raiser.
Primer of the present invention and probe specificity good, be prepared into test kit for the detection sensitivity of III type pig lymphotrophic herpes virus, accuracy high and high specificity; Detection method rapid detection can go out sample whether containing III type pig lymphotrophic herpes virus, and can the copy number content of III type pig lymphotrophic herpes virus in Accurate Determining sample.
Accompanying drawing explanation
Fig. 1 is the typical curve that in embodiment 3, real-time fluorescence quantitative PCR detects III type pig lymphotrophic herpes virus;
Fig. 2 is the result that in embodiment 4, real-time fluorescence quantitative PCR detects III type pig lymphotrophic herpes virus;
Wherein, 1 is the real-time fluorescence quantitative PCR amplification of III type pig lymphotrophic herpes virus in sample, and 2 is the real-time fluorescence quantitative PCR amplification of PRV (Pseudorabies virus), and 3 is the real-time fluorescence quantitative PCR amplification of pig 2 type PCV-II;
Fig. 3 is the sensitivity experiment that in embodiment 5, real-time fluorescence quantitative PCR detects III type pig lymphotrophic herpes virus;
Wherein, the amount of 1 expression III type pig lymphotrophic herpes virus is 8.2 × 10
4copy/μ L, the amount of 2 expression III type pig lymphotrophic herpes virus is 8.2 × 10
3copy/μ L, the amount of 3 expression III type pig lymphotrophic herpes virus is 8.2 × 10
2copy/μ L, the amount of 4 expression III type pig lymphotrophic herpes virus is 8.2 × 10
1copy/μ L, the amount of 5 expression III type pig lymphotrophic herpes virus is 8.2 copies/μ L, and 6 is negative control.
Embodiment
The present invention to be described in further detail below in conjunction with accompanying drawing and embodiment for the ease of it will be appreciated by those skilled in the art that:
The design of embodiment 1 primer and probe and synthesis
Download all Homologous gene sequences of III type pig lymphotrophic herpes virus from GenBank, primer and probe are synthesized by Invitrogen company.By the sequence of type pig lymphotrophic herpes virus type strain (GeneBankNo.AY170315) with
the Homologous gene sequences of its homologous sequence and I type and II type carries out contrasting (I typegeneBankNo.AY177148 and AF478169,
iI typegeneBankNo.AF191043, AY170314 and AY170317
), carry out sequence analysis with MEGA software, with primer Express3.0 software, design primer and TaqMan probe,
Amplification object fragment length is 170bp.
Upstream primer nucleotide sequence is as shown in PLHV3 D F1;
Downstream primer nucleotide sequence is as shown in PLHV3 D R1;
With above-mentioned primer with the use of the nucleotide sequence of probe as shown in PLHV3 D P1.This probe 5 ' end is marked with report FAM fluorescence dye, and another 3 ' end is marked with cancellation TAMRA fluorescence dye.
The extraction step of embodiment 2 STb gene is as follows:
Add the anticoagulation damping fluid of equivalent or gather pig blood sample 3 ~ 5Ml, mixing with anticoagulation blood collection tube, preserving 3 days for 2 ~ 8 DEG C.By centrifugal for blood sample 5000r/min 5min, remove supernatant liquor, draw the white corpuscle on red corpuscle upper strata, move into centrifuge tube that is pollution-free or lifeless matter toxicity for subsequent use.(this test extracts DNA just based on DP315 test kit to extract test kit (DP315) operation instruction extraction viral DNA by Beijing Tian Gen biotechnology company virus genom DNA/RNA, it is all applicable that other commercial viral DNA extracts test kit), directly use or for subsequent use in-20 DEG C of Refrigerator stores.
The foundation of embodiment 3 real-time fluorescence quantitative PCR amplification method
1, real-time fluorescence quantitative PCR reaction system
Take STb gene as template, carry out real-time fluorescence quantitative PCR reaction, namely contain in 20 μ L reaction systems: 10 × quantitative fluorescent PCR damping fluid 2 μ L, 0.5 μ L dNTPs (25mmol/L), 0.5 μ L primer PLHV3 D F1 (10umol/L), 0.5 μ L primer PLHV3 D R1 (10umol/L), 1.2 μ L magnesium ions (25mmol/L), 0.3 μ L fluorescent probe (20umol/L), 1 μ L DNA profiling, 0.2 μ L Taq enzyme (5U), and add to 20 μ L with DEPC process water.
2, real-time fluorescence quantitative PCR reaction conditions
After sample hose being put into ABI company 7500 fluorescent PCR instrument, following condition is set and carries out: 95 DEG C, 2min; 90 DEG C, 10s; 60 DEG C, 40s does 40 circulations altogether.Image data after each loop ends.Reaction terminates rear according to amplification curve and typical curve result of determination.
3. III type pig lymphotrophic herpes virus 116bp gene fragment clone is built into positive plasmid by the foundation of real-time fluorescence quantitative PCR typical curve in PCR2.1-TOPO carrier, adjustment plasmid to 1 × 1010 copy/μ L.Then with the positive plasmid of 10 times of serial dilutions for quantifying positive standard form, set up III type pig lymphotrophic herpes virus real-time fluorescence quantitative PCR reaction.Result is presented at 8.2 ~ 8.2 × 10
4the typical curve that within the scope of copy, III type pig lymphotrophic herpes virus detects has good linear relationship (Fig. 1).
The specificity of embodiment 4 III type pig lymphotrophic herpes virus real-time fluorescence quantitative PCR method is determined
Organize STb gene for template with the live hog of Virus Pollution, with PRV (Pseudorabies virus), pig circular ring virus for contrast, changed by real-time fluorescence quantitative PCR fluorescence intensity.Result shows, the DNA extracted from positive plasmid is template, detected by real-time fluorescence quantitative PCR and can be observed obvious fluorescence intensity change and good positive amplification result, and the DNA profiling fluorescence intensity that PRV (Pseudorabies virus), pig circular ring virus extract does not change.The real-time fluorescence quantitative PCR system that prompting the present invention sets up has good specificity (Fig. 2) to III type pig lymphotrophic herpes virus.
Embodiment 5 sensitivity experiment
Dilute III type pig lymphotrophic herpes virus positive template respectively with DEPC process water, changed by real-time fluorescence quantitative PCR fluorescence intensity.Experimental result shows, real-time fluorescence quantitative PCR detects and can be observed obvious fluorescence intensity change and good positive amplification result, real-time fluorescence quantitative PCR kinetic curve shows, concentration has obvious fluorescence to increase in the above reaction system of 8.2 copy, and increase lower than 8.2 copies and the equal unstressed configuration of negative control, therefore the minimum detectability of this real-time fluorescence quantitative PCR detection system is 8.2 copies, and namely its sensitivity is 8.2 copies/μ L (Fig. 3).
The real-time fluorescence quantitative PCR detection kit of embodiment 6 one kind of III type pig lymphotrophic herpes virus
Described test kit is made up of following reagent: pure water, 10 × PCR reaction buffer, 25mM magnesium ion, 25mM dNTPs mixing solutions, 10 μMs of PLHV3 D F1,10 μMs of PLHV3 D R1,20 μMs of PLHV3 D P1, the Taq enzyme of 5 u/μ L.Described PCR damping fluid can be any one commercially available quantitative fluorescent PCR reaction buffer, and each reagent is all independently divided in reagent bottle, is encapsulated in test kit together with working instructions.
The specific configuration of its 20 μ L reaction system of the quantitative fluorescent PCR of test kit is pure water 13.8 μ L, 10 × PCR reaction buffer 2 μ L, 25mM magnesium ion 1.2 μ L, 25mM dNTPs mixing solutions 0.5 μ L, 10 μMs of PLHV3 D F1 0.5 μ L, 10 μMs of PLHV3 D R1 0.5 μ L, 20 μMs of PLHV3 D P1 0.3 μ L, the Taq enzyme 0.2 μ L of 5 u/μ L, testing sample DNA profiling 1 μ L.
Its reaction conditions is: 95 DEG C, 2min; 95 DEG C, 10s; 60 DEG C, 40s, does 40 circulations altogether, then stops.
Be more than wherein specific implementation of the present invention, it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these apparent replacement forms all belong to protection scope of the present invention.
SEQUENCE LISTING
<110> Huizhou Entry-Exit Inspection and Quarantine Bureau inspection and quarantine complex art center
<120> mono-kind is for detecting III type pig addicted to the PCR primer pair of lymph herpes virus, probe and test kit
<130> 2013
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213> PLHV3 D F1
<400> 1
ttatcaattt ccctcagagc tt 22
<210> 2
<211> 22
<212> DNA
<213> PLHV3 D R1
<400> 2
ttggtgcgag tataagactc tg 22
<210> 3
<211> 22
<212> DNA
<213> PLHV3 D P1
<400> 3
tgcaaatttt gactttccat at 22
<210> 4
<211> 170
<212> DNA
<213> III type pig is addicted to lymph herpes virus DPOL gene conservative fragments
<400> 4
tatcaatttc cctcagagct tgatatgctt tatgcatttt gttcaatgat cagagatttt 60
gatattgaga ttgtgactgg ttataacatt gcaaattttg actttccata tatattacac 120
agagcaaatg tgatatacaa tatgaaggca gagtcttata ctcgcaccaa 170
Claims (7)
1. the primer pair of a specific detection III type pig lymphotrophic herpes virus, comprise upstream primer and downstream primer, it is characterized in that, the nucleotide sequence of described upstream primer PLHV3 D F1 is as shown in SEQ ID NO.1, and the nucleotide sequence of downstream primer PLHV3 D R1 is as shown in SEQ ID NO.2.
2. with primer pair according to claim 1 with the use of fluorescent probe, it is characterized in that, the nucleotide sequence of probe PLHV3 D P1 is as shown in SEQ ID NO.3; 5 ' end of described probe is marked with fluorescent reporter group, and 3 ' end is marked with fluorescent quenching group.
3. fluorescent probe according to claim 2, is characterized in that: described fluorescent reporter group is any one in FAM, VIC; Described fluorescent quenching group is any one in TAMRA, Eclipse.
4. the application of primer pair described in claim 1 in the detection or diagnostic kit of preparation III type pig lymphotrophic herpes virus.
5. a real time fluorescent quantitative detection kit for III type pig lymphotrophic herpes virus, is characterized in that, containing the fluorescent probe described in primer pair described in claim 1 and Claims 2 or 3.
6. test kit according to claim 5, it is characterized in that, the reaction system of described test kit is: the specific configuration of 20 μ L reaction systems is pure water 13.8 μ L, 10 × PCR reaction buffer 2 μ L, 25mM magnesium ion 1.2 μ L, 25mM dNTPs mixing solutions 0.5 μ L, 10 μMs of PLHV3 D F1 0.5 μ L, 10 μMs of PLHV3 D R1 0.5 μ L, 20 μMs of PLHV3 D P1 0.3 μ L, the Taq enzyme 0.2 μ L of 5 u/μ L, testing sample DNA profiling 1 μ L.
7. test kit according to claim 5, it is characterized in that, the fluorescent quantitative PCR condition of described test kit is: 95 DEG C, 2min; 95 DEG C, 10s; 60 DEG C, 40s, does 40 circulations altogether, then stops.
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| Title |
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| 《几种新出现的猪源病毒研究综述》;翁善钢;《广东畜牧兽医科技》;20120831;第37卷(第4期);1-4 * |
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