CN103882149A

CN103882149A - Dual real-time fluorescence PCR detection method of type I and type II of porcine torque teno virus

Info

Publication number: CN103882149A
Application number: CN201410105912.5A
Authority: CN
Inventors: 杨春华; 孙思扬; 胡婷; 石磊
Original assignee: Comprehensive Technology Center Jiangxi Entry And Exit Inspection And Quarantin
Current assignee: Comprehensive Technology Center Jiangxi Entry And Exit Inspection And Quarantin
Priority date: 2014-03-20
Filing date: 2014-03-20
Publication date: 2014-06-25

Abstract

The invention discloses a duel detection primer of type I and type II of a porcine torque teno virus and a duel real-time fluorescence PCR detection method. The invention provides a brand new detection primer for dual detection of the type I and type II porcine torque teno viruses. The primer comprises a specific detection primer of the type I and type II porcine torque teno viruses; the primer is strong in sensitivity and can meet the daily inspection and quarantine requirements of the type I and type II porcine torque teno viruses; the duel detection primer provides technical aid for quarantine of the type I and type II porcine torque teno viruses, the epidemiological investigation and the guidance of the type I and type II porcine torque teno viruses in the production.

Description

The dual real-time fluorescence PCR detection method of pig thin circovirus virus I type and II type

Technical field

The application relates to Molecular Detection field, particularly relates to the dual real-time fluorescence PCR detection method of a boar thin circovirus virus I type and II type.

Background technology

TTV is the negative adopted DNA virus of a kind of uncovered sub-thread ring-type, because be at first separates and obtain in hepatitis, guesses relevantly with hepatitis, is subject to the extensive concern of medical field.The Chinese name of pig source TTV is defined as the thin circovirus virus of pig (Torque Teno Sus Virus, TTSuV).Epidemiological study is found, TTV propagates by modes such as blood transfusion, sexual behaviour, fecal oral routes, blood, saliva, ight soil, milk, bile all can be with poison simultaneously, the result of study of the infection rate of the TTV by different crowd shows: the infection rate of population is many more than 10%, is asymptomatic carrier.Except people can infect TTV, the animals such as cat, dog, pig, ox, chicken, sheep, mouse are also the hosts of TTV.Okamota H etc. are according to the DNA sequence dna design primer of people TTV non-coding region, the TTV gene fragment having increased in pig, dog and cat serum, find in these different generas TTV gene order and difference in length very big, nucleic acid homology is less, has species specificity.The application nested PCR methods such as American scholar Leary detect TTV(Sd-TTV31 from porcine blood serum), genome length is 2878bp.2005, Niel C etc. utilizes multiple boot rolling loop type TRAP (Multiply primed rolling-circle amplification, RCA) the another kind of hypotype of pig TTV detected: Sd-TTV2p, genome length is 2735bp, very low with the homology of the Sd-TTV31 finding before, only have 45.1%, so TTSuV is divided into two kinds of genotype, TTSuV1 type is take Sd-TTV31 as prototype, and TTSuV2 type is take Sd-TTV2p as prototype.At present, the unified thin circovirus virus I of pig type (writing a Chinese character in simplified form TTSuV I) that TTSuV1 type is called, TTSuV2 type is called the thin circovirus virus II of pig type (writing a Chinese character in simplified form TTSuV II).

TTSuV is similar with people TTV genome structure composition, You Feibian district (UTR) and coding region two portions composition, be rich in the region of GC, a promotor and a transcriptional enhancer by one and form in Fei Bian district, the GC region of two ends forms the secondary structure of tool stem ring, in virus replication, may have important regulating and controlling effect; TTV coding region comprises 3 open reading frame (ORF1～ORF3), a PolyA and a TATA box, and aberration rate is higher.The N end of ORF1 is rich in arginine (Arg) protein, and this protein is likely to copy relevant albumen-Rep albumen; ORF2 encodes and copies relevant albumen; ORF3 encodes in conjunction with albumen, is divided into two sections, wherein across a sequence that is similar to intron in eukaryote coding region.When transcribing, coding region produces 3 kinds of different mRNA, and can 6 kinds of different albumen of accurate translation.Two kinds of genotype TTV proteins encoded have similar sequence and function.

External existing many relevant reports, the infection that shows TTSuV is distribution on global is very wide and its prevalence rate is very high.162 parts of porcine blood serum that Segal é s etc. gather Spain pig farm 1985-2005 carry out retrospective investigation, result all detects TTSuV virus in all times, the infection rate of TTSuV1 and TTSuV2 is respectively 33.3%(54/162) and 55.6% (90/162), two kinds of genotype coinfection rates are 23.5%(38/162), the infection that has confirmed TTSuV has just existed before 14 years of this virus of initial discovery (Leary et al., 1999).

In recent years, domesticly also carried out successively the research to TTSuV infection conditions.These studies show that, the infection of TTSuV in swinery is very general at home and abroad, is a new cause of disease can not be ignored.And it is at present also very limited to the understanding of TTSuV, the comprehensive method that detects TTSuV both at home and abroad mainly contains detection of plasma method and molecular biology method, detection of plasma method has Enzyme-linked Immunosorbent Assay (ELISA) method, ELISA is the common method for epidemic disease examination and controlling, but because TTSuV has height genetic variability, between different genes hypotype antibody, there is cross reaction, when genovariation analysis with it, accuracy rate is lower, genovariation situation in antimer, the more important thing is that the loss of ELISA method is higher completely.In addition, because infection time is compared with short and not yet produce antibody and also can cause the false negative of ELISA experimental result.

The molecular biology method detecting for TTSuV at present mainly contains the methods such as regular-PCR, sleeve type PCR, half sleeve type PCR and rolling circle amplification.Conventional PCR and Nested Polymerase Chain Reaction all design primer according to the most conservative non-coding region of at present known TTSuV gene order, and nucleic acid is detected.Rolling circle amplification method is using oligonucleotide arbitrarily as primer, under constant temperature with rolling circle amplification cyclic DNA to be measured.Because TTSuV virus titer in serum is lower, take turns and be difficult to amplify object fragment with regular-PCR one, need to strengthen cycle number, but can make like this primer non-specific binding or polymerase activity decline.Chao Shi PCR has overcome the restriction of single amplification " plateau effect " and has guaranteed the specificity of reaction, but need to carry out PCR for the second time, causes that the probability of crossed contamination is larger.Regular-PCR susceptibility is poor, and Chao Shi PCR, half Chao Shi PCR and rolling circle amplification are consuming time longer, and these traditional round pcrs can only do qualitative analysis to the detection of gene, cannot be accurately quantitative detected gene dosage, greatly restricted the application of round pcr.

Taqman quantitative fluorescent PCR is the nucleic acid detection technique that development in recent years is got up, and can detect fast, sensitively the goal gene in nucleic acid.Adopt primer and probe to combine, gene fragment is increased, its specificity is stronger, and it is more accurate to detect.Utilize the corresponding relation of fluorescent signal and PCR product to carry out qualitative and quantitative analysis to detecting sample, susceptibility is high, the viral nucleic acid of several copy/μ L can be detected.In testing process, fluorescence quantifying PCR method, without carrying out gel electrophoresis, is collected fluorescence with instrument and is carried out judged result, has avoided the impact of artificial subjective factor.And compared with conventional round pcr, its detection speed has more advantage, can in 4-6h, complete.And it is at present less for the research of the high-throughput for detection of TTSuV, quick, sensitive Taqman fluorescent quantitative PCR detection method; Far can not meet the demand of inspection and quarantine application in practice.

Summary of the invention

The application's object is to provide a kind of brand-new dual real-time fluorescence PCR detection method for pig thin circovirus virus I type and II type.

In order to realize above object, the application's one side discloses a kind of primer of the double check for pig thin circovirus virus I type and II type, this primer comprises the first primer pair and the second primer pair, the upstream primer of the first primer pair and downstream primer contain respectively sequence shown in Seq ID No.1 and Seq ID No.2, and the upstream primer of the second primer pair and downstream primer contain respectively sequence shown in Seq ID No.4 and Seq ID No.5;

Seq?ID?No.1：5’-GGTTCAGGAGGCTCAATTTGG-3’

Seq?ID?No.2：5’-TTTGAATTTAACGGTTTTCAGTCTTC-3’

Seq?ID?No.4：5’-TATCGGGCAGGCGGTAATC-3’

Seq?ID?No.5：5’-TACGCTACCGTCAGCCATCTTT-3’。

Wherein, the first primer pair is the primer pair that detects the thin circovirus virus I of pig type, and the second primer pair is the primer pair that detects the thin circovirus virus II of pig type.

The application's another side discloses a kind of probe of the double check for pig thin circovirus virus I type and II type, and this probe comprises the first probe and the second probe, and the first probe contains sequence shown in Seq ID No.3, and the second probe contains sequence shown in Seq ID No.6;

Seq?ID?No.3：5’-TCGCTTCGCTCGCACCACGT-3’

Seq?ID?No.6：5’-AACCGGGCCCCCCTCGATG-3’。

Wherein, the first probe is the probe that detects the thin circovirus virus I of pig type, and the second probe is the probe that detects the thin circovirus virus II of pig type.

Preferably, 5 ' end of the first probe and the second probe is all marked with fluorescence report group, and 3 ' end is all marked with fluorescent quenching group.

Preferred, the fluorescence report group of 5 ' end mark of the first probe is FAM, and the fluorescent quenching group of 3 ' end mark is BHQ1; The fluorescence report group of 5 ' end mark of the second probe is HEX, and the fluorescent quenching group of 3 ' end mark is BHQ1.

The application's one side more also discloses the dual real-time fluorescence PCR detection method of a boar thin circovirus virus I type and II type, comprises and adopts the application's primer and the application's the fluorescently-labeled probe pairing of being with to carry out Taqman fluorescence quantitative PCR detection.

Preferably, the first primer pair and the first probe match in order to detect the thin circovirus virus I of pig type, and the second primer pair and the second probe match in order to detect the thin circovirus virus II of pig type.

The application's one side more also discloses the test kit of the double check of a boar thin circovirus virus I type and II type, contains the primer of the application's design in this test kit.

Preferably, in detection kit, also contain the probe of the application's design.

Owing to adopting above technical scheme, the application's beneficial effect is:

The application provides a kind of brand-new detection primer for the double check of pig thin circovirus virus I type and II type, this primer has comprised the specific detection primer of pig thin circovirus virus I type and II type, and there is very strong sensitivity, can meet the routine check quarantine demand of pig thin circovirus virus I type and II type; For quarantine, the epidemiology survey of pig thin circovirus virus I type and II type, and provide technology help for producing the detection that middle finger leads pig thin circovirus virus I type and II type.

Accompanying drawing explanation

Fig. 1: be nested PCR electrophoresis result figure in the embodiment of the present application, wherein A is sample amplification result, B is the bacterium colony PCR electrophoresis result after clone;

Fig. 2: be that the enzyme that extracts plasmid in the embodiment of the present application is cut result figure;

Fig. 3: be TTSuV1 standard substance (10 in the embodiment of the present application ^-1) annealing temperature optimum result;

Fig. 4: be TTSuV1 standard substance (10 in the embodiment of the present application ^-1-10 ^-6) annealing temperature optimum result;

Fig. 5: be TTSuV2 standard substance (10 in the embodiment of the present application ^-1) annealing temperature optimum result;

Fig. 6: be TTSuV2 standard substance (10 in the embodiment of the present application ^-1-10 ^-6) annealing temperature optimum result;

Fig. 7: the specific test result that is TTSuV1 real-time fluorescence PCR in the embodiment of the present application;

Fig. 8: the specific test result that is TTSuV2 real-time fluorescence PCR in the embodiment of the present application;

Fig. 9: the sensitivity test result that is TTSuV1 real-time fluorescence PCR in the embodiment of the present application;

Figure 10: the sensitivity test result that is TTSuV2 real-time fluorescence PCR in the embodiment of the present application;

Figure 11: the amplification curve that is TTSuV1 standard substance in the embodiment of the present application;

Figure 12: the amplification curve that is TTSuV2 standard substance in the embodiment of the present application;

Figure 13: the specificity result that is dual real-time fluorescence PCR in the embodiment of the present application;

Figure 14: be that in the embodiment of the present application, dual real-time fluorescence PCR detects the sensitivity of TTSuV1;

Figure 15: be that in the embodiment of the present application, dual real-time fluorescence PCR detects the sensitivity of TTSuV2;

Figure 16: be the amplification curve of dual real-time fluorescence PCR to TTSuV1 standard substance in the embodiment of the present application;

Figure 17: be the typical curve of dual real-time fluorescence PCR to TTSuV1 standard substance in the embodiment of the present application;

Figure 18: be the amplification curve of dual real-time fluorescence PCR to TTSuV2 standard substance in the embodiment of the present application;

Figure 19: be the typical curve of dual real-time fluorescence PCR to TTSuV2 standard substance in the embodiment of the present application.

Embodiment

The application studies take the conserved sequence of TTSuV different genotype as object fragment, primer and the Taqman probe of design real-time fluorescence quantitative PCR, final Auele Specific Primer and the Taqman probe that the non-coding region (UTR) for TTSuV1 and TTSuV2 is designed respectively, respectively the substance real-time fluorescence PCR detection method of foundation detection TTSuV1 and TTSuV2 of obtaining.In addition, consider workload and the requirement fast and accurately of inspection and quarantine business of clinical detection, the application, on the basis of the substance real time fluorescent PCR method of setting up, has set up and has detected TTSuV1 and TTSuV2 dual real-time fluorescence PCR method simultaneously.

The application is further research on the basis of existing research, thereby a pair of brand-new detection primer and probe are obtained, for the detection of pig thin circovirus virus I type and the thin circovirus virus II of pig type provides a kind of new scheme, thereby improve quality and the working efficiency of inspection and quarantine, inspection and quarantine and practice production have all been had great importance.It should be noted that, the application's primer and probe all detect and design for real-time fluorescence PCR, and concrete designs for dual real-time fluorescence PCR method especially; Therefore, the application's primer and probe all have good specificity.Be appreciated that on the application's basis, can separately the application's primer be directly used in to the detection that conventional PCR detects or other PCR-based increases completely; Same, during the application's probe also can detect for other of the real time fluorescent PCR method except the application completely, such as genechip detection, or other detection based on nucleotide fragments.Wherein, the detection of PCR-based amplification is as the target fragment amplification of gene chip, clone, order-checking etc.Particularly, in genechip detection, can adopt the application's primer to carry out dual amplification completely, then adopt the application's probe in detecting TTSuV1 and TTSuV2.

Also it should be noted that, when the application's probe is detected for real-time fluorescence PCR, the principle detecting according to probe method real-time fluorescence PCR, 5 ' end of probe needs mark fluorescent reporter group, 3 ' end needs mark fluorescent quenching group, is appreciated that in dual or multiple real time fluorescence PCR detect, existing fluorescence report group and fluorescent quenching group can arbitrarily combine, as long as there is no phase mutual interference between the fluorophor of selecting.In addition, in the application's real-time fluorescence PCR detection method, its key has been to adopt primer and the probe of the application's design, be appreciated that detecting sample can be to cultivate the viral sample of purifying, extract from the biological tissue of doubtful infection or the sample of secretory product or the sample that extracted or preserved by alternate manner.

On the basis of the application's primer, probe and real-time fluorescence PCR detection method, the application has further studied the detection kit for detection of pig thin circovirus virus I type and II type.Be appreciated that directly the solution that the application's primer or probe are prepared into dry powder or high density can be used as kit components and save backup.In addition, as previously mentioned, the application's primer or probe be all can be independent the detection for other use, therefore, in test kit, can only contain equally primer or probe; In addition, for the needed reagent such as conventional PCR detection or real-time fluorescence PCR detection relating to, can directly be positioned in the application's test kit, also can, in the time using test kit, buy corresponding reagent from market, be not specifically limited in this application.

Also by reference to the accompanying drawings the application is described in further detail below by specific embodiment.Following examples are only further detailed the application, should not be construed as the restriction to the application.

Embodiment mono-substance real-time fluorescence PCR detects

One, test materials and equipment

1. main agents and test apparatus

MagNA Pure LC Total Nucleic Acid Isolation Kit is purchased from Roche.Premix Ex TaqTM (Perfect Real Time), Premix Ex Taq, Ex Taq, dNTP Mixture, 2 × GC buffer I, DNA Marker DL2000, DNA Marker DL5000, gel DNA reclaim test kit, pMD18-T Vector, plasmid DNA a small amount of purification kit etc. and are purchased from the precious biotech firm in Dalian.Jm109 intestinal bacteria, IPTG, X-Gal, glycerine, NaCl are analytical pure, purchased from the raw work in Shanghai.Ampicillin, CaCl2 are purchased from Sigma.Agarose is purchased from Promega.Tryptones, yeast extract, agar powder etc. are purchased from Huankai Microbiological Science & Technolgy Co., Guangdong.Glue is red in Biotium.The key instrument using in this experiment comprises: Bio Rad real-time fluorescence PCR instrument CFX96Real-time System, the grads PCR AG22331Hamburg of Eppendorf, the automatic instrument for extracting nucleic acid MagNA of Roche Pure LC2.0, Thermo nucleic acid-protein uv analyzer NANODROP1000, Haier Biohazard Safety Equipment HR40-II A2, Eppendorf whizzer centrifuge5415R, Infors constant-temperature table S-000113769Ecotron, SANYO constant incubator MIR-162, the electrophoresis apparatus ESP601 of Beijing Liu Yichang and electrophoresis chamber DYCP31DN, Eppendorf couveuse Thermomixer compact, Genegenius gel imaging system SYDR2/2144, the electronic balance ALC-201.3 of Beijing Sai Duolisi, SANYO high-pressure sterilizing pot MLS-3780, IKA vortex mixed instrument MS3basic, Eppendorf micropipet comprises: 0.5-10 μ L, 2-20 μ L, 10-100 μ L, 20-200 μ L, 100-1000 μ L.

2. the preparation of solution and substratum

LB substratum: claim peptone (Tryptone) 10g, yeast extract (Yeast extract) 5g, NaCl10g, be dissolved in 800mL deionized water, with NaOH tune pH to 7.0, add deionized water to cumulative volume 1L, autoclaving 20min.4 ℃ of preservations.Solid medium also needs to add the agar of mass fraction 1.5%.Penbritin (Ampicillin) mother liquor: be made into the 100mg/mL aqueous solution ,-20 ℃ save backup.IPTG: dissolve IPTG in distilled water, be mixed with the stock solution of 24mg/mL, with 0.22 μ m strainer degerming, with the packing of l mL EP pipe, be stored in-20 ℃.X-gal: dissolve X-gal with dimethyl formamide, be mixed with the stock solution of 20mg/mL.Be stored in the EP pipe of lucifuge, and be stored in-20 ℃.LB/Amp/X-Gal/IPTG plate culture medium: take peptone (Tryptone) 10g, yeast extract (Yeast extract) 5g, NaCl10g, be dissolved in 800mL deionized water, with NaOH tune pH to 7.0, add 15g agar powder, after autoclave sterilization, be cooled to 60 ℃ of left and right.After adding 1mL Ampicillin (100mg/mL), 1mL IPTG (24mg/mL), 2ml X-Gal (20mg/mL), evenly mix, be down flat plate, 30～35mL substratum/90mm culture dish, 4 ℃ keep in Dark Place.LB/Amp liquid nutrient medium: take yeast extract (Yeast extract) 5g, NaCl10g, is dissolved in 800mL deionized water, with NaOH tune pH to 7.0, adds deionized water to cumulative volume 1L, vapor sterilization 20min under high pressure.Be cooled to room temperature, add the rear evenly mixing of 1mL Ampicillin (100mg/mL), 4 ℃ of preservations.If solid medium also needs to add the agar of mass fraction 1.5%.

3. test sample

220 porcine blood serum samples that this test sample all gathers from each pig farm, the Jiangxi Area third season in 2011.Specificity is tested required virus and is comprised: PRV (Pseudorabies virus) (Pseudorabies virus, PRV), pig parvoviral (Porcine Parvovlirus, PPV), porcine circovirus 2 type (Porcinecircovirus type2, PCV2) provides by Agricultural University Of He'nan.

Two, test method

1. the preparation of pig TTSuV1 and TTSuV2 positive criteria product

TTSuV is as the newfound virus of one, also there is no at present a kind of isolation cultivation method of maturation, so first include in this experiment the fragment of TTSuV Taqman rt-PCR amplification gene with nest-type PRC amplification, then this fragment gene is connected in T carrier, build positive recombinant plasmid, as the positive criteria product of TTSuV Taqman rt-PCR.

(1) sample nucleic acid extraction

Extract full genomic nucleic acids with the automatic instrument for extracting nucleic acid of Roche MagNA Pure LC2.0.Each sample is got 140 μ L serum, by MagNA Pure LC Total Nucleic Acid Isolation Kit description operation, finally uses 100 μ L elution buffer dissolving DNAs, be stored in-20 ℃ frozen for subsequent use.

(2) nest-type PRC amplification

Primer sequence with reference to Kekarainen T report carries out nest-type PRC amplification, increase respectively TTSuV1 and TTSuV2 Partial Fragment, and amplified fragments includes the non-coding region of the high conservative of TTSuV.S1, S2 are the outer primer of amplification TTSuV1, and S3, S4 are the inner primer of amplification TTVSu1; T1, T2 are the outer primer of amplification TTSuV2, and T3, T4 are the inner primer of amplification TTSuV2.Primer entrusts Shanghai Ying Jun Bioisystech Co., Ltd synthetic.Primer sequence and relevant information are in table 1.

The report of Kekarainen T is specifically referring to Kekarainen T, Sibila M, Segales J.Prevalence of swine Torque teno virus in post-weaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS2affected pigs in Spain[J] .J Gen Virol, 2006,87:833-837.

Table 1 nest-type PRC amplimer

TTSuV nest-type PRC first round primer sequence is as shown in table 1 is S1+S2, T1+T2, and TTSuV first round reaction system is: 5U/ μ L TaKaRa Ex Taq0.2 μ L, 5mM Mg ²⁺plus2 × GC Buffer I 10 μ L, 2.5mM dNTP Mixtuer3.2 μ L, 1 μ M Primer10.4 μ L, 1 μ M Primer20.4 μ L, make up water to 20 μ L, prepare PCR premixed liquid, add DNA profiling 4 μ L, carry out respectively the amplification of TTSuV1 and TTSuV2, whole reaction is carried out on grads PCR instrument.TTSuV1 nest-type PRC first round amplification condition is: 94 ℃, and 5min denaturation; Then enter 35 circulations: 94 ℃, 30s sex change, 59 ℃, 30s annealing, 72 ℃, 2min extends; After circulation completes 72 ℃, 5min extends.The condition of the nest-type PRC first round amplification of TTSuV2 is identical with TTSuV1, is just 60 ℃ in the temperature of 30s annealing.

After first round PCR reaction finishes, get respectively the PCR product of TTSuV1 and TTSuV2 as template, with the second cover primer, S3+S4, T3+T4 as shown in table 1, carry out the second amplification of taking turns, second takes turns reaction system is: 5U/ μ L TaKaRa Ex Taq0.2 μ L, 5mM Mg ²⁺plus2 × GC Buffer I 10 μ L, 2.5mM dNTP Mixtuer3.2 μ L, 1 μ M Primer10.6 μ L, 1 μ M Primer20.6 μ L, DNA profiling 4 μ L, make up water to 20 μ L.TTSuV1 nest-type PRC second is taken turns amplification condition: 94 ℃, and 5min denaturation; Then enter 35 circulations: 94 ℃, 30s sex change, 60 ℃, 30s annealing, 72 ℃, 2min extends; After circulation completes 72 ℃, 5min extends.The condition that the nest-type PRC of TTSuV2 second is taken turns amplification is identical with TTSuV1, and just, in working cycle, the temperature of 30s annealing is 62 ℃.

(3) DNA electrophoresis detection

First get 50 × TAE electrophoretic buffer thin up and become 1 × TAE electrophoretic buffer, for subsequent use.Then prepare glue: take 0.3g agarose powder and be placed in 200ml Erlenmeyer flask, add in the electrophoretic buffer of 30ml1 × TAE, be heated to agarose and all melt.Glue plate is put in the glue groove of level, reconstitute product comb, adds 3 microlitre glue red to sepharose solution, to be cooled to 50 ℃ of left and right, gel is poured in glue plate, and between gel thicknesses 3～5mm, cooling 30min left and right takes out gently comb after gel solidifies completely.

The gel making is placed in to electrophoresis chamber, adds 1 × TAE damping fluid and make it not have the about 1mm of glue face.Get 3 μ L DNA samples and 1 μ L6 × loading buffer mixes with micropipette rifle, careful application of sample is in well, and marker adds 3 μ L, adds electrophoresis chamber on sample bonnet.The condition of electrophoresis is: set electrophoresis apparatus voltage 80V, electrophoresis time 30min.After electrophoresis, gel is put into image analysis system observation and take pictures and file.

(4) PCR product reclaims and purifying

The sepharose that contains target DNA is cut under ultraviolet lamp to recovery, operation is carried out in strict accordance with TaKaRa Agrose Gel DNA Purification Kit Ver2.0 (50 amounts) specification sheets, and test kit composition comprises: DR-I Buffer, DR-II Buffer, Rinse A, Rinse B, Elution Buffer, Spin Column, Collection Tube.

Under A, ultraviolet lamp, cut out the sepharose that contains target DNA, exhaust the liquid of gel surface.B, blob of viscose is shredded to accelerate the follow-up blob of viscose thawing time.C, title blob of viscose weight, calculate blob of viscose volume.While calculating blob of viscose volume, calculate with 1mg=1 μ L.D, be 3 gelinite accumulated amounts to adding in blob of viscose blob of viscose melting liquid DR-I Buffer:1.0% gel strength to add DR-I Buffer amount.E, fully mix be placed on 75 ℃ of oscillation incubations with melt blob of viscose.F, to the DR-II Buffer of 1/2 volume that adds DR-I Buffer amount in above-mentioned blob of viscose melting liquid, fully mix.G, the Spin Column in test kit is placed on Collection Tube.H, the solution of step G is shifted in Spin Column, the centrifugal 1min of 12,000rpm, abandons filtrate.I, in Spin Column, add 500 μ L Rinse A, room temperature 12, the centrifugal 30s of 000rpm, abandons filtrate.J, in Spin Column, add 700 μ L Rinse B, room temperature 12, the centrifugal 30s of 000rpm, abandons filtrate.K, repetitive operation step J.L, Spin Column is placed on new 1.5mL centrifuge tube, adds 25 μ l Elution Buffer in the centre of Spin Column film, room temperature leaves standstill 1min.M, room temperature 12, the centrifugal 1min eluted dna of 000rpm.

(5) preparation of escherichia coli jm109 competent cell

A, from-70 ℃ of picking JM109 cells, line in LB solid medium 37 ℃ of overnight incubation.B, from the flat board of incubated overnight single bacterium colony of picking, forward one to and contain in 3-5ml LB liquid nutrient medium, 220rpm shaking culture is about 12 hours, until the logarithmic growth later stage at 37 ℃.This bacteria suspension is inoculated in the ratio of 1:100-1:50 in the 500mL Erlenmeyer flask that contains 100mL LB liquid nutrient medium, 37 ℃ shaking culture 2-3 hour to about OD600=0.5.C, then bacterium liquid is transferred in an aseptic ice-cold 50mL centrifuge tube to ice bath 20min; Make culture be cooled to 0 ℃.The centrifugal 10min of D, 4000rpm, removes supernatant, pipe is inverted to 1min trace nutrient solution is flow to end.The first culture of E, every 50mL adds the 0.1M CaCl2 solution of 30mL precooling, inhales up and down gently moving beating with liquid-transfering gun, makes cell Eddy diffusion.F and then at 4 ℃, the centrifugal 10min of 4000rpm, removes supernatant.Repeating step E, F are once.G, thalline is suspended in to the 0.1M CaCl of 1mL precooling ₂in solution, inhale and beat gently with liquid-transfering gun, make cell Eddy diffusion.H, to add 50% glycerine to make its final concentration be 15% and be sub-packed in-80 ℃ of preservations by 100 μ L/ pipes.

(6) clone of recovery DNA fragmentation

The DNA fragmentation reclaiming being connected with T carrier, and transforming competent escherichia coli cell, operate in strict accordance with pMD18-T Vector(TaKaRa Code:D101A) specification sheets carries out.Kit components comprises: pMD18-T Vector(50ng/ μ L), Control Insert(50ng/ μ L), SolutionI.

A, in Eppendorf tube, prepare following DNA solution, full dose is 5 μ L.While clone, the mol ratio of carrier DNA and insertion DNA is 1:2-1:10.B, add the Solution I of 5 μ l.Linked system is: pMD18-T Vector1 μ L, Insert DNA3 μ L, Solution I5 μ L, supplement ddH ₂o to 10 μ L.C, 16 ℃ of reaction 30min.D, full dose 10 μ L are added in 100 μ L JM109 competent cells, place 30min in ice.E, after 42 ℃ of 45 seconds of heating, then in ice, place 1min.F, add 890 μ l LB substratum, 37 ℃ of shaking culture 60min.G, on the LB Agar Plating that contains X-Gal, IPTG, Amp, cultivate.Counting white, blue colonies.H, select white colony, confirm by PCR method.

(7) bacterium colony PCR checking conversion results

Nest-type PRC second shown in bacterium colony PCR employing table 2 overlaps primer to carry out, i.e. S3+S4, T3+T4.PCR system is: 5U/ μ L TaKaRa Ex Taq0.2 μ L, 5mM Mg ²⁺plus2 × GC Buffer I 10 μ L, 2.5mM dNTP Mixtuer3.2 μ L, 1 μ M Primer10.6 μ L, 1 μ M Primer20.6 μ L, make up water to 20 μ L, preparation bacterium colony PCR premixed liquid, select hickie with the toothpick of sterilizing to PCR premixed liquid from plate culture medium, carry out bacterium colony PCR checking.TTSuV1 bacterium colony PCR reaction conditions: 94 ℃, 5min denaturation; Then 35 circulations: 94 ℃, 30s sex change, 60 ℃, 72 ℃ of 30s annealing, 2min extends; Circulate latter 72 ℃, 5min extends.Get 3 μ l PCR products and carry out 1% agarose gel electrophoresis observation.TTSuV2 bacterium colony PCR reaction conditions is identical with TTSuV1, and just 30s annealing temperature is 62 ℃.

(8) extracting of plasmid

According to bacterium colony PCR result, choose the bacterium liquid extracting plasmid that possibility is correct, use plasmid extraction test kit TaKaRa MiNiBEST Plasmid Purification Kit Ver.2.0 (50 amounts) extracting, operation is carried out in strict accordance with specification sheets, and test kit composition comprises: Solution I, Solution II, Solution III, Rinse A, Rinse B, Elution Buffer.

A, colibacillary cultivation: select in the LB liquid nutrient medium that contains Amp of single colony inoculation to 1～4mL 37 ℃ of incubated overnight from plate culture medium.B, get the bacterium liquid of the overnight incubation of 1～4mL, the centrifugal 2min of 12,000rpm, abandons supernatant.C, containing the Solution I of RNase A, bacterial precipitation is fully suspended with 250 μ L.D, add 250 μ L Solution II up and down gently upset mix, make the abundant cracking of thalline, form transparent solution.E, add the Solution III of 4 ℃ of precoolings of 400 μ L, upset mixes gently, until form the aggegation piece of consolidation, then leaves standstill 2min in room temperature.F, room temperature 12, the centrifugal 10min of 000rpm, gets supernatant.G, Spin Column is placed on new Collection Tube.H, the supernatant liquor of upper step is moved in Spin Column, the centrifugal 1min of 12,000rpm, abandons filtrate.I, to the Rinse A that adds 500 μ L in Spin Column, the centrifugal 30s of 12,000rpm, abandons filtrate.J, to the Rinse B that adds 700 μ L in Spin Column, the centrifugal 30s of 12,000rpm, abandons filtrate.K, repeating step J.L, Spin Column is placed in to Collection Tube, the centrifugal 1min of 12,000rpm, residul liquid-removing.M, SpinColumn is placed on new 1.5mL centrifuge tube, adds sterile purified water or the Elution Buffer of 60 μ l in Spin Column film centre, room temperature leaves standstill 1min.N, 12, the centrifugal 1min eluted dna of 000rpm.

(9) concentration quantitative of plasmid DNA and Purity

Nucleic acid-protein uv analyzer is surveyed the pMD18-TTV1 of extraction and the concentration of pMD18-TTV2 plasmid.

(10) enzyme is cut checking plasmid and is sent order-checking

Recombinant plasmid is carried out to double digestion with restriction enzyme ScaI and XbaI, the enzyme system of cutting is: 10 × Kbuffer2 μ L, plasmid DNA 6 μ L, ScaI1 μ L, XbaI1 μ L, supplement ddH ₂o to 20 μ L.After 37 ℃ of endonuclease reaction 2-3h, get 5 μ L and carry out 1% agarose gel electrophoresis analysis.

(11) order-checking of recombinant plasmid dna and sequential analysis

Enzyme is cut to recombinant plasmid that result is correct and serve Hai Shenggong and check order, and the TTSuV1 of acquisition and TTSuV2 portion gene sequence are logged in to NCBI carry out respectively BLAST and analyze relatively.

(12) dilution of recombinant plasmid

Get 10 μ L recombinant plasmid dna solutions, carry out a series of dilutions by 10 extension rate respectively with DEPC water, be prepared into series of standards product DNA solution, be stored in-20 ℃, for production standard curve.

The foundation of 2.TTSuV Taqman real-time fluorescence PCR condition optimizing and method

(1) TTSuV genomic sequence analysis, design primer and probe

TTSuV1 type is take Sd-TTV31 (accession numbers AB076001) as prototype, TTSuV2 type is take Sd-TTV2p (accession numbers AY823991) as prototype, these two kinds genotypic homology less thaies 50%, for the UTR region of TTV high conservative, according to Taqman primer and probe design principle, and finishing screen is selected the Taqman fluorescent probe of primer also and in the amplification region of this primer, the fluorescence report group of TTV1 probe 5 ' end mark is FAM, the fluorescent quenching group of 3 ' end mark is BHQ1, the fluorescence report group of TTV2 probe 5 ' end mark is HEX, the fluorescent quenching group of 3 ' end mark is BHQ1.BHQ1 is the cancellation reporter group of non-fluorescence dye, but self emitting fluorescence not.Primer and probe sequence are as shown in table 2, and primer and probe entrust Shanghai Ying Jun Bioisystech Co., Ltd synthetic.

Primer and the probe of table 2 the application design

(2) optimization of TTSuV Taqman real-time fluorescence PCR condition

To obtain pMD18-TTV1 and pMD18-TTV2 plasmid as positive criteria product template.Grope respectively Taqman rt-PCR reaction conditions and the system of the suitableeest TTSuV1 and TTSuV2.

In pcr amplification, the height of the variation of temperature, particularly annealing temperature produces larger impact to the amplification of PCR.Improve annealing temperature, the specificity of Taqman rt-PCR reaction improves, but the amount of product reduces; And annealing temperature reduce time, present contrary result.

From the plasmid that pMD18-TTV1 and pMD18-TTV2 have diluted, select 10 respectively ^-1-10 ^-6these 6 gradients, arrange annealing temperature gradient at 53 ℃-65 ℃ and carry out glimmering quantitative PCR, with the time of occurrence of amplification curve, smoothness of curve, plateau the standard such as fluorescence intensity power find out optimum annealing temperature.

The primer sequence of TTSuV1 and TTSuV2 is as shown in table 2, and application of sample, on 96 orifice plates, reacts on real-time fluorescence PCR instrument (Bio Rad, CFX96Real-time System).Every secondary response is all take DEPC water as blank.The preliminary examination reactant of TTV Taqman rt-PCR is: Premix Ex Taq ^tM(2 ×) 10 μ L, V1-F/V2-F (10 μ M) 0.8 μ L, V1-R/V2-R (10 μ M) 0.8 μ L, V1-P/V2-P (10 μ M) 0.4 μ L, DNA profiling 3 μ L, supplement ddH ₂o20 μ L.Taqman rt-PCR reaction conditions: 95 ℃, 90s denaturation; Then enter 40 circulations: 95 ℃, 10s sex change arranges 8 thermogrades between 53-65 ℃, the 30s extension of annealing.In extending, collect fluorescent signal.

Determine after optimum annealing temperature, more respectively with 10 of pMD18-TTV1 and pMD18-TTV2 plasmid ^-3plasmid is template, in the reaction system that is template at the positive plasmid of same concentrations, the primer and the probe optimum concn that adopt the preferred TTV1 of matrix method and TTV2, fluorescent primer concentration is at 0.1 μ M between 0.8 μ M, and concentration and probe concentration is screened between 0.8 μ M at 0.1 μ M.Primer concentration is that 0.1 μ M, 0.2 μ M, 0.4 μ M, 0.6 μ M, 0.8 μ M and concentration and probe concentration are 0.1 μ M, 0.2 μ M, 0.4 μ M, 0.6 μ M, 0.8 μ M combination of two test, determines optimum primer concentration and probe concentration ratio, reads detected result.

The amplification situation of contrast under different condition, follows amplification curve and reaches the optimization principles of minimum Ct value and maximum amplification efficiency, selectes peak optimization reaction system and the amplification condition of pig TTV Taqman rt-PCR.

(3) TTSuV Taqman real-time fluorescence PCR specific test

According to ordinary method, utilize Roche, MagNA Pure LC2.0, to PRV (Pseudorabies virus) (Pseudorabies virus, PRV), pig parvoviral (Porcine Parvovlirus, PPV), the virus such as porcine circovirus 2 type (Porcinecircovirus type2, PCV2) is carried out nucleic acid extraction.

Use respectively TTSuV1Taqman rt-PCR and the TTSuV2Taqman rt-PCR detection method set up, nucleic acid to above-mentioned virus detects, to determine the specificity of the method, simultaneously, recombinant plasmid pMD18-TTV1 and the positive contrast of pMD18-TTV2 to build respectively, the contrast of DEPC water belongs with yin.

(4) TTSuV Taqman real-time fluorescence PCR sensitivity test

Get pMD18-TTV1 and pMD18-TTV2 gradient dilution sample is template, extent of dilution is 10 ⁰-10 ^-10recombinant plasmid, carry out real-time fluorescence amplification by the method after optimizing respectively, detect the sensitivity of the method.

(5) TTSuV Taqman real-time fluorescence PCR replica test

Use respectively TTSuV1Taqman rt-PCR and the TTSuV2Taqman rt-PCR detection method set up, the TTSuV1 to 3 different concns and TTSuV2 positive plasmid carry out independent detection 5 times under same reaction conditions, to determine batch interior repeatability of this method; Identical sample was detected in continuous 5 days, with determine this method batch between repeatability.

(6) drafting of TTSuV Taqman real-time fluorescence PCR typical curve

PMD18-TTV1 and pMD18-TTV2 plasmid are respectively got 6 extent of dilution (10 ^-2-10 ^-7) as standard form, every gradient is done 6 Duplicate Samples, uses respectively this routine TTSuV1 and TTSuV2 substance Taqman real time fluorescence quantifying PCR method, carries out quantitative fluorescent PCR reaction, calculates Ct value drawing standard curve.

Three, results and analysis

1. preparation positive criteria product result

(1) nest-type PRC amplified production electrophoresis result

Carry out nest-type PRC amplification with the primer pair sample of TTSuV1 and TTSuV2, the result obtaining shows, the product size that amplification TTSuV1 fragment obtains is 1397bp, and the product size that amplification TTSuV2 fragment obtains is 1268bp, and electrophoresis result is consistent with expection clip size.Result is as shown in A part in Fig. 1, and wherein, M is DNA Marker DL2000, and 1 is TTV1 positive, and 2 is TTV2 positive.

(2) bacterium colony PCR electrophoresis result

Picking hickie carries out bacterium colony PCR checking, and the result obtaining is as shown in B in Fig. 1, and the product size that amplification TTSuV1 clone positive bacteria obtains is 1397bp; The product size that amplification TTSuV2 clone positive bacteria obtains is 1268bp; Electrophoresis result is consistent with expection clip size.In the B of Fig. 1, M is DNA Marker DL2000, and 1 is TTV1 positive amplified production, and 2 is TTV2 positive amplified production.

(3) plasmid enzyme restriction checking electrophorogram

The recombinant plasmid of extracting is carried out to double digestion checking, and as shown in Figure 2, the product after TTSuV1 recombinant plasmid enzyme is cut is the pMD18-T carrier of about 2692bp and the Insert Fragment of about 1397bp to result.The pMD18-T carrier that product after TTSuV2 recombinant plasmid enzyme is cut is about 2692bp and the Insert Fragment of about 1268bp.Result and expection number of fragments and in the same size, the DNA fragmentation and the test design that show to be connected into T carrier are in full accord.In Fig. 2, M is DNA Marker DL5000, and 1 is TTV1 positive amplified production, and 2 is TTV2 positive amplified production.

(4) plasmid is measured concentration

Recombinant plasmid is carried out to concentration determination with nucleic acid-protein analyser, the concentration of pMD18-TTV1 recombinant plasmid is 40.54ng/ μ L, and the concentration of recombinant plasmid pMD18-TTV2 is 24.24ng/ μ L, the OD of two recombinant plasmids ₂₆₀/ OD ₂₈₀all >1.8, show that the plasmid purity of extracting is better.

(5) sequencing result

Recombinant plasmid pMD18-TTV1 and pMD18-TTV2 are after the raw work order-checking in Shanghai is identified, the TTSuV1 of acquisition and TTSuV2 partial dna sequence are carried out to the homology contrast of DNA sequence dna at the state-run bioinformation of U.S. center NCBI Blast on-line analysis software, whether checking is TTV sequence, result shows that pMD18-TTV1 and Sd-TTV31 (accession numbers AB076001) matching degree are that 97%, pMD18-TTV2Sd-TTV2p (accession numbers AY823991) matching degree is 89%.Primer specificity amplification is described and without mispairing, clone's success has also kept the integrity of sequence, and recombinant plasmid pMD18-TTV1 and pMD18-TTV2 are the expection standard substance in this experiment.

(6) dilution of recombinant plasmid

By DNA concentration and copy number reduction formula its concentration unit that converts, after converting, the concentration of TTSuV1 standard solution is 9.2 × 10 ⁹copies/ μ L.The concentration of TTSuV2 standard solution is 5.6 × 10 ⁸copies/ μ L.

The reduction formula adopting in the application is:

Get 10 μ L TTSuV1 standard solutions, dilute by 10 extension rate respectively with DEPC water, be prepared into 9.2 × 10 ⁹copies/ μ L-9.2 × 10 ⁶copies/ μ L, extent of dilution is 10 ⁰-10 ^-3, get 10 μ L TTSuV2 standard solutions, carry out a series of dilutions by 10 extension rate respectively with DEPC water, be prepared into 5.6 × 10 ⁸copies/ μ L-5.6 × 10 ⁵copies/ μ L, extent of dilution is 10 ⁰-10 ^-3, by 10 ^-3standard substance packing in a small amount, while using standard substance DNA, takes out 1 pipe, presses 1:10 gradient dilution, for production standard curve temporarily.

2.TTSuV Taqman real-time fluorescence PCR result

(1) reaction conditions and the system after the optimization of TTSuV Taqman real-time fluorescence PCR

TTSuV1 standard substance 9.2 × 10 ⁶copies/ μ L concentration recombinant plasmid 10 ^-18 the Gradient annealing temperature of dilution plasmid within the scope of 53 ℃-65 ℃ carry out glimmering quantitative PCR reaction, result shows, temperature is higher, amplification efficiency is lower; 57.7 ℃, 55.4 ℃, 53.8 ℃, 53 ℃ of these 4 annealing temperature amplifications are better, and result is very approaching, as shown in Figure 3.10 of TTSuV1 standard substance ^-1-10 ^-6the result that these 6 gradient plasmids carry out glimmering quantitative PCR reaction within the scope of 53 ℃-65 ℃ shows: at 55.4 ℃ of the highest E=101.9% of amplification efficiency, result as shown in Figure 4.In Fig. 4, be from left to right sequentially 10 ^-1-10 ^-6the detection curve of 6 gradient plasmids.

TTSuV2 standard substance 5.6 × 10 ⁵10 of the recombinant plasmid of copies/ μ L concentration ^-18 different annealing temperature of dilution plasmid within the scope of 53 ℃-65 ℃ carry out glimmering quantitative PCR reaction, result shows, temperature is higher, amplification efficiency is lower, 57.7 ℃, 55.4 ℃, 53.8 ℃, the amplification of 53 ℃ of these 4 annealing temperatures is better, and result is very approaching, as shown in Figure 5.10 of TTSuV2 standard substance ^-1-10 ^-6these 6 gradient plasmids carry out Taqman rt-PCR reaction result and show within the scope of 53 ℃-65 ℃, and at 55.4 ℃ of the highest E=97.5% of amplification efficiency, result as shown in Figure 6, is from left to right sequentially 10 ^-1-10 ^-66 gradient plasmid detection curves.

This example adopts primer and the probe optimum concn of the preferred TTSuV1 of matrix method and TTSuV2, result shows, final definite primer probe final concentration is respectively 0.6 μ mol/L and 0.6 μ mol/L, detect and obtain Ct value minimum, amplified fluorescence curve is the most typical and Δ Rn value is larger, thereby be defined as best primer and concentration and probe concentration.

Reaction system after optimization is: Premix Ex Taq ^tM(2 ×) 10 μ L, V1-F/V2-F (10 μ M) 1.2 μ L, V1-R/V2-R (10 μ M) 1.2 μ L, V1-P/V2-P (10 μ M) 0.6 μ L, DNA profiling 3 μ L, supplement ddH ₂o20 μ L.Reaction conditions after optimization is: 95 ℃, and 90s denaturation; Then carry out 40 circulations: 95 ℃, 10s sex change, 55.4 ℃, the 30s extension of annealing.

(2) TTSuV Taqman real-time fluorescence PCR specific test

Adopt TTSuV1 and the TTSuV2Taqman rt-PCR method that this experiment is set up to detect TTSuV1 and TTSuV2 virus and other pig DNA virus, result shows, TTSuV1Taqman real time fluorescence quantifying PCR method only can obtain specific amplified fluorescence curve to pig TTSuV1, and TTSuV2 and other viruses are not had to amplified fluorescence curve, result is as shown in Figure 7; TTSuV2Taqman rt-PCR method only can obtain specific amplified fluorescence curve to TTSuV2, and TTSuV1 and other viruses are not had to amplified fluorescence curve, and result as shown in Figure 8.Show that TTSuV1Taqman real-time fluorescence PCR and TTSuV2Taqman real-time fluorescence PCR detection method that this experiment is set up have good specificity to TTSuV1 and TTSuV2 virus, with other pig DNA virus no cross reaction.

(3) TTSuV Taqman real-time fluorescence PCR sensitivity test

The detected result of 11 of pMD18-TTV1 gradient plasmids is shown, 9.2 × 10 ⁹within the scope of copies/ μ L-9.2copies/ μ L, the amplification curve of each concentration gradient is level and smooth, and reaction system efficiency is high, plateau,, fluorescence intensity was strong, and concentration and CT value have good linear relationship, as shown in Figure 9, fluorescence curve puts in order, and from left to right template concentrations is followed successively by 9.2 × 10 ⁹copies/ μ L, 9.2 × 10 ⁸copies/ μ L, 9.2 × 10 ⁷copies/ μ L, 9.2 × 10 ⁶copies/ μ L, 9.2 × 10 ⁵copies/ μ L, 9.2 × 10 ⁴copies/ μ L, 9.2 × 10 ³copies/ μ L, 9.2 × 10 ²copies/ μ L, 9.2copies/ μ L.Taqman rt-PCR method detects the genomic detection of TTV1 and is limited to 9.2copies/ μ L, highly sensitive.

The detected result of 11 of pMD18-TTV2 gradient plasmids is shown, 5.6 × 10 ⁸copies/ μ L-5.6 × 10 ¹within the scope of copies/ μ L, the amplification curve of each concentration gradient is level and smooth, and reaction system efficiency is high, plateau,, fluorescence intensity was strong, and concentration and Ct value have good linear relationship, as shown in figure 10, fluorescence curve puts in order, and from left to right template concentrations is followed successively by 5.6 × 10 ⁸copies/ μ L, 5.6 × 10 ⁷copies/ μ L5.6 × 10 ⁶copies/ μ L, 5.6 × 10 ⁵copies/ μ L, 5.6 × 10 ⁴copies/ μ L, 5.6 × 10 ³copies/ μ L, 5.6 × 10 ²copies/ μ L, 5.6 × 10 ¹copies/ μ L.Taqman rt-PCR method detects the genomic detection of TTV2 and is limited to 56copies/ μ L, highly sensitive.

(4) TTSuV Taqman real-time fluorescence PCR replica test

Use TTSuV1Taqman rt-PCR method under same reaction conditions, to do respectively and in 5 batches, repeat and repeat between 5 batches the TTSuV1 standard substance of 3 different concns, result shows that batch interior variation coefficient that repeats is all less than 1%, in batch, repeat the variation coefficient and be all less than 3%, show that the method is reproducible, refer to table 3.

Table 3TTSuV1 real-time fluorescence PCR repeated experiment result

Use TTSuV2 substance Taqman rt-PCR method under same reaction conditions, to do respectively and in 5 batches, repeat and repeat between 5 batches 3 different concns TTSuV2 standard substance, result shows that batch interior variation coefficient that repeats is all less than 1%, in batch, repeat the variation coefficient and be all less than 3%, show that the method is reproducible.Refer to table 4.

Table 4TTSuV2 real-time fluorescence PCR repeated experiment result

(5) TTSuV Taqman real-time fluorescence PCR typical curve

This experiment is take the logarithm of the copy number of plasmid template as X-axis, and Ct value has been drawn TTSuV1Taqman rt-PCR typical curve for Y-axis, and as shown in figure 11, fluorescence curve puts in order the amplification curve of TTSuV1 standard substance, and from left to right template concentrations is followed successively by 9.2 × 10 ⁷copies/ μ L, 9.2 × 10 ⁶copies/ μ L, 9.2 × 10 ⁵copies/ μ L, 9.2 × 10 ⁴copies/ μ L, 9.2 × 10 ³copies/ μ L, 9.2 × 10 ²copies/ μ L.Wherein amplification efficiency E calculates according to the slope meter of typical curve, and equation equation is: E=10-1/ slope.Result shows, 9.2 × 10 ⁷-9.2 × 10 ²within the scope of copies/ μ L, amplification efficiency is 96.9%, and the slope of typical curve is-3.398, and intercept is that 42.685, R2 is 1.000.In each concentration range, have good linear relationship, straight-line equation is: y=-3.398X+42.685, and y is cycle values, x is log copy number/μ L.

Take the logarithm of the copy number of plasmid template as X-axis, Ct value has been drawn TTSuV2Taqman rt-PCR typical curve for Y-axis.As shown in figure 12, fluorescence curve puts in order the amplification curve of TTSuV2 standard substance, and from left to right template concentrations is followed successively by 5.6 × 10 ⁶copies/ μ L, 5.6 × 10 ⁵copies/ μ L, 5.6 × 10 ⁴copies/ μ L, 5.6 × 10 ³copies/ μ L, 5.6 × 10 ²copies/ μ L, 5.6 × 10 ¹copies/ μ L.5.6 × 10 ⁶copies/ μ L-5.6 × 10 ¹within the scope of copies/ μ L, amplification efficiency is 95.3%, and the slope of typical curve is-3.44, and intercept is that 39.511, R2 is 0.998.In each concentration range, have good linear relationship, straight-line equation is: y=-3.44X+39.511, and wherein y is cycle values, x is log copy number/μ L.

In the time that submitted sample is detected, can calculate the contained viral load of this sample according to the Ct value and the typical curve that obtain, but detected be viral DNA copy number, but not there is infective virion.

The application has set up respectively Taqman real-time fluorescence PCR detection method for TTSuV1, TTSuV2, has the advantages such as quick, sensitive, special, qualitative, quantitative, for the quick diagnosis of TTSuV is laid a good foundation.Compare with conventional PCR, present method not only can qualitative detection TTSuV1, TTSuV2, and can detect the pathological material of disease that conventional PCR cannot detect, and the step of also having removed DNA level electrophoresis from, saves time more, and simple to operate.The detection method of utilizing this test to set up, can carry out in early days TTSuV infected pigs, rapid detection and epidemiology survey, also provides new technique means for studying infection and the distribution of TTSuV in pig body simultaneously.

Embodiment bis-dual real-time fluorescence PCRs detect

One, test materials and equipment

This routine material and facility is identical with embodiment mono-.

Two, test method

1. the foundation of dual Taqman real-time fluorescence PCR condition optimizing and method

In order further to improve detection efficiency, reduce experimental procedure, experimental period and the consumption to reagent, on the basis of the substance Taqman real-time fluorescence PCR detection method of the TTSuV1 having set up and TTSuV2, grope the dual Taqman real-time fluorescence PCR detection method of TTSuV1 and TTSuV2.

Because of this example each free best primer probe pair of specificity in theory in the time designing respectively the substance Taqman real-time fluorescence PCR primer probe of TTSuV1 and TTSuV2, by DNAstar software analysis, confirm that two probes, four primers can not form dimer each other.And the reaction system of the substance Taqman real-time fluorescence PCR optimum of TTSuV1 and TTSuV2 is consistent with reaction conditions, therefore, while groping dual Taqman real-time fluorescence PCR, primer in the TTSuV1 having optimized and TTSuV2 substance PCR in real time and the concentration of probe are mixed in reaction system with 1:1, take pMD18-TTV1 and pMD18-TTV2 mixing plasmid as template, in the reaction system and reaction conditions basis of the substance Taqman of TTSuV1 and TTSuV2 real-time fluorescence PCR optimum, by to each factor adjustment, further optimize and make amplification curve reach minimum Ct value and obtain maximum amplification efficiency.

2. not cross reaction of dual Taqman real-time fluorescence PCR

The primer and the probe that in reaction system, contain amplification TTV1 and TTV2, be divided into two groups by reaction: first group of doubling dilution sample take TTV1 standard substance is as template; Second group take TTV2 standard substance doubling dilution sample as template, under the dual Taqman real-time fluorescence PCR of the pig TTV reaction conditions having established, increases.

The dual Taqman real-time fluorescence PCR of 3.TTSuV specific test

The dual Taqman real-time fluorescence PCR detection method of TTSuV that uses the application to set up, to PRV (Pseudorabies virus) (Pseudorabies virus, PRV), pig parvoviral (Porcine Parvovlirus, PPV), porcine circovirus 2 type (Porcinecircovirus type2, PCV2) detect, to determine the specificity of the method, with the positive contrast of mixing plasmid of pMD18-TTV1 and pMD18-TTV2, the contrast of DEPC water belongs with yin.

The dual Taqman real-time fluorescence PCR of 4.TTSuV sensitivity test

Get the gradient plasmid (10 of pMD18-TTV1 and pMD18-TTV2 ⁰-10 ^-10), respectively take it as template, carry out the dual Taqman rt-PCR amplification of pig TTV, detect the sensitivity of the method.

The dual Taqman real-time fluorescence PCR of 5.TTSuV replica test

The dual Taqman rt-PCR detection method that uses this experiment to set up, the pig TTSuV1 to 3 different concns and TTSuV2 positive plasmid carry out independent detection 5 times under same reaction conditions, to determine batch interior repeatability of this method; Continuous 5 days to dilute sample detect, with repeatability between determining batch.

The dual Taqman real-time fluorescence PCR of 6.TTSuV typical curve

PMD18-TTV1 and pMD18-TTV2 plasmid are respectively got 6 gradients (10 ^-2-10 ^-7) as standard form, each dilution template is respectively done 6 Duplicate Samples, with the annealing temperature after optimizing and the reaction density of primer and probe, carries out dual real-time fluorescence PCR reaction respectively, calculates Ct value drawing standard curve.

7. the detection of simultaneous test-additive method to clinical sample

According to document, method by conventional at present detection TTSuV: regular-PCR somatotype detects and not somatotype detection of TTSuV Taqman rt-PCR, 220 samples to the third quarter in 2011 detect, and TTSuV1Taqman real-time fluorescence PCR and the TTSuV2Taqman real-time fluorescence PCR detection method in detected result and this experiment, set up are compared.The primer probe sequence of simultaneous test is as shown in table 5.

The primer that table 5 simultaneous test adopts

It is as shown in table 5 that regular-PCR somatotype detects primer sequence, reaction system is: Premix Ex Taq(2 ×) 10 μ L, TTV1/TTV2-F (10 μ M) 0.5 μ L, TTV1/TTV2-R (10 μ M) 0.5 μ L, DNA profiling 4 μ L, supplement ddH ₂o20 μ L.Reaction conditions is: 94 ℃, and 5min denaturation; Then carry out 50 circulations: 94 ℃, 15s sex change, 54 ℃ (TTSuV1/TTSuV2), and 20s annealing, 72 ℃, 30s extends; After circulation completes, 72 ℃, 5min extends.Whole reaction is at grads PCR instrument, Eppendorf, and AG22331Hamburg, above carries out, and gets 3 μ L PCR products and carry out 1% agarose gel electrophoresis observation after reaction finishes.

The TTV Taqman rt-PCR primer sequence that somatotype does not detect is as shown in table 5, and reaction system is: Premix Ex Taq ^tM(2 ×) 12.5 μ L, QCOM-F (10 μ M) 0.75 μ L, QCOM-R (10 μ M) 0.325 μ L, QCOM-P(10 μ M) 0.75 μ L, DNA profiling 2.5 μ L, supplement ddH ₂o25 μ L, reaction conditions reaction conditions is: 95 ℃, 10min denaturation; Then carry out 40 circulations: 95 ℃, 15s sex change, 60 ℃, the 1min extension of annealing.Application of sample is on 96 orifice plates, and at real-time fluorescence PCR instrument, Bio Rad, reacts on CFX96Real-time System.Every secondary response is all take DEPC water as blank.

Read detected result.Manually threshold line is adjusted to the vertex just above normal negative control product amplification curve.Blank is without Ct value and without specific amplification curve.The Ct value of positive control answers≤38, and occurs specific amplification curve.Otherwise it is invalid that this time experiment is considered as.

Three, test results and analysis

The not cross matching of the dual TTV Taqman of 1.TTSuV real-time fluorescence PCR

Result shows: TTSuV1 standard substance react in the system of only having TTSuV2 primer probe, and without any amplification curve, TTSuV2 standard substance react in the system of only having TTSuV1 primer probe, also without any amplification curve.Illustrate that this two covers primer probe specificity is good, there is no cross reaction.

Reaction conditions and system after the dual Taqman real-time fluorescence PCR of 2.TTSuV is optimized

In the process of optimizing, to add a kind of positive criteria product be template and add TTSuV1 and when two kinds of positive criteria product of TTSuV2 are template simultaneously, the slope of its typical curve and all less in the intercept variation of Y-axis, shows the dual reaction amplification efficiency that do not have a significant effect; But consider that two cover primer probes have competitive effect simultaneously, this experiment is by suitably having adjusted premix amount, make the identical template that increases, the result of the Ct value that dual Taqman rt-PCR method obtains and amplification efficiency and substance Taqman rt-PCR is more approaching.

After optimizing, reaction system is: Premix Ex Taq ^tM(2 ×) 15.2 μ L, V1-F (40 μ M) 0.6 μ L, V1-R (40 μ M) 0.6 μ L, V1-P (40 μ M) 0.3 μ L, V2-F (40 μ M) 0.6 μ L, V2-R (40 μ M) 0.6 μ L, V2-P (40 μ M) 0.3 μ L, DNA profiling 3 μ L, supplement ddH ₂o20 μ L.After optimizing, reaction conditions is: 95 ℃, and 90s sex change; Then 40 circulations: 95 ℃, 10s sex change, 55.4 ℃, the 30s extension of annealing.

The dual Taqman real-time fluorescence PCR of 3.TTSuV specific test

Applying the dual Taqman rt-PCR of the TTSuV method that this experiment sets up detects TTSuV1 and TTSuV2 virus and other pig DNA virus, result shows: remove TTSuV1 and TTSuV2 and can obtain specific amplified fluorescence curve, and other viruses do not have amplified fluorescence curve, as shown in figure 13.Show that the TaqMan rt-PCR detection method that the application sets up has good specificity to TTSuV1 and TTSuV2 virus, with other common pig DNA virus no cross reaction.

The dual Taqman real-time fluorescence PCR of 4.TTSuV sensitivity test

The detected result of 11 of pMD18-TTV1 gradient plasmids is shown, 9.2 × 10 ⁹within the scope of copies/ μ L-9.2copies/ μ L, the amplification curve of each concentration gradient is level and smooth, and reaction system efficiency is high, plateau,, fluorescence intensity was strong, concentration and CT value have good linear relationship as shown in figure 14, and fluorescence curve puts in order, and from left to right template concentrations is followed successively by 9.2 × 10 ⁹, 9.2 × 10 ⁸, 9.2 × 10 ⁷, 9.2 × 10 ⁶, 9.2 × 10 ⁵, 9.2 × 10 ⁴, 9.2 × 10 ³, 9.2 × 10 ², 9.2copies/ μ L.Taqman rt-PCR method detects the genomic detection of TTV1 and is limited to 9.2copies/ μ L, highly sensitive.

The detected result of 11 of pMD18-TTV2 gradient plasmids is shown, 5.6 × 10 ⁸copies/ μ L-5.6 × 10 ¹within the scope of copies/ μ L, each concentration gradient amplification curve is level and smooth, and reaction efficiency is high, plateau fluorescence intensity strong, concentration and CT value have good linear relationship as shown in figure 15, fluorescence curve puts in order, from left to right template concentrations is followed successively by 5.6 × 10 ⁸, 5.6 × 10 ⁷, 5.6 × 10 ⁶, 5.6 × 10 ⁵, 5.6 × 10 ⁴, 5.6 × 10 ³, 5.6 × 10 ², 5.6 × 10 ¹copies/ μ L, Taqman rt-PCR method detects the genomic detection of TTV2 and is limited to 56copies/ μ L, highly sensitive.

The dual Taqman real-time fluorescence PCR of 5.TTSuV replica test

Use the dual Taqman rt-PCR of TTSuV method under same reaction conditions, to do respectively and in 5 batches, repeat and repeat between 5 batches the TTSuV1 standard substance of 3 different concns, result shows that batch interior variation coefficient that repeats is all less than 1%, in batch, repeat the variation coefficient and be all less than 3%, in table 6, show that the method is reproducible.

Table 6 real-time fluorescence quantitative PCR detect TTSuV1 criticize interior and batch between revision test

Use the dual Taqman rt-PCR of TTSuV method under same reaction conditions, to do respectively and in 5 batches, repeat and repeat between 5 batches the TTSuV2 standard substance of 3 different concns, result shows that batch interior variation coefficient that repeats is all less than 1%, in batch, repeat the variation coefficient and be all less than 3%, in table 7, show that the method is reproducible.

Table 7 real-time fluorescence quantitative PCR detect TTSuV2 criticize interior and batch between revision test

The dual Taqman real-time fluorescence PCR of 6.TTSuV typical curve

This example is take plasmid template copy number logarithm as X-axis, and Ct value has been drawn the dual Taqman rt-PCR of TTSuV typical curve for Y-axis, and as shown in figure 16, fluorescence curve puts in order the amplification curve of TTSuV1 standard substance, and from left to right template concentrations is followed successively by 9.2 × 10 ⁷copies/ μ L, 9.2 × 10 ⁶copies/ μ L, 9.2 × 10 ⁵copies/ μ L, 9.2 × 10 ⁴copies/ μ L, 9.2 × 10 ³copies/ μ L, 9.2 × 10 ²copies/ μ L.Wherein amplification efficiency E calculates according to the slope meter of typical curve, and equation equation is: E=10-1/ slope.Result shows: 9.2 × 10 ⁷copies/ μ L-9.2 × 10 ²within the scope of copies/ μ L, amplification efficiency is 97.8%, and the slope of typical curve is-3.377, and intercept is that 42.119, R2 is 1.000.In each concentration range, there is good linear relationship, as Figure 17.Straight-line equation is: y=-3.377x+42.119, and wherein y is cycle values, x is log copy number/μ L.

Take the logarithm of the copy number of plasmid template as X-axis, Ct value has been drawn the dual Taqman rt-PCR of TTV typical curve for Y-axis.As shown in figure 18, fluorescence curve puts in order the amplification curve of TTV2 standard substance, and from left to right template concentrations is followed successively by 5.6 × 10 ⁶copies/ μ L, 5.6 × 10 ⁵copies/ μ L, 5.6 × 10 ⁴copies/ μ L, 5.6 × 10 ³copies/ μ L, 5.6 × 10 ²copies/ μ L, 5.6 × 10 ¹copies/ μ L.5.6 × 10 ⁶copies/ μ L-5.6 × 10 ¹within the scope of copies/ μ L, amplification efficiency is 96.2%, and the slope of typical curve is-3.416, and intercept is that 39.838, R2 is 0.998.In each concentration range, there is good linear relationship, as Figure 19.Straight-line equation is: y=-3.416x+39.838, and wherein y is cycle values, x is log copy number/μ L.

7. comparative test result

QCOM:TTV Taqman rt-PCR carries out not somatotype detection of TTSuV to 220 samples in the third quarter in 2011, and result shows: the infection rate of TTSuV is 80%(176/220).The somatotype that regular-PCR carries out TTV to 220 samples in the third quarter in 2011 detects, and result shows: the infection rate of TTV1 is 59.5%(131/220), the infection rate of TTV2 is 62.3%(137/220), coinfection rate is 40.5%(89/220).

This example is on substance Taqman real-time fluorescence PCR detection method basis, set up the dual Taqman real-time fluorescence PCR detection method that can simultaneously detect TTSuV1 and TTSuV2, there is the advantages such as quick, accurate, special, sensitive, high-throughput, for entry and exit live pig rapid detection provides practical detection means.

Above content is the further description of the application being done in conjunction with concrete embodiment, can not assert that the application's concrete enforcement is confined to these explanations.For the application person of an ordinary skill in the technical field, not departing under the prerequisite of the application's design, can also make some simple deduction or replace, all should be considered as belonging to the application's protection domain.

Claims

1. the primer for the double check of pig thin circovirus virus I type and II type, it is characterized in that: described primer comprises the first primer pair and the second primer pair, the upstream primer of described the first primer pair and downstream primer contain respectively sequence described in Seq ID No.1 and Seq ID No.2, and the upstream primer of described the second primer pair and downstream primer contain respectively sequence described in Seq ID No.4 and Seq ID No.5;

Seq?ID?No.1：5’-GGTTCAGGAGGCTCAATTTGG-3’

Seq?ID?No.2：5’-TTTGAATTTAACGGTTTTCAGTCTTC-3’

Seq?ID?No.4：5’-TATCGGGCAGGCGGTAATC-3’

Seq?ID?No.5：5’-TACGCTACCGTCAGCCATCTTT-3’。

2. primer according to claim 1, is characterized in that: described the first primer pair is the primer pair that detects the thin circovirus virus I of pig type, and described the second primer pair is the primer pair that detects the thin circovirus virus II of pig type.

3. the probe for the double check of pig thin circovirus virus I type and II type, it is characterized in that: described probe comprises the first probe and the second probe, described the first probe contains sequence shown in Seq ID No.3, and described the second probe contains sequence shown in Seq ID No.6;

Seq?ID?No.3：5’-TCGCTTCGCTCGCACCACGT-3’

Seq?ID?No.6：5’-AACCGGGCCCCCCTCGATG-3’。

4. probe according to claim 3, is characterized in that: described the first probe is the probe that detects the thin circovirus virus I of pig type, and described the second probe is the probe that detects the thin circovirus virus II of pig type.

5. probe according to claim 4, is characterized in that: 5 ' end of described the first probe and the second probe is all marked with fluorescence report group, and 3 ' end is all marked with fluorescent quenching group.

6. probe according to claim 5, is characterized in that: the fluorescence report group of 5 ' end mark of described the first probe is FAM, and the fluorescent quenching group of 3 ' end mark is BHQ1; The fluorescence report group of 5 ' end mark of described the second probe is HEX, and the fluorescent quenching group of 3 ' end mark is BHQ1.

7. the dual real-time fluorescence PCR detection method of a boar thin circovirus virus I type and II type, comprises that the primer adopting described in claim 1 or 2 matches and carries out Taqman fluorescence quantitative PCR detection with the probe described in claim 5 or 6.

8. dual real-time fluorescence PCR detection method according to claim 7, it is characterized in that: described the first primer pair and described the first probe match in order to detect the thin circovirus virus I of pig type, and described the second primer pair and described the second probe match in order to detect the thin circovirus virus II of pig type.

9. the test kit of the double check of a boar thin circovirus virus I type and II type, is characterized in that: in described test kit, contain the primer described in claim 1 or 2.

10. test kit according to claim 9, is characterized in that: in described detection kit, also contain the probe described in claim 3-6 any one.