CN109852729A - The thin circovirus virus k2b type real time fluorescent quantitative detection kit of pig - Google Patents

Abstract

The present invention provides the thin circovirus virus k2b type real time fluorescent quantitative detection kit of pig, includes primer and probe, primer and probe sequence is as shown in SEQ ID NO.1-3, primer and probe high specificity provided by the present invention, high sensitivity, identification method is simple, and efficiency and accuracy rate are higher.The detection of TaqMan real-time fluorescence quantitative PCR is carried out to 179 parts of blood serum samples of this clinic inspection using real-time fluorescence quantitative PCR primer and probe provided by the invention, detects 10 parts of positive sample, positive rate 5.59%.

Description

The thin circovirus virus k2b type real time fluorescent quantitative detection kit of pig

Technical field

The invention belongs to epizootiology fields, and in particular to the thin circovirus virus k2b type real time fluorescent quantitative detection examination of pig Agent box.

Background technique

The thin circovirus virus k2b type of pig (Porcine torque teno sus virus k2, PTTSuVk2) is pig in recent years Newfound virus in group, belongs to the thin Circovirus of thin Circoviridae, Kappa.The category has 2 representative species at present, another For the thin circovirus virus k2a type of pig (representative strains are 2p plants, GenBank accession number: AY823991).

The thin circovirus virus of pig (k2a type and k2b type) genome and the thin circovirus virus 1a type of pig (PTTSuV 1a) and 1b type (PTTSuV 1b) (PTTSuV 1a and PTTSuV 1b belong to thin Circoviridae, but are not the thin Circovirus of Kappa but nonyl type Thin Circovirus) composition is similar, and it is the sub-thread cyclic DNA virus of no cyst membrane, viral about 2.8 ~ 3.0 kb of full-length genome size, By the main exploitation reading frame of the end 5' non-translational region (untranslated region, UTR), the end 3' non-translational region (UTR) and two (open reading frame, ORF) ORF1 and ORF2 composition, ORF3 are sheared by ORF2 and ORF1 and are formed.

Currently, the means that detection swinery infects thin circovirus virus are more, mainly there are PCR, real-time fluorescence quantitative PCR (SYBR Green I and TaqMan probe), LAMP etc..But the detection means about the thin circovirus virus k2b type of pig is scarcity, there is not yet real When fluorescence quantifying PCR method correlative study report.Real-time fluorescence quantitative PCR (Quantitative Real-time PCR) It is one kind in DNA amplification reaction, product total amount after each polymerase chain reaction (PCR) recycles is surveyed with fluorescent chemical Method.The method that quantitative analysis is carried out to the specific dna sequence in sample to be tested by internal reference or outer ginseng method.Real-time PCR, by fluorescence signal, is measured in real time to PCR process during PCR amplification.Due in the index of PCR amplification There are linear relationships for the starting copy number of phase, the Ct value of template and the template, so becoming quantitative foundation.Based on TaqMan reality When quantitative fluorescent PCR method a specific fluorescence probe is added when referring to PCR amplification while pair of primers is added, The probe is an oligonucleotides, both ends one reporter fluorescence group of label and a quenching fluorescence group respectively.When probe is complete, The fluorescence signal of reporter group transmitting is quenched group absorptions；When PCR amplification, the 5'-3' 5 prime excision enzyme activity of Taq enzyme is by probe Digestion degradation separates reporter fluorescence group and quenching fluorescence group, so that fluorescence monitoring system can receive fluorescence signal, i.e., As soon as every amplification DNA chain, has a fluorescent molecule to be formed, realizes the accumulation of fluorescence signal and PCR product is formed completely together Step.Based on TaqMan real-time fluorescence quantitative PCR method due to the introducing of probe, specificity is stronger.The present invention is directed to there is not yet base The blank of the thin circovirus virus k2b type of pig is detected in TaqMan real-time fluorescence quantitative PCR method to carry out correlative study.

Summary of the invention

The purpose of the present invention is to provide the thin circovirus virus k2b type real time fluorescent quantitative detection kits of pig.

To achieve the above object, the present invention adopts the following technical scheme:

The thin circovirus virus k2b type real time fluorescent quantitative detection primer of pig and probe, the primer and probe sequence are as follows:

PF1:5 '-AGGACTGCTAGAATCATC-3 '；

PR1:5 '-TGCTCCATGTATAGGTTA-3 '；

Probe pTaqMan-probe:5 '-ACAGTCACATCTATGCCTCCTCC-3 ', wherein 5 ' ends are marked with FAM, 3 ' ends It is marked with BHQ2.

The thin circovirus virus k2b type real time fluorescent quantitative detection kit of pig comprising the primer and probe.

The present invention has the advantages that

Primer and probe high specificity provided by the present invention, high sensitivity, identification method is simple, and efficiency and accuracy rate are higher. TaqMan is carried out to 179 parts of blood serum samples of this clinic inspection using real-time fluorescence quantitative PCR primer and probe provided by the invention Real-time fluorescence quantitative PCR detection, detects 10 parts of positive sample, positive rate 5.59%.

Detailed description of the invention

The amplification kinetic curve of Fig. 1 real-time fluorescence quantitative PCR, wherein 1-8: plasmid concentration is 4.41 × 10⁷~4.41 ×10⁰Copy/μ L.

The standard curve of Fig. 2 real-time fluorescence quantitative PCR.

The specific test of Fig. 3 real-time fluorescence PCR is as a result, 1 be wherein PTTSuV k2b；Experiment contrast is pig common disease Former (such as PTTSuV k2a, PPV, PRV, PCV1, PCV2, PCV3, PRRSV and PEDV).

Specific embodiment

Embodiment 1

1 materials and methods

1.1 material

1.1.1 strain

The thin circovirus virus k2b type of pig (PTTSuV k2b), the thin circovirus virus k2a type of pig (PTTSuV k2a), pig parvoviral (PPV), Porcine pseudorabies virus (PRV), 1 type of pig circular ring virus (PCV1), porcine circovirus 2 type (PCV2), 3 type of pig circular ring virus (PCV3), porcine reproductive and respiratory syndrome virus (PRRSV) and Porcine epidemic diarrhea virus (PEDV) are by Fujian Agricultural section Animal and veterinary research institute, institute identifies and saves.

1.1.2 key instrument and reagent

Sonde method real time fluorescent quantitative reagent THUNDERBIRD Probe qPCR Mix, standard PCR amplification reagent Quick Taq HS Dye Mix spins (Shanghai) Biotechnology Co., Ltd purchased from Japan；Viral nucleic acid extraction reagent box MiniBEST Viral RNA/DNA pMD18-T, DL2000 Marker are purchased from precious bioengineering Dalian Co., Ltd, plastic recovery kit, Small amount plasmid extraction agent box, clone's competent cell are purchased from TIANGEN Biotech (Beijing) Co., Ltd..

The preparation of 1.2 positive criteria products

To specifications using Viral extraction kit MiniBEST Viral RNA/DNA Extraction Kit Ver.4.0 The nucleic acid DNA of the thin circovirus virus k2b type of pig (FJ2017-01 plants) is extracted in operation, is judged with the ORF1 gene for PTTSuV k2b Specific primer (ORF1F:5 '-GGATTACCCGTTGGCTGGATATTT -3 '；ORF1R:5 '- CCTTCCACCCAGTAACTTGT-3′.Expanding fragment length about 777bp.PCR amplification system is 50 μ L systems, is configured as follows: 2 μ 25 μ L of L KOD OneTM PCR Master Mix-Blue, up/down swim primer (ORF1F and ORF1R, 10 pmol) each 1 μ L, 2 μ L extract nucleic acid DNA, and supplement sterile deionized water to final volume is 50 μ L.PCR reaction condition are as follows: after 94 DEG C of 2min PCR cycle (carrying out 35 circulations altogether) is carried out, condition is 94 DEG C of 30s, 55 DEG C of 30s, 68 DEG C of 45 s；PCR cycle terminates Afterwards, in 72 DEG C of 10 min of extension.To target fragment using on plastic recovery kit gel extraction rear clone to carrier T, phase is extracted The plasmid answered, the plasmid after sequencing verifying Pass Test is expected are used as the positive criteria product plasmid of this test (to be denoted as k2b -ORF1).The positive criteria product of acquisition quantifies positive plasmid with ultramicron ultraviolet-uisible spectrophotometer, counts It calculates copy number and carries out 10 times of serial dilutions, it is spare to be placed in -80 DEG C of refrigerators.

The foundation of 1.3 TaqMan real time fluorescent PCR methods

1.3.1 the design of fluorescent quantitation primer

With reference to the ORF1 gene expression characteristics of the PTTSuV k2b logged in GenBank, analysis comparison is carried out using DNAStar software, The highly conserved and special nucleotide region of ORF1 gene order is selected, using 7 software of Oligo, designs the primer of specificity And probe.It is retrieved using BLAST tool, its specificity of preliminary identification, primer is closed by precious bioengineering Dalian Co., Ltd At.PF1:5 '-AGGACTGCTAGAATCATC-3 '；PR1:5 '-TGCTCCATGTATAGGTTA-3 '.Probe sequence PTaqMan-probe:5 '-ACAGTCACATCTATGCCTCCTCC-3 ', wherein 5 ' ends are marked with FAM, and 3 ' ends are marked with BHQ2 Note.

1.3.2 the optimization of reaction condition

Using the THUNDERBIRD Probe qPCR Mix 20 μ L configuration TaqMan real time fluorescence quantifying PCR method recommended Reaction solution.To there is highest fluorescent value (△ Rn), the smallest cycle threshold (Ct value) for index, to annealing temperature (56~64 DEG C), (0.05~0.5 μM) progress condition optimizing of different primers final concentration (0.1~1 μM) and different probe final concentration, set simultaneously Negative control.

1.3.3 the foundation of standard curve and melt curve analysis

To be serially diluted (4.41 × 10 for continuous 10 times of standard positive standard items⁷~4.41 × 10⁰Copy/μ L) plasmid be mould Plate is expanded with the optimal conditions of " 1.3.2 ", obtains amplification kinetic curve.With various concentration standard items (k2b-ORF1 Plasmid) common logarithm of copy number is that abscissa using Ct value as ordinate calculates standard curve (standard linear regression side Journey).

1.3.4 specific detection

The thin circovirus virus k2a type of pig (PTTSuV k2a), pig parvoviral (PPV), pig are detected respectively with the optimal conditions of " 1.3.2 " Pseudorabies virus (PRV), 1 type of pig circular ring virus (PCV1), porcine circovirus 2 type (PCV2), 3 type of pig circular ring virus (PCV3), The nucleic acid of porcine reproductive and respiratory syndrome virus (PRRSV) and Porcine epidemic diarrhea virus (PEDV), what evaluation was established The specificity of TaqMan real time fluorescence quantifying PCR method.

1.3.5 repeatability is assessed with reproducibility

Positive criteria plasmid (k2b-ORF1) is chosen into 3 concentration, sets 3 repetitions, is established with this test real based on TaqMan When fluorescence quantifying PCR method detected, the coefficient of variation in calculating group.The standard positive standard plasmid is placed in -20 DEG C of refrigerators It saves, inspection is primary again every two weeks, and continuous detection 3 times calculates its between-group variation coefficient.

The detection of 1.4 clinical samples

To collected from the 179 parts of progress real-time fluorescence quantitative PCR detections of Fujian various regions pig farm diarrhea pig farm blood sample and analysis.It is right After blood sample extracts its extraction DNA using kit, TaqMan real-time fluorescence quantitative PCR is carried out according to the condition after optimization Detection.Meanwhile being detected against 179 parts of samples with conventional PCR method (primer and condition such as " 1.2 " are shown), calculate two kinds Coincidence rate between detection method.

2 results and analysis

The TaqMan real-time fluorescence quantitative PCR testing conditions of 2.1 optimizations

The TaqMan real-time fluorescence quantitative PCR testing conditions of optimization are as follows: 95 DEG C of initial denaturations 60s, 95 DEG C of 15s, 60 DEG C of 30s, Carry out 40 circulations.20 μ L optimal reaction systems of optimization are system: 10 μ L THUNDERBIRD Probe qPCR Mix, each 1 μ L of up/down trip primer (6 pmol), 1 μ L probe (4 pmol), 1 μ L template DNA, supplement sterile deionized water To 20 μ L of final volume.

2.2 TaqMan real-time fluorescence quantitative PCR amplification curves and standard curve

ORF1 gene based on TaqMan real time fluorescence quantifying PCR method detection PTTSuV k2a is 4.41 × 10⁷~4.41 ×10¹There is good linear relationship within the scope of copy/μ L, the minimum of detection is 44.1 copies/μ L(Fig. 1 the 7th).It is obtained Slope of standard curve is -3.346, Y intercept 43.64, related coefficient (R²) it is 0.977, amplification efficiency 0.99.According to item The kinetic curve that part obtains is shown in Fig. 1, is formed by standard curve and sees Fig. 2.

2.3 TaqMan real-time fluorescence quantitative PCR specific outcomes

That establishes only amplifies positive signal to PTTSuV k2b based on TaqMan real time fluorescence quantifying PCR method, common to pig Amplified signal (figure is not detected in cause of disease (such as PTTSuV k2a, PPV, PRV, PCV1, PCV2, PCV3, PRRSV and PEDV) 3).

The repeatability and reproducibility of 2.4 TaqMan real time fluorescence quantifying PCR methods

Establish the group based on TaqMan real time fluorescence quantifying PCR method examination criteria positive sample in the coefficient of variation be 0.55% ~ 1.76%；The between-group variation coefficient 0.81% ~ 2.49% of the method for foundation.

2.5 clinical sample testing results

Standard PCR detection is carried out to 179 parts of blood serum samples, detects 7 parts of positive sample, positive rate 3.91%.To this 179 parts Blood serum sample carries out the detection of TaqMan real-time fluorescence quantitative PCR, detects 10 parts of positive sample, positive rate 5.59%.Meanwhile 7 Sample of the part through PCR test positive is detected through TaqMan real-time fluorescence quantitative PCR, and result is the positive, coincidence rate 100% (table 1).

The detection of 1 clinical sample of table

The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with repair Decorations, are all covered by the present invention.

SEQUENCE LISTING

<110>Fujian Academy animal and veterinary research institute

<120>the thin circovirus virus k2b type real time fluorescent quantitative detection kit of pig

<130> 5

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 18

<212> DNA

<213>artificial sequence

<400> 1

aggactgcta gaatcatc 18

<210> 2

<211> 18

<212> DNA

<213>artificial sequence

<400> 2

tgctccatgt ataggtta 18

<210> 3

<211> 23

<212> DNA

<213>artificial sequence

<400> 3

acagtcacat ctatgcctcc tcc 23

<210> 4

<211> 24

<212> DNA

<213>artificial sequence

<400> 4

ggattacccg ttggctggat attt 24

<210> 5

<211> 20

<212> DNA

<213>artificial sequence

<400> 5

ccttccaccc agtaacttgt 20

Claims (2)

1. the thin circovirus virus k2b type real time fluorescent quantitative detection primer of pig and probe, it is characterised in that: the primer and probe sequence It is as follows:

PF1:5 '-AGGACTGCTAGAATCATC-3 '；

PR1:5 '-TGCTCCATGTATAGGTTA-3 '；

Probe pTaqMan-probe:5 '-ACAGTCACATCTATGCCTCCTCC-3 ', wherein 5 ' ends are marked with FAM, 3 ' ends It is marked with BHQ2.

2. the thin circovirus virus k2b type real time fluorescent quantitative detection kit of pig comprising primer and probe described in claim 1.