CN109825642A - For detecting real-time fluorescence quantitative PCR primer, probe and its application of the related cyclic annular single-stranded DNA viruses of fur sea dog excrement - Google Patents
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Abstract
The invention discloses a kind of for detecting real-time fluorescence quantitative PCR primer, probe and its application of the related cyclic annular single-stranded DNA viruses (Fur seal feces-associated circular ssDNA virus, FSfaCV) of fur sea dog excrement.The primer is made of upstream primer and downstream primer, wherein, the nucleotide sequence of the upstream primer is as shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.2, and the nucleotide sequence of the probe is as shown in SEQ ID NO.3.Specificity FSfaCV fluorescence quantitative PCR detection primer and probe designed by the invention, viral genome can accurately detect FSfaCV in 1.52 copies/μ L or more, and do not reacted with porcine pseudorabies virus, pig parvoviral, porcine circovirus 2 type, African swine fever virus, highly pathogenic PRRSV, swine fever virus, Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, porcine rotavirus, H3N2 and H1N1 hypotype swine influenza virus, pig reovirus, have the advantages that high sensitivity, specificity it is good, conveniently, it is accurate and efficient.
Description
Technical field
The present invention relates to a kind of real-time fluorescence quantitative PCRs of the related cyclic annular single-stranded DNA viruses of detection fur sea dog excrement to draw
Object, probe and its application.The invention belongs to field of biotechnology.
Background technique
Related cyclic annular single-stranded DNA viruses (the Fur seal feces-associated circular of fur sea dog excrement
SsDNA virus, FSfaCV) it is a kind of novel cyclic single-stranded DNA viruses, belong to circinoviridae, Circovirus.The virus in
It identifies within 2012 and from New Zealand's fur sea dog excrement for the first time, viral (a50 plants) full-length genome is 2925 nucleotide
(nt), Rep replicase and Cap capsid protein including two open reading frame, are separately encoded.Japanese scholars are in 2014 from piglet
The virus is also detected that in fecal sample, identifies a strain virus FSfaCV-J1, full-length genome 2916nt, with new west
The nucleotide homology of blue a50 strain is 92%；Pathogenicity detection discovery, positive rate of the virus in piglet reach 76%
(65/85).So far, only this two relevant reports about the virus.The ecological efiiectiveness of the virus, host's preferendum,
Etiological Characteristics, diagnostic method and rarely known by the people to relevant informations such as the pathogenic properties of pig.
Therefore, the epidemiological study for carrying out FSfaCV is of great significance.And the foundation of diagnostic method is then to carry out to be somebody's turn to do
The basis of viral prevalence disease research.
Summary of the invention
The purpose of the present invention is to provide a kind of for detecting the real-time of the related cyclic annular single-stranded DNA viruses of fur sea dog excrement
Fluorescence quantification PCR primer and probe combinations identify FSfaCV by using Taqman real time fluorescence quantifying PCR method,
To substitute regular-PCR method, make to detect more acurrate and efficient.
In order to achieve the above object, present invention employs following technological means:
Of the invention is a kind of for detecting the real-time fluorescence quantitative PCR of the related cyclic annular single-stranded DNA viruses of fur sea dog excrement
Primer and probe combinations, wherein the primer is made of upstream primer and downstream primer, the nucleotide of the upstream primer
Sequence is as shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.2, the probe
Nucleotide sequence as shown in SEQ ID NO.3,5 ' ends of the probe are marked with fluorescent reporter group, and 3 ' ends are marked with fluorescence and quench
Go out group.
Wherein, it is preferred that 5 ' ends of the probe are marked with fluorescent reporter group Fam, and 3 ' ends are marked with fluorescent quenching group
Tam。
Wherein, it is preferred that the PCR primer and probe is for detecting fur sea dog excrement correlation single-stranded cyclic DNA disease
When malicious, real-time fluorescence quantitative PCR reaction system is 25 μ L, including:
Wherein, it is preferred that viral genome concentration is 1.52 copies/μ L or more in template.
Wherein, it is preferred that the PCR primer and probe is for detecting fur sea dog excrement correlation single-stranded cyclic DNA disease
When malicious, real-time fluorescence quantitative PCR reaction condition are as follows: 95 DEG C 3 minutes；95 DEG C 15 seconds, 60 DEG C 30 seconds, 40 circulation；Prolong in circulation
Fluorescence signal detection is carried out when stretching.
Further, the invention also provides the PCR primer and probe combinations detects fur sea dog excrement in preparation
Application in related ring-type single-stranded DNA viruses reagent.
Compared to the prior art, the beneficial effects of the present invention are:
1, the present invention is by designing a pair of specificity FSfaCV fluorescence quantitative PCR detection primer and probe, can be convenient for pair
The detection of FSfaCV, to improve detection efficiency.
2, specificity FSfaCV fluorescence quantitative PCR detection primer and probe designed by the invention, viral genome exist
Can accurately detect FSfaCV when 1.52 copies/μ L or more, and not with porcine pseudorabies virus, pig parvoviral, pig circular ring virus 2
Malicious 2 types, African swine fever virus, highly pathogenic PRRSV, swine fever virus, Porcine epidemic diarrhea virus,
Transmissible gastro-enteritis virus, porcine rotavirus, H3N2 and H1N1 hypotype swine influenza virus, the reaction of pig reovirus, have spirit
Sensitivity height, specificity advantage good, conveniently, accurate and efficient.
Detailed description of the invention
Fig. 1 is the standard curve of the real-time fluorescence quantitative PCR detection of FAM probe label；
Fig. 2 is real-time fluorescence quantitative PCR sensitivity Detection figure；
Wherein, A:1.52 × 108Copy/μ L；B:1.52 × 107Copy/μ L；C:1.52 × 106Copy/μ L；D:1.52 ×
105Copy/μ L；E:1.52 × 104Copy/μ L；F:1.52 × 103Copy/μ L；G:1.52 × 102Copy/μ L；H:1.52 × 101
Copy/μ L；I:1.52 × 100Copy/μ L；J:1.52 × 10-1Copy/μ L；N: negative control；
Fig. 3 is that Standard PCR detects electrophoretogram；
Wherein, M:DL2000DNA molecular weight standard；A:1.52 × 108Copy/μ L；B:1.52 × 107Copy/μ L；C:
1.52×106Copy/μ L；D:1.52 × 105Copy/μ L；E:1.52 × 104Copy/μ L；F:1.52 × 103Copy/μ L；G:
1.52×102Copy/μ L；H:1.52 × 101Copy/μ L；I:1.52 × 100Copy/μ L；N: negative control；
Fig. 4 is real-time fluorescence quantitative PCR specific detection figure.
Wherein, A: positive control；B: porcine pseudorabies virus；C: pig parvoviral；D: porcine circovirus 2 type；E: African pig
Pestivirus；F: classical porcine reproductive and respiratory syndrome virus；G: highly pathogenic PRRSV；H: hog cholera
Poison；I: three attenuated live vaccines strain of diarrhea of pigs (Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus and porcine rotavirus)；
J:H1N1 hypotype swine influenza virus；K:H3N2 hypotype swine influenza virus；L: pig reovirus；N: negative control.
Specific embodiment
The present invention is further described with attached drawing combined with specific embodiments below, the advantages and features of the present invention will be with
Description and it is apparent.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.This field
Technical staff should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and
Form is modified or is replaced, but these modifications and replacement are fallen within the protection scope of the present invention.
Embodiment 1 is for detecting the related cyclic annular PCR primer of single-stranded DNA viruses of fur sea dog excrement and the design of probe
Referring to J1 plants of FSfaCV of the Rep gene order (No. GenBank: LC133373) that GenBank is included, by life
Object information software carries out sequence alignment to Reference strains, analyzes and obtain the conservative region of FSfaCV Rep gene.Using
GenScript real-time PCR (TaqMan) online primer-design software, design synthesis TaqMan probe and primer.The primer
It is specific as follows including upstream and downstream primer:
Upstream primer qFSfaCV-F:5 '-CGAACCTCGGATGGTCCTAA-3 ', as shown in SEQ ID NO.1；；
Downstream primer qFSfaCV-R:5 '-CAAGAGCATGCCTCTGCAAT-3 ', as shown in SEQ ID NO.2；；
Probe qFSfaCV-P:5 '-Fam-AGCCTCAGCATCAGCAGCACG-Tam-3 ', as shown in SEQ ID NO.3.
The foundation of the related cyclic annular single-stranded DNA viruses fluorescent quantitative PCR detection method of 2 fur sea dog excrement of embodiment
One, the preparation of sample
1. sample acquisition and processing
Clinical health hog snout swab, blood, excrement or tissue sample are acquired, according to a conventional method by the hog snout swab samples of acquisition
It is put into the 1mL PBS containing 50% glycerol, whirlpool concussion, centrifuging and taking supernatant；The blood system of acquisition is from serum；Tissue sample grinding
Homogenate, centrifuging and taking supernatant；The PBS of 5 times of volumes of fecal sample is resuspended, centrifuging and taking supernatant, all samples be placed in -80 DEG C freeze it is standby
With.
2. the extraction of virus genom DNA
The viral genome in sample is extracted according to Axygen body fluid viral DNA/RNA extracts kit operation instructions
DNA is detected or is placed in immediately -20 DEG C of preservations.
Two, the foundation of real-time fluorescence quantitative PCR detection method
Real-time fluorescence quantitative PCR reaction system is 25 μ L；
Real-time fluorescence quantitative PCR reaction condition are as follows: 95 DEG C 3 minutes；95 DEG C 15 seconds, 60 DEG C 30 seconds, 40 circulation；It is recycling
Fluorescence signal detection is carried out when extension.
Three, when fur sea dog excrement related cyclic annular single-stranded DNA viruses quantitative fluorescent PCR standard curve drafting
1, the preparation of standard items
According to China's FSfaCV genomic dna sequence that the macro gene order-checking of previously passed virus obtains, structure is synthesized in vitro
The clone pUC57-FSfaCV containing FSfaCV genomic DNA is built, for constructing plasmid standard.Determined by ultraviolet spectrophotometry
Plasmid DNA concentration calculates copy number, and as a result copy number is 1.52 × 109Copy/μ L, -20 DEG C of preservations.
2, standard items detect
The detection of FSfaCV sonde method real-time fluorescence quantitative PCR is carried out using pUC57-FSfaCV Plasmid DNA as template, is established
Standard curve.
Concrete operations are as follows: the progress of pUC57-FSfaCV Plasmid DNA is serially diluted into 1.52 × 10 for 10 times8Copy/μ L,
1.52×107Copy/μ L, 1.52 × 106Copy/μ L, 1.52 × 105Copy/μ L, 1.52 × 104Copy/μ L, 1.52 × 103It copies
Shellfish/μ L and 1.52 × 102Copy/μ L totally 7 dilutions.
3, the drafting of standard curve
According to the logarithm of gained CT value (cycle threshold) standard items corresponding as ordinate as abscissa, institute
The standard curve of drafting is as shown in Figure 1, slope is y=-3.487x+39.61, R2=0.998.Standard curve is shown: foundation
FSfaCV sonde method real-time fluorescence quantitative PCR detection method has the linear detection range of 7 orders of magnitude, further illustrates the detection
Method sensitivity with higher.
Four, the sensibility of real-time fluorescence quantitative PCR detection method
The progress of pUC57-FSfaCV Plasmid DNA is serially diluted into 1.52 × 10 for 10 times8Copy/μ L, 1.52 × 107Copy/
μL、1.52×106Copy/μ L, 1.52 × 105Copy/μ L, 1.52 × 104Copy/μ L, 1.52 × 103Copy/μ L and 1.52 ×
102Copy/μ L, 1.52 × 101Copy/μ L, 1.52 × 100Copy/μ L and 1.52 × 10-1Copy/μ L totally 10 dilutions, with
Sterile water is negative control, carries out real-time fluorescence quantitative PCR inspection according to the real-time fluorescence quantitative PCR detection method of above-mentioned foundation
It surveys, evaluates the sensibility of reaction system of the present invention.Real-time fluorescence quantitative PCR detection sensitivity amplification curve is as the result is shown: in mould
The fluorescence signal of FSfaCV can be detected when plate concentration is 1.52 copies/μ L, as shown in Figure 2.The FSfaCV that the present invention establishes is real
When fluorescent quantitative PCR detection method have good sensibility.
It according to the diluted concentration of template in real-time fluorescence quantitative PCR, is expanded with regular-PCR, compares verifying.It is general
Lead to PCR result as shown in Figure 3,1.52 × 103The amplifiable purpose band out of copy/μ L genomic DNA, and 1.52 × 102It copies
Shellfish/μ L genomic DNA has no that purpose band expands.The real time fluorescence quantifying PCR method that the present invention establishes is than regular-PCR method
It is 1000 times sensitive.
Five, the specificity of real-time fluorescence quantitative PCR detection method
With porcine pseudorabies virus, pig parvoviral, porcine circovirus 2 type and the DNA of African swine fever virus, and classical poison
Strain and highly pathogenic PRRSV, swine fever virus, three attenuated live vaccines strain (pig epidemic of diarrhea of pigs
Diarrhea virus, transmissible gastro-enteritis virus and porcine rotavirus), H3N2 and H1N1 hypotype swine influenza virus, pig reovirus
CDNA as template according to above-mentioned foundation real-time fluorescence quantitative PCR detection method carry out real-time fluorescence quantitative PCR detection, and with
Water is blank control, 1.52 × 108Copy/μ L pUC57-FSfaCV Plasmid DNA as positive control, evaluation response system
Specificity.
As a result as shown in Figure 4.As the result is shown: real-time fluorescence quantitative PCR only detects the positive control of FSfaCV, and to it
He expands without purpose band pig source virus.As a result illustrate: the FSfaCV real-time fluorescence quantitative PCR that the present invention establishes has good
Specificity.
Detection of 3 real-time fluorescence quantitative PCR detection method of embodiment to clinical sample
With the FSfaCV real time fluorescence quantifying PCR method of foundation to 94 parts of pig clinical serums, respiratory tract swab and tissue sample
Product are detected, wherein 35 parts of detectable FSfaCV special fluorescence signal, recall rate is 37.23% (35/94), and common
The recall rate of PCR is 29.79% (28/94)；As it can be seen that the FSfaCV real time fluorescence quantifying PCR method that the present invention establishes is than common
PCR detection method is sensitiveer, accurate.
Sequence table
<110>Harbin Veterinary Medicine Inst., China Academy of Agriculture is (in China Animal Health and Epidemiology Center Harbin point
The heart)
<120>for detect the real-time fluorescence quantitative PCR primers of the related cyclic annular single-stranded DNA viruses of fur sea dog excrement, probe and
It is applied
<130> KLPI190062
<160> 3
<170> PatentIn version 3.3
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cgaacctcgg atggtcctaa 20
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caagagcatg cctctgcaat 20
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agcctcagca tcagcagcac g 21
Claims (6)
1. a kind of for detecting the real-time fluorescence quantitative PCR primer and probe of the related cyclic annular single-stranded DNA viruses of fur sea dog excrement
Combination, which is characterized in that the primer is made of upstream primer and downstream primer, wherein the nucleosides of the upstream primer
Acid sequence is as shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.2, the spy
For the nucleotide sequence of needle as shown in SEQ ID NO.3,5 ' ends of the probe are marked with fluorescent reporter group, and 3 ' ends are marked with fluorescence
Quenching group.
2. PCR primer according to claim 1 and probe combinations, which is characterized in that 5 ' ends of the probe are marked with glimmering
Light reporter group Fam, 3 ' ends are marked with fluorescent quenching group Tam.
3. PCR primer according to claim 1 or 2 and probe combinations, which is characterized in that the PCR primer and probe
When for detecting fur sea dog excrement correlation ring-type single-stranded DNA viruses, real-time fluorescence quantitative PCR reaction system is 25 μ L,
In include:
4. PCR primer according to claim 3 and probe combinations, which is characterized in that viral genome concentration is in template
1.52 copies/μ L or more.
5. PCR primer according to claim 1 and probe combinations, which is characterized in that the PCR primer and probe is used for
When detecting the related cyclic annular single-stranded DNA viruses of fur sea dog excrement, real-time fluorescence quantitative PCR reaction condition are as follows: 95 DEG C 3 minutes；95
DEG C 15 seconds, 60 DEG C 30 seconds, 40 circulations；Fluorescence signal detection is carried out when recycling and extending.
6. the described in any item PCR primers of claim 1-5 and probe combinations are related cyclic annular in preparation detection fur sea dog excrement
Application in single-stranded DNA viruses reagent.
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| CN110607404A (en) * | 2019-10-30 | 2019-12-24 | 广西大学 | Real-time fluorescent quantitative PCR (polymerase chain reaction) detection primer for porcine enterovirus G type and kit thereof |
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