CN109097495A - Senecan virus and foot and mouth disease virus dual real-time fluorescence quantitative PCR detection kit - Google Patents
Senecan virus and foot and mouth disease virus dual real-time fluorescence quantitative PCR detection kit Download PDFInfo
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Abstract
The present invention provides a kind of dual real-time fluorescence quantitative PCR detection kits and its primer special, TaqMan probe for identifying Senecan virus (SVA) and foot and mouth disease virus (FMDV).Contain two pairs of primers and two probes in kit of the invention, primer and probe is directed to Senecan viral (SVA) and the design of foot and mouth disease virus (FMDV) conservative gene area respectively.Kit and detection method of the invention be easy to operate, high specificity, sensibility are high, reproducible, it may be implemented to Senecan virus and foot and mouth disease virus accurate quantitative analysis, by the quality monitoring in the detection and production of vaccine, such as vaccine production process of Senecan virus and foot and mouth disease virus and rationalize with playing a significant role in seedling, has a extensive future.
Description
Technical field
The invention belongs to the veterinary animal Pathogen tests in technical field of biological, in particular to a kind of for in plug
Card virus and foot and mouth disease virus carry out the dual real-time fluorescence quantitative PCR detection kit of qualitative and quantitative analysis and its dedicated draw
Object and TaqMan probe.
Background technique
Senecan virus (Seneca Virus A, SVA) and foot and mouth disease virus (Foot-and-Mouth Disease
Virus, FMDV) Picornaviridae (Picornaviridae) is belonged to, it is all a kind of single-stranded positive RNA of no cyst membrane
Virus, Senecan virus belong to the Typical Representative of Seneca Virus Tobamovirus, most connect with cardiovirus (Cardio Virus)
Closely, and foot and mouth disease virus belong to Hostis virus.
Senecan virus is to take place mostly in a kind of infectious disease of pig, faces and examines feature as infection pig nose and oral cavity shape
At ulcer, anorexia, limping, lead to newborn piglet acute death.Early stage Senecan case takes place mostly in growing and fattening pigs, nose, mouth
The visible blister symptom of the coronal band portion of portion and hoof, but some Senecan positive cases found recently betide newborn piglet,
Symptom is diarrhea, no blister lesion, these cases are mainly by carrying out PCR detection discovery to serum, excrement and different tissues
's.A company in Maryland, USA in 2002 finds that 2007, a batch transported Minnesota ,USA to from Canada for the first time
The similar blister disease symptoms such as bubble occurs in the hog snout mirror in state, hoof coronary band festers, exclude aftosa, vesicular stomatitis through detection
Disease and pig blisters are determined as Senecan virus-positive eventually by PCR detection.2014 later so far the U.S., China, plus
It puts on airs, Brazil, Thailand, clinical case caused by the multinational discovery SVA virus such as Colombia, and further determines that it is by SVA
Cause to cause a disease.Before 2007, SVA is studied mainly as oncolytic, can effectively treat some neuroendocrine tumors.
One kind that foot and mouth disease virus can cause the artiodactyls such as ox, pig and sheep to infect is acute, hot, high degree in contact infects
Disease, characterized by mucous membrane of mouth, lingual surface, lip, asoscope, hoof and skin of breast occur blister and fester.Aftosa median lethal
Rate is only 1%, but infected animal 100% falls ill, and Communication results are high, and actual animal yield is made to fall sharply.FMD is once quick-fried
Hair will cause huge economic loss, therefore the research of the disease is extremely paid attention in countries in the world.It is big in view of its harm, influence model
It encloses wide, is classified as first of A class deadly infectious disease by World Organization for Animal Health (OIE).
In recent years, due to the discovery of Senecan virus, SVA infection and FMDV infection only cannot be distinguished from clinical symptoms,
Difficulty is brought to clinical definite, needs to be determined with laboratory detection technology.How to be detected with quick, simple method,
It distinguishes Senecan virus and foot and mouth disease virus is some pig farm urgent problems.
On the other hand, vaccine is the most effective measure of pre- anti-virus, and wherein inactivated vaccine plays in the prevention of infectious disease
Important function, prepare inactivated vaccine be prevent Senecan virus and foot and mouth disease virus prevalence main effective means.In epidemic disease
In seedling preparation, cause of disease is effectively inactivated, it is possible to provide safe vaccine mainly passes through the viral, hoof-and-mouth disease by Senecan
It is inactivated after malicious in vitro culture, is then mixed and made into immune vaccine with emulsifier.Preparation polyvaccine for example Senecan virus and
When foot and mouth disease virus bivalent vaccine, accurately, special, rapidly detection Senecan virus and the horizontal of foot-and-mouth disease virus antigen exist
Production of vaccine monitoring etc. is of great significance.
Summary of the invention
The first purpose of the invention is to provide double for carrying out to Senecan viral (SVA) and foot and mouth disease virus (FMDV)
The primer and TaqMan probe of weight real-time fluorescence quantitative PCR detection, and realize Senecan viral (SVA) and foot and mouth disease virus
(FMDV) qualitative and quantitative analysis.
Primer provided by the present invention for the progress real-time fluorescence quantitative PCR detection of Senecan virus are as follows: draw upstream
The nucleotide sequence of object (SVA-F) is as shown in SED ID NO:1 in sequence table, and the nucleotide sequence of downstream primer (SVA-R) is such as
In sequence table shown in SEQ ID NO:2.
The primer sequence as derived from above-mentioned primer also belongs to the content of present invention.The derived sequence refers in SEQ ID NO:
The primer sequence replaced, missed or added on the basis of 1 and/or SEQ ID NO:2 Jing Guo one to ten base.
Primer provided by the present invention for foot and mouth disease virus progress real-time fluorescence quantitative PCR detection are as follows: draw upstream
The nucleotide sequence of object (FMDV-F) is as shown in SED ID NO:5 in sequence table, the nucleotide sequence of downstream primer (FMDV-R)
As shown in SEQ ID NO:6 in sequence table.
The primer sequence as derived from above-mentioned primer also belongs to the content of present invention.The derived sequence refers in SEQ ID NO:
The primer sequence replaced, missed or added on the basis of 5 and/or SEQ ID NO:6 Jing Guo one to ten base.
TaqMan probe provided by the present invention for the progress real-time fluorescence quantitative PCR detection of Senecan virus are as follows:
The nucleotide sequence of TaqMan probe (SVA-P) is as shown in SED ID NO:3 in sequence table；The probe is by fluorescent marker
, 5 ' ends are marked with reporter fluorescence group, and 3 ' ends are marked with quenching fluorescence group.
TaqMan probe provided by the present invention for foot and mouth disease virus progress real-time fluorescence quantitative PCR detection are as follows:
The nucleotide sequence of TaqMan probe (FMDV-P) is as shown in SED ID NO:7 in sequence table；The probe is by fluorescence mark
Note, 5 ' ends are marked with reporter fluorescence group, and 3 ' ends are marked with quenching fluorescence group.
The reporter fluorescence group marked to above two viral diagnosis probe is not identical.
The content of present invention is also belonged to by the derived sequence of above-mentioned TaqMan probe sequence.The derived sequence refers in SEQ
On the basis of ID NO:3/SEQ ID NO:7, sequence 5 ' end and/or 3 ' end add again, reduces one or more bases obtain
The sequence arrived.
The TaqMan for carrying out dual real-time fluorescence quantitative PCR detection to Senecan virus and foot and mouth disease virus is visited
5 ' end reporter fluorescence groups of needle (SVA-P) are FAM, and 3 ' end fluorescent quenching groups are TAMRA；TaqMan probe (FMDV-P)
5 ' end reporter fluorescence groups are ROX, and 3 ' end fluorescent quenching groups are BHQ2.
It is extended when to prevent PCR amplification, phosphatizing treatment is held in the 3 ' of the TaqMan probe.
A second object of the present invention is to provide double for carrying out to Senecan viral (SVA) and foot and mouth disease virus (FMDV)
Weight real-time fluorescence quantitative PCR detection kit.
Dual real-time fluorescence quantitative PCR detection kit provided by the present invention, including it is above-mentioned for Senecan virus
The primer and TaqMan probe of dual real-time fluorescence quantitative PCR detection are carried out with foot and mouth disease virus.
Dual real-time fluorescence quantitative PCR detection system when specifically, using the kit are as follows: real time fluorescent quantitative
One-step method PCR reaction solution 2 × One Step RT-PCR Buffer III 12.5 μ L (being purchased from TakaRa company), TaKaRa Ex
0.5 μ L of 0.5 μ L of Taq HS (being purchased from TakaRa company), PrimeScript RT Enzyme Mix II (is purchased from TakaRa public affairs
Department), SVA-F (10 μM) 1 μ L, SVA-R (10 μM) 1 μ L, SVA-P (10 μM) 1 μ L, FMDV-F (10 μM) 1 μ L, FMDV-R (10 μM)
1 1.5 μ L, RNA-free H of μ L, FMDV-P (10 μM)23 μ L of O, 2 μ L of template.
Third object of the present invention is to provide the primer, probes in the inspection for preparing Senecan virus and foot and mouth disease virus
Application in test agent.
Fourth object of the present invention is to provide the kit to Senecan viral (SVA) and foot and mouth disease virus
(FMDV) application in the detection of non-disease diagnostic purpose is carried out.
One of above-mentioned application is qualitative, quantitative for carrying out to Senecan viral (SVA) and foot and mouth disease virus (FMDV)
Detection.The dual real-time fluorescence quantitative PCR detecting method the following steps are included:
1) it establishes standard curve: choosing specific and conserved sequence (the Senecan virus of the 3C regional gene of Senecan virus
From SED ID NO:4 in 5 ' end 6564-6820 bit bases, sequence table, gene is detected) sequence, design are constructed as standard items
Standard items primer (the SED ID NO:9 and SED ID in sequence table of real-time fluorescence quantitative PCR detection is carried out to Senecan virus
NO:10), using the geneome RNA of Senecan virus as template, Senecan virus 3C region detection is expanded with standard items primer PCR
Gene connects in carrier pCR-4 TOPO, and building carries the recombinant plasmid pCR4- of Senecan virus 3C region detection gene
TOPO-SVA carries out concentration mensuration to the correct recombinant plasmid of identification；Choose the specificity of the 3C regional gene of foot and mouth disease virus
Conserved sequence (foot and mouth disease virus detects gene from SED ID NO:8 in 5 ' end 5986-6553 bit bases, sequence table) conduct
Standard items construct sequence, design and carry out the standard items primer of real-time fluorescence quantitative PCR detection (in sequence table to foot and mouth disease virus
SED ID NO:11 and SED ID NO:12), using the geneome RNA of foot and mouth disease virus as template, expanded with standard items primer PCR
Foot and mouth disease virus 3C region detection gene, connects in carrier pCR-4 TOPO, and building carries foot and mouth disease virus 3C region detection
The recombinant plasmid pCR4-TOPO-FMDV of gene carries out concentration mensuration to the correct recombinant plasmid of identification.SVA core will be carried respectively
Recombinant plasmid pCR-4 TOPO-SVA and the pCR-4 TOPO-FMDV of nucleotide sequence and FMDV nucleotide sequence as standard items,
Recombinant plasmid pCR-4 TOPO-SVA10 times gradient dilution is at 1 × 108、1×107、1×106、1×105、1×104、1×103、1
×102、2.5×101Copy (copies)/μ L；10 times of gradient dilutions of recombinant plasmid pCR-4 TOPO-FMDV are at 1 × 108、1×
107、1×106、1×105、1×104、1×103、1×102、1×101(copies)/μ L is copied, with the standard items of various concentration
As template, under the guidance of primer SVA-F, SVA-R, FMDV-F, FMDV-R and TaqMan probe SVA-P, FMDV-P respectively
Real-time fluorescence quantitative PCR detection is carried out, after detection, with the concentration Log value (X-axis) of each standard items to its corresponding Ct value (Y
Axis) mapping, draw standard curve；
2) geneome RNA for extracting sample to be tested, using the geneome RNA of extraction as template, in above-mentioned primer and TaqMan
Dual real-time fluorescence quantitative PCR detection is carried out under the guidance of probe；
3) it is realized with the variation of obtained respective CT value or fluorescence signal and Senecan virus and foot and mouth disease virus is determined
Property detection, Senecan virus and/or foot and mouth disease virus correspondence fluorescence channel occur " S " type amplification curve then show to test sample
Contain corresponding virus in product, that is, is determined as the positive；
4) to positive sample to be tested is determined as in step 3), further according to the standard in the intensity and step 1) of fluorescence signal
Curve obtains the copy number of contained Senecan virus and foot and mouth disease virus in sample to be tested, realizes quantitative detection.
In the dual real-time fluorescence quantitative PCR detecting method of above-mentioned Senecan virus (SVA) and foot and mouth disease virus (FMDV)
In, the sample to be tested in the step 2) can be raw material serum, the vaccine semi-finished product for picking up from pig for production of vaccine, and pig farm is sent
Sample product detect it for non-diagnostic purpose.By determining Senecan virus and foot and mouth disease virus in such sample to be tested
Amount detection provides objective data to realize the monitoring to product and its raw material based on pig for the disposition of subsequent raw material.
Dual real-time fluorescence quantitative PCR detection system in the step 1) and step 2) can include: 2 μ L of template, in real time
12.5 μ L (being purchased from TakaRa company) of fluorescent quantitation one-step method PCR reaction solution 2 × One Step RT-PCR Buffer III,
0.5 μ L of TaKaRa Ex Taq HS 0.5 μ L (being purchased from TakaRa company), PrimeScript RT Enzyme Mix II (purchase
In TakaRa company), 1111 μ L of μ L, FMDV-F (10 μM) of μ L, SVA-P (10 μM) of μ L, SVA-R (10 μM) of SVA-F (10 μM),
1 1.5 μ L, RNA-free H of μ L, FMDV-P (10 μM) of FMDV-R (10 μM)2O 3μL。
Dual real-time fluorescence quantitative PCR detection condition in the step 1) and step 2) can are as follows: first 42 DEG C of reverse transcriptions
20min, 95 DEG C of initial denaturation 30s；Then 94 DEG C of denaturation 10s, 55 DEG C of annealing 30s, 45 circulations (PCR amplification).In each circulation
Annealing at the end of carry out fluorescence signal detection.
Specific result judgement method can in the step 3) are as follows: if (not including the 37th in 37 circulations in the channel FAM
A circulation) there is " S " type amplification curve, then it is confirmed as Senecan virus-positive (containing Senecan virus in sample), if ROX is logical
There is " S " type amplification curve in (not including the 37th circulation) in 37 circulations in road, then is confirmed as the positive (sample of foot and mouth disease virus
Contain foot and mouth disease virus in product)；If (including the 39th circulation) does not go out more than 39 circulations in the channel FAM and/or the channel ROX
It is negative (without containing Senecan virus and/or foot and mouth disease virus in sample) to be then confirmed as corresponding virus for existing " S " type amplification curve；
In any channel between 37-39 circulation (including the 37th circulation and do not include the 39th circulation) amplification of " S " type occur bent
Line is then determined as suspicious, need to examine again.
The present invention provides a kind of for detecting the dual real-time glimmering of Senecan viral (SVA) and foot and mouth disease virus (FMDV)
Fluorescent Quantitative PCR detection kit and its primer special, TaqMan probe, can be real to Senecan virus and foot and mouth disease virus using it
Quick differentiation and detection are applied, can also match seedling (as assessment preparation vaccine resists with rationalization for the quality monitoring in vaccine production process
Former exact level, provides data basis for vaccine antigen content) strong foundation is provided, it is ensured that the safety and conjunction of vaccine inoculation
Rationality has directive function to the production of Senecan virus and foot and mouth disease virus vaccine.Kit and detection method of the invention
Easy to operate, high specificity, sensibility are high, reproducible, may be implemented to determine Senecan virus and the accurate of foot and mouth disease virus
Amount, can Senecan virus and foot and mouth disease virus detection (including in pathological material of disease or culture Senecan virus and foot and mouth disease virus
Accurate detection) and production of vaccine in play a significant role, have a extensive future.
The present invention is described in further details combined with specific embodiments below.
Detailed description of the invention
Fig. 1 is that primer screening of the present invention for carrying out real-time fluorescence quantitative PCR detection to Senecan viral (SVA) expands
Curve；
Fig. 2 is that the present invention expands for carrying out the primer screening of real-time fluorescence quantitative PCR detection to foot and mouth disease virus (FMDV)
Increase curve；
Fig. 3 is that the amplification of standard items of the present invention for carrying out dual real-time fluorescence quantitative PCR detection to SVA and FMDV is bent
Line；
Fig. 4 is the present invention for carrying out the standard curve of dual real-time fluorescence quantitative PCR detection to SVA and FMDV；
Fig. 5 is the specific detection result that SVA of the present invention and FMDV carries out dual real-time fluorescence quantitative PCR detecting method；
Fig. 6 is the amplification curve inspection that SVA of the present invention and FMDV carries out dual real-time fluorescence quantitative PCR detecting method repeatability
Survey result.
Specific embodiment
Method therefor is conventional method unless otherwise instructed in following embodiments, and specific steps can be found in: " molecular cloning
Experiment guide " (" Molecular Cloning:A Laboratory Manual " Sambrook, J., Russell, David
W., Molecular Cloning:A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring
Harbor)。
The percent concentration is mass/mass (W/W, unit g/100g) percent concentration, matter unless otherwise instructed
Amount/volume (W/V, unit g/100mL) percent concentration or volume/volume (V/V, Unit/mL/100mL) percent concentration.
The acquirement approach of various biomaterials described in embodiment is to provide a kind of approach of experiment acquisition only to reach
To specifically disclosed purpose, the limitation to biological material source of the present invention should not be become.In fact, used biomaterial
Source be it is extensive, it is any keep on the right side of the law the biomaterial that can be obtained with moral ethics can be according to mentioning in embodiment
Show and is replaced.
The primer is synthesized by Beijing Hua Da gene Co., Ltd；Probe used is synthesized by TAKARA genome company.
Embodiment is implemented under the premise of the technical scheme of the present invention, gives detailed embodiment and specific
Operating process, embodiment will be helpful to understand the present invention, but protection scope of the present invention is not limited to following embodiments.
Biological genome is a direct most objective index for reacting biological essential information, contained by different virus
Genomic information is different, can be mutual using genome base by different virus taxis to different groups by genomic information
The principle that recruits pair, it can be achieved that specific site gene order massive amplification.
The present invention is based on the above basic principles to devise 2 pairs of specific primers and 2 oligonucleotide probes, mutual using base
The principle recruited pair establishes the dual real-time of a kind of specific detection Senecan viral (SVA) and foot and mouth disease virus (FMDV)
Fluorescent quantitative PCR detection method.
Embodiment 1, design detect Senecan viral (SVA) and foot and mouth disease virus with Real-Time Fluorescent Quantitative PCR Technique
(FMDV) primer and TaqMan probe
It is retrieved from the nucleic acid database GenBank (http://www.ncbi.nlm.nih.gov) of NCBI and obtains Senecan
Viral full-length genome sequence (GeneBank serial number: NC_011349, KT321458, KX173339, KX173338,
KX173340、KX751943、KX751944、KY747510、KY038016、KY747511、KY747512、KX751945、
KX751946、KX377924、KY419132、DQ641257、KU051392、KT757280、KU051391、KY486158、
KY486165, KC667560, KR063109, KR063107, KY368743) and foot and mouth disease virus full-length genome sequence
(GeneBank serial number: AF506822.2, KX712091.1, HQ412603.1, AJ539141.1, AY390432.1,
AY304994.1, GQ406249.1, KT968663.1, HQ632773.1), after being compared with DNA Star software, according to drawing
Object, TaqMan probe design principle choose the nucleotide sequence (area 3C relatively conservative between the different strains of Senecan virus respectively
Domain, shown in SED ID NO:4) and foot and mouth disease virus difference strain between relatively conservative nucleotide sequence (region 3C, SED ID
Shown in NO:8), and using preferred specific and conserved sequence as detection sequence.
About the 3C regional gene of Senecan virus, document " Complete genome that Hales L M etc. is delivered
sequence analysis of Seneca Valley virus-001,a novel oncolytic picornavirus
2008,89 (5): [J] .Journal of General Virology has illustrated that the 3C gene is conservative in 1265-1275. "
Region；" the separation identification and Study on Pathogenicity of pig Sai Neijia paddy viral (SVA) " master thesis that Zhao Xiaoya has been delivered
At this in (Agricultural University Of South China, 2016 master's thesis (24-33)) (identical as document 1CN201610379248 content)
4763-4879 bit base section is chosen as target sequence and establishes Seneca Valleyvirus Taq Man fluorescent quantitation in the region 3C
PCR detection method, Unfortunately, this method sensibility are only 1 × 102Copy/μ L, is not able to satisfy the needs of detection.
The present invention preferably goes out specific fragment as detection sequence from the 3C conservative region of Senecan virus, and final true
The fixed detection sequence holds 6564-6820 bit base (SED ID NO:4 in sequence table) from 5 ' for Senecan virus.According to selected
Detection sequence, design to Senecan virus carry out real-time fluorescence quantitative PCR detection primer and TaqMan probe.
Gene region is detected about foot and mouth disease virus, lot of domestic and international research and utilization 3D gene is carried out as target sequence at present
Fluorescence quantitative RT-RCR technology detects aftosa, and a few studies do target sequence progress using foot and mouth disease virus 2B gene and 5 ' UTR
Fluorescence quantitative RT-RCR technology detects aftosa, and the present invention finds highly conserved and special in foot and mouth disease virus 3C gene region
Design primer and probe are come in nucleotide region.
The present invention preferably goes out specific fragment as detection sequence from the 3C conservative region of foot and mouth disease virus, and final true
The fixed detection sequence holds 5986-6553 bit base (SED ID NO:8 in sequence table) from 5 ' for foot and mouth disease virus.According to selected
Detection sequence, design to foot and mouth disease virus carry out real-time fluorescence quantitative PCR detection primer and TaqMan probe.
From the numerous primers and probe obtained based on detection sequence determined above, the present invention design multiple groups group merge from
In filter out group 1 be used as preferred group:
1 (preferably group) of group:
SVA-F (upstream primer): 5 '-TATCTCAGATCCCTGGCTGTC-3 ' (sequence location: Senecan virus from 5 ' end
6634-6654 bit base, SED ID NO:1 in sequence table)；
SVA-R (downstream primer): 5 '-CCTGATGATCACATTGTTGAGC-3 ' (sequence location: Senecan virus is from 5 '
6741-6762 bit base is held, SED ID NO:2 in sequence table)；
(the sequence positions SVA-P (TaqMan probe): 5 '-FAM-CACGCTTACGGCGAGCGTCGCATCAAG-TAMRA-3 '
Set: Senecan virus holds 6661-6687 bit bases, SED ID NO:3 in sequence table from 5 ')；
FMDV-F (upstream primer): 5 '-YGCCTMCCTHGTDCCTCGTCAYCTYT-3 ' (sequence locations: foot and mouth disease virus
From SED ID NO:5 in 5 ' end 6158-6179 bit bases, sequence table, wherein Y=C, T, M=A, C, H=A, T, C, D=G,
A,T)；
FMDV-R (downstream primer): 5 '-GAGAGCATGTCCTGTCCTYTYACTTT-3 ' (sequence locations: foot and mouth disease virus
From SED ID NO:6 in 5 ' end 6260-6281 bit bases, sequence table, wherein Y=C, T)；
FMDV-P (TaqMan probe): 5 '-ROX-CNGAGAAGTAYGACAAGATCATGYT-BHQ2-3 ' (sequence location:
Foot and mouth disease virus holds 6183-6207 bit bases, SED ID NO:7 in sequence table from 5 ', wherein N=A, G, C, T, Y=C,
T)；
For compared with group 1, by the primer obtained based on other viral conservative region detection sequences of Senecan and probe with
Other foot and mouth disease virus primers and probe combinations are classified as group 2 as a control group:
2 (control groups) of group:
SVA-F1 (upstream primer): 5 '-TATAAGATGACTCCTGCCAAC-3 ' (sequence location: Senecan virus is from 5 '
Hold 6898-6918 bit base)；
SVA-R1 (downstream primer): 5 '-AGAATTTGGAAGCCATGCTCTC-3 ' (sequence location: Senecan virus is from 5 '
Hold 7025-7046 bit base)；
(the sequence positions SVA-P1 (TaqMan probe): 5 '-FAM-TTCTGTCTTCCCTCCGACTTCCTCTC-TAMRA-3 '
Set: Senecan virus holds 6924-6949 bit bases from 5 ')；
FMDV-F1 (upstream primer): 5 '-AAGATCATGTTGGACGGCAGAGCCAT-3 ' (sequence locations: hoof-and-mouth disease
Poison holds 6198-6223 bit bases from 5 ')；
FMDV-R1 (downstream primer): 5 '-ATGTCCCGCACGCGATTCCCACGGT-3 ' (sequence locations: foot and mouth disease virus
From 5 ' end 6310-6334 bit bases)；
FMDV-P1 (TaqMan probe): 5 '-ROX-CAGTGACTACAGAGTGTTTGAGTTTGAG-BHQ2-3 ' (sequence
Position: foot and mouth disease virus holds 6230-6257 bit bases from 5 ')；
In above-mentioned TaqMan probe, 5 ' ends have had reporter fluorescence group FAM or ROX, are directed to two kinds of diseases in the same set
The group of the probe institute band of poison is not identical；3 ' ends have had fluorescent quenching group corresponding with 5 ' end reporter fluorescence groups
TAMRA or BHQ2.
It is extended when to prevent PCR amplification, the 3 ' phosphorylated processing in end of above-mentioned TaqMan probe.
The primed probe of FMDV and SVA quantitative fluorescent PCR screens and condition optimizing experiment: expanding the above designed PCR
Increase primer and the most suitable annealing temperature of each primer is screened, five gradients, respectively 53 DEG C, 55 DEG C, 57 are arranged in annealing temperature
DEG C, 59 DEG C, 61 DEG C, each temperature gradient does 2 repetitions, shown in the following Tables 1 and 2 of reaction system, reaction condition are as follows: first 42 DEG C
Reverse transcription 20min, 94 DEG C of initial denaturations, 30s；{ 94 DEG C of denaturation, 10s, 53 DEG C of -61 DEG C of annealing, 30s } × 45 circulations.
Compare amplification curve screening primer and annealing temperature after reaction, each group primer and probe is compared, with
The upper listed result of two groups of primers and probe in 55 DEG C of annealing temperature is as depicted in figs. 1 and 2, organizes 1 FMDV and SVA fluorescence
Quantitative pcr amplification curve is " S " type curve of standard, organizes 2 FMDV and SVA fluorescent quantitative PCR curve fluorescence threshold not
Such as organize 1.The primer of 1 design of group is determined by amplification curve and probe is preferred.
Embodiment 2, Senecan virus and foot and mouth disease virus are carried out with the primer of group 1 and TaqMan probe it is dual real-time glimmering
Fluorescent Quantitative PCR detection
One, the geneome RNA of Senecan virus and foot and mouth disease virus is extracted
By Senecan virocyte culture (the cell passaged virus of this laboratory separation identification, for obtaining standard items
And positive reference substance), foot and mouth disease virus cell culture (Jin Yu Bao Ling company MYA98 vaccine strain, for obtain standard items and
Positive reference substance) and sample to be tested as sample to be extracted, extract the geneome RNA of sample to be extracted, specific extracting method
Referring to AXYGEN kit (AxyprepTMBody Fluid Viral DNA/RNA Miniprep Kit, AXYGEN company)
It introduces, comprising the following steps:
(1) press kit specification, prepare isopropanol containing 1% glacial acetic acid in advance and in reagent Buffer W1A and
The dehydrated alcohol of prescribed concentration is added in Buffer W2；
(2) sample to be extracted of 200 μ L is added in 1.5mL centrifuge tube, and 200 μ L Buffer V-L are added, whirlpool shake
After swinging mixing, 5min is stood；
(3) add 75 μ L Buffer V-N, whirlpool shake in the 1.5mL centrifuge tube for being mixed with sample and reagent of step (2)
Mixing is swung, 12000g is centrifuged 5min；
(4) supernatant is transferred in 2mL centrifuge tube (providing in kit), adds 300 μ L isopropanols (1% glacial acetic acid), on
It is lower to be inverted 6-8 times, it is uniformly mixed；
(5) preparation pipe (providing in kit) is placed in another 2mL centrifuge tube, the mixed liquor of step (4) is taken to move into system
In standby pipe, 6000g is centrifuged 1min；
(6) filtrate is abandoned, pipe will be prepared and put back into the 2mL centrifuge tube of step (5), 500 μ L Buffer W1A, room temperature are added
1min is stood, 12000g is centrifuged 1min；
(7) filtrate is abandoned, pipe will be prepared and put back into the 2mL centrifuge tube of step (5), 800 μ L Buffer W2,12000g are added
It is centrifuged 1min；
(8) filtrate is abandoned, pipe will be prepared and put back into the 2mL centrifuge tube of step (5), 1min is directly centrifuged with 12000g；
(9) pipe will be prepared to be placed in the 1.5mL centrifuge tube (providing in kit) of another cleaning, is added preparing periosteum center
40 μ L are stored at room temperature 1min without enzyme water, and 12000g is centrifuged 1min, affords RNA.
Using the RNA extracted from sample to be tested using above method as detection sample；From Senecan virocyte culture
The RNA of extraction is as Senecan virus positive control product；The RNA extracted from foot and mouth disease virus cell culture is as hoof-and-mouth disease
Malicious positive reference substance；It is according to following two method that the RNA and foot and mouth disease virus extracted from Senecan virocyte culture is thin
Standard items are prepared in the RNA extracted in born of the same parents' culture.
Two, the standard curve of Senecan virus and foot and mouth disease virus dual real-time fluorescence quantitative PCR detection is established
1, PCR amplification Senecan virus and foot and mouth disease virus detect gene
The geneome RNA and foot-and-mouth disease virus genome RNA for the Senecan virus extracted using step 1 is templates, in primer
PCR amplification Senecan viral nucleotide detection sequence under the guidance of SVA-standard-F and SVA-standard-R, in primer
PCR amplification foot and mouth disease virus nucleotide detection sequence under the guidance of FMDV-standard-F and FMDV-standard-R, 25 μ L
PCR amplification system is as shown in Table 1 and Table 2, PCR amplification condition are as follows: first 42 DEG C of reverse transcriptions 20min, 95 DEG C of initial denaturation 30s；Then
94 DEG C of denaturation 10s, 55 DEG C of annealing 30s, 72 DEG C of extension 45s, 30 circulations (PCR amplification), 72 DEG C of extension 10min.Amplification terminates
Afterwards, it recycles, purify pcr amplification product, obtain Senecan virus 3C region detection gene (sequence SED ID NO:4 in sequence table)
With foot and mouth disease virus 3C region detection gene (sequence SED ID NO:8 in sequence table).
The PCR amplification system of 1 Senecan viral nucleotide of table detection gene
The PCR amplification system of 2 foot and mouth disease virus nucleotide of table detection gene
2, standard items are prepared
The Senecan viral nucleotide detection gene and foot and mouth disease virus nucleotide detection gene difference gram that step 1 is obtained
It is grand into pCR-4 TOPO carrier (be purchased from Invitrogen company), screening positive recombinant plasmid send Huada gene company to be sequenced.
Sequencing result shows to obtain sequence and correctly carries Senecan viral nucleotide detection gene (sequence SED in sequence table respectively
ID NO:4) and foot and mouth disease virus nucleotide detection gene (sequence SED ID NO:8 in sequence table) recombinant plasmid, order respectively
Entitled pCR-4 TOPO-SVA and pCR-4 TOPO-FMDV, i.e. standard items.
3, real-time fluorescence quantitative PCR standard curve is established
Gene is detected so that correctly carrying Senecan viral nucleotide detection gene and foot and mouth disease virus nucleotide is sequenced
Recombinant plasmid pCR-4 TOPO-SVA and pCR-4 TOPO-FMDV measures concentration as standard items, with Qubit3.0, and calculates each
SVA standard items (pCR-4 TOPO-SVA) are diluted to 1 × 10 according to 10 times of gradients by copy (copies) number of standard items8、1
×107、1×106、1×105、1×104、1×103、1×102、2.5×101copies/μL；FMDV is marked according to 10 times of gradients
Quasi- product (pCR-4 TOPO-FMDV) are diluted to 1 × 108、1×107、1×106、1×105、1×104、1×103、1×102、1×
101Copies/ μ L, each sample do 2 repetitions, using the standard items of each viral various concentration as template (DNA profiling),
Primer SVA-F, SVA-R, FMDV-F (specially FMDV-F ': 5 '-shown in group 1
TGCCTCCCTTGTTCCTCGTCATCTTT-3 ', sequence SED ID NO:13 in sequence table), FMDV-R (specially FMDV-R ':
5 '-GAGAGCATGTCCTGTCCTTTCACTTT-3 ', sequence SED ID NO:14 in sequence table) and TaqMan probe SVA-P and
TaqMan probe FMDV-P (specially FMDV-P ': 5 '-CAGAGAAGTATGACAAGATCATGTT-3 ', sequence in sequence table
SED ID NO:15) guidance under carry out dual real-time fluorescence quantitative PCR detection, the detection body of 25 μ L real-time fluorescence quantitative PCRs
As shown in table 3, real-time fluorescence quantitative PCR testing conditions are (quantitative PCR apparatus, model C FX96 are purchased from U.S. Bole): first 42 for system
DEG C reverse transcription 20min, 95 DEG C of initial denaturation 30s；Then 94 DEG C of denaturation 10s, 55 DEG C of annealing 30s, 45 circulations (PCR amplification).
3 Senecan virus of table and foot and mouth disease virus dual real-time fluorescence quantitative PCR detection system
| Reagent name | Volume (μ L) |
| 2 × One Step RT-PCR Buffer III (is purchased from TaKaRa company) | 12.5 |
| TaKaRa Ex Taq HS (is purchased from TakaRa company) | 0.5 |
| PrimeScript RT Enzyme Mix II (is purchased from TakaRa company) | 0.5 |
| SVA-F(10μM) | 1.0 |
| SVA-R(10μM) | 1.0 |
| FMDV-F′(10μM) | 1.0 |
| FMDV-R′(10μM) | 1.0 |
| SVA-P(10μM) | 1.0 |
| FMDV-P′(10μM) | 1.5 |
| DNA profiling | 2.0 |
| RNA-free H2O | 3.0 |
The real-time fluorescence quantitative PCR amplification curve of standard items is as shown in figure 3, standard items amplification curve is smooth serpentine
Curve (positive), corresponding standard concentration is respectively as follows: SVA standard items (pCR-4 to eight groups of black lines from left to right in Fig. 3
TOPO-SVA) it is diluted to 1 × 108、1×107、1×106、1×105、1×104、1×103、1×102、2.5×101copies/μ
L；Corresponding standard concentration is respectively as follows: FMDV standard items (pCR-4 TOPO-FMDV) to eight groups of grey lines from left to right in Fig. 3
It is diluted to 1 × 108、1×107、1×106、1×105、1×104、1×103、1×102、1×101copies/μL.Detection terminates
Afterwards, it is mapped with the concentration Log value (X-axis) of each standard items to its corresponding Ct value (Y-axis), draws standard curve, standard curve such as Fig. 4
Shown, related coefficient is respectively R2=0.997 (SVA) and R2=0.997 (FMDV), error is smaller, and standard curve is available, by marking
The linear equation that directrix curve obtains is respectively as follows: y=-3.299x+40.793 (SVA)；Y=-3.180x+40.371 (FMDV).
Three, dual real-time fluorescence quantitative PCR detection is carried out to Senecan passaged virus and aftosa passaged virus
With dual real-time fluorescence quantifying PCR method to from 10 parts of cell cultures, (cell of this laboratory separation identification is passed
Generation virus, sample to be tested) in the geneome RNA (detection sample) that extracts detected, with Senecan viral cultures and mouth hoof
The geneome RNA of epidemic disease viral cultures is positive reference substance, using no enzyme water as negative controls, according to real-time fluorescence quantitative PCR
Whether testing result qualitatively judges containing Senecan virus and/or foot and mouth disease virus in detection sample, and establishing criteria is bent
Line quantifies the copy number of virus.
It is specific that detection method includes the following steps:
1) extract sample to be tested geneome RNA, using the geneome RNA of extraction as template, primer SVA-F, SVA-R,
Dual real-time fluorescence quantitative PCR detection, 25 μ L are carried out under the guidance of FMDV-F, FMDV-R and TaqMan probe SVA-P, FMDV-P
The detection architecture of real-time fluorescence quantitative PCR includes: 2 μ L of template, real time fluorescent quantitative one-step method 2 × One of PCR reaction solution Step
0.5 μ L of 12.5 μ L of RT-PCR Buffer III (being purchased from TakaRa company), TaKaRa Ex Taq HS (is purchased from TakaRa public affairs
Department), PrimeScript RT Enzyme Mix II 0.5 μ L (being purchased from TakaRa company), SVA-F (10 μM) 1 μ L, SVA-R
111 1.5 μ L of μ L, FMDV-P (10 μM) of μ L, FMDV-R (10 μM) of μ L, FMDV-F (10 μM) of (10 μM) 1 μ L, SVA-P (10 μM),
RNA-free H2O 3μL.Real-time fluorescence quantitative PCR reaction condition are as follows: first 42 DEG C of reverse transcription 20min, 95 DEG C of 30s initial denaturations；So
94 DEG C of denaturation 10s afterwards, 55 DEG C of annealing 30s, 45 circulations (PCR amplification).Fluorescence letter is carried out at the end of the annealing of each circulation
Number detection.
2) it is realized with the variation of obtained respective CT value or fluorescence signal viral to Senecan and/or foot and mouth disease virus
Qualitative detection is confirmed as if " S " type amplification curve occurs in (not including the 37th circulation) in 37 circulations in the channel FAM
Senecan virus-positive (contains Senecan virus) in sample, if (not including the 37th to follow in 37 circulations in the channel ROX
Ring) there is " S " type amplification curve, then it is positive (containing foot and mouth disease virus in sample) to be confirmed as foot and mouth disease virus.
3) to positive sample to be tested is determined as in step 2), further according to the standard in the intensity and step 1) of fluorescence signal
Curve obtains the copy number of contained Senecan virus and/or foot and mouth disease virus in sample to be tested, realizes quantitative detection.
If 39 circulations are above in the channel FAM and/or the channel ROX in step 2) does not occur the expansion of " S " type (containing 39 circulations)
Increase curve, then it is negative (without containing Senecan virus and/or foot and mouth disease virus in sample) to be confirmed as corresponding virus；In any channel
37-39 circulation between (containing 37 circulation and without 39 circulation) occur " S " type amplification curve be then determined as it is suspicious, need weight
Inspection.
As shown in table 4 for the testing result of 10 parts of cell cultures, the testing result of No. 1-3 and No. 5 sample is FMDV
Positive (infection foot and mouth disease virus), the testing result of No. 4 samples is that FMDV is suspicious, is shown in No. 1-3 and No. 5 samples containing aftosa
Virus, No. 4 samples need to be examined again；The testing result of 7-9 sample is SVA positive (infection Senecan virus), No. 6 and No. 10 samples
Testing result be that SVA is suspicious, show that No. 6 and No. 10 samples need to be examined again containing Senecan virus in 7-9 sample.
4 10 parts of cell cultures of table carry out Senecan virus and foot and mouth disease virus dual real-time fluorescence quantitative PCR detection knot
Fruit
Test one, detection Senecan of the present invention be viral and foot and mouth disease virus dual real-time fluorescence quantitative PCR detecting method
Sensibility
The recombinant plasmid of Senecan viral nucleotide detection gene and foot and mouth disease virus nucleotide detection gene will be carried
PCR-4 TOPO-SVA and pCR-4 TOPO-FMDV are as standard items, according to 10 times of gradients by SVA standard items (pCR-4 TOPO-
SVA) it is diluted to 1 × 108、1×107、1×106、1×105、1×104、1×103、1×102、2.5×101copies/μL；It presses
FMDV standard items (pCR-4 TOPO-FMDV) are diluted to 1 × 10 according to 10 times of gradients8、1×107、1×106、1×105、1×
104、1×103、1×102、1×101Copies/ μ L, using the standard items of various concentration as template, in primer SVA-F, SVA-
R, it is fixed that dual real-time fluorescence is carried out under the guidance of FMDV-F, FMDV-R and TaqMan probe SVA-P and TaqMan probe FMDV-P
PCR detection, PCR detection architecture and testing conditions are measured referring to embodiment 2, Senecan virus of the present invention is detected and foot and mouth disease virus is double
The sensibility of weight real-time fluorescence quantitative PCR detection method.As a result with shown in Fig. 3.
Senecan virus of the present invention and foot and mouth disease virus dual real-time fluorescence quantitative PCR detecting method can detecte to plug
Card virus 2.5 × 101Copies/ μ L and foot and mouth disease virus 1 × 101Copies/ μ L (sensibility), and amplification curve is specific
" S " type curve, illustrate primer and TaqMan probe of the invention in conjunction with Senecan virus and foot and mouth disease virus RNA be suitable for.
Test two, detection Senecan of the present invention be viral and foot and mouth disease virus dual real-time fluorescence quantitative PCR detecting method
Specificity
To bovine viral diarrhoea bovine diarrhoea virus (BVDV), swine fever virus (CSFV), bovine parainfluenza virus (BPIV) and ox
Epizootic fever virus (BEFV) extracts RNA, to pseudorabies (PRV), rhinotracheitis virus (IBRV), porcine circovirus 2 type
(PCV2), parvovirus (PPV) extracts DNA, using the RNA of normal PK-15 cell as negative controls, while with Senecan virus
The RNA of culture and O-shaped foot and mouth disease virus (O-shaped FMDV), A type foot and mouth disease virus (A type FMDV), Asia-1 type foot and mouth disease virus
(FMDV Asia-1) is positive reference substance, using no enzyme water as blank control, in primer SVA-F, SVA-R, FMDV-F, FMDV-R
And dual real-time fluorescence quantitative PCR detection is carried out under the guidance of TaqMan probe SVA-P and TaqMan probe FMDV-P respectively,
PCR detection architecture and testing conditions detect Senecan virus of the present invention and foot and mouth disease virus dual real-time fluorescence referring to embodiment 2
The specificity of quantitative PCR detecting method.
Testing result is as shown in figure 5, only Senecan viral (SVA) and O-shaped foot and mouth disease virus (O-shaped FMDV), A type aftosa
There is specific " S " type amplification curve (result sun in viral (A type FMDV), Asia-1 type foot and mouth disease virus (FMDV Asia-1)
Property), other samples do not occur specific " S " type amplification curve (result is negative), and testing result shows can with method of the invention
Specifically detect Senecan virus and O-shaped foot and mouth disease virus, A type foot and mouth disease virus, Asia-1 type foot and mouth disease virus.
Test three, detection Senecan of the present invention be viral and foot and mouth disease virus dual real-time fluorescence quantitative PCR detecting method
Repeatability
SVA standard items (pCR-4 TOPO-SVA) are diluted to 1 × 10 according to 10 times of gradients8、1×107、1×106、1×
105、1×104、1×103、1×102、2.5×101copies/μl；According to 10 times of gradients by FMDV standard items (pCR-4 TOPO-
FMDV) it is diluted to 1 × 108、1×107、1×106、1×105、1×104、1×103、1×102、1×101Copies/ μ L, and make
For template, 2 repetitions of each gradient, in primer SVA-F, SVA-R, FMDV-F, FMDV-R and TaqMan probe SVA-P and
Dual real-time fluorescence quantitative PCR detection, PCR detection architecture and testing conditions reference are carried out under the guidance of TaqMan probe FMDV-P
Embodiment 2 detects the repeatability of Senecan virus and foot and mouth disease virus dual real-time fluorescence quantitative PCR detecting method of the present invention.
Testing result is as shown in Fig. 6 and table 5, and the amplification curve of each concentration gradient is relatively gathered as seen from the figure, recurring number
Without obvious gap, show the repetition of Senecan virus of the present invention and foot and mouth disease virus dual real-time fluorescence quantitative PCR detecting method
Property it is preferable, recurring number standard deviation difference only up to 0.40.
The repeatability examination of the Senecan virus of the present invention of table 5 and foot and mouth disease virus dual real-time fluorescence quantitative PCR detecting method
Test result
Embodiment 3 prepares the real-time fluorescence quantitative PCR detection kit that Senecan is viral and foot and mouth disease virus is dual
Based on embodiment 1 and embodiment 2, Senecan virus of the present invention and foot and mouth disease virus dual real-time fluorescence quantitative PCR
Detection kit includes the primer for carrying out dual real-time fluorescence quantitative PCR detection to Senecan virus and foot and mouth disease virus
(SVA-F, SVA-R, FMDV-F and FMDV-R) and TaqMan probe (SVA-P and FMDV-P).
25 μ L dual real-time fluorescence quantitative PCR detection systems when using kit are as follows: real time fluorescent quantitative one-step method
12.5 μ L, TaKaRa Ex Taq HS of PCR reaction solution 2 × One Step RT-PCR Buffer III, 0.5 μ L,
0.5 1.0 1.0 μ L, SVA-P (10 of μ L, SVA-R (10 μM) of μ L, SVA-F (10 μM) of PrimeScript RT Enzyme Mix II
μM) 1 μ L, FMDV-F (10 μM) 1.0 μ L, FMDV-R (10 μM) 1.0 μ L, FMDV-P (10 μM) 1.5 μ L, 2.0 μ L of template ribonucleic acid,
RNA-free H2O 3.0μL。
For convenience of detection, it may also include positive reference substance and negative controls in kit, positive reference substance is Senecan
Viral RNA and foot and mouth disease virus RNA, negative controls are the reaction system without Senecan virus and foot and mouth disease virus, such as H2O
(distilled water, aseptic deionized water etc.).
The application method of each reagent can refer to the content of embodiment 2 in kit.
The real-time fluorescence quantitative PCR detection of embodiment 4, pig farm doubtful Senecan disease and hoof-and-mouth disease pathological material of disease
With Senecan virus and foot and mouth disease virus dual real-time fluorescence quantitative PCR detection kit to 14 portions of pigs from collection
The geneome RNA (detection sample) extracted in the doubtful Senecan virus in field and foot and mouth disease virus sample (sample to be tested) is detected,
Using Senecan virus genome RNA and foot-and-mouth disease virus genome RNA as positive reference substance, using no enzyme water as negative controls,
According to dual real-time fluorescence quantitative PCR detection as a result, in test sample whether containing Senecan virus and foot and mouth disease virus into
Row qualitatively judges, and quantifies to the copy number of virus.
Specific detection method is same as Example 2.
Testing result is as shown in table 6, and the testing result of 1-3 sample is SVA positive (infection Senecan virus), shows 1-
Containing Senecan virus in No. 3 samples；The testing result of 4-12 sample is FMDV positive (infection foot and mouth disease virus), shows 4-12
Contain foot and mouth disease virus in number sample；13, the testing result of No. 14 samples is feminine gender, is shown in 13 and No. 14 samples without Senecan
Virus and foot and mouth disease virus.
The doubtful Senecan virus in 6 14 parts of pig farms of table and foot and mouth disease virus sample dual real-time fluorescence quantitative PCR detection result
It compares compared to the prior art, present invention has an advantage that
FMDV and SVA double fluorescent quantitative PCR detection method has the latent invention of CN107326100A Yan Ruo in existing invention
" aftosa and the double real-time fluorescence quantitative PCR detection kit of Senecan virus ", detection method, this method is first will be viral
RNA reverse transcription is that template carries out real-time fluorescence quantitative PCR amplification at cDNA, then by cDNA, and experimental procedure is cumbersome.With it is above-mentioned existing
Method is compared, and it is directly template addition by RNA that the present invention, which is provided, which does not have to first reverse transcription into cDNA to FMDV and SVA RNA sample,
One-step method PCR system carry out real-time fluorescence quantitative PCR detection, advantage be the present invention in not first by reverse transcription of viral RNA at
The step of cDNA, and amplification curve is specific " S " type curve, illustrates the primed probe of this method in conjunction with viral RNA most
It is suitable for.Spy optimizes relevant primer and probe to the present invention thus, establishes a kind of Senecan virus and the dual reality of foot and mouth disease virus
When fluorescent quantitative PCR detection method, can quickly simultaneously detect difference Senecan virus and foot and mouth disease virus, be clinical sample inspection
It surveys, plays a significant role in strain identification and production of vaccine.
" a kind of reagent identified for FMDV and SVA, side of CN201711339422.1 flower group's justice invention in existing invention
Method and application ", FMDV and SVA detection target sequence is of the invention all at the 3D gene of the virus full-length genome in the detection method
For the detection FMDV and SVA target sequence of offer at the 3C gene of the virus full-length genome, the target sequence of the two selection is different；The hair
Bright middle susceptibility is 10-6Dilution, and do not know original content in the invention, so can not be compared.Sensibility of the present invention
It is 101Copy/μ L.
In existing invention CN201711324308.1 congratulate eastern hair tonic it is bright " for detecting swine foot-and-mouth disease virus and Sai Neijia paddy
Double PCR primer, detection method and the kit of virus ", 2C base of the FMDV detection target sequence in the virus in the detection method
Because of place, SVA detects target sequence at the VP4 gene of the virus, and the target sequence of the two selection is also different；Susceptibility is in the invention
102Copy/μ L, the present invention in detection sensitivity be 101Copy/μ L, the two sensibility compare, and sensibility of the present invention is higher than
CN201711324308.1；The invention be qualitative detection, no standard measure, and the present invention can it is qualitative can be with quantitative detection.
Sequence table
<110>Jinyu Baoling Biology Drugs Co., Ltd
<120>Senecan virus and foot and mouth disease virus dual real-time fluorescence quantitative PCR detection kit
<130> CGCNB185095W
<141> 2018-08-16
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>SVA upstream primer (Seneca Virus A)
<400> 1
tatctcagat ccctggctgt c 21
<210> 2
<211> 22
<212> DNA
<213>SVA downstream primer (Seneca Virus A)
<400> 2
cctgatgatc acattgttga gc 22
<210> 3
<211> 27
<212> DNA
<213>SVA Taqman probe (Seneca Virus A)
<400> 3
cacgcttacg gcgagcgtcg catcaag 27
<210> 4
<211> 257
<212> DNA
<213>SVA detects gene (Seneca Virus A)
<400> 4
tggctccttc gaggctctca tctctcactt tttcaccgtg gacaatggtt ttagccctgc 60
gctgggaccg tatctcagat ccctggctgt ctcggtgcac gcttacggcg agcgtcgcat 120
caagattacc ggtggcctcc cctccggttg tgccgcgacc agcctgctga acacagtgct 180
caacaatgtg atcatcagga ctgctctggc attgacttac aaggaatttg aatatgacat 240
ggttgatatc atcgcct 257
<210> 5
<211> 26
<212> DNA
<213>FMDV upstream primer (Foot-and-Mouth Disease Virus)
<400> 5
ygcctmccth gtdcctcgtc ayctyt 26
<210> 6
<211> 26
<212> DNA
<213>FMDV downstream primer (Foot-and-Mouth Disease Virus)
<400> 6
gagagcatgt cctgtcctyt yacttt 26
<210> 7
<211> 25
<212> DNA
<213>FMDV Taqman probe (Foot-and-Mouth Disease Virus)
<220>
<221> misc_feature
<222> (2)..(2)
<223> n is a, c, g, or t
<400> 7
cngagaagta ygacaagatc atgyt 25
<210> 8
<211> 568
<212> DNA
<213>FMDV detects gene (Foot-and-Mouth Disease Virus)
<400> 8
gaagaaacct gtcgctttga aagtgaaagc aaagaacttg atcgtcactg agagtggtgc 60
tcccccgact gacttgcaaa agatggtcat gggtaacacc aagcctgttg agctcatcct 120
cgacgggaag acggtggcca tctgctgcgc cactggagtg tttggtactg cctaccttgt 180
tcctcgtcat cttttcgcag agaagtatga caagatcatg ttggacggca gagccatgac 240
agacagtgac tacagagtgt ttgagtttga gattaaagtg aaaggacagg acatgctctc 300
agacgccgcg ctcatggtgc ttcaccgtgg gaatcgcgtg cgggacatca cgaagcactt 360
ccgtgatgtg gcaagaatga agaaaggcac ccccgtcgtc ggcgtgatca acaacgctga 420
tgttgggaga ctgatcttct ctggtgaggc ccttacctac aaggacattg tagtgtgcat 480
ggacggagac accatgcccg gtctcttcgc ctacaaagcc gccaccaagg cgggttactg 540
tggaggagcc gttcttgcaa aggacgga 568
<210> 9
<211> 22
<212> DNA
<213>SVA expands upstream primer (Seneca Virus A)
<400> 9
tggctccttc gaggctctca tc 22
<210> 10
<211> 22
<212> DNA
<213>SVA expands downstream primer (Seneca Virus A)
<400> 10
aggcgatgat atcaaccatg tc 22
<210> 11
<211> 23
<212> DNA
<213>FMDV expands upstream primer (Foot-and-Mouth Disease Virus)
<400> 11
gaagaaacct gtcgctttga aag 23
<210> 12
<211> 23
<212> DNA
<213>FMDV expands downstream primer (Foot-and-Mouth Disease Virus)
<400> 12
tccgtccttt gcaagaacgg ctc 23
<210> 13
<211> 26
<212> DNA
<213>FMDV upstream primer (Foot-and-Mouth Disease Virus)
<400> 13
tgcctccctt gttcctcgtc atcttt 26
<210> 14
<211> 26
<212> DNA
<213>FMDV downstream primer (Foot-and-Mouth Disease Virus)
<400> 14
gagagcatgt cctgtccttt cacttt 26
<210> 15
<211> 25
<212> DNA
<213>FMDV Taqman probe (Foot-and-Mouth Disease Virus)
<400> 15
cagagaagta tgacaagatc atgtt 25
Claims (10)
1. primer and Taqman spy for carrying out dual real-time fluorescence quantitative PCR detection to Senecan virus and foot and mouth disease virus
Needle characterized by comprising
For detecting the upstream primer (SVA-F) and downstream primer (SVA-R) of Senecan virus, the core of upstream primer (SVA-F)
Nucleotide sequence is as shown in SED ID NO:1 in sequence table, SEQ ID in the nucleotide sequence of downstream primer (SVA-R) such as sequence table
Shown in NO:2；
For carrying out the TaqMan probe (SVA-P) of real-time fluorescence quantitative PCR detection, TaqMan probe to Senecan virus
(SVA-P) nucleotide sequence is as shown in SED ID NO:3 in sequence table；
For detecting the upstream primer (FMDV-F) and downstream primer (FMDV-R) of foot and mouth disease virus, upstream primer (FMDV-F)
Nucleotide sequence is as shown in SED ID NO:5 in sequence table, SEQ in the nucleotide sequence of downstream primer (FMDV-R) such as sequence table
Shown in ID NO:6；With
For carrying out the TaqMan probe (FMDV-P) of real-time fluorescence quantitative PCR detection, TaqMan probe to foot and mouth disease virus
(FMDV-P) nucleotide sequence is as shown in SED ID NO:7 in sequence table；
The probe is by fluorescent marker, and 5 ' ends are marked with reporter fluorescence group, and 3 ' ends are marked with quenching fluorescence group；
And the reporter fluorescence group marked to two kinds of viral diagnosis probes is not identical.
2. primer according to claim 1 and TaqMan probe, it is characterised in that:
It reports at the 5 ' ends for carrying out the TaqMan probe (SVA-P) of real-time fluorescence quantitative PCR detection to Senecan virus
Fluorophor is FAM, and 3 ' end fluorescent quenching groups are TAMRA；
For carrying out 5 ' end reporter fluorescences of the TaqMan probe (FMDV-P) of real-time fluorescence quantitative PCR detection to foot and mouth disease virus
Group is ROX, and 3 ' end fluorescent quenching groups are BHQ2；
3 ' phosphorylated the processing in end of the probe.
3. feature exists for carrying out dual real-time fluorescence quantitative PCR detection kit to Senecan virus and foot and mouth disease virus
In:
Including primer of any of claims 1 or 2 and TaqMan probe.
4. kit according to claim 3, it is characterised in that:
Dual real-time fluorescence quantitative PCR detection system when using the kit are as follows: real time fluorescent quantitative one-step method PCR reaction
12.5 μ L, TaKaRa Ex Taq HS of liquid 2 × One Step RT-PCR Buffer III, 0.5 μ L, PrimeScript RT
Enzyme Mix II 0.5 μ L, SVA-F (10 μM) 1.0 μ L, SVA-R (10 μM) 1.0 μ L, SVA-P (10 μM) 1 μ L, FMDV-F
1.0 1.5 μ L, RNA-free H of μ L, FMDV-P (10 μM) of (10 μM) 1.0 μ L, FMDV-R (10 μM)23.0 μ L of O, template ribonucleic acid
2.0μL。
5. primer of any of claims 1 or 2 and TaqMan probe are in the detection examination for preparing Senecan virus and foot and mouth disease virus
Application in agent.
6. kit described in claim 3 or 4 is carrying out non-disease diagnostic purpose to Senecan virus and foot and mouth disease virus
Application in detection.
7. application according to claim 5 or 6, it is characterised in that:
For carrying out qualitative and quantitative analysis to Senecan virus and foot and mouth disease virus, comprising the following steps:
1) it establishes standard curve: the recombinant plasmid pCR4- of the detection gene of Senecan virus and foot and mouth disease virus will be carried respectively
TOPO-SVA and pCR4-TOPO-FMDV dilutes SVA standard items (pCR-4TOPO-SVA) as standard items, according to 10 times of gradients
To 1 × 108、1×107、1×106、1×105、1×104、1×103、1×102、2.5×101copies/μL；According to 10 times of ladders
FMDV standard items (pCR-4TOPO-FMDV) are diluted to 1 × 10 by degree8、1×107、1×106、1×105、1×104、1×103、1
×102、1×101Copies/ μ L, using the standard items of various concentration as template, in primer of any of claims 1 or 2 and
Dual real-time fluorescence quantitative PCR detection is carried out under the guidance of TaqMan probe, after detection, with the concentration Log of each standard items
It is worth (X-axis) to map to its corresponding Ct value (Y-axis), draws standard curve；
2) geneome RNA for extracting sample to be tested draws using the geneome RNA of extraction as template of any of claims 1 or 2
Dual real-time fluorescence quantitative PCR detection is carried out under the guidance of object and TaqMan probe；
3) the qualitative inspection to Senecan virus and foot and mouth disease virus is realized with the variation of obtained respective CT value or fluorescence signal
It surveys；
4) to positive sample to be tested is determined as in step 3), further according to the standard song in the intensity and step 1) of fluorescence signal
Line obtains copy number viral contained in the sample, realizes quantitative detection.
8. application according to claim 7, it is characterised in that:
Sample to be tested in the step 2) can be raw material serum, vaccine semi-finished product and the pig farm for picking up from pig for production of vaccine
Submitted sample.
9. application according to claim 7 or 8, it is characterised in that:
Dual real-time fluorescence quantitative PCR detection system in the step 1) and step 2) includes: 2 μ L of template, and real-time fluorescence is fixed
2 × One of one-step method PCR reaction solution Step RT-PCR Buffer III, 12.5 μ L, TaKaRa Ex Taq HS, 0.5 μ L is measured,
0.5 1.0 1.0 μ L, SVA-P (10 of μ L, SVA-R (10 μM) of μ L, SVA-F (10 μM) of PrimeScript RT Enzyme Mix II
μM) 1 1.0 1.0 1.5 μ L, RNA-free H of μ L, FMDV-P (10 μM) of μ L, FMDV-R (10 μM) of μ L, FMDV-F (10 μM)2O 3.0
μL；
Dual real-time fluorescence quantitative PCR detection condition in the step 1) and step 2) are as follows: first 42 DEG C of reverse transcriptions 20min, 95
DEG C initial denaturation 30s；Then 94 DEG C of denaturation 10s, 55 DEG C of annealing 30s, 45 circulations (PCR amplification).
10. the application according to any one of claim 7-9, it is characterised in that:
Determination method in the step 3) are as follows:
If " S " type amplification curve occurs to sample in (not including the 37th circulation) in 37 circulations in the channel FAM, it is confirmed as
Senecan virus-positive, if " S " type amplification song occurs to sample in (not including the 37th circulation) in 37 circulations in the channel ROX
Line is then confirmed as the foot and mouth disease virus positive；If sample is in the channel FAM and/or the channel ROX (including the more than 39 circulations
39 circulations) do not occur " S " type amplification curve, then it is confirmed as Senecan virus and/or foot and mouth disease virus is negative；In any channel
37-39 circulation between (including the 37th circulation and do not include the 39th circulation) occur " S " type amplification curve be then determined as
It is suspicious, it need to examine again.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810933044.8A CN109097495A (en) | 2018-08-16 | 2018-08-16 | Senecan virus and foot and mouth disease virus dual real-time fluorescence quantitative PCR detection kit |
| PCT/CN2018/107963 WO2020034317A1 (en) | 2018-08-16 | 2018-09-27 | Dual real-time fluorescent quantitative pcr detection reagent and reagent kit for seneca virus a and foot-and-mouth disease virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810933044.8A CN109097495A (en) | 2018-08-16 | 2018-08-16 | Senecan virus and foot and mouth disease virus dual real-time fluorescence quantitative PCR detection kit |
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| Publication Number | Publication Date |
|---|---|
| CN109097495A true CN109097495A (en) | 2018-12-28 |
Family
ID=64849796
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|---|---|---|---|
| CN201810933044.8A Pending CN109097495A (en) | 2018-08-16 | 2018-08-16 | Senecan virus and foot and mouth disease virus dual real-time fluorescence quantitative PCR detection kit |
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| WO (1) | WO2020034317A1 (en) |
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| WO2020034317A1 (en) | 2020-02-20 |
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