WO2020034317A1 - 塞内卡病毒和口蹄疫病毒双重实时荧光定量pcr检测试剂及试剂盒 - Google Patents

塞内卡病毒和口蹄疫病毒双重实时荧光定量pcr检测试剂及试剂盒 Download PDF

Info

Publication number
WO2020034317A1
WO2020034317A1 PCT/CN2018/107963 CN2018107963W WO2020034317A1 WO 2020034317 A1 WO2020034317 A1 WO 2020034317A1 CN 2018107963 W CN2018107963 W CN 2018107963W WO 2020034317 A1 WO2020034317 A1 WO 2020034317A1
Authority
WO
WIPO (PCT)
Prior art keywords
virus
fmdv
sva
foot
mouth disease
Prior art date
Application number
PCT/CN2018/107963
Other languages
English (en)
French (fr)
Inventor
王秀明
陈君彦
张竞
魏学峰
刘国英
关平原
范秀丽
张贵刚
武瑾贤
王艳杰
刘建奇
Original Assignee
金宇保灵生物药品有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 金宇保灵生物药品有限公司 filed Critical 金宇保灵生物药品有限公司
Publication of WO2020034317A1 publication Critical patent/WO2020034317A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the invention belongs to the technical field of biological detection, and relates to veterinary animal pathogen detection, in particular to a dual real-time fluorescent quantitative PCR detection special primer, a TaqMan probe and a detection kit for qualitative and quantitative detection of Seneca virus and foot-and-mouth disease virus. .
  • Seneca Virus A SVA
  • FMDV Foot-and-Mouth Disease Virus
  • SVA SVA
  • FMDV Foot-and-Mouth Disease Virus
  • Seneca virus belongs to the typical representative of the Seneca Virus genus, which is closest to Cardio Virus
  • the foot-and-mouth disease virus belongs to the virus of the foot-and-mouth disease virus.
  • Seneca virus is an infectious disease that mainly occurs in pigs. Its clinical features are infection of pigs with ulcers, anorexia, and lameness in the nose and mouth, which causes acute death of newborn piglets. Early Seneca cases mainly occurred in fattening pigs, and vesicular symptoms were seen in the coronal bands of the nose, mouth and hoof. However, some recently found Seneca positive cases occurred in newborn piglets with symptoms of diarrhea and no vesicular disease. These cases were mainly detected by PCR testing of serum, feces and different tissues.
  • Foot-and-mouth disease virus is an acute, thermal, and high-contact infectious disease that can cause cloven-hoofed animals such as cattle, pigs, and sheep. It is characterized by blisters and ulceration on the oral mucosa, tongue, lips, nose, hoof, and breast skin. .
  • the average lethal rate of foot-and-mouth disease is only 1%, but 100% of infected animals develop disease, and the transmission efficiency is extremely high, which causes the actual livestock output to decrease sharply.
  • Foot-and-Mouth Disease FMD is the most severe and infectious disease in the world, and is ranked first by the World Organisation for Animal Health (OIE).
  • Seneca virus due to the discovery of Seneca virus, SVA infection and FMDV infection cannot be distinguished from clinical symptoms alone, which makes clinical diagnosis difficult and needs to be determined by laboratory testing techniques. How to detect and distinguish between Seneca virus and foot-and-mouth disease virus by a fast and simple method is an urgent problem for some pig farms.
  • vaccines are the most effective measures to prevent viruses.
  • inactivated vaccines play an important role in the prevention of infectious diseases.
  • Preparation of inactivated vaccines is the main effective means to prevent the epidemic of Seneca virus and foot-and-mouth disease virus.
  • effective inactivation of pathogens can provide a safe vaccine.
  • the vaccine is mainly produced by inactivating Seneca virus and foot-and-mouth disease virus in vitro, and then mixing it with an emulsifier to prepare an immune vaccine.
  • the first object of the present invention is to provide primers and TaqMan probes for dual real-time fluorescent quantitative PCR detection of Seneca virus (SVA) and foot-and-mouth disease virus (FMDV) to achieve Seneca virus (SVA) and foot-and-mouth disease. Qualitative and quantitative detection of virus (FMDV).
  • the primers used for real-time quantitative PCR detection of Seneca virus are: the nucleotide sequence of the upstream primer (SVA-F) is shown as SED ID NO: 1 in the sequence listing, and the core of the downstream primer (SVA-R) The nucleotide sequence is shown as SEQ ID NO: 2 in the sequence listing.
  • Primer sequences derived from the aforementioned primers also belong to the present invention.
  • the derived sequence refers to a primer sequence obtained by substitution, deletion, or addition of one to ten bases on the basis of SEQ ID NO: 1 and / or SEQ ID NO: 2.
  • the primers used for real-time quantitative PCR detection of foot-and-mouth disease virus are: the nucleotide sequence of the upstream primer (FMDV-F) is shown in the sequence table as SED ID NO: 5, and the nucleotide of the downstream primer (FMDV-R) The sequence is shown as SEQ ID NO: 6 in the sequence listing.
  • Primer sequences derived from the aforementioned primers also belong to the present invention.
  • the derived sequence refers to a primer sequence obtained by substitution, deletion, or addition of one to ten bases on the basis of SEQ ID NO: 5 and / or SEQ ID NO: 6.
  • the TaqMan probe used for real-time quantitative PCR detection of Seneca virus is:
  • the nucleotide sequence of the TaqMan probe (SVA-P) is shown in SED ID NO: 3 in the sequence listing; the probe is Fluorescently labeled with a reporter fluorophore at the 5 'end and a quencher fluorophore at the 3' end.
  • the TaqMan probe used for real-time fluorescent quantitative detection of foot-and-mouth disease virus is:
  • the nucleotide sequence of the TaqMan probe (FMDV-P) is shown in the sequence table as SED ID NO: 7; the probe is fluorescently labeled , Its 5 ′ end is labeled with a reporter fluorophore, and its 3 ′ end is labeled with a quencher fluorophore.
  • Derived sequences from the above TaqMan probe sequences also belong to the present invention.
  • the derived sequence refers to a sequence obtained by adding or subtracting one or more bases at the 5 ′ end and / or the 3 ′ end of the sequence based on SEQ ID NO: 3 / SEQ ID NO: 7.
  • the reporter fluorophores labeled by the two virus detection probes SVA-P and FMDV-P are different.
  • the 5′-end reporting fluorescent group of the TaqMan probe is FAM
  • the 3′-end fluorescent quenching group is TAMRA
  • the 5′-end reporting fluorescent group of the TaqMan probe is a fluorescent group.
  • the group is ROX
  • the 3'-end fluorescence quenching group is BHQ2.
  • the 3 'end of the TaqMan probe has been phosphorylated.
  • a second object of the present invention is to provide a dual real-time fluorescent quantitative PCR detection kit for Seneca virus (SVA) and foot-and-mouth disease virus (FMDV).
  • SVA Seneca virus
  • FMDV foot-and-mouth disease virus
  • the dual real-time quantitative PCR detection kit includes the above primers and a TaqMan probe.
  • the kit may further include a standard product, the standard product is a recombinant plasmid pCR4-TOPO-SVA with a Seneca virus detection gene and a recombinant plasmid pCR4-TOPO-FMDV with a foot-and-mouth disease virus detection gene.
  • the standard product is a recombinant plasmid pCR4-TOPO-SVA with a Seneca virus detection gene and a recombinant plasmid pCR4-TOPO-FMDV with a foot-and-mouth disease virus detection gene.
  • the dual real-time quantitative PCR detection system (using a 25 ⁇ L system as an example) when using the kit is: real-time quantitative one-step PCR reaction solution 2 ⁇ One Step RT-PCR Buffer III 12.5 ⁇ L (purchased from TakaRa Company), TaKaRa Ex Taq HS 0.5 ⁇ L (purchased from TakaRa), PrimeScript RT Enzyme Mix II 0.5 ⁇ L (purchased from TakaRa), SVA-F (10 ⁇ M) 1 ⁇ L, SVA-R (10 ⁇ M) 1 ⁇ L, SVA-P ( 10 ⁇ M) 1 ⁇ L, FMDV-F (10 ⁇ M) 1 ⁇ L, FMDV-R (10 ⁇ M) 1 ⁇ L, FMDV-P (10 ⁇ M) 1.5 ⁇ L, RNA-free H 2 O 3 ⁇ L, and template 2 ⁇ L.
  • the template is either the standard DNA or the genomic RNA of the test sample.
  • a third object of the present invention is to provide applications of the primers and probes in the preparation of detection reagents for Seneca virus and foot-and-mouth disease virus.
  • a fourth object of the present invention is to provide the application of the kit in the detection of Seneca virus (SVA) and foot-and-mouth disease virus (FMDV).
  • SVA Seneca virus
  • FMDV foot-and-mouth disease virus
  • the detection is for the detection of viruses in the vaccine production process, or for pathogens. Detection for non-disease diagnostic purposes, or detection of pathogens in disease diagnosis.
  • the test samples can be samples sent from pig farms, or raw material serum and semi-finished vaccine products in vaccine production.
  • Recombinant plasmids pCR-4TOPO-SVA and pCR-4TOPO-FMDV carrying the SVA nucleotide sequence and the FMDV nucleotide sequence were used as standards, and the recombinant plasmid pCR-4TOPO-SVA was diluted 10-fold into 1 ⁇ 10 8 and 1 ⁇ 10 7 ⁇ 1 ⁇ 10 6 ⁇ 1 ⁇ 10 5 ⁇ 1 ⁇ 10 4 ⁇ 1 ⁇ 10 3 ⁇ 1 ⁇ 10 2 ⁇ 2.5 ⁇ 10 1 copy (copies) / ⁇ L; recombinant plasmid pCR-4TOPO-FMDV 10-fold gradient dilution 1 ⁇ 10 8 , 1 ⁇ 10 7 , 1 ⁇ 10 6 , 1 ⁇ 10 5 , 1 ⁇ 10 4 , 1 ⁇ 10 3 , 1 ⁇ 10 2 , 1 ⁇ 10 1 copies (copies) / ⁇ L, at different concentrations
  • the standard DNA was used as a template, and real-time quantitative
  • Plasmid pCR4-TOPO-FMDV Select the specific conserved sequence of the 3C region gene of foot-and-mouth disease virus (foot-and-mouth disease virus at base 5986-65553 at the 5 ′ end, SED ID NO: 8 in the sequence list, detection gene) as Standards were used to construct sequences, and standard primers (SED ID NO: 11 and SED ID NO: 12 in the sequence listing) for real-time fluorescent quantitative PCR detection of foot-and-mouth disease virus were designed. The genomic RNA of foot-and-mouth disease virus was used as a template, and standard primers were used for PCR.
  • the 3C region detection gene of foot-and-mouth disease virus was amplified and ligated into the vector pCR-4TOPO to construct a recombinant plasmid pCR4-TOPO-FMDV carrying the 3C region detection gene of foot-and-mouth disease virus, and the concentration of the correctly identified recombinant plasmid was determined.
  • the qualitative detection of Seneca virus and foot-and-mouth disease virus is achieved by using the respective CT values or changes in the fluorescence signal obtained.
  • the "S" type amplification curve appears in the corresponding fluorescence channel of Seneca virus and / or foot-and-mouth disease virus. It indicates that the test sample contains the corresponding virus, that is, it is determined to be positive;
  • step 3 For the test sample determined to be positive in step 3), according to the intensity of the fluorescent signal and the standard curve in step 1), obtain the copy numbers of Seneca virus and foot-and-mouth disease virus contained in the test sample to achieve Quantitative detection.
  • the sample to be tested in step 2) may be raw serum collected from pigs used for vaccine production, semi-finished vaccine products, and samples sent from pig farms for inspection. And foot and mouth disease virus quantitative detection to monitor pig-based products and their raw materials, and provide objective data for subsequent raw material disposal.
  • the dual real-time quantitative PCR detection system in steps 1) and 2) may include: template 2 ⁇ L, real-time fluorescent one-step PCR reaction solution 2 ⁇ One Step RT-PCR Buffer III 12.5 ⁇ L (purchased from TakaRa), TaKaRa Ex Taq HS 0.5 ⁇ L (purchased from TakaRa), PrimeScript RT Enzyme Mix II 0.5 ⁇ L (purchased from TakaRa), SVA-F (10 ⁇ M) 1 ⁇ L, SVA-R (10 ⁇ M) 1 ⁇ L, SVA-P (10 ⁇ M) 1 ⁇ L , 1 ⁇ L of FMDV-F (10 ⁇ M), 1 ⁇ L of FMDV-R (10 ⁇ M), 1.5 ⁇ L of FMDV-P (10 ⁇ M), and 3 ⁇ L of RNA-free H 2 O.
  • the conditions of the dual real-time quantitative PCR detection in the steps 1) and 2) may be: reverse transcription at 42 ° C for 20 min, pre-denaturation at 95 ° C for 30s; then denaturation at 94 ° C for 10s, annealing at 55 ° C for 30s, 45 cycles ( PCR amplification). Fluorescent signal detection is performed at the end of each cycle of annealing.
  • the specific method for determining the result in step 3) may be: if an "S" type amplification curve appears in the FAM channel within 37 cycles (excluding the 37th cycle), then it is confirmed that the Seneca virus is positive (sample Seneca virus is included), if the “S” type amplification curve appears in the ROX channel within 37 cycles (excluding the 37th cycle), then it is confirmed to be positive for FMD virus (the sample contains FMD virus); if FAM Channels and / or ROX channels do not appear in the "S" type amplification curve at 39 cycles or more (including the 39th cycle), then it is confirmed that the corresponding virus is negative (the sample does not contain Seneca virus and / or foot-and-mouth disease virus ), "S" type amplification curve appeared in 39 or more cycles, also confirmed as corresponding virus negative; between 37-39 cycles in any channel (including the 37th cycle and not including the 39th cycle) (That is, 37 or 38 cycles) when the "S" type amplification curve appears, it is determined to be
  • the invention provides a dual real-time fluorescent quantitative PCR detection kit for detecting Seneca virus (SVA) and foot-and-mouth disease virus (FMDV), and its special primers and TaqMan probes, which can be used to implement Seneca virus and foot-and-mouth disease virus. Rapid differentiation and detection can also provide a strong basis for quality monitoring and rationalization of vaccine production (such as assessing the accurate content of formulated vaccine antigens and providing a data basis for vaccine antigen content) to ensure the safety and rationality of vaccination , Has a guiding role in the production of Seneca virus and foot-and-mouth disease virus vaccines.
  • the kit and the detection method of the invention have simple operation, strong specificity, high sensitivity, and good reproducibility, can realize accurate quantification of Seneca virus and foot-and-mouth disease virus, and can detect Seneca virus and foot-and-mouth disease virus (including (Accurate detection of Seneca virus and foot-and-mouth disease virus in disease materials or cultures) and vaccine production play an important role in broad application prospects.
  • FIG. 1 is a primer screening amplification curve for detecting Seneca virus (SVA);
  • FIG. 2 is a primer screening amplification curve for detecting foot-and-mouth disease virus (FMDV);
  • Figure 3 is an amplification curve of a standard for dual detection of SVA and FMDV;
  • Figure 4 is a standard curve for dual detection of SVA and FMDV
  • Figure 5 shows the specific detection results of SVA and FMDV dual real-time quantitative PCR detection
  • Figure 6 is an amplification curve of a SVA and FMDV dual real-time quantitative PCR detection repeatability experiment.
  • the percentage concentrations are mass / mass (W / W, unit g / 100g) percentage concentration, mass / volume (W / V, unit g / 100mL) percentage concentration or volume / volume (V / V, Units mL / 100 mL) percentage concentration.
  • the primers used in the examples were synthesized by Beijing Huada Gene Co., Ltd .; the probes used were synthesized by TAKARA Gene Company.
  • Biological genome is a most objective indicator that directly reflects basic biological information.
  • Different viruses contain different genomic information. Different types of viruses can be classified into different groups through genomic information. The principle of complementary pairing of genomic bases can be used to achieve Massive amplification of specific site gene sequences.
  • the present invention designed two specific primers and two oligonucleotide probes. Based on the principle of complementary base pairing, a specific detection of Seneca virus (SVA) and foot-and-mouth disease virus (FMDV) was established. ) Dual real-time quantitative PCR detection method.
  • SVA Seneca virus
  • FMDV foot-and-mouth disease virus
  • Example 1 Design of primers and TaqMan probes for detection of Seneca virus (SVA) and foot-and-mouth disease virus (FMDV) using real-time quantitative PCR technology
  • a specific fragment is selected from the 3C conserved region of the Seneca virus as a detection sequence, and the detection sequence is finally determined to be the base 6564-6820 of the Seneca virus from the 5 ′ end (SED ID in the sequence listing NO : 4). Based on the selected detection sequence, primers and TaqMan probes for real-time quantitative PCR detection of Seneca virus were designed.
  • FMD virus detection gene many studies have used 3D genes as target sequences for fluorescent quantitative RT-PCR technology to detect FMD, and a few studies have used FMD virus 2B gene and 5′UTR as target sequences for fluorescent quantitative RT-PCR technology to detect FMD.
  • the invention found a highly conserved and specific nucleotide region in the 3C gene region of foot-and-mouth disease virus to design primers and probes.
  • a specific fragment is preferably selected as a detection sequence from the 3C conserved region of the foot-and-mouth disease virus, and the detection sequence is finally determined to be the base 5986-6553 of the foot-and-mouth disease virus from the 5 ′ end (SED ID NO: 8 in the sequence listing). Based on the selected detection sequences, primers and TaqMan probes for FMD virus detection were designed.
  • the present invention designs multiple sets of combinations and selects group 1 as a preferred group from them:
  • SVA-F upstream primer: 5′-TATCTCAGATCCCTGGCTGTC-3 ′ (sequence position: Seneca virus from the 5 ′ end at positions 6634-6654, SED ID in the sequence listing NO: 1);
  • SVA-R downstream primer: 5′-CCTGATGATCACATTGTTGAGC-3 ′ (sequence position: Seneca virus from base 6741-6762 at 5 ′ end, SED ID NO in sequence list: 2);
  • SVA-P (TaqMan probe): 5′-FAM-CACGCTTACGGCGAGCGTCGC ATCAAG-TAMRA-3 ′ (sequence position: 6661-6687 bases of Seneca virus from 5 ′ end, SED ID NO in sequence list: 3) ;
  • group 2 the primers and probes obtained based on the detection sequences of other conserved regions of Seneca virus and other foot-and-mouth disease virus primers and probes are listed as group 2 as a control group:
  • SVA-F1 upstream primer: 5′-TATAAGATGACTCCTGCCAAC-3 ′ (sequence position: Seneca virus bases 6898-6918 from the 5 ′ end);
  • SVA-R1 downstream primer: 5′-AGAATTTGGAAGCCATGCTCTC-3 ′ (sequence position: Seneca virus bases 7025-7046 from the 5 ′ end);
  • SVA-P1 (TaqMan probe): 5′-FAM-TTCTGTCTTCCCTCCGACTTC CTCTC-TAMRA-3 ′ (sequence position: Seneca virus bases 6924-6949 from the 5 ′ end);
  • FMDV-F1 upstream primer: 5′-AAGATCATGTTGGACGGCAGAG CCAT-3 ′ (sequence position: foot and mouth disease virus from base 5198 to 6198-6223);
  • FMDV-R1 downstream primer: 5′-ATGTCCCGCACGCGATTCCCACGGT-3 ′ (sequence position: 6310-6334 bases of FMD virus from 5 ′ end);
  • FMDV-P1 (TaqMan probe): 5′-ROX-CAGTGACTACAGAGTG TTTGAGTTTGAG-BHQ2-3 ′ (sequence position: foot and mouth disease virus from base 5230-6257 of 5 ′ end);
  • the 5 ′ end has a reporter fluorescent group FAM or ROX, and the probes for the two viruses in the same group have different groups; the 3 ′ end has a report that is the same as the 5 ′ end.
  • the 3 'end of the TaqMan probe is phosphorylated.
  • Primer probe screening and condition optimization experiments for FMDV and SVA fluorescence quantitative PCR The PCR amplification primers and the optimal annealing temperature of each primer designed above are screened. Five gradients of annealing temperature are set, which are 53 ° C and 55 ° C. , 57 ° C, 59 ° C, 61 ° C, each temperature gradient is repeated twice.
  • the reaction system is shown in Table 1 and Table 2 below.
  • the reaction conditions are: reverse transcription at 42 ° C for 20 min, pre-denaturation at 94 ° C, 30s; ⁇ Denaturation at 94 ° C, 10s, 53 ° C-61 ° C annealing, 30s ⁇ ⁇ 45 cycles.
  • the primers and the annealing temperature are compared by comparing the amplification curve, and the primers and the probes are compared.
  • the results of the two sets of primers and probes at the annealing temperature of 55 ° C are shown in Figures 1 and 2.
  • the FMDV and SVA quantitative PCR amplification curves of group 1 are standard “S” -type curves, and the fluorescence thresholds of the FMDV and SVA quantitative PCR amplification curves of group 2 are not as good as those of group 1.
  • the primers and probes designed by Group 1 were identified as the dual real-time quantitative PCR detection reagents for Seneca virus (SVA) and foot-and-mouth disease virus (FMDV).
  • Seneca virus cell culture (a cell-passage virus isolated and identified from the hoof vesicular fluid of a diseased pig in a pig farm in Henan by Jinyu Pauling Company for the use of standard and positive controls), foot-and-mouth disease virus cell culture (Jinyu Pauling company MYA98 vaccine strain, used to obtain standards and positive controls) and test samples as samples to be extracted, to extract the genomic RNA of the samples to be extracted, the specific extraction method refer to the AXYGEN kit (Axyprep TM Body Fluid Viral DNA / RNA Miniprep Kit (AXYGEN), including the following steps:
  • step (3) Add 75 ⁇ L Buffer V-N to the 1.5 mL centrifuge tube mixed with sample and reagent in step (2), mix by vortexing, and centrifuge at 12000 g for 5 min;
  • RNA extracted from the test sample using the above method is used as the detection sample; the RNA extracted from the Seneca virus cell culture is used as the Seneca virus positive control; the RNA extracted from the foot and mouth disease virus cell culture is used as the foot and mouth disease virus positive control Standards were prepared from RNA extracted from Seneca virus cell culture and RNA extracted from foot-and-mouth disease virus cell culture according to the following two methods.
  • the genomic RNA of Seneca virus extracted in step 1 (corresponding to the specific conserved sequence of the gene of the 3C region of Seneca virus, that is, Seneca virus from the 5 ′ end of base 6564-6820, SED ID in the sequence table NO: 4, SVA detection gene) and foot-and-mouth disease virus genomic RNA (corresponding to the specific conserved sequence of the gene of foot-and-mouth disease virus 3C region, that is, foot-and-mouth disease virus from the 5 ′ end of base 5986-6553, SED ID in the sequence table , FMDV detection gene) as template, the primers SVA-standard-F and SVA-standard-R (see Table 1 for the sequence, SED ID NO: 9 and SED ID NO: 10 in the sequence listing, SED ID NO: 4 as Sena standard virus sequence primers designed for real-time quantitative PCR detection of Seneca virus primers) PCR amplification of Seneca virus nucleotide detection sequences, primers FMDV-standard-F and FMDV-standard -
  • Foot and mouth disease virus Nucleotide detection sequence, 25 ⁇ L PCR amplification system is shown in Tables 1 and 2.
  • the PCR amplification conditions are: reverse transcription at 42 ° C for 20min, pre-denaturation at 95 ° C for 30s; then denaturation at 94 ° C for 10s, and 55 ° C annealing for 30s. Extension at 72 ° C for 45s, 30 cycles (PCR amplification), extension at 72 ° C for 10min.
  • the PCR amplification product was recovered and purified, and the Seneca virus 3C region detection gene (sequence SED ID NO: 4 in the sequence listing) and the foot and mouth disease virus 3C region detection gene (sequence SED ID NO: 8 in the sequence listing) were obtained. ).
  • SVA standard primers SED ID NO: 9 and SED ID NO: 10 in the Sequence Listing
  • FMDV standard primers SED ID NO: 11 in the Sequence Listing
  • Seneca virus nucleotide detection gene (sequence SED ID NO: 4) obtained in step 1 and foot-and-mouth disease virus nucleotide detection gene (sequence SED in the sequence list) ID NO: 8) were cloned into pCR-4TOPO vector (purchased from Invitrogen), and the positive recombinant plasmids were screened and sent to Huada Gene for sequencing.
  • Sequencing results showed that the correct sequences were obtained respectively carrying the Seneca virus nucleotide detection gene (sequence SED ID NO: 4 in the sequence list) and foot-and-mouth disease virus nucleotide detection gene (sequence SED ID NO: 8 in the sequence list).
  • Recombinant plasmids were named pCR-4TOPO-SVA and pCR-4TOPO-FMDV, namely SVA standard and FMDV standard.
  • Recombinant plasmids pCR-4TOPO-SVA and pCR-4TOPO-FMDV carrying the Seneca virus nucleotide detection gene and foot-and-mouth disease virus nucleotide detection gene that were sequenced correctly were used as standards, and the concentrations were determined using Qubit 3.0, and each was calculated. Number of copies of standards (copies). Dilute SVA standards (pCR-4TOPO-SVA) to 1 ⁇ 10 8 , 1 ⁇ 10 7 , 1 ⁇ 10 6 , 1 ⁇ 10 5 , 1 ⁇ 10 4 according to a 10-fold gradient.
  • the detection system of 25 ⁇ L real-time quantitative PCR is shown in Table 3.
  • the real-time quantitative PCR detection conditions were (quantitative PCR instrument, model CFX96, purchased from the United States).
  • Bole reverse transcription at 42 ° C for 20 min, pre-denaturation at 95 ° C for 30s; then denaturation at 94 ° C for 10s, annealing at 55 ° C for 30s, 45 cycles (PCR amplification).
  • the real-time quantitative PCR amplification curve of the standard is shown in Figure 3.
  • the standard amplification curve is a smooth "S" -shaped curve (positive).
  • the eight groups of black lines in Figure 3 correspond to the concentration of the SVA standard from left to right.
  • SVA standard pCR-4TOPO-SVA
  • concentrations of the FMDV standards corresponding to the eight groups of gray lines from left to right in Figure 3 are: FMDV standard (pCR-4TOPO-FMDV) diluted to 1 ⁇ 10 8 , 1 ⁇ 10 7 , 1 ⁇ 10 6 , 1 ⁇ 10 5 , 1 ⁇ 10 4 , 1 ⁇ 10 3 , 1 ⁇ 10 2 , 1 ⁇ 10 1 copies / ⁇ L.
  • the concentration log value (X-axis) of each standard is plotted against its corresponding Ct value (Y-axis) to draw a standard curve.
  • the standard curve is shown in Figure 4.
  • R 2 0.997 (FMDV)
  • Genomic RNA (detection samples) extracted from 10 cell cultures (samples to be tested, hog blister skin or nasal scope blister skin and blister fluid from unknown virus in pig farms) were detected by a dual real-time quantitative PCR method to The genomic RNA of the Seneca virus culture and the foot-and-mouth disease virus culture was used as the positive control, and the enzyme-free water was used as the negative control. Based on the real-time fluorescent quantitative PCR test results, whether the test samples contained Seneca virus and / or foot-and-mouth disease The virus was qualitatively judged, and the copy number of the virus was quantified according to a standard curve.
  • the specific detection method includes the following steps:
  • the real-time PCR reaction conditions were: reverse transcription at 42 ° C for 20 min, pre-denaturation at 95 ° C for 30s; then denaturation at 94 ° C for 10s, annealing at 55 ° C for 30s, and 45 cycles (PCR amplification). Fluorescent signal detection is performed at the end of each cycle of annealing.
  • Seneca virus and / or foot-and-mouth disease virus is achieved by using the respective CT values or changes in the fluorescence signal. If “S” appears in the FAM channel within 37 cycles (excluding the 37th cycle) Type amplification curve, it is confirmed that it is positive for Seneca virus (the sample contains Seneca virus), and if the "S" type amplification curve appears in the ROX channel within 37 cycles (excluding the 37th cycle), then It was confirmed to be positive for FMD virus (the sample contained FMD virus).
  • step 3 For the test sample determined as positive in step 2), according to the intensity of the fluorescence signal and the standard curve in step 1), obtain the copy number of Seneca virus and / or foot-and-mouth disease virus contained in the test sample To achieve quantitative detection.
  • Table 4 shows the results of dual real-time quantitative PCR of Seneca virus and foot-and-mouth disease virus on 10 cell cultures.
  • the results of samples 1-3 and 5 are FMDV-positive (infected with foot-and-mouth disease virus), and number 4
  • the test result of the sample is suspicious of FMDV, which indicates that the samples 1-3 and 5 contain foot-and-mouth disease virus, and the sample 4 needs to be re-examined;
  • the test results of samples 7-9 are positive for SVA (infected with Seneca virus), and the 6
  • the test results of samples 10 and 10 were suspicious of SVA, indicating that samples 7-9 contained Seneca virus, and samples 6 and 10 required retesting.
  • Recombinant plasmids pCR-4TOPO-SVA and pCR-4TOPO-FMDV carrying the Seneca virus nucleotide detection gene and foot-and-mouth disease virus nucleotide detection genes were used as standards, and the SVA standard (pCR-4TOPO- (SVA) diluted to 1 ⁇ 10 8 , 1 ⁇ 10 7 , 1 ⁇ 10 6 , 1 ⁇ 10 5 , 1 ⁇ 10 4 , 1 ⁇ 10 3 , 1 ⁇ 10 2 , 2.5 ⁇ 10 1 copies / ⁇ L; 10 times Gradient dilutes FMDV standard (pCR-4TOPO-FMDV) to 1 ⁇ 10 8 , 1 ⁇ 10 7 , 1 ⁇ 10 6 , 1 ⁇ 10 5 , 1 ⁇ 10 4 , 1 ⁇ 10 3 , 1 ⁇ 10 2 , 1 ⁇ 10 1 copies / ⁇ L, using standards of different concentrations as templates, under the guidance of primers SVA-F, SVA-R, FMDV-F, FM
  • Seneca virus and foot-and-mouth disease virus dual real-time fluorescence quantitative PCR detection method can detect Seneca virus 2.5 ⁇ 10 1 copies / ⁇ L and foot-and-mouth disease virus 1 ⁇ 10 1 copies / ⁇ L (sensitivity), and the amplification curve is specific
  • the "S" curve indicates that the primers and TaqMan probes identified by the present invention are suitable for binding to Seneca virus and foot-and-mouth disease virus RNA.
  • Dilute SVA standard (pCR-4TOPO-SVA) to 1 ⁇ 10 8 , 1 ⁇ 10 7 , 1 ⁇ 10 6 , 1 ⁇ 10 5 , 1 ⁇ 10 4 , 1 ⁇ 10 3 , 1 ⁇ 10 2.
  • dilute FMDV standard (pCR-4TOPO-FMDV) to 1 ⁇ 10 8 , 1 ⁇ 10 7 , 1 ⁇ 10 6 , 1 ⁇ 10 5 , 1 ⁇ 10 4 , 1 ⁇ 10 3 , 1 ⁇ 10 2 , 1 ⁇ 10 1 copies / ⁇ L, and as a template, each gradient is repeated 2 times.
  • the Seneca virus and foot-and-mouth disease virus dual real-time quantitative PCR detection kit includes primers (SVA-F, SVA for double-time real-time PCR detection of Seneca virus and foot-and-mouth disease virus). -R, FMDV-F and FMDV-R) and TaqMan probes (SVA-P and FMDV-P).
  • the kit may further include a standard product, the standard product being the aforementioned recombinant plasmid pCR4-TOPO-SVA with the Seneca virus detection gene and the recombinant plasmid pCR4-TOPO-FMDV with the foot and mouth disease virus detection gene.
  • the 25 ⁇ L dual real-time quantitative PCR detection system when using the kit is: real-time quantitative one-step PCR reaction solution 2 ⁇ One Step RT-PCR Buffer III 12.5 ⁇ L, TaKaRa Ex Taq HS 0.5 ⁇ L, PrimeScript RT Enzyme Mix II 0.5 ⁇ L , SVA-F (10 ⁇ M) 1.0 ⁇ L, SVA-R (10 ⁇ M) 1.0 ⁇ L, SVA-P (10 ⁇ M) 1 ⁇ L, FMDV-F (10 ⁇ M) 1.0 ⁇ L, FMDV-R (10 ⁇ M) 1.0 ⁇ L, FMDV-P (10 ⁇ M ) 1.5 ⁇ L, template 2.0 ⁇ L, RNA-free H 2 O 3.0 ⁇ L.
  • the template for the receipt of the standard curve is the standard DNA
  • the template for the detection of the sample is the genomic RNA of the sample to be tested.
  • the kit can also include positive and negative controls.
  • the positive controls are Seneca virus RNA and foot-and-mouth disease virus RNA (positive samples can verify whether there is a problem in the template extraction process. Bands from positive samples indicate nucleic acids.
  • the negative control is a reaction system without Seneca virus and foot-and-mouth disease virus, such as H 2 O (double distilled water, sterile deionized water, etc.).
  • genomic RNA detection sample
  • samples of suspected Seneca virus and foot-and-mouth disease virus samples samples to be tested
  • the Seneca virus and foot-and-mouth disease virus dual real-time quantitative PCR detection kit Using Seneca virus genomic RNA and foot-and-mouth disease virus genomic RNA as a positive control and enzyme-free water as a negative control, based on the results of the dual real-time fluorescent quantitative PCR test, determine whether the test samples contain Seneca virus and foot-and-mouth disease virus. Determine and quantify the copy number of the virus.
  • the specific detection method is the same as in Example 2.
  • Seneca virus and foot-and-mouth disease virus samples are shown in Table 6.
  • the results of the dual real-time quantitative PCR test are shown in Table 6.
  • Samples 1-3 are positive for SVA (infected with Seneca virus), indicating that samples 1-3 contain sera.
  • Existing inventions include FMDV and SVA dual fluorescence quantitative PCR detection methods including CN107326100A Yan Ruo Qian's "Foot-and-Mouth Disease and Seneca Virus Dual Real-Time Fluorescent Quantitative PCR Detection Kit", which is a detection method that first reverse-transcribes viral RNA The cDNA is used as a template, and real-time fluorescent quantitative PCR is used for amplification. The experimental steps are complicated. Compared with the above-mentioned existing methods, the present invention provides that FMDV and SVA RNA samples are not first transcribed into cDNA, and RNA is directly added as a template to a one-step PCR system for real-time quantitative PCR detection.
  • the advantage is that the The step of reverse transcription of viral RNA into cDNA, and the amplification curve is a specific "S" type curve, indicating that the primers of this method are most suitable for binding to viral RNA.
  • the invention specifically optimizes relevant primers and probes for this purpose, and establishes a dual real-time fluorescent quantitative PCR detection method for Seneca virus and foot-and-mouth disease virus, which can quickly and simultaneously detect and distinguish between Seneca virus and foot-and-mouth disease virus, which is a clinical sample detection Play an important role in the identification of strains and vaccine production.
  • the target sequences of FMDV and SVA detection are both at the 3D gene of the entire genome of the virus.
  • the FMDV and SVA target sequences provided by the present invention are at the 3C gene of the entire genome of the virus, and the target sequences selected by the two are different; the sensitivity in the present invention is 10-6 dilution, and the concentration of the protozoan in the present invention is unclear, so it cannot be detected. Compare.
  • the sensitivity of the present invention is 10 1 copies / ⁇ L.
  • CN201711324308.1 He Dongsheng invented a "dual PCR primer, detection method and kit for detecting foot-and-mouth disease virus and Seneca virus", in which the target sequence for FMDV detection is at the 2C gene of the virus , the SVA detecting a target sequence of the VP4 gene of the virus, two different selected target sequence; sensitivity of the invention, 10 2 copies / ⁇ L, in the present invention, the detection sensitivity of 101 copies / ⁇ L, both sensitive By comparison, the sensitivity of the present invention is higher than CN201711324308.1; the invention is qualitative detection and cannot be quantified, and the present invention can be qualitatively and quantitatively detected.
  • the invention provides a dual real-time fluorescent quantitative PCR detection kit for detecting Seneca virus (SVA) and foot-and-mouth disease virus (FMDV), a special primer, and a TaqMan probe, which can be used as a detection reagent for SVA and FMDV against Seneca.
  • SVA Seneca virus
  • FMDV foot-and-mouth disease virus
  • TaqMan probe a TaqMan probe

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

提供一种鉴别塞内卡病毒和口蹄疫病毒的双重实时荧光定量PCR检测试剂盒，所述试剂盒包含两对引物和两条探针，所述引物和探针分别针对塞内卡病毒和口蹄疫病毒的3C区域基因设计。

Description

塞内卡病毒和口蹄疫病毒双重实时荧光定量PCR检测试剂及试剂盒 技术领域
本发明属于生物检测技术领域，涉及兽医动物病原检测，特别涉及一种用于对塞内卡病毒和口蹄疫病毒进行定性、定量检测的双重实时荧光定量PCR检测专用引物和TaqMan探针以及检测试剂盒。
背景技术
塞内卡病毒(Seneca Virus A，SVA)和口蹄疫病毒(Foot-and-Mouth Disease Virus，FMDV)同属于小核糖核酸病毒科(Picornaviridae)，都是一种无囊膜的单股正链RNA病毒，塞内卡病毒属于Seneca Virus病毒属的典型代表，与心病毒属(Cardio Virus)最接近，而口蹄疫病毒属于口蹄疫病毒属病毒。
塞内卡病毒是主要发生于猪的一种传染病，其临诊特征为感染猪只鼻部和口腔形成溃疡、厌食、跛行，导致新生仔猪急性死亡。早期塞内卡病例主要发生于育肥猪，鼻部、口部和蹄部的冠状带部可见水疱症状，但最近发现的一些塞内卡阳性病例发生于新生仔猪，症状为腹泻，无水疱病变，这些病例主要是通过对血清、粪便和不同组织进行PCR检测发现的。2002年在美国马里兰州的一家公司首次发现，2007年，一批从加拿大运往美国明尼苏达州的猪鼻镜出现水泡、蹄部冠状带溃烂等类似水疱病症状，经检测排除口蹄疫、水泡性口炎病和猪水疱病，最终通过PCR检测确定为塞内卡病毒阳性。之后2014年至今在美国、中国、加拿大、巴西、泰国、哥伦比亚等多国均发现SVA病毒导致的临床病例，并进一步确定是由SVA导致致病。
口蹄疫病毒可引起牛、猪和羊等偶蹄动物感染的一种急性、热性、高度接触性传染病，以口腔黏膜、舌面、唇、鼻镜、蹄部和乳房皮肤发生水疱和溃烂为特征。口蹄疫平均致死率仅为1％，但是被感染动物100％发病，且传播效力极高，使实际的畜产量锐减。由于口蹄疫病(FMD)危害大，影响范围广，被世界动物卫生组织(OIE)列为A类烈性传染病之首。
近几年，由于塞内卡病毒的发现，仅从临床症状上无法区分SVA感染和FMDV感染，给临床确诊带来了难度，需要用实验室检测技术加以确定。如何用快速、简单的方法检测、区分塞内卡病毒和口蹄疫病毒是一些猪场急需解决的问题。
另一方面，疫苗是预防病毒的最有效措施，其中灭活疫苗在传染病的预防中发挥着重要作用，制备灭活疫苗是预防塞内卡病毒以及口蹄疫病毒流行的主要有效手段。在疫苗制备中，对病原进行有效的灭活，可提供安全的疫苗，主要通过 将塞内卡病毒、口蹄疫病毒体外培养后灭活，然后与乳化剂混合制成免疫疫苗。在制备多价疫苗例如塞内卡病毒和口蹄疫病毒双价疫苗时，准确、特异、快速地检测塞内卡病毒以及口蹄疫病毒抗原的水平在疫苗生产监控等方面具有重要意义。
发明内容
本发明的第一个目的是提供用于对塞内卡病毒(SVA)和口蹄疫病毒(FMDV)进行双重实时荧光定量PCR检测的引物和TaqMan探针，以实现塞内卡病毒(SVA)和口蹄疫病毒(FMDV)的定性、定量检测。
用于对塞内卡病毒进行实时荧光定量PCR检测的引物为：上游引物(SVA-F)的核苷酸序列如序列表中SED ID NO：1所示，下游引物(SVA-R)的核苷酸序列如序列表中SEQ ID NO：2所示。
由上述引物衍生的引物序列也属于本发明内容。所述衍生序列是指在SEQ ID NO：1和/或SEQ ID NO：2的基础上经过一至十个碱基的取代、缺失或添加得到的引物序列。
用于对口蹄疫病毒进行实时荧光定量PCR检测的引物为：上游引物(FMDV-F)的核苷酸序列如序列表中SED ID NO：5所示，下游引物(FMDV-R)的核苷酸序列如序列表中SEQ ID NO：6所示。
由上述引物衍生的引物序列也属于本发明内容。所述衍生序列是指在SEQ ID NO：5和/或SEQ ID NO：6的基础上经过一至十个碱基的取代、缺失或添加得到的引物序列。
用于对塞内卡病毒进行实时荧光定量PCR检测的TaqMan探针为：TaqMan探针(SVA-P)的核苷酸序列如序列表中SED ID NO：3所示；所述探针为经过荧光标记的，其5′端标记有报告荧光基团，3′端标记有淬灭荧光基团。
用于对口蹄疫病毒进行实时荧光定量PCR检测的TaqMan探针为：TaqMan探针(FMDV-P)的核苷酸序列如序列表中SED ID NO：7所示；所述探针为经过荧光标记的，其5′端标记有报告荧光基团，3′端标记有淬灭荧光基团。
由上述TaqMan探针序列的衍生序列也属于本发明内容。所述衍生序列是指在SEQ ID NO：3/SEQ ID NO：7的基础上，在序列的5′端和/或3′端再添加、减少一个或多个碱基得到的序列。
对上述两种病毒检测探针SVA-P和FMDV-P所标记的报告荧光基团不相同。
一个实施例中，TaqMan探针(SVA-P)的5′端报告荧光基团为FAM，3′端荧光淬灭基团为TAMRA；TaqMan探针(FMDV-P)的5′端报告荧光基团为ROX， 3′端荧光淬灭基团为BHQ2。
为防止PCR扩增时被延伸，所述TaqMan探针的3′端已经磷酸化处理。
本发明的第二个目的是提供一种用于对塞内卡病毒(SVA)和口蹄疫病毒(FMDV)进行双重实时荧光定量PCR检测试剂盒。
该双重实时荧光定量PCR检测试剂盒，包括上述引物和TaqMan探针。
所述试剂盒中还可以包括标准品，所述标准品为带有塞内卡病毒检测基因的重组质粒pCR4-TOPO-SVA和带有口蹄疫病毒检测基因的重组质粒pCR4-TOPO-FMDV。
具体来讲，使用所述试剂盒时的双重实时荧光定量PCR检测体系(以25μL体系为例)为：实时荧光定量一步法PCR反应液2×One Step RT-PCR Buffer III12.5μL(购于TakaRa公司)，TaKaRa Ex Taq HS 0.5μL(购于TakaRa公司)，PrimeScript RT Enzyme Mix II 0.5μL(购于TakaRa公司)，SVA-F(10μM)1μL，SVA-R(10μM)1μL，SVA-P(10μM)1μL，FMDV-F(10μM)1μL，FMDV-R(10μM)1μL，FMDV-P(10μM)1.5μL，RNA-free H 2O 3μL，模板2μL。模板为标准品DNA或待测样品基因组RNA。
本发明的第三个目的是提供所述引物、探针在制备塞内卡病毒和口蹄疫病毒的检测试剂中的应用。
本发明的第四个目的是提供所述试剂盒在对塞内卡病毒(SVA)和口蹄疫病毒(FMDV)进行检测中的应用，该检测为针对疫苗生产过程中病毒的检测，或是针对病原的非疾病诊断目的检测，或是疾病诊断中病原的检测。检测样品可以是猪场送检样品，或疫苗生产中的原料血清、疫苗半成品等。
对塞内卡病毒(SVA)和口蹄疫病毒(FMDV)进行定性、定量检测的双重实时荧光定量PCR检测方法，包括以下步骤：
1)建立标准曲线：
将分别携带SVA核苷酸序列和FMDV核苷酸序列的重组质粒pCR-4TOPO-SVA和pCR-4TOPO-FMDV作为标准品，重组质粒pCR-4TOPO-SVA10倍梯度稀释成1×10 8、1×10 7、1×10 6、1×10 5、1×10 4、1×10 3、1×10 2、2.5×10 1拷贝(copies)/μL；重组质粒pCR-4TOPO-FMDV 10倍梯度稀释成1×10 8、1×10 7、1×10 6、1×10 5、1×10 4、1×10 3、1×10 2、1×10 1拷贝(copies)/μL，以不同浓度的标准品DNA作为模板，在引物SVA-F、SVA-R、FMDV-F、FMDV-R及TaqMan探针SVA-P、FMDV-P的引导下分别进行实时荧光定量PCR检测；检测结束后，以各标准品的浓度Log值(X轴)对其相应Ct值(Y轴)作图，绘制标准曲线；
重组质粒pCR4-TOPO-SVA的构建：选取塞内卡病毒的3C区域基因的特异性保守序列(塞内卡病毒自5′端第6564-6820位碱基，序列表中SED ID NO：4，检测基因)作为标准品构建序列，设计对塞内卡病毒进行实时荧光定量PCR检测的标准品引物(序列表中SED ID NO：9和SED ID NO：10)，以塞内卡病毒的基因组RNA为模板，用标准品引物PCR扩增塞内卡病毒3C区域检测基因，连接入载体pCR-4TOPO中，构建携带塞内卡病毒3C区域检测基因的重组质粒pCR4-TOPO-SVA，对鉴定正确的重组质粒进行浓度测定；
重组质粒pCR4-TOPO-FMDV的构建：选取口蹄疫病毒的3C区域基因的特异性保守序列(口蹄疫病毒自5′端第5986-6553位碱基，序列表中SED ID NO：8，检测基因)作为标准品构建序列，设计对口蹄疫病毒进行实时荧光定量PCR检测的标准品引物(序列表中SED ID NO：11和SED ID NO：12)，以口蹄疫病毒的基因组RNA为模板，用标准品引物PCR扩增口蹄疫病毒3C区域检测基因，连接入载体pCR-4TOPO中，构建携带口蹄疫病毒3C区域检测基因的重组质粒pCR4-TOPO-FMDV，对鉴定正确的重组质粒进行浓度测定。
2)提取待测样品的基因组RNA，以提取的基因组RNA为模板，在上述引物和TaqMan探针的引导下进行双重实时荧光定量PCR检测；
3)用得到的各自的CT值或荧光信号的变化实现对塞内卡病毒和口蹄疫病毒的定性检测，在塞内卡病毒和/或口蹄疫病毒的对应荧光通道出现“S”型扩增曲线则表明待测样品中含有对应的病毒，即确定为阳性；
4)对步骤3)中确定为阳性的待测样品，再根据荧光信号的强度和步骤1)中的标准曲线，得出待测样品中所含塞内卡病毒和口蹄疫病毒的拷贝数，实现定量检测。
在上述检测方法中，所述步骤2)中的待测样品可以为采自猪用于疫苗生产的原料血清、疫苗半成品，猪场送检样品，通过对此类待测样品中塞内卡病毒和口蹄疫病毒的定量检测以实现对基于猪的产品及其原料的监控，并为后续原料处置提供客观数据。
所述步骤1)和步骤2)中的双重实时荧光定量PCR检测体系可包括：模板2μL，实时荧光定量一步法PCR反应液2×One Step RT-PCR Buffer III 12.5μL(购于TakaRa公司)，TaKaRa Ex Taq HS 0.5μL(购于TakaRa公司)，PrimeScript RT Enzyme Mix II 0.5μL(购于TakaRa公司)，SVA-F(10μM)1μL，SVA-R(10μM)1μL，SVA-P(10μM)1μL，FMDV-F(10μM)1μL，FMDV-R(10μM)1μL，FMDV-P(10μM)1.5μL，RNA-free H 2O 3μL。
所述步骤1)和步骤2)中的双重实时荧光定量PCR检测条件可为：先42℃反转录20min，95℃预变性30s；然后94℃变性10s，55℃退火30s，45个循环(PCR扩增)。在每个循环的退火结束时进行荧光信号检测。
所述步骤3)中具体的结果判定方法可为：若FAM通道中在37个循环内(不包括第37个循环)出现“S”型扩增曲线，则确认为塞内卡病毒阳性(样品中含有塞内卡病毒)，若ROX通道中在37个循环内(不包括第37个循环)出现“S”型扩增曲线，则确认为口蹄疫病毒阳性(样品中含有口蹄疫病毒)；若FAM通道和/或ROX通道中在39个循环或以上(包括第39个循环)未出现“S”型扩增曲线，则确认为对应病毒阴性(样品中不含有塞内卡病毒和/或口蹄疫病毒)，在39个或以上循环中出现“S”型扩增曲线，也确认为对应病毒阴性；任一通道中在37-39个循环之间(包括第37个循环且不包括第39个循环，即37个或38个循环)出现“S”型扩增曲线则判定为可疑，需重检。
本发明提供了用于检测塞内卡病毒(SVA)和口蹄疫病毒(FMDV)的双重实时荧光定量PCR检测试剂盒及其专用引物、TaqMan探针，利用其能对塞内卡病毒和口蹄疫病毒实施快速区分和检测，还可为疫苗生产过程中的质量监控和合理化配苗(如评估配制疫苗抗原的准确含量，为疫苗抗原含量提供数据基础)提供有力依据，确保疫苗接种的安全性和合理性，对塞内卡病毒和口蹄疫病毒疫苗的生产具有指导作用。本发明的试剂盒和检测方法操作简便、特异性强、敏感性高、重复性好，可以实现对塞内卡病毒和口蹄疫病毒的准确定量，能在塞内卡病毒和口蹄疫病毒的检测(包括病料或培养物中塞内卡病毒和口蹄疫病毒的准确检测)及疫苗生产中发挥重要作用，应用前景广阔。
下面结合具体实施例对本发明做进一步详细说明。
附图说明
图1为用于对塞内卡病毒(SVA)进行检测的引物筛选扩增曲线；
图2为用于对口蹄疫病毒(FMDV)进行检测的引物筛选扩增曲线；
图3为用于对SVA和FMDV进行双重检测的标准品的扩增曲线；
图4为用于对SVA和FMDV进行双重检测的标准曲线；
图5为SVA和FMDV双重实时荧光定量PCR检测的特异性检测结果；
图6为SVA和FMDV双重实时荧光定量PCR检测重复性实验的扩增曲线。
具体实施方式
下述实施例中所用方法如无特别说明均为常规方法，具体步骤可参见：《分子克隆实验指南》(《Molecular Cloning:A Laboratory Manual》Sambrook，J.， Russell，David W.，Molecular Cloning:A Laboratory Manual，3rd edition，2001，NY，Cold Spring Harbor)。
所述百分比浓度如无特别说明均为质量/质量(W/W，单位g/100g)百分比浓度、质量/体积(W/V，单位g/100mL)百分比浓度或体积/体积(V/V，单位mL/100mL)百分比浓度。
实施例中描述到的各种生物材料的取得途径仅是提供一种实验获取的途径以达到具体公开的目的，不应成为对本发明生物材料来源的限制。事实上，所用到的生物材料的来源是广泛的，任何不违反法律和道德伦理能够获取的生物材料都可以按照实施例中的提示替换使用。
实施例中所用引物由北京华大基因有限公司合成；所用探针由TAKARA基因公司合成。
实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，实施例将有助于理解本发明，但是本发明的保护范围不限于下述的实施例。
生物基因组是直接反应生物基本信息的一个最客观的指标，不同的病毒所含有的基因组信息不同，通过基因组信息可将不同的病毒分类至不同的族群，利用基因组碱基互补配对的原则，可实现特异位点基因序列的大量扩增。
本发明基于以上基本原理设计了2对特异性引物和2条寡核苷酸探针，利用碱基互补配对的原理，建立了一种特异性检测塞内卡病毒(SVA)和口蹄疫病毒(FMDV)的双重实时荧光定量PCR检测方法。
实施例1、设计用实时荧光定量PCR技术检测塞内卡病毒(SVA)和口蹄疫病毒(FMDV)的引物和TaqMan探针
从NCBI的核酸数据库GenBank(http://www.ncbi.nlm.nih.gov)检索获得塞内卡病毒全基因组的序列(GeneBank序号：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)和口蹄疫病毒全基因组的序列(GeneBank序号：AF506822.2、KX712091.1、HQ412603.1、AJ539141.1、AY390432.1、AY304994.1、GQ406249.1、KT968663.1、HQ632773.1)，用DNA Star软件进行比对后，根据引物、TaqMan探针设计原则，分别选取塞内卡病毒不同毒株之间相对保守的核苷酸序列(3C区域，SED ID NO：4所示)和口蹄疫病毒不同毒株之间相对保守的核苷酸序列 (3C区域，SED ID NO：8所示)，并将优选的特异性保守序列(SED ID NO：4和SED ID NO：8)作为检测序列。
关于塞内卡病毒的3C区域基因，Hales L M等发表的文献“Complete genome sequence analysis of Seneca Valley virus-001,a novel oncolytic picornavirus[J].Journal of General Virology,2008,89(5):1265-1275.”中阐述3C基因为保守的区域；赵晓亚已发表的“猪塞内加谷病毒(SVA)的分离鉴定及致病性研究”硕士学位论文(华南农业大学，2016硕士毕业论文(24-33))(与文献1CN201610379248内容相同)中在3C区域选取4763-4879位碱基段作为靶序列建立了Seneca Valleyvirus Taq Man荧光定量PCR检测方法，然而遗憾的是，该方法敏感性仅为1×10 2拷贝/μL，不能满足检测的需要。
本发明从塞内卡病毒的3C保守区域中优选出特异性片段作为检测序列，并最终确定该检测序列为塞内卡病毒自5′端第6564-6820位碱基(序列表中SED ID NO：4)。依据选定的检测序列，设计对塞内卡病毒进行实时荧光定量PCR检测的引物及TaqMan探针。
关于口蹄疫病毒检测基因区域，很多研究利用3D基因作为靶序列进行荧光定量RT-PCR技术检测口蹄疫，少数研究利用口蹄疫病毒2B基因和5′UTR做靶序列进行荧光定量RT-PCR技术检测口蹄疫，本发明在口蹄疫病毒3C基因区域找到高度保守且特异的核苷酸区域，来设计引物和探针。
本发明从口蹄疫病毒的3C保守区域中优选出特异性片段作为检测序列，并最终确定该检测序列为口蹄疫病毒自5′端第5986-6553位碱基(序列表中SED ID NO：8)。依据选定的检测序列，设计对口蹄疫病毒进行实时荧光定量PCR检测的引物及TaqMan探针。
从基于以上确定的检测序列获得的众多引物及探针中，本发明设计多组组合并从中筛选出组1作为优选组：
组1(优选组)：
SVA-F(上游引物)：5′-TATCTCAGATCCCTGGCTGTC-3′(序列位置：塞内卡病毒自5′端第6634-6654位碱基，序列表中SED ID NO：1)；
SVA-R(下游引物)：5′-CCTGATGATCACATTGTTGAGC-3′(序列位置：塞内卡病毒自5′端第6741-6762位碱基，序列表中SED ID NO：2)；
SVA-P(TaqMan探针)：5′-FAM-CACGCTTACGGCGAGCGTCGC ATCAAG-TAMRA-3′(序列位置：塞内卡病毒自5′端第6661-6687位碱基，序列表中SED ID NO：3)；
FMDV-F(上游引物)：5′-YGCCTMCCTHGTDCCTCGTCAYC TYT-3′(序列位置：口蹄疫病毒自5′端第6158-6179位碱基，序列表中SED ID NO：5，其中，Y＝C、T，M＝A、C，H＝A、T、C，D＝G、A、T)；
FMDV-R(下游引物)：5′-GAGAGCATGTCCTGTCCTYTYA CTTT-3′(序列位置：口蹄疫病毒自5′端第6260-6281位碱基，序列表中SED ID NO：6，其中，Y＝C、T)；
FMDV-P(TaqMan探针)：5′-ROX-CNGAGAAGTAYGACAAGAT CATGYT-BHQ2-3′(序列位置：口蹄疫病毒自5′端第6183-6207位碱基，序列表中SED ID NO：7，其中，N＝A、G、C或T，Y＝C或T)；
为与组1相比较，将基于塞内卡病毒其他保守区域检测序列获得的引物及探针与其它口蹄疫病毒引物及探针组合列为组2作为对照组：
组2(对照组)：
SVA-F1(上游引物)：5′-TATAAGATGACTCCTGCCAAC-3′(序列位置：塞内卡病毒自5′端第6898-6918位碱基)；
SVA-R1(下游引物)：5′-AGAATTTGGAAGCCATGCTCTC-3′(序列位置：塞内卡病毒自5′端第7025-7046位碱基)；
SVA-P1(TaqMan探针)：5′-FAM-TTCTGTCTTCCCTCCGACTTC CTCTC-TAMRA-3′(序列位置：塞内卡病毒自5′端第6924-6949位碱基)；
FMDV-F1(上游引物)：5′-AAGATCATGTTGGACGGCAGAG CCAT-3′(序列位置：口蹄疫病毒自5′端第6198-6223位碱基)；
FMDV-R1(下游引物)：5′-ATGTCCCGCACGCGATTCCCACGGT-3′(序列位置：口蹄疫病毒自5′端第6310-6334位碱基)；
FMDV-P1(TaqMan探针)：5′-ROX-CAGTGACTACAGAGTG TTTGAGTTTGAG-BHQ2-3′(序列位置：口蹄疫病毒自5′端第6230-6257位碱基)；
上述TaqMan探针中，5′端已带有报告荧光基团FAM或ROX，在同一组中针对两种病毒的探针所带的基团不相同；3′端已带有与5′端报告荧光基团相对应的荧光淬灭基团TAMRA或BHQ2。
为防止PCR扩增时被延伸，上述TaqMan探针的3′端经磷酸化处理。
FMDV和SVA荧光定量PCR的引物探针筛选及条件优化实验：对以上所设计的PCR扩增引物及各引物最适的退火温度进行筛选，退火温度设置五个梯度，分别为53℃、55℃、57℃、59℃、61℃，每个温度梯度做2个重复，反应体系如下表1和表2所示，反应条件为：先42℃反转录20min，94℃预变性，30s；{94℃ 变性，10s，53℃-61℃退火，30s}×45个循环。
反应结束后比较扩增曲线筛选引物及退火温度，对各组引物和探针进行比较，以上所列两组引物及探针在55℃的退火温度时的结果如图1和图2所示，组1的FMDV和SVA荧光定量PCR扩增曲线为标准的“S”型曲线，组2的FMDV和SVA荧光定量PCR扩增曲线荧光阈值不如组1。通过扩增曲线确定了组1设计的引物与探针作为塞内卡病毒(SVA)和口蹄疫病毒(FMDV)的双重实时荧光定量PCR检测试剂。
实施例2、用组1的引物及TaqMan探针对塞内卡病毒和口蹄疫病毒进行双重实时荧光定量PCR检测
一、提取塞内卡病毒和口蹄疫病毒的基因组RNA
将塞内卡病毒细胞培养物(金宇保灵公司从位于河南某猪场发病猪的蹄子水泡液中分离并鉴定的细胞传代病毒，用于获得标准品和阳性对照品)、口蹄疫病毒细胞培养物(金宇保灵公司MYA98疫苗毒株，用于获得标准品和阳性对照品)以及待测样品作为待提取样品，提取待提取样品的基因组RNA，具体提取方法参照AXYGEN试剂盒(Axyprep TMBody Fluid Viral DNA/RNA Miniprep Kit，AXYGEN公司)的介绍，包括以下步骤：
(1)按试剂盒说明书，预先配制含1％冰乙酸的异丙醇和在试剂Buffer W1A和Buffer W2中添加指定浓度的无水乙醇；
(2)在1.5mL离心管中加入200μL的待提取样品，并加入200μL Buffer V-L，漩涡震荡混匀后，静置5min；
(3)在步骤(2)的混合有样品及试剂的1.5mL离心管中加75μL Buffer V-N，漩涡震荡混匀，12000g离心5min；
(4)将上清转移至2mL离心管(试剂盒内提供)中，加300μL异丙醇(1％冰乙酸)，上下倒置6-8次，混合均匀；
(5)将制备管(试剂盒内提供)置于另一2mL离心管中，取步骤(4)的混合液移入制备管中，6000g离心1min；
(6)弃滤液，将制备管置回至步骤(5)的2mL离心管中，加500μL Buffer W1A，室温静置1min，12000g离心1min；
(7)弃滤液，将制备管置回至步骤(5)的2mL离心管中，加800μL Buffer W2，12000g离心1min；
(8)弃滤液，将制备管置回到步骤(5)的2mL离心管中，直接以12000g离心1min；
(9)将制备管置于另一洁净的1.5mL离心管(试剂盒内提供)中，在制备管膜中央加40μL无酶水，室温静置1min，12000g离心1min，洗脱得到RNA。
将利用以上方法从待测样品中提取的RNA作为检测样；从塞内卡病毒细胞培养中提取的RNA作为塞内卡病毒阳性对照品；从口蹄疫病毒细胞培养中提取的RNA作为口蹄疫病毒阳性对照品；按照下述二的方法将从塞内卡病毒细胞培养中提取的RNA和口蹄疫病毒细胞培养中提取的RNA制备得到标准品。
二、建立塞内卡病毒和口蹄疫病毒双重实时荧光定量PCR检测的标准曲线
1、PCR扩增塞内卡病毒和口蹄疫病毒检测基因
以步骤一提取的塞内卡病毒的基因组RNA(对应塞内卡病毒的3C区域基因的特异性保守序列，即塞内卡病毒自5′端第6564-6820位碱基，序列表中SED ID NO：4，SVA检测基因)和口蹄疫病毒基因组RNA(对应口蹄疫病毒的3C区域基因的特异性保守序列，即口蹄疫病毒自5′端第5986-6553位碱基，序列表中SED ID NO：8，FMDV检测基因)为模板，在引物SVA-standard-F和SVA-standard-R(序列参见表1，序列表中SED ID NO：9和SED ID NO：10，是以SED ID NO：4作为SVA标准品序列设计得到的对塞内卡病毒进行实时荧光定量PCR检测的标准品引物)的引导下PCR扩增塞内卡病毒核苷酸检测序列，在引物FMDV-standard-F和FMDV-standard-R(序列参见表2，序列表中SED ID NO：11和SED ID NO：12，是以SED ID NO：8作为FMDV标准品序列设计得到的对口蹄疫病毒进行实时荧光定量PCR检测的标准品引物)的引导下PCR扩增口蹄疫病毒核苷酸检测序列，25μL PCR扩增体系如表1和表2所示，PCR扩增条件为：先42℃反转录20min，95℃预变性30s；然后94℃变性10s、55℃退火30s、72℃延伸45s，30个循环(PCR扩增)，72℃延伸10min。扩增结束后，回收、纯化PCR扩增产物，得到塞内卡病毒3C区域检测基因(序列表中序列SED ID NO：4)和口蹄疫病毒3C区域检测基因(序列表中序列SED ID NO：8)。
表1 塞内卡病毒核苷酸检测基因的PCR扩增体系
Figure PCTCN2018107963-appb-000001
Figure PCTCN2018107963-appb-000002
表2 口蹄疫病毒核苷酸检测基因的PCR扩增体系
Figure PCTCN2018107963-appb-000003
2、制备标准品
分别以塞内卡病毒和口蹄疫病毒的基因组RNA为模板，用SVA标准品引物(序列表中SED ID NO：9和SED ID NO：10)和FMDV标准品引物(序列表中SED ID NO：11和SED ID NO：12)PCR扩增，将步骤1获得的塞内卡病毒核苷酸检测基因(序列表中序列SED ID NO：4)和口蹄疫病毒核苷酸检测基因(序列表中序列SED ID NO：8)分别克隆至pCR-4TOPO载体(购自Invitrogen公司)中，筛选阳性重组质粒送华大基因公司测序。测序结果表明获得了序列正确的分别携带塞内卡病毒核苷酸检测基因(序列表中序列SED ID NO：4)和口蹄疫病毒核苷酸检测基因(序列表中序列SED ID NO：8)的重组质粒，分别命名为pCR-4TOPO-SVA和pCR-4TOPO-FMDV，即SVA标准品和FMDV标准品。
3、建立实时荧光定量PCR标准曲线
以测序正确的携带塞内卡病毒核苷酸检测基因和口蹄疫病毒核苷酸检测基因的重组质粒pCR-4TOPO-SVA和pCR-4TOPO-FMDV作为标准品，用Qubit3.0测定浓度，并计算各标准品的拷贝(copies)数，按照10倍梯度将SVA标准品(pCR-4TOPO-SVA)稀释至1×10 8、1×10 7、1×10 6、1×10 5、1×10 4、1×10 3、1×10 2、2.5×10 1copies/μL；按照10倍梯度将FMDV标准品(pCR-4TOPO-FMDV)稀释至1×10 8、1×10 7、1×10 6、1×10 5、1×10 4、1×10 3、1×10 2、1×10 1copies/μL，每个样品做2个重复，以各病毒不同浓度的标准品分别作为模板(DNA模板)，在组1所示的引物SVA-F、SVA-R、FMDV-F(具体为FMDV-F′：5′-TGCCTC CCTTGTTCCTCGTCATCTTT-3′，序列表中序列SED ID NO：13)、FMDV-R(具体为FMDV-R′：5′-GAGAGCATGTCCTGTCCTTTCACT TT-3′，序列表中序列SED ID NO：14)及TaqMan探针SVA-P和TaqMan探针FMDV-P(具体为FMDV-P′：5′-CAGAGAAGTATGACAAGATC ATGTT-3′，序列表中序列SED ID NO：15)的引导下进行双重实时荧光定量PCR检测，25μL实时荧光定量PCR的检测体系如表3所示，实时荧光定量PCR检测条件为(定量PCR仪，型号CFX96，购自美国伯乐)：先42℃反转录20min，95℃预变性30s；然后94℃变性10s，55℃退火30s，45个循环(PCR扩增)。
表3 塞内卡病毒和口蹄疫病毒双重实时荧光定量PCR检测体系
试剂名称 体积(μL)
2×One Step RT-PCR Buffer III(购自TaKaRa公司) 12.5
TaKaRa Ex Taq HS(购自TakaRa公司) 0.5
PrimeScript RT Enzyme Mix II(购自TakaRa公司) 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模板 2.0
RNA-free H 2O 3.0
标准品的实时荧光定量PCR扩增曲线如图3所示，标准品扩增曲线为平滑的“S”形曲线(阳性)，图3中八组黑色线条从左向右对应的SVA标准品浓度分别为：SVA标准品(pCR-4TOPO-SVA)稀释至1×10 8、1×10 7、1×10 6、1×10 5、1×10 4、1×10 3、1×10 2、2.5×10 1copies/μL；图3中八组灰色线条从左向右对应的FMDV标准品浓度分别为：FMDV标准品(pCR-4TOPO-FMDV)稀释至1×10 8、1×10 7、1×10 6、1×10 5、1×10 4、1×10 3、1×10 2、1×10 1copies/μL。检测结束后，以各标准品的浓度Log值(X轴)对其相应Ct值(Y轴)作图，绘制标准曲线，标准曲线如图4所示，相关系数分别为R 2＝0.997(SVA)和R 2＝0.997(FMDV)，误差较小，标准曲线可用，由标准曲线得到的线性方程分别为：y＝-3.299x+40.793(SVA)；y＝-3.180x+40.371(FMDV)。
三、对塞内卡传代病毒和口蹄疫传代病毒进行双重实时荧光定量PCR检测
用双重实时荧光定量PCR方法对从10份细胞培养物(待测样品，来自猪场未知病毒的猪蹄或鼻镜的水疱皮和水疱液)中提取的基因组RNA(检测样)进行检测，以塞内卡病毒培养物以及口蹄疫病毒培养物的基因组RNA为阳性对照品，以无酶水为阴性对照品，根据实时荧光定量PCR检测结果，对检测样中是否含有塞内卡病毒和/或口蹄疫病毒进行定性判定，并依据标准曲线对病毒的拷贝数进行定量。
具体检测方法包括以下步骤：
1)提取待测样品的基因组RNA，以提取的基因组RNA为模板，在引物SVA-F、SVA-R、FMDV-F、FMDV-R及TaqMan探针SVA-P、FMDV-P的引导下进行双重实时荧光定量PCR检测，25μL实时荧光定量PCR的检测体系包括：RNA模板2μL，实时荧光定量一步法PCR反应液2×One Step RT-PCR Buffer III 12.5μL(购于TakaRa公司)，TaKaRa Ex Taq HS 0.5μL(购于TakaRa公司)，PrimeScript RT Enzyme Mix II 0.5μL(购于TakaRa公司)，SVA-F(10μM)1μL，SVA-R(10μM)1μL，SVA-P(10μM)1μL，FMDV-F(10μM)1μL，FMDV-R(10μM)1μL，FMDV-P(10μM)1.5μL，RNA-free H 2O 3μL。实时荧光定量PCR反应条件为：先42℃反转录20min，95℃30s预变性；然后94℃变性10s，55℃退火30s，45个循环(PCR扩增)。在每个循环的退火结束时进行荧光信号检测。
2)用得到的各自的CT值或荧光信号的变化实现对塞内卡病毒和/或口蹄疫病毒的定性检测，若FAM通道中在37个循环内(不包括第37个循环)出现“S”型扩增曲线，则确认为塞内卡病毒阳性(样品中含有塞内卡病毒)，若ROX通道中在37个循环内(不包括第37个循环)出现“S”型扩增曲线，则确认为口蹄疫 病毒阳性(样品中含有口蹄疫病毒)。
3)对步骤2)中确定为阳性的待测样品，再根据荧光信号的强度和步骤1)中的标准曲线，得出待测样品中所含塞内卡病毒和/或口蹄疫病毒的拷贝数，实现定量检测。
若步骤2)中FAM通道和/或ROX通道中39个循环以上(含39个循环)未出现“S”型扩增曲线，则确认为对应病毒阴性(样品中不含有塞内卡病毒和/或口蹄疫病毒)；任一通道中在37-39个循环之间(含37个循环且不含39个循环)出现“S”型扩增曲线则判定为可疑，需重检。
针对10份细胞培养物进行塞内卡病毒和口蹄疫病毒双重实时荧光定量PCR检测的结果如表4所示，1-3号及5号样品的检测结果为FMDV阳性(感染口蹄疫病毒)，4号样品的检测结果为FMDV可疑，表明1-3号及5号样品中含口蹄疫病毒，4号样品需重检；7-9号样品的检测结果为SVA阳性(感染塞内卡病毒)，6号和10号样品的检测结果为SVA可疑，表明7-9号样品中含塞内卡病毒，6号和10号样品需重检。
表4. 10份细胞培养物检测结果
Figure PCTCN2018107963-appb-000004
Figure PCTCN2018107963-appb-000005
以下对实施例2建立的对塞内卡病毒和口蹄疫病毒进行双重实时荧光定量PCR检测方法进行效果验证。
试验一、敏感性实验
将携带塞内卡病毒核苷酸检测基因和口蹄疫病毒核苷酸检测基因的重组质粒pCR-4TOPO-SVA和pCR-4TOPO-FMDV作为标准品，按照10倍梯度将SVA标准品(pCR-4TOPO-SVA)稀释至1×10 8、1×10 7、1×10 6、1×10 5、1×10 4、1×10 3、1×10 2、2.5×10 1copies/μL；按照10倍梯度将FMDV标准品(pCR-4TOPO-FMDV)稀释至1×10 8、1×10 7、1×10 6、1×10 5、1×10 4、1×10 3、1×10 2、1×10 1copies/μL，以不同浓度的标准品作为模板，在引物SVA-F、SVA-R、FMDV-F、FMDV-R及TaqMan探针SVA-P和TaqMan探针FMDV-P的引导下进行双重实时荧光定量PCR检测，PCR检测体系及检测条件参照实施例2，检测塞内卡病毒和口蹄疫病毒双重实时荧光定量PCR检测方法的敏感性。结果如图3所示。
塞内卡病毒和口蹄疫病毒双重实时荧光定量PCR检测方法可以检测至塞内卡病毒2.5×10 1copies/μL和口蹄疫病毒1×10 1copies/μL(敏感性)，并且扩增曲线为特定的“S”型曲线，说明本发明确定的引物和TaqMan探针与塞内卡病毒和口蹄疫病毒RNA结合适宜。
试验二、特异性实验
对牛病毒性腹泻粘膜病病毒(BVDV)、猪瘟病毒(CSFV)、牛副流感病毒(BPIV)和牛流行热病毒(BEFV)提取RNA，对猪伪狂犬(PRV)、鼻气管炎病毒(IBRV)、猪圆环病毒2型(PCV2)、细小病毒(PPV)提取DNA，以正常PK-15细胞的RNA为阴性对照品，同时以塞内卡病毒培养物的RNA和O型口蹄疫病毒(O型FMDV)、A型口蹄疫病毒(A型FMDV)、Asia-1型口蹄疫病毒(FMDV Asia-1)为阳性对照品，以无酶水为空白对照，在引物SVA-F、SVA-R、FMDV-F、FMDV-R及TaqMan探针SVA-P和TaqMan探针FMDV-P的引导下分别进行双重实时荧光定量PCR检测，PCR检测体系及检测条件参照实施例2，检测塞内卡病毒和口蹄疫病毒双重实时荧光定量PCR检测方法的特异性。
检测结果如图5所示，仅塞内卡病毒(SVA)和O型口蹄疫病毒(O型FMDV)、A型口蹄疫病毒(A型FMDV)、Asia-1型口蹄疫病毒(FMDV Asia-1)出现特定的“S”型扩增曲线(结果阳性)，其它样品未出现特定的“S”型扩增曲线(结果阴性)，检测结果表明用本发明的方法可特异性地检测出塞内卡病毒和O型口蹄疫病毒、A型口蹄疫病毒、Asia-1型口蹄疫病毒。
试验三、重复性实验
按照10倍梯度将SVA标准品(pCR-4TOPO-SVA)稀释至1×10 8、1×10 7、1×10 6、1×10 5、1×10 4、1×10 3、1×10 2、2.5×10 1copies/μl；按照10倍梯度将FMDV标准品(pCR-4TOPO-FMDV)稀释至1×10 8、1×10 7、1×10 6、1×10 5、1×10 4、1×10 3、1×10 2、1×10 1copies/μL，并作为模板，每个梯度2个重复，在引物SVA-F、SVA-R、FMDV-F、FMDV-R及TaqMan探针SVA-P和TaqMan探针FMDV-P的引导下进行双重实时荧光定量PCR检测，PCR检测体系及检测条件参照实施例2，检测塞内卡病毒和口蹄疫病毒双重实时荧光定量PCR检测方法的重复性。
检测结果如图6和表5所示，从图中可见每个浓度梯度的扩增曲线较聚拢，循环数无明显差距，表明塞内卡病毒和口蹄疫病毒双重实时荧光定量PCR检测方法的重复性较好，循环数标准偏差相差最高仅为0.40。
表5.重复性试验结果
Figure PCTCN2018107963-appb-000006
Figure PCTCN2018107963-appb-000007
实施例3、塞内卡病毒和口蹄疫病毒双重的实时荧光定量PCR检测试剂盒
基于实施例1和实施例2，塞内卡病毒和口蹄疫病毒双重实时荧光定量PCR检测试剂盒包括用于对塞内卡病毒和口蹄疫病毒进行双重实时荧光定量PCR检测的引物(SVA-F、SVA-R、FMDV-F和FMDV-R)和TaqMan探针(SVA-P和FMDV-P)。
试剂盒中还可以包括标准品，标准品为前述带有塞内卡病毒检测基因的重组质粒pCR4-TOPO-SVA和带有口蹄疫病毒检测基因的重组质粒pCR4-TOPO-FMDV。
在使用试剂盒时的25μL双重实时荧光定量PCR检测体系为：实时荧光定量一步法PCR反应液2×One Step RT-PCR Buffer III 12.5μL，TaKaRa Ex Taq HS0.5μL，PrimeScript RT Enzyme Mix II 0.5μL，SVA-F(10μM)1.0μL，SVA-R(10μM)1.0μL，SVA-P(10μM)1μL，FMDV-F(10μM)1.0μL，FMDV-R(10μM)1.0μL，FMDV-P(10μM)1.5μL，模板2.0μL，RNA-free H 2O 3.0μL。回执标准曲线时的模板为标准品DNA，检测样品时模板为待测样品基因组RNA。
为方便检测，试剂盒中还可包括阳性对照品和阴性对照品，阳性对照品为塞内卡病毒RNA和口蹄疫病毒RNA(阳性样品可以验证模板提取过程是否有问题，阳性样品出来条带说明核酸提取过程没问题)，阴性对照品为不含塞内卡病毒和口蹄疫病毒的反应体系，如H 2O(双蒸水、无菌去离子水等)。
试剂盒中各试剂的使用方法可参考实施例2的内容。
实施例4、猪场疑似塞内卡病和口蹄疫病病料的实时荧光定量PCR检测
用塞内卡病毒和口蹄疫病毒双重实时荧光定量PCR检测试剂盒对从收集的14份猪场疑似塞内卡病毒和口蹄疫病毒样品(待测样品)中提取的基因组RNA(检测样)进行检测，以塞内卡病毒基因组RNA和口蹄疫病毒基因组RNA为阳性对照品，以无酶水为阴性对照品，根据双重实时荧光定量PCR检测结果，对检测样品中是否含有塞内卡病毒和口蹄疫病毒进行定性判定，并对病毒的拷贝数进行定量。
具体检测方法与实施例2相同。
塞内卡病毒和口蹄疫病毒样品双重实时荧光定量PCR检测结果如表6所示，1-3号样品的检测结果为SVA阳性(感染塞内卡病毒)，表明1-3号样品中含塞内卡病毒；4-12号样品的检测结果为FMDV阳性(感染口蹄疫病毒)，表明4-12号样品中含口蹄疫病毒；13、14号样品的检测结果为阴性，表明13和14号样品中不含塞内卡病毒和口蹄疫病毒。
表6. 14份猪场疑似塞内卡病毒和口蹄疫病毒样品检测结果
Figure PCTCN2018107963-appb-000008
相较于现有技术相比，本发明的优势在于：
现有发明中FMDV和SVA双重荧光定量PCR检测方法有CN107326100A闫若潜发明的“口蹄疫和塞内卡病毒二重实时荧光定量PCR检测试剂盒”，其检测方法，该方法为先将病毒RNA反转录成cDNA，再将cDNA为模板进行实时荧光定量PCR扩增，实验步骤繁琐。与上述现有方法相比，本发明提供对FMDV和SVA RNA样品不用先反转录成cDNA，直接将RNA为模板加入一步法PCR体系进行实时荧光定量PCR检测，优点为本发明中没有先将病毒RNA反转录成cDNA的步骤，并且扩增曲线为特定的“S”型曲线，说明本方法的引物探针与病毒RNA 结合最适宜。本发明为此特优化了相关引物和探针，建立了一种塞内卡病毒和口蹄疫病毒双重实时荧光定量PCR检测方法，可快速同时检测区别塞内卡病毒和口蹄疫病毒，为临床样品的检测，毒株鉴定及疫苗生产中发挥重要作用。
现有发明中CN201711339422.1花群义发明的“一种用于FMDV和SVA鉴别的试剂、方法及应用”，该检测方法中FMDV和SVA检测靶序列都在该病毒全基因组的3D基因处，本发明提供的检测FMDV和SVA靶序列在该病毒全基因组的3C基因处，两者选择的靶序列不同；该发明中敏感度为10 -6稀释度，并且不清楚该发明中原浓度，所以无法进行比较。本发明敏感性为10 1拷贝/μL。
现有发明中CN201711324308.1贺东生发明的“用于检测猪口蹄疫病毒与塞内加谷病毒的双重PCR引物、检测方法及试剂盒”，该检测方法中FMDV检测靶序列在该病毒的2C基因处，SVA检测靶序列在该病毒的VP4基因处，两者选择的靶序列也不同；该发明中敏感度为10 2拷贝/μL，本发明中检测敏感性为10 1拷贝/μL，两者敏感性比较，本发明敏感性高于CN201711324308.1；该发明为定性检测，无法定量，而本发明可以定性又可以定量检测。
工业应用性
本发明提供了用于检测塞内卡病毒(SVA)和口蹄疫病毒(FMDV)的双重实时荧光定量PCR检测试剂盒及其专用引物、TaqMan探针，能作为SVA和FMDV的检测试剂对塞内卡病毒和口蹄疫病毒实施快速区分和检测，用于疫苗生产和病料检测，能够工业生产和在产业上使用，具备工业应用性。

Claims (18)

  1. 用于对塞内卡病毒和口蹄疫病毒进行双重实时荧光定量PCR检测的引物和Taqman探针，包括：
    用于检测塞内卡病毒的上游引物(SVA-F)和下游引物(SVA-R)，SVA-F的核苷酸序列如序列表中SED ID NO：1所示，SVA-R的核苷酸序列如序列表中SEQ ID NO：2所示；
    用于对塞内卡病毒进行检测的TaqMan探针(SVA-P)，SVA-P的核苷酸序列如序列表中SED ID NO：3所示；
    用于检测口蹄疫病毒的上游引物(FMDV-F)和下游引物(FMDV-R)，FMDV-F的核苷酸序列如序列表中SED ID NO：5所示，FMDV-R的核苷酸序列如序列表中SEQ ID NO：6所示；和
    用于对口蹄疫病毒进行检测的TaqMan探针(FMDV-P)，FMDV-P的核苷酸序列如序列表中SED ID NO：7所示；
    所述探针为经过荧光标记的，其5′端标记有报告荧光基团，3′端标记有淬灭荧光基团；且对两种病毒检测的探针SVA-P和FMDV-P所标记的报告荧光基团不相同。
  2. 根据权利要求1所述的引物和TaqMan探针，
    探针SVA-P的5′端报告荧光基团为FAM，3′端荧光淬灭基团为TAMRA；
    探针FMDV-P的5′端报告荧光基团为ROX，3′端荧光淬灭基团为BHQ2；
    所述探针的3′端经磷酸化处理。
  3. 用于对塞内卡病毒和口蹄疫病毒进行双重实时荧光定量PCR检测试剂盒，
    包括权利要求1或2所述的引物和TaqMan探针。
  4. 根据权利要求3所述的试剂盒，所述试剂盒中还包括标准品，所述标准品为带有塞内卡病毒检测基因的重组质粒pCR4-TOPO-SVA和带有口蹄疫病毒检测基因的重组质粒pCR4-TOPO-FMDV。
  5. 根据权利要求3或4所述的试剂盒，试剂盒中还包括阳性对照品和阴性对照品，阳性对照品为塞内卡病毒RNA和口蹄疫病毒RNA，阴性对照品为不含塞内卡病毒和口蹄疫病毒的反应体系，如H 2O(双蒸水、无菌去离子水等)。
  6. 根据权利要求3或4或5所述的试剂盒，使用所述试剂盒时的25μL双重实时荧光定量PCR检测体系为：实时荧光定量一步法PCR反应液2×One Step RT-PCR Buffer III 12.5μL，TaKaRa Ex Taq HS 0.5μL，PrimeScript RT Enzyme Mix  II 0.5μL，SVA-F(10μM)1.0μL，SVA-R(10μM)1.0μL，SVA-P(10μM)1.0μL，FMDV-F(10μM)1.0μL，FMDV-R(10μM)1.0μL，FMDV-P(10μM)1.5μL，RNA-free H 2O 3.0μL，模板2.0μL。
  7. 权利要求1或2所述的引物和TaqMan探针在制备塞内卡病毒和口蹄疫病毒的检测试剂中的应用。
  8. 根据权利要求7所述的应用，用于对塞内卡病毒和口蹄疫病毒进行非疾病诊断的定性、定量检测，包括以下步骤：
    1)建立标准曲线：将分别携带塞内卡病毒和口蹄疫病毒的检测基因的重组质粒pCR4-TOPO-SVA和pCR4-TOPO-FMDV作为标准品，按照10倍梯度将SVA标准品pCR-4TOPO-SVA稀释至1×10 8、1×10 7、1×10 6、1×10 5、1×10 4、1×10 3、1×10 2、2.5×10 1copies/μL，按照10倍梯度将FMDV标准品pCR-4TOPO-FMDV稀释至1×10 8、1×10 7、1×10 6、1×10 5、1×10 4、1×10 3、1×10 2、1×10 1copies/μL，以不同浓度的标准品DNA作为模板，在权利要求1或2所述的引物和TaqMan探针的引导下进行双重实时荧光定量PCR检测，检测结束后，以各标准品的浓度Log值(X轴)对其相应Ct值(Y轴)作图，绘制标准曲线；
    2)提取待测样品的基因组RNA，以提取的基因组RNA为模板，在权利要求1或2所述的引物和TaqMan探针的引导下进行双重实时荧光定量PCR检测；
    3)用步骤2)得到的塞内卡病毒和口蹄疫病毒各自的CT值或荧光信号的变化分别判定待测样品中是否存在塞内卡病毒和口蹄疫病毒完成定性检测；
    4)对步骤3)中确定为塞内卡病毒和/或口蹄疫病毒阳性的待测样品，再根据荧光信号的强度和步骤1)中的标准曲线，得出该样品中所含的病毒的拷贝数，完成定量检测。
  9. 根据权利要求8所述的应用，
    所述步骤2)中的待测样品采自猪，包括：猪场送检样品，用于疫苗生产的原料血清、疫苗半成品。
  10. 根据权利要求8或9所述的应用，
    所述步骤1)和步骤2)中的双重实时荧光定量PCR检测25μL体系包括：模板2μL，实时荧光定量一步法PCR反应液2×One Step RT-PCR Buffer III 12.5μL，TaKaRa Ex Taq HS 0.5μL，PrimeScript RT Enzyme Mix II 0.5μL，SVA-F(10μM)1.0μL，SVA-R(10μM)1.0μL，SVA-P(10μM)1.0μL，FMDV-F(10μM)1.0μL，FMDV-R(10μM)1.0μL，FMDV-P(10μM)1.5μL，RNA-free H 2O 3.0μL；
    所述步骤1)和步骤2)中的双重实时荧光定量PCR检测条件为：先42℃反转录20min，95℃预变性30s；然后94℃变性10s，55℃退火30s，45个循环(PCR扩增)。
  11. 根据权利要求8-10中任一项所述的应用，
    所述步骤3)中的判定规则为：
    若样品在FAM通道中在37个循环内(不包括第37个循环)出现“S”型扩增曲线，则确认样品为塞内卡病毒阳性；若样品在ROX通道中在37个循环内(不包括第37个循环)出现“S”型扩增曲线，则确认样品为口蹄疫病毒阳性；
    若样品在FAM通道和/或ROX通道中在39个或以上循环(包括第39个循环)出现或未出现“S”型扩增曲线，则确认样品为塞内卡病毒和/或口蹄疫病毒阴性；
    任一通道中在37或38个循环出现“S”型扩增曲线则判定为可疑，需重检。
  12. 对塞内卡病毒和口蹄疫病毒进行检测的方法，使用权利要求1或2所述的引物和TaqMan探针或权利要求3至6任一所述的试剂盒对送件样品进行检测。
  13. 根据权利要求12所述方法，为塞内卡病毒和口蹄疫病毒的双重实时荧光PCR定量检测，包括以下步骤：
    1)建立标准曲线：将分别携带塞内卡病毒和口蹄疫病毒的检测基因的重组质粒pCR4-TOPO-SVA和pCR4-TOPO-FMDV作为标准品，按照10倍梯度将SVA标准品pCR-4TOPO-SVA稀释至1×10 8、1×10 7、1×10 6、1×10 5、1×10 4、1×10 3、1×10 2、2.5×10 1copies/μL，按照10倍梯度将FMDV标准品pCR-4TOPO-FMDV稀释至1×10 8、1×10 7、1×10 6、1×10 5、1×10 4、1×10 3、1×10 2、1×10 1copies/μL，以不同浓度的标准品作为模板，在用于对塞内卡病毒和口蹄疫病毒进行双重实时荧光定量PCR检测的引物和Taqman探针的引导下进行双重实时荧光定量PCR检测，检测结束后，以各标准品的浓度Log值(X轴)对其相应Ct值(Y轴)作图，绘制标准曲线；
    2)提取待测样品的基因组RNA，以提取的基因组RNA为模板，在用于对塞内卡病毒和口蹄疫病毒进行双重实时荧光定量PCR检测的引物和Taqman探针的引导下进行双重实时荧光定量PCR检测；
    3)用步骤2)得到的塞内卡病毒和口蹄疫病毒各自的CT值或荧光信号的变化分别判定待测样品中是否存在塞内卡病毒和口蹄疫病毒完成定性检测；
    4)对步骤3)中确定为塞内卡病毒和/或口蹄疫病毒阳性的待测样品，再根据荧光信号的强度和步骤1)中的标准曲线，得出该样品中所含的病毒的拷贝数， 完成定量检测。
  14. 根据权利要求13所述的方法，步骤1)和步骤2)中所述用于对塞内卡病毒和口蹄疫病毒进行双重实时荧光定量PCR检测的引物和Taqman探针包括：
    用于检测塞内卡病毒的上游引物(SVA-F)和下游引物(SVA-R)，SVA-F的核苷酸序列如序列表中SED ID NO：1所示，SVA-R的核苷酸序列如序列表中SEQ ID NO：2所示；
    用于对塞内卡病毒进行检测的TaqMan探针(SVA-P)，SVA-P的核苷酸序列如序列表中SED ID NO：3所示；
    用于检测口蹄疫病毒的上游引物(FMDV-F)和下游引物(FMDV-R)，FMDV-F的核苷酸序列如序列表中SED ID NO：5所示，FMDV-R的核苷酸序列如序列表中SEQ ID NO：6所示；和
    用于对口蹄疫病毒进行检测的TaqMan探针(FMDV-P)，FMDV-P的核苷酸序列如序列表中SED ID NO：7所示；
    所述探针为经过荧光标记的，其5′端标记有报告荧光基团，3′端标记有淬灭荧光基团；且对两种病毒检测的探针SVA-P和FMDV-P所标记的报告荧光基团不相同；
    步骤2)中所述待测样品采自猪，包括：猪场送检样品、猪病料，用于疫苗生产的原料血清、疫苗半成品。
  15. 根据权利要求14所述的方法，
    探针SVA-P的5′端报告荧光基团为FAM，3′端荧光淬灭基团为TAMRA；
    探针FMDV-P的5′端报告荧光基团为ROX，3′端荧光淬灭基团为BHQ2；
    所述探针的3′端经磷酸化处理。
  16. 根据权利要求13所述的方法，
    所述步骤1)和步骤2)中的双重实时荧光定量PCR检测25μL体系包括：模板2μL，实时荧光定量一步法PCR反应液2×One Step RT-PCR Buffer III 12.5μL，TaKaRa Ex Taq HS 0.5μL，PrimeScript RT Enzyme Mix II 0.5μL，SVA-F(10μM)1.0μL，SVA-R(10μM)1.0μL，SVA-P(10μM)1.0μL，FMDV-F(10μM)1.0μL，FMDV-R(10μM)1.0μL，FMDV-P(10μM)1.5μL，RNA-free H 2O 3.0μL。
  17. 根据权利要求13所述的方法，
    所述步骤1)和步骤2)中的双重实时荧光定量PCR检测条件为：先42℃反转录20min，95℃预变性30s；然后94℃变性10s，55℃退火30s，45个循环(PCR 扩增)。
  18. 根据权利要求17所述的检测方法，所述步骤3)中的判定规则为：
    若样品在FAM通道中在37个循环内(不包括第37个循环)出现“S”型扩增曲线，则确认样品为塞内卡病毒阳性；若样品在ROX通道中在37个循环内(不包括第37个循环)出现“S”型扩增曲线，则确认样品为口蹄疫病毒阳性；
    若样品在FAM通道和/或ROX通道中在39个或以上循环(包括第39个循环)出现或未出现“S”型扩增曲线，则确认样品为塞内卡病毒和/或口蹄疫病毒阴性；
    任一通道中在37或38个循环出现“S”型扩增曲线则判定为可疑，需重检。
PCT/CN2018/107963 2018-08-16 2018-09-27 塞内卡病毒和口蹄疫病毒双重实时荧光定量pcr检测试剂及试剂盒 WO2020034317A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810933044.8A CN109097495A (zh) 2018-08-16 2018-08-16 塞内卡病毒和口蹄疫病毒双重实时荧光定量pcr检测试剂盒
CN201810933044.8 2018-08-16

Publications (1)

Publication Number Publication Date
WO2020034317A1 true WO2020034317A1 (zh) 2020-02-20

Family

ID=64849796

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/107963 WO2020034317A1 (zh) 2018-08-16 2018-09-27 塞内卡病毒和口蹄疫病毒双重实时荧光定量pcr检测试剂及试剂盒

Country Status (2)

Country Link
CN (1) CN109097495A (zh)
WO (1) WO2020034317A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111979356A (zh) * 2020-08-31 2020-11-24 福建农林大学 一种大黄鱼虹彩病毒的双重TaqMan探针法荧光定量PCR检测试剂盒及制备方法

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110305986A (zh) * 2019-07-18 2019-10-08 金宇保灵生物药品有限公司 Sva、o型fmdv和a型fmdv的一步法三重实时荧光定量pcr检测引物和探针
CN110358864A (zh) * 2019-07-24 2019-10-22 金宇保灵生物药品有限公司 Sva、fmdv、svdv和vsv的一步法多重实时荧光定量pcr检测引物和探针
CN111996202B (zh) * 2020-03-24 2021-09-10 中国农业科学院兰州兽医研究所 一种重组o型口蹄疫病毒vp1基因的塞内卡重组病毒、重组疫苗株及其制备方法和应用
CN117304276B (zh) * 2023-11-30 2024-04-12 江门海关技术中心 含口蹄疫病毒o型和塞内卡病毒核酸的病毒样颗粒

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105925728A (zh) * 2016-06-01 2016-09-07 华南农业大学 一种塞内加谷病毒实时荧光定量pcr检测引物及试剂盒
CN107326100A (zh) * 2017-07-17 2017-11-07 河南省动物疫病预防控制中心 口蹄疫和塞尼卡谷病毒二重实时荧光定量pcr检测试剂盒
CN107955840A (zh) * 2017-12-13 2018-04-24 华南农业大学 用于检测猪口蹄疫病毒与塞内加谷病毒的双重pcr引物、检测方法及试剂盒
CN108034761A (zh) * 2017-12-14 2018-05-15 深圳出入境检验检疫局动植物检验检疫技术中心 一种用于fmdv和sva鉴别的试剂、方法及应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070065814A1 (en) * 2005-09-21 2007-03-22 Engelhard Eric K Detecting foot-and-mouth disease virus
CN103063838B (zh) * 2012-10-11 2015-03-11 广东出入境检验检疫局检验检疫技术中心 一种鉴别进境动物口蹄疫感染与免疫的试剂盒
WO2017181070A1 (en) * 2016-04-15 2017-10-19 Kansas State University Research Foundation Vaccine against seneca valley virus
CN108384893A (zh) * 2018-05-03 2018-08-10 中国农业科学院兰州兽医研究所 用于检测口蹄疫病毒和塞尼卡谷病毒的实时荧光定量rt-pcr试剂盒及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105925728A (zh) * 2016-06-01 2016-09-07 华南农业大学 一种塞内加谷病毒实时荧光定量pcr检测引物及试剂盒
CN107326100A (zh) * 2017-07-17 2017-11-07 河南省动物疫病预防控制中心 口蹄疫和塞尼卡谷病毒二重实时荧光定量pcr检测试剂盒
CN107955840A (zh) * 2017-12-13 2018-04-24 华南农业大学 用于检测猪口蹄疫病毒与塞内加谷病毒的双重pcr引物、检测方法及试剂盒
CN108034761A (zh) * 2017-12-14 2018-05-15 深圳出入境检验检疫局动植物检验检疫技术中心 一种用于fmdv和sva鉴别的试剂、方法及应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BO, QINGRU ET AL.: "Establishment of a Method for Differential Diagnosis of Type O and Asial foot-and-mouth Disease Virus by Fluorescent RT-PCR", JOURNAL OF AGRICULTURAL BIOTECHNOLOGY, vol. 17, no. 1, 31 December 2009 (2009-12-31), pages 24 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111979356A (zh) * 2020-08-31 2020-11-24 福建农林大学 一种大黄鱼虹彩病毒的双重TaqMan探针法荧光定量PCR检测试剂盒及制备方法

Also Published As

Publication number Publication date
CN109097495A (zh) 2018-12-28

Similar Documents

Publication Publication Date Title
WO2020034317A1 (zh) 塞内卡病毒和口蹄疫病毒双重实时荧光定量pcr检测试剂及试剂盒
CN107299155A (zh) 一种鹅星状病毒实时荧光定量pcr检测的引物和探针
CN110760620A (zh) 一种猪瘟病毒与非洲猪瘟病毒双重荧光pcr检测试剂、试剂盒和检测方法
CN107034309B (zh) 快速检测猪伪狂犬病毒的实时荧光rpa试剂盒、试纸条rpa试剂盒及其用途
CN108866243B (zh) 一种猪肠道冠状病毒4重荧光定量pcr检测试剂盒
WO2022179494A1 (zh) 一种伤寒沙门氏菌的检测试剂盒、其制备方法及其应用
CN106244725B (zh) 一种诊断猪脐带血伪狂犬病毒野毒株的Taqman实时荧光PCR试剂盒及其用途
CN107236825B (zh) 用于real-time RPA快速检测和区分PRV野毒和疫苗毒的核酸及方法
CN112795704A (zh) 检测猪伪狂犬病病毒的raa引物对和探针、试剂盒及其应用
CN112739833A (zh) 利用巢式RPA技术检测SARS-CoV-2的引物对、探针、试剂盒及其应用
CN108504778A (zh) 一种同时检测猪圆环病毒2型和猪伪狂犬病毒的试剂盒及应用
CN109913591A (zh) 基于TaqMan探针法的A型塞尼卡病毒荧光定量RT-PCR检测方法和试剂盒
CN113913559B (zh) 一种荧光定量检测prrsv的试剂、其应用于prrsv分型的检测方法
CN113249517A (zh) 一种牛疫病实时荧光定量pcr检测用引物和探针及试剂盒
CN107699635B (zh) 猪流行性腹泻病毒荧光rpa检测方法
Zhang et al. Development of a loop-mediated isothermal amplification for visual detection of the HCLV vaccine against classical swine fever in China
CN113046482B (zh) 鸽腺病毒b型环介导等温扩增检测引物组及试剂盒
Qi et al. Development and application of a TaqMan-based real-time PCR method for the detection of the ASFV MGF505-7R gene
CN110747293B (zh) 用于鉴别猪水泡病毒、口蹄疫病毒与塞尼卡谷病毒的三重荧光rt-pcr检测试剂盒
CN110305986A (zh) Sva、o型fmdv和a型fmdv的一步法三重实时荧光定量pcr检测引物和探针
CN105907894B (zh) 一种检测仔猪脐带血中圆环病毒Ⅱ型的Taqman实时荧光PCR试剂盒及其应用
CN113151586B (zh) 一种用于检测、鉴别猪伪狂犬病毒ⅰ型和ⅱ型的引物组合、试剂盒及方法
CN115786587A (zh) 一种用于同时检测4种病原体的多重pcr的引物组及其检测方法、试剂盒
CN104388593A (zh) 一种用于检测猪圆环病毒的Taqman Real-time PCR试剂盒
Ren et al. Establishment and application of a TaqMan-based multiplex real-time PCR for simultaneous detection of three porcine diarrhea viruses

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18930148

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18930148

Country of ref document: EP

Kind code of ref document: A1