WO2023082865A1 - 鉴别羊痘病毒和牛结节性皮肤病病毒的单引物双重荧光检测试剂盒及其专用引物和探针 - Google Patents

鉴别羊痘病毒和牛结节性皮肤病病毒的单引物双重荧光检测试剂盒及其专用引物和探针 Download PDF

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WO2023082865A1
WO2023082865A1 PCT/CN2022/121193 CN2022121193W WO2023082865A1 WO 2023082865 A1 WO2023082865 A1 WO 2023082865A1 CN 2022121193 W CN2022121193 W CN 2022121193W WO 2023082865 A1 WO2023082865 A1 WO 2023082865A1
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virus
skin disease
probe
value
sample
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French (fr)
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王鹏
辛俊利
黄海碧
宋庆庆
赵丽霞
刘玉梅
刘建奇
刘东霞
张彦婷
范文霞
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金宇保灵生物药品有限公司
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    • 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
    • C12Q1/6851—Quantitative amplification
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the invention belongs to the technical field of biology, and in particular relates to a single-primer dual fluorescence detection kit for distinguishing goat pox virus (including goat pox virus and sheep pox virus) and bovine nodular skin disease virus, as well as special primers and probes thereof, and A non-disease diagnostic method for the identification of sheep pox virus and bovine nodular skin disease virus.
  • Capripox virus belongs to the family Poxviridae and the subfamily Chordatepoxviridae, which includes goat poxvirus (GTPV), sheep poxvirus (SPPV) and bovine nodular skin disease virus (LSDV). It is a DNA virus with a genome length of about 150kb, encoding 147 open reading frames.
  • the disease symptoms caused by goat pox virus are mainly skin papule-pustules type pox.
  • Goat pox virus can infect goats of all breeds, sexes and ages, among which lambs are the most susceptible, with an infection rate of 100%. It infects other goats mainly through contact with damaged skin, inhalation of the respiratory tract or transmission of vectors, and spreads between groups fast.
  • Sheep pox also known as sheep smallpox, is a contagious disease caused by sheep pox virus. Other characteristics are specific pox rashes on the skin and mucous membranes of the whole body. Sheep of different breeds, genders, and ages are susceptible, and sick sheep have fever and have a higher mortality rate.
  • Bovine nodular skin disease is an acute, subacute or chronic infectious disease characterized by fever, skin mucous membranes, and extensive nodules on the surface of organs caused by bovine nodular skin disease virus.
  • the surface lymph nodes of sick cattle are enlarged, mastitis may occur in lactating cows, orchitis in bulls may cause permanent or temporary infertility, etc., and the animals may die in severe cases.
  • the above three goat poxviruses have caused serious economic losses to the dairy and beef cattle breeding industry.
  • Vaccine prevention is the main means of virus prevention and control. Although there is a certain cross-protection between goat pox virus, sheep pox virus and bovine nodular skin disease virus, goat pox vaccine can be used as a preventive measure for bovine nodular skin disease, but for sexual vaccine prevention will bring better prevention and control effect.
  • goat pox vaccine can be used as a preventive measure for bovine nodular skin disease, but for sexual vaccine prevention will bring better prevention and control effect.
  • the small differences in genome sequences between members of the capapoxvirus genus increase the difficulty of identifying members of the capapoxvirus genus, such as the identification of capipoxviruses (including goatpoxviruses and ovinepoxviruses) and bovine nodular skin disease virus Difficulty in distinguishing between.
  • patent document CN112126717A discloses a double fluorescent PCR primer, probe, method and kit for distinguishing goatpox virus and bovine nodular skin disease virus , but the method disclosed in this document 1 cannot directly differentiate and detect sheep pox virus and bovine nodular skin disease virus.
  • one aspect of the present invention provides a single-primer dual fluorescence detection kit for distinguishing goat pox virus and bovine nodular skin disease virus, and the goat pox virus includes goat pox virus and Sheep pox virus, the detection kit includes:
  • the universal PCR amplification primer set that is used to detect goat poxvirus genus comprises the forward primer LSD13F shown in SEQ ID NO:1 and the reverse primer LSD13R shown in SEQ ID NO:2;
  • the probe GaPvP that is used to detect sheeppox virus its nucleotide sequence is as shown in SEQ ID NO:3;
  • the probe LSDVP that is used for detecting bovine nodular skin disease virus its nucleotide sequence is shown in SEQ ID NO:4.
  • the 5' ends of the probe GaPvP and the probe LSDVP are labeled with different fluorescent reporter groups, and the 3' ends are both labeled with fluorescent quenching groups.
  • the detection kit also includes standard products: CDS17 standard product and CDS21 standard product; each standard product is a single package or equal concentration mixed package; When performing dual real-time fluorescent quantitative PCR detection of single primers for viral viruses, mix single-packaged standard products at equal concentrations or use mixed-packaged standard products directly.
  • Another aspect of the present invention provides a general PCR amplification primer for detecting goat poxvirus, which is used in the above-mentioned detection kit, including forward primer LSD13F shown in SEQ ID NO: 1 and SEQ ID NO: Reverse primer LSD13R indicated in 2.
  • Another aspect of the present invention provides a probe GaPvP for detecting sheep pox virus, which is used in the above-mentioned detection kit, the nucleotide sequence of the probe GaPvP is shown in SEQ ID NO: 3, its 5' The end is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.
  • Another aspect of the present invention provides a kind of probe LSDVP that is used for detecting bovine nodular skin disease virus, and it is used in above-mentioned detection kit, and the nucleotide sequence of this probe LSDVP is as shown in SEQ ID NO:4 , the 5' end is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.
  • Another aspect of the present invention provides a non-disease diagnosis method for distinguishing between sheep pox virus and bovine nodular skin disease virus, which comprises the following steps:
  • step S2 Using the DNA extracted in step S1 as a template, use the general PCR amplification primer set, probe GaPvP and probe LSDVP in the above-mentioned kit to perform single-primer double fluorescent PCR amplification to obtain an amplification curve;
  • step S3 Analyzing the amplification curve obtained in step S2 to determine whether the sample to be tested contains sheeppox virus and/or bovine nodular skin disease virus;
  • the judging criteria are:
  • the fluorescence channel corresponding to the probe GaPvP has an amplification curve, and the Ct value is less than or equal to 38, it is determined that the sample to be tested contains goat pox virus; if the Ct value>40, it is determined to be negative, that is, the sample to be tested does not contain sheeppox virus; if 38 ⁇ Ct value ⁇ 40, re-tested as Ct value ⁇ 40, it is determined that the sample to be tested contains sheeppox virus; if there is no amplification curve or the Ct value of retesting>40 , it is determined that the sample to be tested does not contain sheeppox virus;
  • the fluorescence channel corresponding to the probe LSDVP has an amplification curve, and the Ct value ⁇ 38, it is determined that the sample to be tested contains bovine nodular skin disease virus; if the Ct value>40, it is determined as negative, that is, the The sample to be tested does not contain bovine nodular skin disease virus; if 38 ⁇ Ct value ⁇ 40, it is detected as Ct value ⁇ 40 again, then it is determined that the sample to be tested contains bovine nodular skin disease virus; if If there is no amplification curve or the Ct value of the retest is >40, it is determined that the sample to be tested does not contain bovine nodular skin disease virus.
  • the 25-30 ⁇ L system for double fluorescent PCR amplification in step S2 includes: forward primer LSD13F (10 ⁇ M) 0.25-0.75 ⁇ L, reverse primer LSD13R (10 ⁇ M) 0.25-0.75 ⁇ L, probe GaPvP ( 10 ⁇ M) 0.50-1.00 ⁇ L, probe LSDVP (10 ⁇ M) 0.50-1.00 ⁇ L, DNA template 1-3 ⁇ L, qPCR Mix 10-15 ⁇ L, ddH 2 O supplement 25-30 ⁇ L.
  • the procedure for performing dual fluorescent PCR amplification in step S2 includes: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 10-15 seconds, annealing and extension at 55°C for 30-34 seconds, and 40-45 cycles.
  • the fluorescent reporter group labeled at the 5' end of the probe GaPvP is VIC, and the quencher group labeled at the 3' end is MGB; the fluorescent reporter group labeled at the 5' end of the probe LSDVP The group is FAM, and the quenching group labeled at the 3' end is MGB; the judgment criteria are:
  • the sample to be tested contains bovine nodular skin disease virus; if the Ct value>40, the sample to be tested does not contain bovine nodular skin disease virus. Nodular skin disease virus; if 38 ⁇ Ct value ⁇ 40, if the Ct value ⁇ 40 is detected again, it is determined that the sample to be tested contains bovine nodular skin disease virus; if there is no amplification curve or Ct value detected again >40, it is determined that the test sample does not contain bovine nodular skin disease virus.
  • the single primer dual fluorescence detection kit for distinguishing goatpox virus and bovine nodular skin disease virus provided based on the above technical scheme includes general PCR primers for detecting goat poxvirus genus, and respectively for detecting goat pox virus (comprising goat pox virus and sheep pox virus) and the fluorescent probe of bovine nodular skin disease virus, can realize the purpose of directly distinguishing and detecting sheep pox virus and bovine nodular skin disease virus by only using a pair of general PCR primers and two fluorescent probes, Compared with the method disclosed in the above document 1, the operation is simpler and more efficient.
  • kit provided by the present invention and method all can reach 1 copy/ ⁇ L to the detection sensitivity of sheeppox virus and bovine nodular skin disease virus, obviously higher than above-mentioned document 1 differential detection sheeppox virus genus and bovine nodular dermatosis virus.
  • Sensitivity of nodular dermatosis virus (10 copies/ ⁇ L). Therefore, the method and kit for distinguishing goat pox virus and bovine nodular skin disease virus provided by the present invention can directly distinguish and distinguish goat pox virus and bovine nodular skin disease virus in one operation, and have higher sensitivity.
  • Figure 1 is a schematic diagram of the partial sequence alignment of the CDS17 gene of goat pox vaccine strain AV41 strain (MH381810.1) and the CDS21 gene of bovine nodular skin disease virus China/GD01/2020 strain (MW355944.1);
  • Fig. 2 is to use the amplification curve of CDS17 standard substance and CDS21 standard substance of series concentration of group 1 primer and probe in embodiment 1;
  • Fig. 3 is to use the amplification curve of the CDS17 standard substance of series concentration on ABI qPCR instrument using group 1 primer and probe among the embodiment 1;
  • Fig. 4 is a standard curve drawn according to the amplification curve shown in Fig. 3;
  • Fig. 5 is to use the amplification curve of the CDS21 standard substance of series concentration on ABI qPCR instrument using group 1 primer and probe in embodiment 1;
  • FIG. 6 is a standard curve drawn according to the amplification curve shown in FIG. 5 .
  • the present invention aims to provide a single-primer dual fluorescent PCR for the differential detection of goat pox virus and bovine nodular skin disease virus
  • the detection method can be used to directly distinguish between goatpox virus and bovine nodular skin disease virus, and provide technical support for the vaccine prevention and control of goatpox virus.
  • the present invention also provides a single-primer double fluorescence detection kit for distinguishing sheeppox virus and bovine nodular skin disease virus, its special primers and probes, and a detection method.
  • the gene sequences of sheep pox virus, goat pox virus, and bovine nodular skin disease virus were respectively retrieved from NCBI's nucleic acid database GenBank (http://www.ncbi.nlm.nih.gov), and a large number of retrieved Comparing the gene sequences, it was found that the CDS17 gene (the 14911-15231 base sequence of MH381810.1 of the goat pox virus (such as goat pox vaccine strain AV41 strain (MH381810.1)) was found, and the SEQ ID NO Shown in: 5) and the CDS21 gene of bovine nodular skin disease virus (such as China/GD01/2020 strain (MW355944.1)) (the 15264-15602 base sequence of MW355944.1, SEQ ID in the sequence listing NO: 6) in comparison, there is a region of 12 base deletions in the CDS17 gene (as shown in the box marked in Figure 1), so the CDS17 gene of sheep pox virus and bovine nodular skin
  • the inventors Based on the determined target sequences, the inventors used primer probe design software to design universal PCR primers for detecting capapoxvirus, and detection probes for capapoxvirus and bovine nodular skin disease virus respectively. As verified by the inventors, there can be multiple combinations of universal PCR primers for the detection of goatpoxvirus that can achieve a distinguishing effect, but there are only two detection probes that can distinguish between goatpox virus and bovine nodular skin disease virus.
  • the only group (the probe GaPvP (5'-AG--------------GC-3' for sheep pox virus shown in SEQ ID NO: 3) and shown in SEQ ID NO: 4
  • the probe LSDVP (5'-TG--------------AA-3') for bovine nodular skin disease virus, the four main difference bases of the two specially designed probes
  • the base must be evenly distributed at the 5' end and 3' end of the probe to achieve the discrimination effect, while the probes with other structural forms at this position (for example, the main difference bases are only distributed at the 5' end or 3' end of the probe).
  • the present invention designs multiple sets of primers and probe combinations and screens out the following groups 1 and 2, and the sequence information is as follows:
  • Universal PCR primers for the detection of cappoxviruses include:
  • LSD13F 5'-TCATTATCMTCACTACTAACRGTAT-3' (SEQ ID NO: 1);
  • LSD13R 5'-AAAGCAATATGAAAMMRGCA-3' (SEQ ID NO:2);
  • Probe GaPvP for sheeppox virus 5'-AGATGGCGATGGTGC-3' (SEQ ID NO: 3), the fluorescent reporter group labeled at the 5' end is VIC, and the quencher group labeled at the 3' end is MGB;
  • Probe LSDVP for bovine nodular skin disease virus 5'-TGATGGTGATGGTAA-3' (SEQ ID NO: 4), the fluorescent reporter group labeled at the 5' end is FAM, and the quencher group labeled at the 3' end for MGB.
  • Universal PCR primers for the detection of cappoxviruses include:
  • LSD13F1 5'-CATTATCATCACTACTAACGGTATC-3' (SEQ ID NO: 7);
  • LSD13R1 5'-AATATCTATTGAACGTGTTACATTG-3' (SEQ ID NO:8);
  • the PCR model is BioRad, wherein the curve marked with ⁇ is the amplification curve of bovine nodular skin disease virus standard substance, and the curve is from left to right respectively expressed as 10 6 copies/ ⁇ L, 10 5 copies/ ⁇ L, 10 4 copies/ ⁇ L, 10 3 copies/ ⁇ L, 10 2 copies/ ⁇ L, 10 1 copies/ ⁇ L, 10 0 copies/ ⁇ L; the other group expressed as sheep Amplification curves of poxvirus standards, the curves from left to right represent 10 6 copies/ ⁇ L, 10 5 copies/ ⁇ L, 10 4 copies/ ⁇ L, 10 3 copies/ ⁇ L, 10 2 copies/ ⁇ L, 10 1 copies / ⁇ L, 10 0 copies/ ⁇ L), it can be seen that the detection limit of the primers and probes of group 1 to the standard samples of sheeppox virus and bovine nodular skin disease virus can reach 1 copy/ ⁇ L.
  • the detection limits of the primers and probes of group 2 to the standard samples of sheeppox virus and bovine nodular skin disease virus can only reach 10 2 copies/ ⁇ L (not shown). Therefore, the primers and probes of group 1 are preferred in the present invention as the combination of primers and probes for single-primer double real-time fluorescence quantitative PCR for the identification and detection of sheep pox virus and bovine nodular skin disease virus.
  • Example 2 The establishment of a single primer double real-time fluorescent quantitative PCR standard curve of sheep pox virus and bovine nodular skin disease virus
  • Example 1 the group 1 primer and probe combination obtained in Example 1 was used to perform single-primer dual real-time fluorescent quantitative PCR detection on sheeppox virus and bovine nodular skin disease virus and establish a standard curve.
  • Goatpoxvirus universal primers LSD13F and LSD13R in group 1 obtained in Example 1 were used to extract the genomic DNA from the goatpox vaccine strain AV41 strain culture respectively (using the QIAGEN kit to operate according to the instructions to carry out the extraction of genomic DNA, The same below) and genomic DNA extracted from bovine nodular skin disease virus China/GD01/2020 strain culture were amplified by PCR to obtain the CDS17 gene fragment of sheep pox virus and the CDS21 gene fragment of bovine nodular skin disease virus respectively , the 25 ⁇ L reaction system used is shown in Table 1 below.
  • the PCR reaction conditions are: 95°C pre-denaturation for 3 minutes; 95°C denaturation for 10 seconds, 55°C annealing extension for 30 seconds, a total of 45 cycles; extension at 72°C for 7 minutes.
  • Table 1 PCR amplification system of CDS17 gene of sheeppox virus and CDS21 gene of bovine nodular skin disease virus
  • the purified and recovered CDS17 gene fragment and CDS21 gene fragment were respectively cloned into the pCE2-TA/Blunt-Zero vector vector (purchased from Invitrogen Company) to construct recombinant plasmids, and the positive recombinant plasmids were screened and sent to Shenggong Bioengineering (Shanghai) Incorporated sequencing to verify the success of the plasmid construction.
  • the sequencing results showed that two recombinant plasmids carrying the CDS17 gene fragment and the CDS21 gene fragment with the correct sequence were obtained, which were named pCE2-CDS17 and pCE2-CDS21 recombinant plasmids respectively.
  • CDS21 standard The sequencing results showed that two recombinant plasmids carrying the CDS17 gene fragment and the CDS21 gene fragment with the correct sequence were obtained, which were named pCE2-CDS17 and pCE2-CDS21 recombinant plasmids respectively
  • the single primer dual real-time fluorescent quantitative PCR amplification curve of CDS17 standard product (qPCR machine model is ABI) as shown in Figure 3, the standard product amplification curve is smooth " S " shape curve (positive), 10 in Fig. 3
  • concentration of the standard corresponding to the group of lines from left to right is 1 ⁇ 10 9 , 1 ⁇ 10 8 , 1 ⁇ 10 7 , 1 ⁇ 10 6 , 1 ⁇ 10 5 , 1 ⁇ 10 4 , 1 ⁇ 10 3 , 1 ⁇ 10 2 , 1 ⁇ 10 1 , 1 ⁇ 10 0 copies/ ⁇ L.
  • the dual real-time fluorescent quantitative PCR amplification curve of the CDS21 standard product (qPCR machine model is ABI) as shown in Figure 5, the standard product amplification curve is a smooth "S" shaped curve (positive), 10 in Figure 5
  • the concentration of the standard corresponding to the group of lines from left to right is 1 ⁇ 10 9 , 1 ⁇ 10 8 , 1 ⁇ 10 7 , 1 ⁇ 10 6 , 1 ⁇ 10 5 , 1 ⁇ 10 4 , 1 ⁇ 10 3 , 1 ⁇ 10 2 , 1 ⁇ 10 1 , 1 ⁇ 10 0 copies/ ⁇ L.
  • Embodiment 3 carry out single primer double real-time fluorescent quantitative PCR detection to sheep pox virus and bovine nodular skin disease virus
  • the genomic DNA (detection sample) extracted from the sample to be tested (such as virus culture or disease tissue obtained from sheep) is detected by a one-step single-primer double real-time fluorescent quantitative PCR detection method, wherein the method prepared in Example 2
  • the mixed solution of CDS17 standard substance and CDS21 standard substance is used as positive control, and enzyme-free water is used as negative control.
  • Qualitative detection then according to the Ct value and the standard curve in the above-mentioned embodiment 2, draw the copy number of the target gene of sheep pox virus and/or bovine nodular skin disease virus contained in the sample to be tested, realize the quantitative detection of virus .
  • the specific detection method includes the following steps:
  • test result is judged to be valid; otherwise, this test deemed invalid.
  • Ct value ⁇ 38 If there is an amplification curve in the FAM channel of the test sample and the Ct value is ⁇ 38, it is judged to be positive for bovine nodular skin disease virus nucleic acid, that is, the sample to be tested contains bovine nodular skin disease virus; Ct value ⁇ 40 is judged as positive, no amplification curve or retested Ct value > 40, judged as negative, that is, the sample to be tested does not contain bovine nodular skin disease virus.
  • the copy number of the corresponding viral target gene contained in the sample to be tested can be measured.
  • Example 4 Specificity, sensitivity and repeatability tests of single primer dual real-time fluorescent quantitative PCR detection method for differential detection of sheep pox virus and bovine nodular skin disease virus
  • the CDS17 standard and CDS21 standard were mixed at equal concentrations and diluted to 1 ⁇ 10 9 , 1 ⁇ 10 8 , 1 ⁇ 10 7 , 1 ⁇ 10 6 , 1 ⁇ 10 5 , 1 ⁇ 10 4 , 1 ⁇ 10 3 , 1 ⁇ 10 2 , 1 ⁇ 10 1 , 1 ⁇ 10 0 copies/ ⁇ L of the standard mixture, using different concentrations of the standard mixture as templates, obtained in Example 1
  • real-time fluorescent quantitative PCR detection was carried out, and the PCR reaction system and reaction conditions were referred to Example 2 to verify the detection sensitivity of the method of the present invention.
  • Figure 3 represents the single primer dual real-time fluorescent quantitative PCR amplification curve of the CDS17 standard product
  • Figure 5 represents the single primer dual real-time fluorescent quantitative PCR amplification curve of the CDS21 standard product
  • This Invention of a single-primer dual real-time fluorescent quantitative PCR detection method for the differential detection of sheep pox virus and bovine nodular skin disease virus can reach 1 copy/ ⁇ L for the detection of CDS17 and CDS21 target genes, that is, the differential detection of sheep pox virus and bovine nodule provided by the present invention
  • the sensitivity of the dual real-time fluorescence quantitative PCR method with single primer for dermatosis virus can reach 1 copy/ ⁇ L.
  • the CDS17 standard and CDS21 standard were mixed at equal concentrations and diluted to 1 ⁇ 10 9 , 1 ⁇ 10 8 , 1 ⁇ 10 7 , 1 ⁇ 10 6 , 1 ⁇ 10 5 , 1 ⁇ 10 4 , 1 ⁇ 10 3 , 1 ⁇ 10 2 , 1 ⁇ 10 1 , 1 ⁇ 10 0 copies/ ⁇ L, each gradient was repeated three times, and each gradient standard mixture was used as a template.
  • each gradient standard mixture was used as a template.
  • real-time fluorescent quantitative PCR detection was carried out, and the PCR reaction system and reaction conditions were referred to Example 2 to verify the detection repeatability of the method of the present invention.
  • the results show that the single-primer dual real-time fluorescent quantitative PCR detection method provided by the present invention for the identification and detection of sheeppox virus and bovine nodular skin disease virus has good repeatability, and the standard deviation of the number of cycles is at most no more than 0.5.
  • Example 5 Single-primer dual real-time fluorescent quantitative PCR kit for differential detection of sheep pox virus and bovine nodular skin disease virus
  • the real-time fluorescent quantitative PCR detection kit includes primers for double real-time fluorescent quantitative PCR detection of sheep pox virus and bovine nodular skin disease virus (embodiment LSD13F and LSD13R of group 1 in 1, this pair of primers is the universal PCR amplification primer for detecting goat poxvirus) and probe (LSDP and CaPvP of group 1 in embodiment 1, probe GaPvP and probe LSDVP
  • the 5' end can be labeled with different fluorescent reporter groups, and the 3' end can be labeled with a fluorescent quencher group.
  • the probe GaPvP is used to detect sheeppox virus, and the probe LSDVP is used to detect bovine nodular skin disease virus. ).
  • the reagents used for the 25-30 ⁇ L real-time fluorescent quantitative PCR reaction system include: forward primer LSD13F (10 ⁇ M) 0.25-0.75 ⁇ L, reverse primer LSD13R (10 ⁇ M) 0.25-0.75 ⁇ L, 0.50-1.00 ⁇ L of probe GaPvP (10 ⁇ M), 0.50-1.00 ⁇ L of probe LSDVP (10 ⁇ M), 1-3 ⁇ L of DNA template, 10-15 ⁇ L of qPCR Mix, and 25-30 ⁇ L of ddH 2 O.
  • a positive control substance and a negative control substance may also be included in the test kit, and the positive control substance is the genomic DNA of sheeppox virus and bovine nodular skin disease virus, and each positive control substance is a single package, and the negative control substance
  • the product is a reaction system that does not contain Clostridium perfringens types A, B, C, and D, such as H 2 O (double distilled water, sterile deionized water, etc.).
  • test kit can also include standard products: CDS17 standard product and CDS21 standard product, each standard product is a single package or an equal-concentration mixed package; When single-primer dual real-time fluorescent quantitative PCR detection, mix the equal concentrations of each standard in a single package.
  • the standard curve and instructions obtained in Example 2 can also be included in the kit.
  • the contents of the instructions include the PCR reaction program: 95°C pre-denaturation for 3 minutes; 40-45 times.
  • Embodiment 6 clinical sample detection
  • This embodiment utilizes the single-primer double real-time fluorescent quantitative PCR kit provided in Example 5 to identify and detect sheep pox virus and bovine nodular skin disease virus to five copies provided by Jinyu Baoling Biopharmaceutical Co., Ltd. Animal Routine Disease and Pathogen Research Center Clinical cattle and sheep tissues, virus cultures obtained from sheep disease materials and other samples were tested qualitatively and quantitatively by PCR, and the test results are shown in Table 3 below.
  • the T1 and T2 samples were positive for sheeppox virus, that is, contained sheeppox virus; the T3 sample was negative; the T4 sample was positive for bovine nodular skin disease virus, that is, contained bovine pox virus. Nodular skin disease virus; T5 sample is double positive for sheep pox virus and bovine nodular skin disease virus, that is, contains both sheep pox virus and bovine nodular skin disease virus.
  • the results in Table 3 also show the viruses contained in T1-T5 The copy number of the gene of interest. It can be seen that the kit provided by the present invention can be used to identify and detect sheeppox virus and bovine nodular skin disease virus, and can also realize quantitative detection while realizing qualitative detection. Therefore, the kit and method provided by the present invention can provide technical support and assistance for the identification and distinction of goatpoxviruses, epidemiological investigations, and virus prevention and control, and also contribute to the purification and screening of vaccine raw materials in vaccine production.

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Abstract

本发明公开一种鉴别羊痘病毒和牛结节性皮肤病病毒的单引物双重荧光检测试剂盒及其专用引物和探针，属于生物学技术领域。本发明提供的试剂盒包括用于检测山羊痘病毒属的通用PCR扩增引物、用于检测羊痘病毒的探针和用于检测牛结节性皮肤病病毒的探针，可以用于对山羊痘病毒属中的羊痘病毒和牛结节性皮肤病病毒直接进行定性鉴别检测，具有灵敏度高、特异性强、重复性好的优点，还可以实现准确定量的目的。本发明可以为山羊痘病毒属病毒的鉴别区分、流行病学调查、病毒防控，疫苗原料的净化与筛选等提供技术支持与帮助。

Description

鉴别羊痘病毒和牛结节性皮肤病病毒的单引物双重荧光检测试剂盒及其专用引物和探针 技术领域
本发明属于生物学技术领域，具体涉及一种鉴别羊痘病毒(包括山羊痘病毒和绵羊痘病毒)和牛结节性皮肤病病毒的单引物双重荧光检测试剂盒及其专用引物和探针，以及鉴别区分羊痘病毒和牛结节性皮肤病病毒的非疾病诊断方法。
背景技术
山羊痘病毒属(Capripox virus)属于痘病毒科、脊索动物痘病毒亚科，该属包括山羊痘病毒(GTPV)、绵羊痘病毒(SPPV)和牛结节性皮肤病病毒(LSDV)，该属病毒为DNA病毒，基因组全长约150kb，编码147个开放阅读框。
山羊痘病毒引发的疾病症状主要是皮肤丘疹-脓包型痘疹。山羊痘病毒可以感染所有品种、性别和年龄的山羊，其中羔羊最易感，感染率达100％，主要通过接触破损后的皮肤、呼吸道的吸入或者媒介的传播从而感染其他山羊，同群间传播迅速。绵羊痘，又名绵羊天花是由绵羊痘病毒引起的一种接触性传染病，其他的特征是在全身皮和粘膜上发生特异的痘疹。不同品种、性别、年龄的绵羊均易感，病羊发热并有较高的死亡率。牛结节皮肤病是由牛结节皮肤病病毒引起的以患牛发热，皮肤黏膜、器官表面广泛性结节为特征的急性、亚急性或慢性传染病。病牛体表淋巴结肿大，泌乳牛可发生乳房炎，公牛睾丸炎造成永久或暂时性不育等，严重时导致动物死亡。以上三种山羊痘病毒属病毒给奶牛和肉牛养殖业造成了严重的经济损失。
疫苗预防是防控病毒的主要手段，虽然山羊痘病毒、绵羊痘病毒和牛结节性皮肤病病毒间存在一定的交叉保护性，可以利用山羊痘疫苗作为牛结节皮肤病的预防措施，但是针对性的疫苗预防将会带来更好的防控效果。然而，山羊痘病毒属成员间基因组序列差异较小，这增加了对山羊痘病毒属成员的鉴别难度，例如对羊痘病毒(包括山羊痘病毒和绵羊痘病毒)与牛结节性皮肤病病毒之间的鉴别难度。目前虽然已有鉴别山羊痘病毒属成员的方法，例如专利文献CN112126717A(以下称文献1)公开一种鉴别羊痘病毒和牛结节性皮肤病病毒的双重荧光PCR引物、探针、方法及试剂盒，但是该文献1公开的方法并不能直接对羊痘病毒和牛结节性皮肤病病毒进行鉴别检测。
发明内容
针对现有技术中存在的一个或多个问题，本发明一个方面提供一种鉴别羊痘病毒和牛结节性皮肤病病毒的单引物双重荧光检测试剂盒，所述羊痘病毒包括山羊痘病毒和绵羊痘病毒，所述检测试剂盒包括：
用于检测山羊痘病毒属的通用PCR扩增引物组，该通用PCR扩增引物组包括SEQ ID NO:1所示的正向引物LSD13F和SEQ ID NO:2所示的反向引物LSD13R；
用于检测羊痘病毒的探针GaPvP，其核苷酸序列如SEQ ID NO:3所示；和
用于检测牛结节性皮肤病病毒的探针LSDVP，其核苷酸序列如SEQ ID NO:4所示。
在一些实施方式中，所述探针GaPvP和探针LSDVP的5’端标记有不同的荧光报告基团，3’端均标记有荧光淬灭基团。
在一些实施方式中，所述检测试剂盒还包括标准品：CDS17标准品和CDS21标准品；各标准品为单一包装或等浓度混合包装；使用所述试剂盒对羊痘病毒和牛结节性皮肤病病毒进行单引物双重实时荧光定量PCR检测时，将单一包装的标准品等浓度混合或直接使用混合包装的标准品。
本发明另一方面提供一种用于检测山羊痘病毒属的通用PCR扩增引物，其用于上述的检测试剂盒中，包括SEQ ID NO：1所示的正向引物LSD13F和SEQ ID NO:2所示的反向引物LSD13R。
本发明另一方面提供一种用于检测羊痘病毒的探针GaPvP，其用于上述的检测试剂盒中，该探针GaPvP的核苷酸序列如SEQ ID NO:3所示，其5’端标记有荧光报告基团，3’端标记有荧光淬灭基团。
本发明另一方面提供一种用于检测牛结节性皮肤病病毒的探针LSDVP，其用于上述的检测试剂盒中，该探针LSDVP的核苷酸序列如SEQ ID NO:4所示，其5’端标记有荧光报告基团，3’端标记有荧光淬灭基团。
本发明又一方面提供一种鉴别区分羊痘病毒和牛结节性皮肤病病毒的非疾病诊断方法，其包括以下步骤：
S1：提取待测样品的DNA；
S2：以步骤S1提取的DNA为模板，用上述提及的试剂盒中的通用PCR扩增引物组、探针GaPvP和探针LSDVP进行单引物双重荧光PCR扩增，获得扩增曲线；
S3：对步骤S2获得的扩增曲线进行分析，判定待测样品中是否含有羊痘病毒和/或牛结节性皮肤病病毒；
判定标准为：
若探针GaPvP对应的荧光通道出现扩增曲线，且Ct值≤38，则判定为所述待测样品中含有羊痘病毒；若Ct值>40，则判定为阴性，即所述待测样品中不含有羊痘病毒；若38＜Ct值≤40，再次检测为Ct值≤40，则判定为所述待测样品中含有羊痘病毒；若无扩增曲线或再 次检测的Ct值>40，则判定为所述待测样品中不含有羊痘病毒；
若探针LSDVP对应的荧光通道出现扩增曲线，且Ct值≤38，则判定为所述待测样品中含有牛结节性皮肤病病毒；若Ct值>40，则判定为阴性，即所述待测样品中不含有牛结节性皮肤病病毒；若38＜Ct值≤40，再次检测为Ct值≤40，则判定为所述待测样品中含有牛结节性皮肤病病毒；若无扩增曲线或再次检测的Ct值>40，则判定为所述待测样品中不含有牛结节性皮肤病病毒。
在一些实施方式中，步骤S2中进行双重荧光PCR扩增的25～30μL体系包含：正向引物LSD13F(10μM)0.25～0.75μL、反向引物LSD13R(10μM)0.25～0.75μL、探针GaPvP(10μM)0.50～1.00μL、探针LSDVP(10μM)0.50～1.00μL、DNA模板1～3μL、qPCR Mix10～15μL、ddH 2O补足25～30μL。
在一些实施方式中，步骤S2中进行双重荧光PCR扩增的程序包括：95℃预变性3min；95℃变性10～15s，55℃退火延伸30～34s，循环40-45次。
在一些实施方式中，所述探针GaPvP的5’端标记的荧光报告基团为VIC，3’端标记的淬灭基团为MGB；所述探针LSDVP的5’端标记的荧光报告基团为FAM，3’端标记的淬灭基团为MGB；所述判定标准为：
若VIC荧光通道出现扩增曲线，且Ct值≤38，则判定为所述待测样品中含有羊痘病毒；若Ct值>40，则所述待测样品中不含有羊痘病毒；若38＜Ct值≤40，再次检测为Ct值≤40则判定为所述待测样品中含有羊痘病毒；若无扩增曲线或再次检测的Ct值>40，则判定为所述待测样品中不含有羊痘病毒；
若FAM荧光通道出现扩增曲线，且Ct值≤38，则判定为所述待测样品中含有牛结节性皮肤病病毒；若Ct值>40，则所述待测样品中不含有牛结节性皮肤病病毒；若38＜Ct值≤40，再次检测为Ct值≤40则判定为所述待测样品中含有牛结节性皮肤病病毒；若无扩增曲线或再次检测的Ct值>40，则判定为所述待测样品中不含有牛结节性皮肤病病毒。
基于以上技术方案提供的鉴别羊痘病毒和牛结节性皮肤病病毒的单引物双重荧光检测试剂盒包括用于检测山羊痘病毒属的通用PCR引物、以及分别用于检测羊痘病毒(包括山羊痘病毒和绵羊痘病毒)和牛结节性皮肤病病毒的荧光探针，可以实现仅使用一对通用PCR引物和两条荧光探针便直接鉴别检测羊痘病毒和牛结节性皮肤病病毒的目的，相对于上述文献1公开的方法操作更加简便高效。经灵敏度试验结果表明，本发明提供的试剂盒和方法对羊痘病毒和牛结节性皮肤病病毒的检测灵敏度均可以达到1拷贝/μL，明显高于上述文献1鉴别检测羊痘病毒属和牛结节性皮肤病病毒的灵敏度(10拷贝/μL)。因此，本发明提供的鉴别羊痘病毒和牛结节性皮肤病病毒的方法和试剂盒能够一次操作直接鉴别区分羊痘病毒和牛结节性皮肤病病毒，并具有较高的灵敏度，可以为山羊痘病毒属病毒的鉴别区分、流行病学调查、病毒防控提供技术支持与帮助，也有助于疫苗生产中疫苗原料的净化与筛选。
附图说明
图1为山羊痘疫苗毒株AV41毒株(MH381810.1)CDS17基因和牛结节性皮肤病病毒China/GD01/2020毒株(MW355944.1)CDS21基因的部分序列比对示意图；
图2为使用实施例1中组1引物和探针对系列浓度的CDS17标准品和CDS21标准品的扩增曲线；
图3为使用实施例1中组1引物和探针在ABI qPCR仪上对系列浓度的CDS17标准品的扩增曲线；
图4为根据图3所示的扩增曲线绘制的标准曲线；
图5为使用实施例1中组1引物和探针在ABI qPCR仪上对系列浓度的CDS21标准品的扩增曲线；
图6为根据图5所示的扩增曲线绘制的标准曲线。
具体实施方式
针对现有技术中还没有同时鉴别检测羊痘病毒和牛结节性皮肤病病毒的方法的缺陷，本发明旨在提供一种鉴别检测羊痘病毒和牛结节性皮肤病病毒的单引物双重荧光PCR检测方法，以用于直接区分羊痘病毒和牛结节性皮肤病病毒，为山羊痘属病毒的疫苗防控等提供技术支持。基于此，本发明还提供了鉴别羊痘病毒和牛结节性皮肤病病毒的单引物双重荧光检测试剂盒及其专用引物和探针，以及检测方法。
以下结合具体实施例，对本发明进一步阐述。应当理解的是，具体实施例仅用于进一步说明本发明，而不是用于限制本发明的内容。
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)。
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。
实施例中涉及的序列均可以用已有技术合成。
实施例1：引物和探针的设计和优化
从NCBI的核酸数据库GenBank(http://www.ncbi.nlm.nih.gov)分别检索获得绵羊痘病毒、山羊痘病毒、牛结节性皮肤病病毒的基因序列，并对检索所获得的大量基因序列进行比对，发现将羊痘病毒(例如山羊痘疫苗毒株AV41毒株(MH381810.1))的CDS17基因(MH381810.1的第14911-15231位碱基序列，序列表中SEQ ID NO:5所示)与牛结节性皮肤病病毒(例如 China/GD01/2020毒株(MW355944.1))的CDS21基因(MW355944.1的第15264-15602位碱基序列，序列表中SEQ ID NO:6所示)相比较，在CDS17基因中存在一处12个碱基缺失的区域(如图1中标记的框中所示)，因此确定了羊痘病毒的CDS17基因和牛结节性皮肤病病毒的CDS21基因分别作为鉴别两者的靶标序列。基于确定的靶标序列，发明人利用引物探针设计软件设计了用于检测山羊痘病毒属的通用PCR引物，以及分别针对羊痘病毒和牛结节性皮肤病病毒的检测探针。经发明人验证，可以实现区分效果的用于检测山羊痘病毒属的通用PCR引物可以有多种组合，但是分别针对羊痘病毒和牛结节性皮肤病病毒且有区分效果的检测探针仅有唯一一组(SEQ ID NO:3所示的针对羊痘病毒的探针GaPvP(5’-AG-----------GC-3’)和SEQ ID NO:4所示的针对牛结节性皮肤病病毒的探针LSDVP(5’-TG-----------AA-3’))，该特别设计的两个探针的4个主要差异碱基必须均匀分布在探针的5’端和3’端，才能够实现区分鉴别效果，而该位置的其他结构形式的探针(例如主要差异碱基仅分布在探针的5’端或3’端，或者不均匀分布在探针的两端(例如5’端存在3个差异碱基，3’端存在1个差异碱基等))都会导致探针丧失区分羊痘病毒和牛结节性皮肤病病毒的能力。
从基于以上确定的靶标序列设计的众多通用PCR引物和探针中，本发明设计多组引物和探针组合并筛选出以下组1和组2，序列信息如下所示：
组1：
用于检测山羊痘病毒属的通用PCR引物包括：
LSD13F：5’-TCATTATCMTCACTACTAACRGTAT-3’(SEQ ID NO:1)；
LSD13R：5’-AAAGCAATATGAAAMMRGCA-3’(SEQ ID NO:2)；
针对羊痘病毒的探针GaPvP：5’-AGATGGCGATGGTGC-3’(SEQ ID NO:3)，其5’端标记的荧光报告基团为VIC，3’端标记的淬灭基团为MGB；
针对牛结节性皮肤病病毒的探针LSDVP：5’-TGATGGTGATGGTAA-3’(SEQ ID NO:4)，其5’端标记的荧光报告基团为FAM，3’端标记的淬灭基团为MGB。
组2：
用于检测山羊痘病毒属的通用PCR引物包括：
LSD13F1：5’-CATTATCATCACTACTAACGGTATC-3’(SEQ ID NO:7)；
LSD13R1：5’-AATATCTATTGAACGTGTTACATTG-3’(SEQ ID NO:8)；
SEQ ID NO:3所示的针对羊痘病毒的探针GaPvP；
SEQ ID NO:4所示的针对牛结节性皮肤病病毒的探针LSDVP。
利用上述组1和组2的引物和探针分别对阳性标准品(羊痘病毒和牛结节性皮肤病病毒阳性标准品等拷贝混合后的10倍稀释系列标准品溶液，其中各标准品的浓度分别为：10 6copies/μL、10 5copies/μL、10 4copies/μL、10 3copies/μL、10 2copies/μL、10 1copies/μL、10 0 copies/μL，以下实施例2中详细描述)和阴性对照进行单引物双重实时荧光PCR检测(PCR检测体系如下述实施例2中描述)，以从组1和组2中筛选出优选的引物和探针组合。图2示出了组1的引物和探针的扩增曲线(PCR机型为BioRad，其中标记有○的曲线为牛结节性皮肤病病毒标准品的扩增曲线，曲线从左到右分别表示为10 6copies/μL、10 5copies/μL、10 4copies/μL、10 3copies/μL、10 2copies/μL、10 1copies/μL、10 0copies/μL；另一组表示为羊痘病毒标准品的扩增曲线，曲线从左到右分别表示为10 6copies/μL、10 5copies/μL、10 4copies/μL、10 3copies/μL、10 2copies/μL、10 1copies/μL、10 0copies/μL)，可见组1的引物和探针对羊痘病毒和牛结节性皮肤病病毒标准品的检测最低限均能达到1拷贝/μL。而组2的引物和探针对羊痘病毒和牛结节性皮肤病病毒标准品的检测最低限均只能达到10 2copies/μL(未图示)。因此本发明优选组1的引物和探针作为鉴别检测羊痘病毒和牛结节性皮肤病病毒的单引物双重实时荧光定量PCR的引物和探针组合。
实施例2：羊痘病毒和牛结节性皮肤病病毒的单引物双重实时荧光定量PCR标准曲线的建立
该实施例采用实施例1获得的组1引物和探针组合对羊痘病毒和牛结节性皮肤病病毒进行单引物双重实时荧光定量PCR检测并建立标准曲线。
2.1、PCR扩增羊痘病毒的CDS17基因和牛结节性皮肤病病毒的CDS21基因
使用实施例1获得的组1中山羊痘病毒属通用引物LSD13F和LSD13R，分别对从山羊痘疫苗毒株AV41毒株培养物提取的基因组DNA(利用QIAGEN试剂盒按照说明书操作进行基因组DNA的提取，下同)和从牛结节性皮肤病病毒China/GD01/2020毒株培养物提取的基因组DNA进行PCR扩增，分别获得羊痘病毒的CDS17基因片段和牛结节性皮肤病病毒的CDS21基因片段，使用的25μL反应体系如下表1所示，PCR反应条件为：95℃预变性3min；95℃变性10s，55℃退火延伸30s，共计45个循环；延伸72℃7min。
表1：羊痘病毒的CDS17基因和牛结节性皮肤病病毒的CDS21基因的PCR扩增体系
Figure PCTCN2022121193-appb-000001
2.2、标准品制备
将纯化回收的CDS17基因片段和CDS21基因片段，分别克隆至pCE2-TA/Blunt-Zero vector载体(购自Invitrogen公司)中，构建成重组质粒，并筛选阳性重组质粒送生工生物工程(上海)股份有限公司测序，验证质粒构建是否成功。测序结果表明获得了序列正确的携 带CDS17基因片段和CDS21基因片段的2个重组质粒，分别命名为pCE2-CDS17和pCE2-CDS21重组质粒，提取重组质粒DNA即标准品，分别命名为CDS17标准品和CDS21标准品。
2.3、实时荧光定量PCR标准曲线的建立
对上述提取的CDS17标准品和CDS21标准品，分别用Nanodrop测定浓度，并计算各标准品的拷贝数后等浓度混合，按照10倍梯度稀释至1×10 9、1×10 8、1×10 7、1×10 6、1×10 5、1×10 4、1×10 3、1×10 2、1×10 1、1×10 0copies/μL的标准品混合液，以不同浓度的标准品混合液作为模板，按照下表2中反应体系(27μL)，在实施例1中组1中的引物和探针的引导下进行单引物双重实时荧光定量PCR检测，PCR反应条件包括：qPCR仪机型为ABI时为：95℃预变性3min；95℃变性15s，55℃退火延伸34s，共计45个循环。qPCR仪机型为BioRad时为：95℃预变性3min；95℃变性10s，55℃退火延伸30s，共计45个循环。
表2：单引物双重实时荧光定量PCR反应体系
Figure PCTCN2022121193-appb-000002
CDS17标准品的单引物双重实时荧光定量PCR扩增曲线(qPCR仪机型为ABI)如图3所示，标准品扩增曲线为平滑的“S”形曲线(阳性)，图3中的10组线条从左向右对应的标准品浓度分别为1×10 9、1×10 8、1×10 7、1×10 6、1×10 5、1×10 4、1×10 3、1×10 2、1×10 1、1×10 0copies/μL。检测结束后，以各标准品的浓度(X轴)对其相应Ct值(Y轴)作图，绘制标准曲线，结果如图4所示，相关系数R 2＝0.973，误差较小，标准曲线可用。CDS21标准品的单引物双重实时荧光定量PCR扩增曲线(qPCR仪机型为ABI)如图5所示，标准品扩增曲线为平滑的“S”形曲线(阳性)，图5中的10组线条从左向右对应的标准品浓度分别为1×10 9、1×10 8、1×10 7、1×10 6、1×10 5、1×10 4、1×10 3、1×10 2、1×10 1、1×10 0copies/μL。检测结束后，以各标准品的浓度(X轴)对其相应Ct值(Y轴)作图，绘制标准曲线，结果如图6所示，相关系数R 2＝0.987，误差较小，标准曲线可用。
实施例3、对羊痘病毒和牛结节性皮肤病病毒进行单引物双重实时荧光定量PCR检测
用一步法单引物双重实时荧光定量PCR检测方法对从待测样品(例如病毒培养物或获自羊的病料组织)中提取的基因组DNA(检测样品)进行检测，其中以实施例2制备的CDS17 标准品和CDS21标准品的混合液作为阳性对照，以无酶水为阴性对照，根据实时荧光定量PCR检测结果，对待测样品中是否含有羊痘病毒和/或牛结节性皮肤病病毒进行定性检测，再根据Ct值和上述实施例2中的标准曲线，得出待测样品中所含羊痘病毒和/或牛结节性皮肤病病毒的目的基因的拷贝数，实现病毒的定量检测。
具体检测方法包括以下步骤：
1)提取待测样品的基因组DNA，以提取的基因组DNA为模板，在引物(实施例1中组1的LSD13F、LSD13R)和探针(实施例1中组1的LSDP和CaPvP)的引导下进行单引物双重实时荧光定量PCR检测，27μL实时荧光定量PCR的检测体系如上表2所示，实时荧光定量PCR(qPCR仪机型为BioRad)反应条件为：95℃预变性3min；95℃变性10s，55℃退火延伸30s，共计45个循环。在每个循环的退火结束时进行荧光信号检测，牛结节性皮肤病病毒选择FAM通道和羊痘病毒选择VIC通道，获得扩增曲线。
2)用得到的扩增曲线的Ct值或荧光信号的变化实现对羊痘病毒和牛结节性皮肤病病毒的定性检测，判定标准为：
2.1、试验有效性判定
两个阳性对照在FAM和VIC通道均有典型扩增曲线且Ct值≤40，且阴性对照在FAM和VIC通道均无Ct值、无扩增曲线，则判试验结果有效；否则，此次试验视为无效。
2.2、待测样品判定
若检测样品FAM通道出现扩增曲线且Ct值≤38，判定为牛结节皮肤病病毒核酸阳性，即待测样品中含有牛结节皮肤病病毒；若38＜Ct值≤40，再次检测为Ct值≤40则判为阳性，无扩增曲线或再次检测的Ct值>40，判为阴性，即待测样品中不含有牛结节皮肤病病毒。若检测样品VIC通道出现扩增曲线且Ct值≤38，判定为羊痘病毒核酸阳性，即待测样品中含有羊痘病毒；若38＜Ct值≤40，再次检测为Ct值≤40则判为阳性，无扩增曲线或再次检测的Ct值>40，判为阴性，即待测样品中不含有羊痘病毒。无论是FAM通道，还是VIC通道，若Ct值>40，均判定为阴性，即待测样品中不含有牛结节皮肤病病毒和/或羊痘病毒。
随后可根据扩增曲线的Ct值和实施例2中确定的相应标准曲线，测得待测样品中所含的对应病毒目的基因的拷贝数。
实施例4、鉴别检测羊痘病毒和牛结节性皮肤病病毒的单引物双重实时荧光定量PCR检测方法的特异性、灵敏度和重复性试验
4.1、特异性试验
按照QIAGEN试剂盒说明书操作提取羊链球菌、羊丝状支原体、牛多杀性巴氏杆菌、传染性牛鼻气管炎病毒、牛溶血性曼氏杆菌、牛大肠杆菌(申请人实验室保存)的基因组DNA作为检测样品，以实施例2制备的CDS17标准品和CDS21标准品的混合液作为阳性对照，以无酶水为阴性对照，在本发明实施例1获得的组1所示的引物和探针的引导下进行单引物 双重实时荧光定量PCR检测，PCR反应体系及反应条件参照实施例2，以验证本发明方法的特异性。
结果显示，只有阳性对照出现扩增曲线，而各检测样品所对应的通道均没有扩增曲线，表明各检测样品均为阴性，证明本发明提供的方法特异性良好。
4.2、灵敏度试验
按照实施例2所述，将CDS17标准品和CDS21标准品等浓度混合，按照10倍梯度稀释至1×10 9、1×10 8、1×10 7、1×10 6、1×10 5、1×10 4、1×10 3、1×10 2、1×10 1、1×10 0copies/μL的标准品混合液，以不同浓度的标准品混合液作为模板，在实施例1获得的组1的引物和探针的引导下进行实时荧光定量PCR检测，PCR反应体系及反应条件参照实施例2，以验证本发明方法的检测灵敏度。
检测结果如图3和图5所示，其中图3表示CDS17标准品的单引物双重实时荧光定量PCR扩增曲线，图5表示CDS21标准品的单引物双重实时荧光定量PCR扩增曲线，可见本发明鉴别检测羊痘病毒和牛结节性皮肤病病毒的单引物双重实时荧光定量PCR检测方法检测CDS17和CDS21目的基因均能达到1拷贝/μL，即本发明提供的鉴别检测羊痘病毒和牛结节性皮肤病病毒的单引物双重实时荧光定量PCR方法的灵敏度可达到1拷贝/μL。
4.3、重复性试验
按照实施例2所述，将CDS17标准品和CDS21标准品等浓度混合，按照10倍梯度稀释至1×10 9、1×10 8、1×10 7、1×10 6、1×10 5、1×10 4、1×10 3、1×10 2、1×10 1、1×10 0copies/μL，每个梯度进行3个重复，以每个梯度标准品混合液作为模板，在实施例1获得的组1的引物和探针的引导下进行实时荧光定量PCR检测，PCR反应体系及反应条件参照实施例2，以验证本发明方法的检测重复性。
结果显示，本发明提供的鉴别检测羊痘病毒和牛结节性皮肤病病毒的单引物双重实时荧光定量PCR检测方法的重复性较好，循环数标准偏差相差最高不超过0.5。
实施例5：鉴别检测羊痘病毒和牛结节性皮肤病病毒的单引物双重实时荧光定量PCR试剂盒
基于实施例1和实施例2，本发明所提供的实时荧光定量PCR检测试剂盒，包括用于对羊痘病毒和牛结节性皮肤病病毒进行单引物双重实时荧光定量PCR检测的引物(实施例1中组1的LSD13F和LSD13R，该对引物为用于检测山羊痘病毒属的通用PCR扩增引物)和探针(实施例1中组1的LSDP和CaPvP，探针GaPvP和探针LSDVP的5’端可标记有不同的荧光报告基团，3’端均可标记有荧光淬灭基团，其中探针GaPvP用于检测羊痘病毒，探针LSDVP用于检测牛结节性皮肤病病毒)。
具体来讲，该试剂盒在使用时，用于25～30μL实时荧光定量PCR反应体系的试剂包含：正向引物LSD13F(10μM)0.25～0.75μL、反向引物LSD13R(10μM)0.25～0.75μL、探针 GaPvP(10μM)0.50～1.00μL、探针LSDVP(10μM)0.50～1.00μL、DNA模板1～3μL、qPCR Mix 10～15μL、ddH 2O补足25～30μL。
为方便检测，试剂盒中还可包括阳性对照品和阴性对照品，所述阳性对照品为羊痘病毒和牛结节性皮肤病病毒的基因组DNA，各阳性对照品为单一包装，所述阴性对照品为不含A、B、C、D型产气荚膜梭菌的反应体系，如H 2O(双蒸水、无菌去离子水等)。
为方便检测，试剂盒中还可包括标准品：CDS17标准品和CDS21标准品，各标准品为单一包装或等浓度混合包装；使用所述试剂盒对羊痘病毒和牛结节性皮肤病病毒进行单引物双重实时荧光定量PCR检测时，将单一包装的各标准品等浓度混合。
为方便检测，试剂盒中还可包括实施例2获得的标准曲线和说明书，该说明书内容包括PCR反应程序：95℃预变性3min；95℃变性10～15s，55℃退火延伸30～34s，循环40-45次。
实施例6、临床样品检测
该实施例利用实施例5提供的鉴别检测羊痘病毒和牛结节性皮肤病病毒的单引物双重实时荧光定量PCR试剂盒对金宇保灵生物药品有限公司动物常规疫病与病原研究中心提供的5份临床牛羊组织、获自羊病料组织的病毒培养物等样品进行PCR定性和定量检测，检测结果如下表3所示。
根据表3结果，在检测的5份临床样品中，T1和T2样品为羊痘病毒阳性，即含有羊痘病毒；T3样品为阴性；T4样品为牛结节性皮肤病病毒阳性，即含有牛结节性皮肤病病毒；T5样品为羊痘病毒和牛结节性皮肤病病毒双阳性，即同时含有羊痘病毒和牛结节性皮肤病病毒，表3结果同时显示了T1-T5中所含病毒目的基因的拷贝数。可见本发明提供的试剂盒可以用于鉴别检测羊痘病毒和牛结节性皮肤病病毒，并且在实现定性检测的同时，还可以实现定量检测。因此，本发明提供的试剂盒和方法可以为山羊痘病毒属病毒的鉴别区分、流行病学调查、病毒防控提供技术支持与帮助，也有助于疫苗生产中疫苗原料的净化与筛选。
表3：5份临床样品的单引物双重实时荧光定量PCR检测结果
Figure PCTCN2022121193-appb-000003
最后应说明的是：以上所述仅为本发明的优选实施例，并不用于限制本发明。尽管参照 前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种鉴别羊痘病毒和牛结节性皮肤病病毒的单引物双重荧光检测试剂盒，所述羊痘病毒包括山羊痘病毒和绵羊痘病毒，所述检测试剂盒包括：
    用于检测山羊痘病毒属的通用PCR扩增引物组，该通用PCR扩增引物组包括SEQ ID NO:1所示的正向引物LSD13F和SEQ ID NO:2所示的反向引物LSD13R；
    用于检测羊痘病毒的探针GaPvP，其核苷酸序列如SEQ ID NO:3所示；和
    用于检测牛结节性皮肤病病毒的探针LSDVP，其核苷酸序列如SEQ ID NO:4所示。
  2. 根据权利要求1所述的检测试剂盒，其中所述探针GaPvP和探针LSDVP的5’端标记有不同的荧光报告基团，3’端均标记有荧光淬灭基团。
  3. 根据权利要求1或2所述的检测试剂盒，其还包括标准品：CDS17标准品和CDS21标准品；各标准品为单一包装或等浓度混合包装；使用所述试剂盒对羊痘病毒和牛结节性皮肤病病毒进行单引物双重实时荧光定量PCR检测时，将单一包装的标准品等浓度混合或直接使用混合包装的标准品。
  4. 一种用于检测山羊痘病毒属的通用PCR扩增引物，其用于权利要求1-3中任一项所述的检测试剂盒中，包括SEQ ID NO：1所示的正向引物LSD13F和SEQ ID NO:2所示的反向引物LSD13R。
  5. 一种用于检测羊痘病毒的探针GaPvP，其用于权利要求1-3中任一项所述的检测试剂盒中，该探针GaPvP的核苷酸序列如SEQ ID NO:3所示，其5’端标记有荧光报告基团，3’端标记有荧光淬灭基团。
  6. 一种用于检测牛结节性皮肤病病毒的探针LSDVP，其用于权利要求1-3中任一项所述的检测试剂盒中，该探针LSDVP的核苷酸序列如SEQ ID NO:4所示，其5’端标记有荧光报告基团，3’端标记有荧光淬灭基团。
  7. 一种鉴别区分羊痘病毒和牛结节性皮肤病病毒的非疾病诊断方法，其包括以下步骤：
    S1：提取待测样品的DNA；
    S2：以步骤S1提取的DNA为模板，用权利要求1-3中任一项提及的试剂盒中的通用PCR扩增引物组、探针GaPvP和探针LSDVP进行单引物双重荧光PCR扩增，获得扩增曲线；
    S3：对步骤S2获得的扩增曲线进行分析，判定待测样品中是否含有羊痘病毒和/或牛结 节性皮肤病病毒；
    判定标准为：
    若探针GaPvP对应的荧光通道出现扩增曲线，且Ct值≤38，则判定为所述待测样品中含有羊痘病毒；若Ct值>40，则判定为阴性，即所述待测样品中不含有羊痘病毒；若38＜Ct值≤40，再次检测为Ct值≤40，则判定为所述待测样品中含有羊痘病毒；若无扩增曲线或再次检测的Ct值>40，则判定为所述待测样品中不含有羊痘病毒；
    若探针LSDVP对应的荧光通道出现扩增曲线，且Ct值≤38，则判定为所述待测样品中含有牛结节性皮肤病病毒；若Ct值>40，则判定为阴性，即所述待测样品中不含有牛结节性皮肤病病毒；若38＜Ct值≤40，再次检测为Ct值≤40，则判定为所述待测样品中含有牛结节性皮肤病病毒；若无扩增曲线或再次检测的Ct值>40，则判定为所述待测样品中不含有牛结节性皮肤病病毒。
  8. 根据权利要求7所述的方法，其中步骤S2中进行双重荧光PCR扩增的25～30μL体系包含：正向引物LSD13F(10μM)0.25～0.75μL、反向引物LSD13R(10μM)0.25～0.75μL、探针GaPvP(10μM)0.50～1.00μL、探针LSDVP(10μM)0.50～1.00μL、DNA模板1～3μL、qPCR Mix 10～15μL、ddH 2O补足25～30μL。
  9. 根据权利要求7或8所述的方法，其中步骤S2中进行双重荧光PCR扩增的程序包括：95℃预变性3min；95℃变性10～15s，55℃退火延伸30～34s，循环40-45次。
  10. 根据权利要求7-9中任一项所述的方法，其中所述探针GaPvP的5’端标记的荧光报告基团为VIC，3’端标记的淬灭基团为MGB；所述探针LSDVP的5’端标记的荧光报告基团为FAM，3’端标记的淬灭基团为MGB；所述判定标准为：
    若VIC荧光通道出现扩增曲线，且Ct值≤38，则判定为所述待测样品中含有羊痘病毒；若Ct值>40，则所述待测样品中不含有羊痘病毒；若38＜Ct值≤40，再次检测为Ct值≤40则判定为所述待测样品中含有羊痘病毒；若无扩增曲线或再次检测的Ct值>40，则判定为所述待测样品中不含有羊痘病毒；
    若FAM荧光通道出现扩增曲线，且Ct值≤38，则判定为所述待测样品中含有牛结节性皮肤病病毒；若Ct值>40，则所述待测样品中不含有牛结节性皮肤病病毒；若38＜Ct值≤40，再次检测为Ct值≤40则判定为所述待测样品中含有牛结节性皮肤病病毒；若无扩增曲线或再次检测的Ct值>40，则判定为所述待测样品中不含有牛结节性皮肤病病毒。
PCT/CN2022/121193 2021-11-10 2022-10-08 鉴别羊痘病毒和牛结节性皮肤病病毒的单引物双重荧光检测试剂盒及其专用引物和探针 WO2023082865A1 (zh)

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