Primer group, primer probe and fluorescent quantitative qPCR (quantitative polymerase chain reaction) detection method for detecting camel contagious pustular dermatitis virus

Abstract

The invention relates to the technical field of virus detection, in particular to a primer group, a primer probe and a fluorescent quantitative qPCR (quantitative polymerase chain reaction) detection method for detecting camel contagious pustular dermatitis virus. The primer group comprises an upstream primer and a downstream primer which are used for detecting a CCEV-99 gene segment in camel contagious pustular dermatitis virus DNA, the nucleotide sequence of the CCEV-99 gene segment is as shown in SEQ ID NO.1, and the nucleotide sequence of a primer probe is as shown in SEQ ID NO.4. The method has good specificity, sensitivity, stability and repeatability, is suitable for detecting a solution containing the camel contagious pustular dermatitis virus and a recombinant plasmid containing a CCEV-99 gene segment in a laboratory, and lays a foundation for development of related vaccines and preparations.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of virus detection, in particular to a primer set, a primer probe and a fluorescent quantitative qPCR detection method for detecting camel contagious ecthyma virus. BACKGROUND

[0002] Camel contagious ecthyma virus (CCEV) can cause camel contagious ecthyma, which is an enveloped double-stranded DNA virus belonging to Parapoxvirus of Orthopoxvirus of Poxviridae. Camel contagious ecthyma caused by CCEV was first discovered in Kazakhstan in 1968, and then confirmed in some regions of the Middle East, Asia and Africa.

[0003] With the continuous expansion of global camel breeding scale and the frequent trade of camel-related livestock products, and there is no commercial vaccine for prevention and control, and no specific drug for treatment, CCEV has been widely prevalent in North Africa and the Middle East, causing serious economic losses. The virus can infect camels of different ages and genders, but mainly harms young camels. Infected young camels have difficulty eating and insufficient intake of nutrients, which affects their growth and development, and severe cases can die due to secondary infection, so the mortality rate of young camels is significantly higher than that of adult camels. The disease is mainly transmitted through contact, so once a camel in a breeding group is infected with the disease, it is easy to spread to other camels quickly, causing greater economic losses.

[0004] In addition, the virus can be transmitted to humans, causing single or multiple skin lesions on the fingers, hands or forearms

[0005] At present, there is little research on the pathogenic mechanism of CCEV, but as a virus of the same genus as ORFV, they have high similarity in pathogenic mechanism, etc. ORFV mainly binds to receptors such as sulfated heparan sulfate proteoglycan on the surface of host cells through the glycoprotein on the surface of the virus.

[0006] The epidemic disease caused by camel contagious ecthyma virus has caused great harm in the field of camel breeding, seriously restricting the healthy development of the camel industry. Timely and accurate detection of CCEV is the key to preventing and controlling camel contagious ecthyma. Early diagnosis can help breeders quickly take isolation, treatment and control measures, effectively prevent the spread of the epidemic and reduce economic losses.

[0007] However, there are many viruses similar to CCEV, such as ORFV, camel pox virus (CMPV), sheep pox virus (SPPV), goat pox virus (GTPV) and lumpy skin disease virus (LSDV). ORFV and CCEV are members of the subgenus of parapoxvirus; CMPV is a member of the orthopoxvirus genus; GTPV, SPPV and LSDV are members of the genus of capripoxvirus. These viruses show both similarities and significant differences in the range of infected hosts, the site of disease and clinical symptoms. This makes it difficult for the prior art to accurately and sensitively detect CCEV.

[0008] In addition, the difficulty in accurately detecting CCEV also limits the development and research of related vaccines and preparations. SUMMARY

[0009] The technical problem to be solved by the present application is to provide a primer set, a primer probe and a fluorescent quantitative qPCR detection method for detecting camel contagious pustular disease virus.

[0010] The technical solution of the present application to solve the above technical problem is as follows:

[0011] The present application provides a primer set for detecting camel contagious pustular disease virus, which comprises an upstream primer and a downstream primer for detecting a CCEV-99 gene fragment in the DNA of camel contagious pustular disease virus, and the nucleotide sequence of the CCEV-99 gene fragment is shown in SEQ ID NO. 1.

[0012] On the basis of the above technical solution, the present application can also be improved as follows.

[0013] Further, the nucleotide sequence of the upstream primer is shown in SEQ ID NO. 2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 3.

[0014] The present application also provides a primer probe for detecting camel contagious pustular disease virus, and the nucleotide sequence of the qPCR probe is shown in SEQ ID NO. 4.

[0015] The present application also provides a recombinant plasmid containing a CCEV-99 gene fragment shown in SEQ ID NO. 1.

[0016] The present application also provides a kit for detecting camel contagious pustular disease virus, which comprises the primer set as described above, and the primer probe as described above; and further comprises a standard plasmid solution containing the recombinant plasmid as described above.

[0017] The application also provides a fluorescent quantitative qPCR detection method for camel contagious ecthyma virus, which detects a to-be-detected sample by using the kit.

[0018] Further, the method comprises the following steps:

[0019] A to-be-detected sample is obtained, and viral DNA in the to-be-detected sample is extracted;

[0020] The viral DNA is used as a template, and a fluorescent quantitative PCR is performed by using the qPCR primer group and the qPCR primer probe, so that a Ct value of the to-be-detected sample is obtained;

[0021] According to the Ct value of the to-be-detected sample and a standard curve, a fluorescent quantitative result of the camel contagious ecthyma virus in the to-be-detected sample is obtained.

[0022] Further, the reaction procedure of the fluorescent quantitative PCR is as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 5 s; 60℃ annealing and elongation for 30 s, 40 cycles.

[0023] Further, the reaction system of the PCR fluorescent quantitative PCR is as follows:

[0024] System components Volume / μM 2x SuperReal PreMix (Probe) 10 Upstream primer 1 Downstream primer 1 Probe 0.4 DNA template 1 ddH2O 6.6

[0025] Further, the standard curve is established in the following manner: the Ct values of standard plasmid solutions with multiple concentration gradients are detected respectively, the copy number of the standard plasmid is calculated according to the concentration of the standard plasmid solution, and a standard curve of the copy number and the Ct value is obtained.

[0026] The application has the following beneficial effects:

[0027] The application takes the CCEV-99 gene fragment in the camel contagious ecthyma virus genome as a detection target, designs primers and probes, and establishes a qPCR detection method, so that the method has good specificity, sensitivity, stability and repeatability. Meanwhile, the detection method of the application is also applicable to detection of solutions containing camel contagious ecthyma virus and recombinant plasmids containing the CCEV-99 gene fragment in a laboratory, and lays a foundation for development of related vaccines and preparations. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is an electrophoretogram obtained in Example 1 of the application;

[0029] Figure 2 It is a double enzyme digestion electrophoretogram obtained in Example 3 of the application for pET32a-99;

[0030] Figure 3Figure of fluorescence intensity curve for different final concentrations of primer probe combination in Example 5 of the present application;

[0031] Figure 4 Figure of fluorescence intensity curve for fluorescence quantitative PCR under optimized reaction conditions in Example 5 of the present application;

[0032] Figure 5 Standard curve obtained in Example 5 of the present application;

[0033] Figure 6 Fluorescence quantitative qPCR amplification curve of each virus obtained in Example 6 of the present application;

[0034] Figure 7 Nucleic acid agarose electrophoresis map obtained by general PCR method in Example 7 of the present application;

[0035] Figure 8 PCR amplification curve of fluorescence quantitative qPCR method in Example 7 of the present application;

[0036] Figure 9 Electrophoresis map of 10 samples detected by fluorescence quantitative qPCR method in Example 8 of the present application. DETAILED DESCRIPTION

[0037] The principles and features of the present application are described below in conjunction with the accompanying drawings, and the examples are only used to explain the present application and not to limit the scope of the present application.

[0038] The primer set for detecting camel infectious pustular disease virus of the present application comprises an upstream primer and a downstream primer for detecting a CCEV-99 gene fragment in camel infectious pustular disease virus DNA, and the nucleotide sequence of the CCEV-99 gene fragment is shown in SEQ ID NO. 1.

[0039] The present application takes the CCEV-99 gene fragment in the camel infectious pustular disease virus genome as a detection target, designs primers and probes, and establishes a fluorescence quantitative qPCR detection method, so that the method has good specificity, sensitivity, stability and repeatability. In the detection of clinical samples for non-therapeutic purposes, it also shows good practicability. At the same time, the detection method of the present application is also suitable for detecting solutions containing camel infectious pustular disease virus and recombinant plasmids containing CCEV-99 gene fragments in the laboratory, and lays a foundation for the development of related vaccines and preparations.

[0040] Preferably, the nucleotide sequence of the upstream primer is shown in SEQ ID NO. 2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 3.

[0041] Preferably, the nucleotide sequence of the qPCR probe is shown as SEQ ID NO. 4, and preferably, the 5' end of the probe is labeled with FAM and the 3' end is labeled with MGB.

[0042] The recombinant plasmid of the application, wherein the CCEV-99 gene fragment is shown as SEQ ID NO. 1.

[0043] The kit for detecting camel contagious pustular disease virus of the application comprises the primer set as described above, and the primer probe as described above; and further comprises a standard plasmid solution, wherein the standard plasmid solution contains the recombinant plasmid as described above.

[0044] The fluorescence quantitative qPCR detection method of the camel contagious pustular disease virus of the application uses the kit as described above to detect the sample to be tested.

[0045] Preferably, the method comprises the following steps:

[0046] The sample to be tested is obtained, and the viral DNA in the sample to be tested is extracted.

[0047] The viral DNA is used as a template, and the fluorescence quantitative PCR is performed using the qPCR primer set and the qPCR primer probe to obtain the Ct value of the sample to be tested.

[0048] According to the Ct value of the sample to be tested and the standard curve, the fluorescence quantitative result of the camel contagious pustular disease virus in the sample to be tested is obtained.

[0049] Preferably, the reaction program of the fluorescence quantitative PCR is as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 5 s; 60℃ annealing and extension for 30 s, 40 cycles.

[0050] Preferably, the reaction system of the PCR fluorescence quantitative PCR is as follows:

[0051] System components Volume / μM 2x SuperReal PreMix (Probe) 10 Upstream primer 1 Downstream primer 1 Probe 0.4 DNA template 1 ddH2O 6.6

[0052] Preferably, the standard curve is established in the following manner: the Ct values of a plurality of concentration gradient standard plasmid solutions are detected respectively, the copy number of the standard plasmid is calculated according to the concentration of the standard plasmid solution, and the standard curve of the copy number and the Ct value is obtained.

[0053] The application further provides a CCEV-99 gene fragment detection method of the camel contagious pustular disease virus, which uses the above-described primer, probe and fluorescence quantitative qPCR detection method to detect whether the sample to be tested contains the CCEV-99 gene fragment and the content thereof. In the method, the sample to be tested can be a recombinant plasmid solution containing the CCEV-99 gene fragment.

[0054] The CCEV nucleic acid samples used in the following examples were laboratory-previously extracted and verified samples; ORFV was preserved in the laboratory of the Herbivore Bacterial Disease Team of the Lanzhou Animal Husbandry Institute of the Chinese Academy of Agricultural Sciences; LSDV, MAV, and WR were provided by Professor Peng Cheng of the College of Animal Medicine, China Agricultural University.

[0055] The E. coli DH5a competent cells and pET-28a empty plasmid used in the following examples were preserved in the laboratory, and the main reagents and main instruments are shown in Tables 1 and 2.

[0056] Table 1 Main reagents

[0057]

[0058]

[0059] Table 2 Main instruments

[0060]

[0061] Example 1 Extraction of viral genomes

[0062] In this example, the CCEV virus in the obtained tissue sample was extracted, and the specific experimental steps are as follows:

[0063] Tissue sample processing: Take a small amount of 0.5 g pathological tissue, grind into powder in liquid nitrogen, add a small amount of sterile 0.01 mM PBS and repeat freeze-thawing 3-5 times, centrifuge at 5,000 rpm for 5 min. Take 200 μL of supernatant for subsequent experiments. The pathological tissue in this example is specifically the lip papules or scabs of sick camels.

[0064] Add 20 μL of Proteinase K solution and mix well. Add 200 μL of buffer GB and mix well by inverting, place at 70°C for 10 min, and the solution should be clear and short centrifugation to remove water droplets on the inner wall of the tube cap. Add 200 μL of anhydrous ethanol and mix well by shaking for 15 s, at which time a flocculent precipitate may appear, and short centrifugation to remove water droplets on the inner wall of the tube cap.

[0065] Add the obtained solution and flocculent precipitate to an adsorption column CB3 (the adsorption column is placed in a collection tube) and centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and place the adsorption column CB3 back into the collection tube. Add 500 μL of buffer GD (check whether anhydrous ethanol has been added before use) to the adsorption column CB3, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and place the adsorption column CB3 into the collection tube. Add 600 μL of rinse solution PW (check whether anhydrous ethanol has been added before use) to the adsorption column CB3, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and place the adsorption column CB3 into the collection tube, and repeat the operation of this step.

[0066] Put the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 min, and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material.

[0067] Transfer the adsorption column CB3 into a clean centrifuge tube, and add 60 μL of elution buffer TE to the middle of the adsorption membrane. Place it at room temperature for 2 min, centrifuge at 12,000 rpm for 2 min, collect the solution into the centrifuge tube, and obtain the CCEV virus solution.

[0068] Extract the DNA in the CCEV virus solution and perform whole genome sequencing. The sequencing results are compared with the known German isolate CCEV strain. The DNA sequence of the virus solution has the highest homology with the German PCPV isolate (PP565896), which is greater than 96.1%, which proves that the virus solution is CCEV virus.

[0069] Screening and amplification of target genes in Example 2

[0070] Through physicochemical property analysis, a fragment in the sequence of CCEV is selected as a candidate target gene for the fluorescent quantitative qPCR detection method, which is CCEV-99.

[0071] The nucleotide sequence of CCEV-99 is shown in SEQ ID NO. 1, and CCEV-99 is located at positions 105235 to 103767 of the CCEV virus.

[0072] The predicted isoelectric point (pI) of CCEV-99 is 8.19, the instability index (II) is 30.93, and the GRAVY value is about -0.213, indicating that the protein as a whole tends to be slightly hydrophilic, relatively stable, has a high surface polarity, has no signal peptide, and no transmembrane structure, and may be a potential antigen epitope.

[0073] In this embodiment, the CCEV virus genome extracted in Example 1 is used as a template to amplify the CCEV-99 gene using amplification primers.

[0074] The design and synthesis of the amplification primers are as follows: for the CCEV-99 gene fragment in the CCEV genome, the SnapGene software (SnapGene 6.0.2) is used to design the primers, and Sac I and Hind III enzyme digestion sites are added before the upstream and downstream primers. The primers shown in Table 3 are synthesized by Xinjiang Youkang Biological Co., Ltd. The CCEV-99 gene is amplified using the primers, and the size of the CCEV-99 gene fragment is 1557 bp.

[0075] The amplification primers are shown in Table 3, the PCR amplification reaction system is shown in Table 4, and the PCR amplification procedure of the CCEV-99 gene is shown in Table 5, respectively.

[0076] Table 3 Primer sequence

[0077]

[0078] Table 4 PCR amplification reaction system

[0079]

[0080] Table 5 PCR amplification procedure of CCEV-99 gene

[0081]

[0082] After the amplification is completed, the CCEV-99 amplification product is recovered and identified, and the specific experimental steps are as follows:

[0083] (1) After the PCR product is subjected to agarose gel electrophoresis, it is verified whether the size of the target fragment is correct, and the single target DNA band is cut from the agarose gel (as much as possible to remove the excess part) and placed in a clean centrifuge tube, and the weight is weighed.

[0084] (2) 3 times the volume of gel solution PE is added to the gel block (if the gel weighs 0.1 g, the volume can be considered as 100 μL, and 300 μL of gel solution PE is added) and the gel is dissolved at room temperature for 10 min, and the centrifuge tube is constantly and gently turned upside down to ensure that the gel block is fully dissolved.

[0085] (3) The solution obtained in the previous step is added to an adsorption column CA5 (the adsorption column is placed in a collection tube), and is placed at room temperature for 2 min, centrifuged at 12,000 rpm for 45 s, and the waste liquid in the collection tube is discarded, and the adsorption column CA5 is placed in the collection tube.

[0086] (4) 600 μL of rinse solution PW is added to the adsorption column CA5, and is placed for 2 min, centrifuged at 12,000 rpm for 45 s, and the waste liquid in the collection tube is discarded, and the adsorption column CA5 is placed in the collection tube. Repeat step 4.

[0087] (5) The adsorption column CA5 is placed back into the collection tube, and is centrifuged at 12,000 rpm for 2 min to remove as much rinse solution as possible. The adsorption column CA5 is placed at room temperature for several minutes, and is completely dried to prevent residual rinse solution from affecting the next experiment.

[0088] (6) Put the adsorption column CA5 into a clean centrifuge tube, and add 30 μL elution buffer TB to the middle of the adsorption membrane. Let it stand at room temperature for 2 min. Centrifuge at 12,000 rpm for 2 min to collect the DNA solution. In order to improve the recovery of DNA, the solution obtained by centrifugation can be added back to the centrifugal adsorption column, and let it stand at room temperature for 2 min. Centrifuge at 12,000 rpm for 2 min, and collect the DNA solution into the centrifuge tube to complete the recovery.

[0089] The amplified fragments were identified, and the obtained nucleic acid agarose electrophoresis chart is shown in Figure 1 Figure 1 Bands 1 and 2 are the amplified CCEV-99 fragments, and "-" is the negative control. Figure 1 It can be seen that the CCEV-99 primer amplified fragment is 1557 bp, and the size of the amplified product is consistent with the expectation.

[0090] Example 3 Construction and verification of recombinant plasmid standard

[0091] In this embodiment, the CCEV-99 gene amplified in Example 2 is connected with an expression vector to construct a recombinant plasmid standard, and the specific process is as follows:

[0092] (1) Extraction of expression vector

[0093] Resuscitate the bacterial solution of pET-32a empty plasmid, and inoculate into LB liquid medium at a volume ratio of 1:100 (the working concentration of ampicillin sodium is 100 μg / mL). The resuscitated bacterial solution is used to extract the plasmid, and the steps are as follows:

[0094] Column equilibration step: add 500 μL of equilibration solution BL to the adsorption column CP3 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.

[0095] Take 3 mL of the bacterial solution of the overnight culture, and add it into a centrifuge tube. Use a conventional benchtop centrifuge to centrifuge at 12,000 rpm for 1 min, and try to aspirate the supernatant (when the bacterial solution is more, the bacterial precipitate can be collected into one centrifuge tube by multiple centrifugations).

[0096] Add 250 μL of solution P1 (please check whether RNase A has been added) to the centrifuge tube containing the bacterial precipitate, and use a pipette or vortex shaker to thoroughly suspend the bacterial precipitate.

[0097] Add 250 μL of solution P2 to the centrifuge tube, and gently turn it up and down for 6-8 times to fully lyse the bacteria.

[0098] ​Add 350 μL solution P3 to a centrifuge tube, immediately gently flip up and down 6-8 times, mix well, at this time a white flocculent precipitate will appear, centrifuge at 12,000 rpm for 10 min.

[0099] Transfer the supernatant collected in the previous step to the adsorption column CP3 with a pipette (the adsorption column is placed in the collection tube), try not to suck out the precipitate. Centrifuge at 12,000 rpm for 45 s, discard the waste liquid in the collection tube, and place the adsorption column CP3 in the collection tube.

[0100] Add 600 μL rinse solution PW to the adsorption column CP3 (check if anhydrous ethanol has been added first), centrifuge at 12,000 rpm for 45 s, discard the waste liquid in the collection tube, and place the adsorption column CP3 in the collection tube, repeat the operation steps.

[0101] Place the adsorption column CP3 in the collection tube, centrifuge at 12,000 rpm for 2 min, the purpose is to remove the residual rinse solution in the adsorption column. After centrifugation, open the adsorption column CP3, and place it at room temperature for a few minutes to completely dry the residual rinse solution in the adsorption material.

[0102] Place the adsorption column CP3 in a clean centrifuge tube, add 50 μL elution buffer EB to the middle of the adsorption membrane, and place it at room temperature for 2 min. After centrifugation at 12,000 rpm for 2 min, collect the plasmid solution in the centrifuge tube. In order to increase the recovery rate of the plasmid, the obtained solution can be re-added to the adsorption column, placed at room temperature for 2 min, and centrifuged at 12,000 rpm for 2 min, and the plasmid solution is collected in the centrifuge tube.

[0103] (2) Connection of target gene and expression vector and transformation

[0104] Use BamH I and Hind III fast-cut enzymes to cut the recovered CCEV-99 target fragment and pET-32a empty plasmid at 37°C for 45 min. After enzyme cutting, perform gel recovery. The enzyme cutting system is shown in Table 6.

[0105] Table 6 Enzyme cutting system of CCE V-99 and expression vector pET-32a(+)

[0106]

[0107] Add T4 DNA ligase to the enzyme cutting product and enzyme cutting vector at 4°C overnight to connect and construct a prokaryotic expression vector, and obtain pET32a-CCEV-99 recombinant plasmid respectively. The connection system is shown in Table 7.

[0108] Table 7 Connection system

[0109]

[0110] The connected product was transformed into E. coli BL21 (DE3) competent cells, and the steps were as follows:

[0111] The BL21 competent cells were dissolved in a low temperature environment, the overnight connected product was added thereto and mixed gently, and ice bath was performed for 30 min. After ice bath, the above solution was subjected to water bath at 42°C for 80 s, and after the end, ice bath was performed for 5 min. 900 μL of LB medium was added, and culture was performed at 180 rpm and 37°C for 1 h. The culture tube was taken out, centrifuged at 3,500 rpm for 3 min, 500 μL of supernatant was discarded, the rest was blown and mixed, and then 100 μL was taken and inoculated on an LB (50 μg / mL kan+) solid plate, and incubation was performed in a 37°C constant temperature incubator overnight to obtain pET32a-99 recombinant plasmid bacterial liquid.

[0112] (3) Verification of recombinant plasmid standard

[0113] Bacterial liquid PCR identification: the culture dish was taken out, and single colonies in the recombinant plasmid bacterial liquid were picked and added to a 1.5 mL centrifuge tube containing 30 μL of sterile ddH2O. 10 μL of the single colony mixture was taken and added to another clean 1.5 mL centrifuge tube, and a label was made. 4-5 single colonies were picked from each transformed plate according to the above operation. The second 1.5 mL centrifuge tube was boiled for 10 min as a DNA template, and primer identification was performed using the pET-32a vector. The primer sequence is shown in Table 8, and the PCR system and amplification program are shown in Tables 9 and 10, respectively.

[0114] The PCR product was added to a 1% agarose gel, and electrophoresis was performed at 120 V for 30 min. The bacterial liquid in the inner opening tube corresponding to the positive band was amplified with LB liquid medium (containing 100 μg / mL of Amp).

[0115] Table 8 Primer sequence of pET-32a vector

[0116]

[0117] Table 9 PCR amplification reaction system

[0118]

[0119] Table 10 PCR amplification program of gene

[0120]

[0121] Sequencing of recombinant plasmid: the recombinant plasmid bacterial liquid was extracted according to the plasmid extraction steps in the expression vector extraction of the present embodiment, and the extracted recombinant plasmid was sent to Xinjiang Youkang Biological Co., Ltd. for sequencing.

[0122] The constructed pET32a-99 plasmid was double enzyme-digested using Sac I and Hind III fast enzyme for verification, and the electrophoretogram is shown in Figure 2 As can be seen, the pET32a-99 recombinant plasmid has a target band at 1557 bp, and the target band is consistent with the expected result. Compared with the sequencing result, there is no mutation and deletion, proving that the two recombinant plasmids are successfully constructed.

[0123] Determination of recombinant plasmid concentration: The recombinant plasmid with correct sequencing result was determined for concentration using a single drop spectrophotometer, and the copy number calculation and plasmid dilution were performed according to the following formula.

[0124]

[0125] Example 4 Design and synthesis of fluorescent quantitative qPCR primer probe and primer

[0126] The qPCR primer probe and fluorescent quantitative qPCR primer were designed according to the design principle and synthesized by Xinjiang Youkang Biological Co., Ltd. The qPCR primer probe and fluorescent quantitative qPCR primer are shown in Table 11.

[0127] Table 11 CCEV-99 primer and probe sequence information

[0128]

[0129] The qPCR primer probe design principle is as follows:

[0130] a. The probe sequence should be specifically complementary to the target gene sequence, and should avoid hybridization with other non-target nucleic acid sequences.

[0131] b. The probe length is usually between 15-30 bases. Too short probe may have poor specificity and easily bind to non-target sequences; while too long probe will increase the synthesis cost and may affect the hybridization efficiency due to the formation of its own secondary structure.

[0132] c. When designing, the probe itself should be avoided to form secondary structures such as hairpin structure, stem-loop structure, etc.

[0133] d. The G-C content of the probe is generally between 40% and 60%.

[0134] e. The probe should be as close to the upstream primer as possible, but cannot overlap with the primer. Generally speaking, the distance between the probe and the upstream primer is about 5-30 bases, which is relatively appropriate.

[0135] f. Select appropriate fluorescent groups and quenching groups. The fluorescent group should have high fluorescence quantum yield, light stability and high affinity for nucleic acids. The quenching group should be able to efficiently quench the fluorescence signal of the fluorescent group.

[0136] The principles for designing the fluorescence quantitative qPCR primers are as follows:

[0137] a. The product length is 80-200 bp, preferably 80-150 bp.

[0138] b. The primer length is 18-25 nt, and the difference between the upstream and downstream primers is preferably no more than 3 bp.

[0139] c. Avoid continuous structures of G / C or A / T; avoid the presence of reverse repeat sequences or self-complementary sequences with more than 3 bp; and avoid high GC / high AT content regions at the 3' end.

[0140] d. The G-C content is generally between 40% and 60%, and the four bases are evenly distributed.

[0141] e. The Tm values of the forward and reverse primers preferably differ by no more than 1°C, and the primer Tm values are adjusted to be close to 60°C.

[0142] Example 5: Establishment of a fluorescence quantitative qPCR detection method for camel contagious pustular dermatitis virus

[0143] Screening of optimal primer and probe concentrations: To determine the optimal working concentrations of the primers and probes designed in Example 5, the primers were set to 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, and 1.0 μM, and the probes were set to 0.2 μM, 0.4 μM, and 0.6 μM, as shown in Table 12. Different concentrations of primers and probes were orthogonally combined to obtain 15 groups of primer and probe combinations, as shown in Table 13. The standard plasmid solution with a concentration of 366.51 ng / μL and a copy number of 2.08 x 10 11 copies / mL was subjected to fluorescence quantitative qPCR, 3 replicates were set for each group, and a negative control was set. The reaction program is shown in Table 10, and the combination with the lowest Ct value was selected as the optimal primer and probe final concentration.

[0144] Table 12: Screening of optimal primer and probe concentrations

[0145]

[0146]

[0147] As Figure 3As shown in the reaction results of Table 13, the 15 different combinations of primer probe final concentrations all specifically detected the CCEV standard positive sample. The primer final concentration was proportional to the fluorescence intensity but had a small effect, and regardless of the primer final concentration, the fluorescence intensity was the highest when the probe final concentration was 0.4 μM. When the primer final concentration was 1 μM and the probe final concentration was 0.4 μM (combination 14), the Ct value was the smallest and the fluorescence intensity was higher, and thus the combination of 1 μM primer final concentration and 0.4 μM probe final concentration was used to establish the standard curve.

[0148] Table 13 Determination of optimal primer probe final concentrations for fluorescence quantitative PCR detection of camel contagious ecthyma virus

[0149]

[0150] Establishment of standard curve: the recombinant plasmid obtained in Example 3 was diluted by a factor of 2 to obtain standard plasmid solutions of different concentrations. Standard plasmid solutions of 10 2 ~ 10 9 copies / mL concentration were selected, 3 replicates were set for each concentration, and a negative control was set, and the reaction system was as shown in Table 14, and the reaction procedure was 95°C pre-denaturation for 10 min; 95°C denaturation for 5 s; 60°C annealing and extension for 30 s, 40 cycles. Amplification was performed using a fluorescence quantitative PCR instrument, and the results were analyzed to construct a standard curve.

[0151] Table 14 PCR amplification reaction system

[0152]

[0153] The concentration of the pET32a-99 standard plasmid was 366.51 ng / μL, and the copy number was calculated to be 2.08 x 10 11 copies / mL according to the formula in Example 3.

[0154] The fluorescence quantitative PCR was performed according to the optimized reaction conditions, and the reaction results are shown in Figure 4 According to the reaction results, the concentrations of the 8 recombinant plasmids were calculated, and the logarithm of the copy number was taken as the abscissa, and the CT value corresponding to the copy number was taken as the ordinate to draw the standard curve as shown in Figure 5 The slope of the standard curve was -3.321, the intercept was 45.603, the standard curve equation was y = -3.3216x + 45.603, the correlation coefficient R 2 = 0.9984, which met R 2 > 0.99, 110% > E > 90%, indicating that there was a good linear relationship between the concentrations of the recombinant plasmids and the Ct.

[0155] Example 6 Specificity test

[0156] The camel infectious pustular virus fluorescence quantitative qPCR method established in the application uses the optimized primer and probe concentration, uses the nucleic acid samples of ORFV, LSDV, MAV and WR as templates, uses the nucleic acid sample of CCEV as a positive control, sets a negative control, performs the fluorescence quantitative qPCR reaction with 3 repeats in each group, and evaluates the specificity according to the test results.

[0157] The virus fluorescence quantitative amplification results of the embodiment are shown in Figure 6 Figure 6 It can be seen that the ORFV, LSDV, MAV, WR and the negative control do not appear amplification, and only the CCEV and the positive control appear good amplification curves, which indicates that the camel infectious pustular virus fluorescence quantitative qPCR established in the application has no cross reaction to other pathogens and has good specificity.

[0158] Example 7 Sensitivity test

[0159] The camel infectious pustular virus fluorescence quantitative qPCR method established in the application uses the optimized primer and probe concentration, dilutes the standard plasmid by 100-10 5 copies / mL as templates. A negative control is set, fluorescence quantitative qPCR reaction is performed with 3 repeats in each group, the sensitivity is evaluated according to the test results, and the ordinary PCR method is used for detection.

[0160] The nucleic acid agarose electrophoresis chart detected by the ordinary PCR method is shown in Figure 7 The PCR amplification curve of the fluorescence quantitative qPCR method of the application is shown in Figure 8 Figure 7 and Figure 8 It can be seen that the minimum limit of the ordinary PCR detection is 1x10 5 copies / mL, and the minimum limit of the fluorescence quantitative PCR detection is 1x10 0 copies / mL. Therefore, the sensitivity of the established fluorescence quantitative PCR is 10 5 times of that of the ordinary PCR. This indicates that the camel infectious pustular virus fluorescence quantitative PCR of the application has good sensitivity.

[0161] Example 8 Reproducibility test

[0162] The camel infectious pustular virus fluorescence quantitative qPCR method established in the application uses the optimized primer and probe concentration, dilutes the standard plasmid by 100-10 3 ~10 5 ​​Copies / mL were used as templates. A negative control was set up with 3 replicates per group. Intra-batch and batch-to-batch repeatability tests were performed. Stability was evaluated based on the test results, and the coefficient of variation was calculated using the formula: Coefficient of Variation (CV) = Standard Deviation (SD) / Mean × 100%. The results of quantitative real-time PCR amplification are shown in Table 15.

[0163] Table 15 Results of Repeatability Experiments

[0164]

[0165] In theory, a coefficient of variation of 0-15% is considered small variation, a coefficient of variation of 16-35% is considered medium variation, and a coefficient of variation greater than 35% is considered high variation.

[0166] As shown in Table 15, the coefficient of variation within each batch was less than 0.252%, the coefficient of variation between batches was less than 0.22%, and both the intra-batch and inter-batch coefficients of variation were less than 0.3%. This indicates that the quantitative real-time qPCR for camel infectious pustular virus established in this experiment has good stability and repeatability.

[0167] Electrophoresis images of 10 amplified samples are shown below. Figure 9 As shown, according to Figure 9 As can be seen, all samples were able to amplify the gene to the target size, and the bands were clear, which also proves that the present invention has good stability and reproducibility.

[0168] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A primer set for detecting camel infectious pustular virus, characterized in that, The primer set includes an upstream primer and a downstream primer for detecting the CCEV-99 gene fragment in camel infectious pustular virus DNA, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The primer set for detecting camel infectious pustular virus according to claim 1, characterized in that, The nucleotide sequence of the upstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

3.

3. A primer probe for detecting camel infectious pustular virus, characterized in that, The nucleotide sequences of the primers and probes are shown in SEQ ID NO.

4.

4. A recombinant plasmid, characterized in that, The recombinant plasmid contains the CCEV-99 gene fragment as shown in SEQ ID NO.

1.

5. A kit for detecting camel infectious pustular virus, characterized in that, It includes the primer set as described in claim 1 or 2, and the primer probe as described in claim 3; it also includes a standard plasmid solution containing the recombinant plasmid as described in claim 4.

6. A real-time quantitative qPCR method for detecting camel infectious pustular virus, characterized in that, The test sample was tested using the kit described in claim 5.

7. The qPCR detection method for camel infectious pustular virus according to claim 6, characterized in that, Includes the following steps: Obtain the sample to be tested and extract viral DNA from the sample; Using the viral DNA as a template, real-time quantitative PCR was performed using the qPCR primer set and the qPCR primer probe to obtain the Ct value of the sample to be tested. Based on the Ct value of the sample to be tested and the standard curve, the fluorescence quantitative results of camel infectious pustular virus in the sample to be tested were obtained.

8. The method for detecting camel infectious pustular virus by real-time quantitative qPCR according to claim 7, characterized in that, The reaction procedure for the quantitative real-time PCR was as follows: pre-denaturation at 95℃ for 10 min; denaturation at 95℃ for 5 s; annealing and extension at 60℃ for 30 s, for 40 cycles.

9. The method for detecting camel infectious pustular virus by real-time quantitative qPCR according to claim 8, characterized in that, The reaction system for the quantitative PCR is as follows: 。 10. The method for detecting camel infectious pustular virus by real-time quantitative qPCR according to claim 7, characterized in that, The standard curve is established by detecting the Ct values ​​of standard plasmid solutions at multiple concentration gradients, calculating the copy number of the standard plasmid based on the concentration of the standard plasmid solution, and obtaining a standard curve of the copy number versus the Ct value.

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