CN114350848A - Dual fluorescent probe primer combination and kit for identifying African swine fever type I strain and type II strain and application of dual fluorescent probe primer combination and kit - Google Patents
Dual fluorescent probe primer combination and kit for identifying African swine fever type I strain and type II strain and application of dual fluorescent probe primer combination and kit Download PDFInfo
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Abstract
The invention discloses a dual fluorescent probe primer combination for identifying an African swine fever I-type strain and an African swine fever II-type strain, a kit and application thereof, and belongs to the technical field of molecular biology. The dual fluorescent probe primer combination comprises a primer probe set for detecting an I-type strain of the African swine fever virus and a primer probe set for detecting a general P72 gene of the African swine fever virus. The invention analyzes and compares the genome in the conserved region of the African swine fever virus, finally screens out the gene B117L, selects the fragment with larger difference between the I-type strain and the II-type strain, designs the probe primer and optimizes the reaction condition, leads the amplification efficiency of the two virus genes to be similar, develops the kit convenient for diagnosis, can detect the I-type infection and the II-type infection of the African swine fever virus or the common infection of the two viruses at one time, has the advantages of high sensitivity, strong specificity and good repeatability, and provides technical support for the prevention and control of the African swine fever in a pig farm.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a dual fluorescent probe primer combination for identifying an African swine fever type I strain and a type II strain, a kit and application thereof.
Background
African Swine Fever (ASF) is an acute, febrile, highly contagious disease in pigs caused by African Swine Fever Virus (ASFV). Domestic pigs and wild pigs are generally susceptible, and soft ticks are the storage host and the medium of ASFV. The African swine fever virus has only one serotype, and the genome of ASFV can be divided into 24 different genotypes according to the B646L gene (coding P72 protein) of a conserved region. In 1957, African swine fever virus genotype I was first transmitted from the African continent, and was epidemic in Europe, Latin America, and still has endemic prevalence in Africa and Italian island, etc. In 2007, the African swine fever virus gene II spreads from the African continent to other continents for the first time, and is epidemic in Europe and Asia. In 2018, the first African swine fever epidemic situation is reported in China and is a gene II African swine fever virus. Only the type I and the type II of 24 genotypes of the African swine fever virus are rapidly spread in other continents outside Africa, and the epidemic source and the possible transmission mode thereof can be traced by identifying the genotypes, so that prevention and control measures can be taken in time.
The African swine fever virus type I OURT88/3 strain is a natural type I low virulent strain separated from the soft tick in a pig farm. The research shows that: the OURT88/3 strain lacks 10L, 11L, 12L, 13L, 14L and MGF 505/5301R, 2R, 3R genes of a multigene family MGF360, so that the replication efficiency of the virus in macrophages or soft ticks is reduced, and simultaneously, the host is stimulated to produce high-level IFN (interferon), and the deleted strain can provide certain virus attack protection capability for certain genotypes. In addition, the frame shift of OURT88/3 in genes such as EP402R and EP153R results in the failure to express virulence protein CD2v, and thus clinical symptoms and viremia are not induced after infection of domestic pigs.
The vaccine developed in China at present mainly takes an African swine fever II strain as a parent, the constructed attenuated live vaccine with MGF360-505R gene deletion and CD2V (EP402R) and MGF360-505R double gene combined deletion is constructed, the main deleted virulence genes comprise multigene families MGF360-505(MGF360-9L, MGF360-10L, MGF360-11L, MGF360-12L, MGF360-13L, MGF360-14L, 505-1R, 505-2R, 505-3R) and EP402R, so that the vaccinated pigs only infected with the I type attenuated strain have no obvious clinical symptoms and viremia, which bring great difficulty to clinical diagnosis and prevention and control of epidemic situation, in addition, the current PCR detection kit designs probe primers by the vaccine deleted gene fragment so as to identify African wild strains and vaccine strains, however, the deleted fragment of the natural African swine fever I type attenuated strain and the deleted vaccine strain deletion have a large amount of coincided regions, the method cannot identify the infection of the I-type natural low virulent strain of the African swine fever, so that the establishment of a method for quickly, sensitively and accurately identifying the I-type virulent strain and the II-type virulent strain of the African swine fever is particularly important.
Disclosure of Invention
The invention aims to provide a dual fluorescent probe primer combination for identifying an African swine fever I-type strain and an African swine fever II-type strain, a kit and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a dual fluorescent probe primer combination for identifying an African swine fever type I strain and a type II strain comprises the following components: a primer probe group for detecting the I-type strain of the African swine fever virus and a primer probe group for detecting the general P72 gene of the African swine fever virus;
the primer probe group for detecting the African swine fever virus type I strain has the following sequence:
an upstream primer ASFV-I-F: shown as SEQ ID NO.1
Downstream primer ASFV-I-R: shown as SEQ ID NO.2
Fluorescent probe ASFV-I-P: as shown in SEQ ID NO. 3;
the primer probe set for detecting the general P72 gene of the African swine fever virus has the following sequence:
the upstream primer P72-F: shown as SEQ ID NO.4
The downstream primer P72-R: shown as SEQ ID NO.5
Fluorescent probe P72-P: as shown in SEQ ID NO. 6.
Further, the fluorescent probe is a hydrolysis probe, and a fluorescent reporter group is marked at the 5 'end of the probe and a quenching group is marked at the 3' end of the probe.
Further, the fluorescent reporter group is selected from any one of FAM, HEX, Cy5 and ROX; the quenching group is selected from any one of BHQ1, MGB and BHQ 2.
Further, the 5' end of the different specific probes are labeled with different fluorescent reporter groups.
A kit for identifying an African swine fever I-type strain and an African swine fever II-type strain comprises the probe primer combination.
Further, the kit also comprises a positive control and a negative control. Further, the positive control is a recombinant plasmid containing an African swine fever type I B117L gene fragment and a P72 gene fragment; the negative control was double distilled water.
A method for identifying an African swine fever virus I type strain and an African swine fever virus II type strain is characterized in that the primer probe combination or the kit is used for carrying out real-time fluorescence quantitative PCR, a fluorescence signal is collected, and whether a tested sample contains the African swine fever virus I type strain and/or the African swine fever virus II type strain is determined.
The reaction conditions of the real-time fluorescent quantitative PCR are as follows: 2min at 37 ℃, 30s at 95 ℃, 10s at 95 ℃ and 30s at 58 ℃ in 45 cycles.
Compared with the prior art, the invention has the following advantages:
1) the invention analyzes and compares the genomes of the I type strain and the II type strain in a conserved region of the African swine fever virus, finally screens out the gene B117L, selects a fragment with larger difference between the I type strain and the II type strain, designs a probe primer, and simultaneously improves the specificity of the probe by using a quenching group MGB, thereby distinguishing the gene sequences of the I type strain and the II type strain.
2) The double fluorescence quantitative experiment is a quantitative experiment by amplifying 2 target genes simultaneously in one reaction tube, so that the amplification of one target gene inevitably affects the amplification of the other target gene, and the amplification efficiency is synchronously required by the design of primers and the optimization of reaction conditions, rather than simply mixing all the primers and templates in the same reaction tube. According to the invention, through the design of primers and probes, a proper primer probe sequence is found in a highly conserved sequence, the amplification efficiency is close, the process is difficult, the result is difficult to control, multiple experiments are required, and simultaneously, the reaction conditions are optimized by adjusting the proportion of the primers and the fluorescent probes, so that the amplification efficiency of two virus genes in a sample is consistent, and the sensitivity of each single reaction is consistent.
3) The kit has stronger specificity, has no non-specific amplification curve for classical swine fever virus, porcine circovirus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine parvovirus and porcine encephalitis B virus, and ensures the accuracy of detection;
4) after one-time detection operation, the invention can identify whether the African swine fever virus is infected with type I or type II or is infected with both viruses in a sample, has the advantages of high sensitivity, strong specificity and good repeatability, and provides technical support for prevention and control of African swine fever in a pig farm.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows the result of the gene sequence alignment of the African swine fever virus type I strain and type II strain.
FIG. 2 is a graph showing fluorescence amplification of African swine fever virus type I.
FIG. 3 is a standard curve of African swine fever virus type I.
FIG. 4 is a graph showing fluorescence amplification of the African swine fever virus P72 gene.
FIG. 5 is a standard graph of the African swine fever virus P72 gene.
FIG. 6 is a graph showing fluorescence amplification curves obtained in a specific experiment.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
On one hand, the vaccine developed in China at present mainly takes an African swine fever type II strain as a parent, and the vaccinated pigs and the infected type I low virulent strains have no obvious clinical symptoms and viremia, so that great difficulty is brought to clinical diagnosis and prevention and control of epidemic situation; on the other hand, the deletion fragment of the natural low virulent strain of the African swine fever type I and the deletion fragment of the vaccine strain have a large number of overlapped regions, and the infection of the natural low virulent strain of the African swine fever type I cannot be identified by the method. The invention analyzes and compares the genome in the conserved region of African swine fever virus, finally screens out gene B117L and selects the fragment with larger difference between the I type strain and the II type strain as shown in figure 1, designs the probe primer, thereby identifying the I type strain and the II type strain, develops the kit convenient for diagnosis and establishes the corresponding detection method.
The probe primer combination provided by the invention comprises:
(1) a primer probe set for detecting an African swine fever virus type I strain, comprising:
upstream primer ASFV-I-F: 5'-TCTGCGACCACATCCC-3' (SEQ ID NO.1)
Downstream primer ASFV-I-R: 5'-AATAAAGCCCAAACCA-3' (SEQ ID NO.2)
Fluorescent probe ASFV-I-P FAM-CGTGGCCGCACCTCAT-MGB (SEQ ID NO.3)
The target sequence is as follows:
AATAAAGCCCAAACCATTAAAAATATTTTTATCTGTTAGATTTAATTTAATAAATGGCTCATGGAATGTGTGGTGCGCCGCTGCATGAGGTGCGGCCACGGCCGCATGGGATGTGGTCGCATA(SEQ ID NO.7)；
(2) a primer probe set for detecting the general P72 gene of African swine fever virus, the primer probe set comprising:
upstream primer P72-F: 5'-CCACGGGAGGAATACCAA-3' (SEQ ID NO.4)
The downstream primer P72-R: 5'-GCAGATGCCGATACCACA-3' (SEQ ID NO.5)
Fluorescent probe P72-P HEX-TCATATTAACGTATCCAGAGCAAGA-BHQ 1(SEQ ID NO.6)
The target sequence is as follows:
GCAGATGCCGATACCACAAGATCAGCCGTAGTGATAGACCCCACGTAATCCGTGTCCCAACTAATATAAAATTCTCTTGCTCTGGATACGTTAATATGACCACTGGGTTGGTATTCCTCCCGTGG(SEQ ID NO.8)。
the kit provided by the invention comprises the following reagents:
positive control, negative control, primer premix, probe premix and PCR amplification solution;
wherein:
the positive control is a recombinant plasmid containing an African swine fever type I B117L gene fragment and a recombinant plasmid containing an African swine fever type I P72 gene fragment;
negative control is double distilled water;
the primer premix comprises: a premix of 10 μ M ASFV-I-F, ASFV-I-R, P72-F, P72-R in a molar ratio of 1:1:1: 1;
the probe premix liquid comprises: a premix of 10 μ M ASFV-I-P, P72-P at a molar ratio of 1: 1;
the PCR amplification solution comprises: fluorescence quantification 2 × Animal Detection Probe Master Mix (Novowed).
The dual fluorescence PCR method for identifying the I type strain and the II type strain by using the probe primer or the kit comprises the following steps:
1) extracting the total DNA of the sample to be detected for later use;
2) preparing a reaction system:
3) Amplification program
At 37 ℃ for 2min, at 95 ℃ for 30s, and at 95 ℃ for 10s and at 58 ℃ for 30s (collecting fluorescence signals), for a total of 45 cycles; 2 fluorescence channels, namely a reporter group 'FAM', a quenching group 'MGB', a reporter group 'HEX' and a quenching group 'BHQ 1' respectively;
4) and the result is judged
The positive control 2 channels have Ct values less than 30 and show specific S-shaped amplification curves, the negative control has no Ct value and no specific amplification curve, and the establishment of the positive control and the negative control can determine the establishment of the experimental result.
The criteria are as follows:
the Ct value of the detected sample is less than or equal to 35, and a specific S-shaped amplification curve appears, so that the virus nucleic acid is judged to be positive; the CT value of the detected sample is more than 35 and less than or equal to 40, a specific amplification curve appears, the virus nucleic acid is judged to be suspicious, the suspicious sample needs to be sampled again to extract DNA for rechecking, the Ct value is less than or equal to 40, the virus nucleic acid is judged to be positive, and the virus nucleic acid is judged to be negative otherwise. The virus nucleic acid is judged to be negative if the CT value is more than 40 or no Ct value and no specific amplification curve exists.
The technical solution of the present invention will be described in detail below. The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
Example 1
1. Preparation of Positive Standard
Positive standard of African swine fever type I B117L and P72 DNA was extracted from known African swine fever type I positive samples, total DNA was extracted according to Tiangen DNA virus extraction kit, target fragments of B117L and P72 were amplified respectively, 25. mu.L of reaction system was used, 2 XPCR Mix 12.5. mu.L, upstream primer 1. mu.L, downstream primer 1. mu. L, DNA 2. mu.L, TRUEscript Enzyme Mix 0.8. mu.L and RNase free H2O (nuclease free water) 7.7. mu.L. PCR amplification procedure: 5min at 94 ℃; circulating at 94 ℃ for 30s, at 55 ℃ for 30s and at 72 ℃ for 30s for 30 cycles; extension was then carried out at 72 ℃ for 10 min. After amplification was complete, all products were identified by electrophoresis on a 1% agarose gel. And (3) purifying and recovering the PCR product identified as positive by using a rubber recovery kit of Tiangen, connecting the PCR product to a pEASY-T1 vector, transforming the PCR product to DH5 alpha competent cells, selecting positive clones, carrying out shake bacteria amplification by using LB culture solution, and sending the bacterial solution to Shanghai biological engineering Limited company for sequencing.
2. Dual fluorescent quantitative PCR condition optimization
Extracting standard plasmid with plasmid extraction kit of Tiangen, detecting plasmid template concentration with NanoDrop 2000 nucleic acid concentration determinator, diluting two standard plasmids with 10 times gradient respectively, and taking concentration 104Mixing copise/. mu.L at a ratio of 1:1 to obtain a total system of 25. mu.L, and addingThe primers (upstream and downstream) and the corresponding probes are respectively added with 0.5 muL, 0.25 muL (200nM:100nM), 0.5 muL (200nM:200nM), 1 muL, 0.5 muL (400nM:200nM), 1 muL (400nM:200nM), and 1 muL (400nM:400nM), all the primer probe application concentrations are 10 muM, the amplification program provided by the invention is used for amplification at 37 ℃ for 2min, at 95 ℃ for 30s, at 95 ℃ for 10s, at 58 ℃ for 30s (collecting fluorescence signals), and the total number is 45 cycles, and PCR amplification is carried out to obtain the optimal primer probe concentration ratio when the CT value is lowest and the fluorescence intensity increase value is higher.
The results of the ratio of primer probe concentrations are given in the following table:
an optimal reaction system groping experiment is carried out by using different combined probes and primer concentrations, and the ASFV-I type and P72 amplification efficiencies in the double qPCR reaction are the closest when the final concentration of the probe is 200nM and the final concentration of the primer is 400nM, and the CT value is lower.
Two standard plasmids are respectively diluted by 10 times of gradient and used as templates, wherein the concentration is 107、106、105、104、103、102、101、100Sensitivity assay was performed using copise/. mu.L using a single qPCR reaction system: DNA template 5. mu.L, upstream and downstream primers 1. mu.L (400nM), probe 0.5. mu.L (200nM), PCR amplification solution 12.5. mu.L, ddH2O6. mu.L, all primer probes were applied at a concentration of 10. mu.M. The amplification procedure was carried out at 37 ℃ for 2min, 95 ℃ for 30s, at 95 ℃ for 10s, at 58 ℃ for 30s (fluorescence signal acquisition), for a total of 45 cycles, PCR amplification was carried out, and the standard curve was analyzed. As shown in FIGS. 2-5, the results show that in the current diluted concentration range, the template amount and the corresponding Ct value are in a better linear relationship, and the correlation coefficient R2The detection quantity is respectively 0.999 and 1.000, and the minimum detection quantity of the fluorescence quantitative PCR is 10 copise/mu L, so the fluorescence quantitative PCR established by the invention has higher sensitivity.
3. Repeatability test
Respectively using 10 times of gradient diluted positive standard substance with 8 concentrations as template, and respectively having final concentration of 107、106、105、104、103、102、101、100copise/. mu.L, fluorescent quantitative PCR was performed according to the reaction system and procedure providing fluorescent quantitation, 3 replicates were set for each gradient, and the reproducibility of the method was verified. The results show that the coefficient of variation (CV value) of the repeated experiments of the invention is below 0.5 percent, which shows that the invention has good repeatability.
4. Specificity test
The positive samples of classical swine fever virus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine parvovirus and porcine encephalitis B virus stored by the inventor are used as templates, the primers and the probes in the invention are used for carrying out fluorescence quantitative PCR amplification, as shown in figure 6, the detection results of different signal channels in the system are all negative, and the method is proved to have strong specificity and have no cross reaction with other main infectious pathogens.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
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Claims (10)
1. A dual fluorescent probe primer combination for identifying an African swine fever type I strain and a type II strain is characterized in that: the method comprises the following steps: a primer probe group for detecting the I-type strain of the African swine fever virus and a primer probe group for detecting the general P72 gene of the African swine fever virus;
the primer probe group for detecting the African swine fever virus type I strain has the following sequence:
an upstream primer ASFV-I-F: shown as SEQ ID NO.1
Downstream primer ASFV-I-R: shown as SEQ ID NO.2
Fluorescent probe ASFV-I-P: as shown in SEQ ID NO. 3;
the primer probe set for detecting the general P72 gene of the African swine fever virus has the following sequence:
the upstream primer P72-F: shown as SEQ ID NO.4
The downstream primer P72-R: shown as SEQ ID NO.5
Fluorescent probe P72-P: as shown in SEQ ID NO. 6.
2. The dual fluorescent probe primer combination for identifying the African swine fever type I strain and the African swine fever type II strain according to claim 1, which is characterized in that: the fluorescent probe is a hydrolysis probe, and a fluorescent reporter group is marked at the 5 'end of the probe and a quenching group is marked at the 3' end of the probe.
3. The dual fluorescent probe primer combination for identifying the African swine fever type I strain and the African swine fever type II strain according to claim 2, which is characterized in that: the fluorescent reporter group is selected from any one of FAM, HEX, Cy5 and ROX; the quenching group is selected from any one of BHQ1, MGB and BHQ 2.
4. The dual fluorescent probe primer combination for identifying the African swine fever type I strain and the African swine fever type II strain according to claim 2, which is characterized in that: the 5' end of different specificity probes is marked with different fluorescent reporter groups.
5. A kit for identifying an African swine fever type I strain and a type II strain is characterized in that: comprising the probe-primer combination of any one of claims 1 to 4.
6. The kit of claim 5, wherein: the kit also includes a positive control and a negative control.
7. The kit of claim 6, wherein: the positive control is a recombinant plasmid containing an African swine fever type I B117L gene fragment and a P72 gene fragment; the negative control was double distilled water.
8. Use of the probe-primer combination of any one of claims 1 to 4 or the kit of any one of claims 5 to 7 for the identification of African swine fever type I and type II strains for non-disease diagnostic purposes.
9. A detection method for identifying an African swine fever type I strain and a type II strain is characterized by comprising the following steps: the method comprises the steps of carrying out real-time fluorescence quantitative PCR by using the probe primer combination of any one of claims 1 to 4 or the kit of any one of claims 5 to 7, collecting fluorescence signals, and determining whether the tested sample contains African swine fever type I strain and/or type II strain.
10. The detection method according to claim 9, characterized in that: the reaction conditions of the real-time fluorescent quantitative PCR are as follows: 2min at 37 ℃, 30s at 95 ℃, 10s at 95 ℃ and 30s at 58 ℃ in 45 cycles.
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