CN114350848B - Dual fluorescent probe primer combination for identifying African swine fever type I strain and type II strain, kit and application thereof - Google Patents
Dual fluorescent probe primer combination for identifying African swine fever type I strain and type II strain, kit and application thereof Download PDFInfo
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Abstract
The invention discloses a dual fluorescent probe primer combination for identifying African swine fever type I strain and type II strain, a kit and application thereof, and belongs to the technical field of molecular biology. The double fluorescent probe primer combination comprises a primer probe group for detecting the African swine fever virus I-type strain and a primer probe group for detecting the African swine fever virus general P72 gene. The genome is analyzed and compared in a conserved region of African swine fever virus, the gene B117L is finally screened out, a fragment with larger difference between the I type strain and the II type strain is selected, the design of a probe primer and the optimization of reaction conditions are carried out, so that the amplification efficiency of two virus genes is similar, a kit convenient to diagnose is developed, the I type infection and the II type infection of the African swine fever virus or the common infection of the two viruses can be detected at one time, and the kit 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.
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 African swine fever type I strain and type II strain, a kit and application thereof.
Background
African swine fever (AFRICAN SWINE FEVER, ASF) is an acute, febrile, highly contagious disease in pigs caused by African swine fever virus (AFRICAN SWINE FEVER virus, ASFV). Pigs and wild boars are generally susceptible, and soft ticks are the storage hosts and mediums of ASFV. 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 (encoding P72 protein) of the conserved region. In 1957, african swine fever virus genotype I was first transmitted from the African continent and was in epidemic form in Europe and Latin America, and thus far was still endemic in African and Italian islands of Latin. In 2007, african swine fever virus genotype II was first spread from the African continent to other continents, and was prevalent in Europe and Asia. In 2018, china reports the first African swine fever epidemic situation, which is a gene II type African swine fever virus. Only the genotype I and the genotype II of 24 genotypes of the African swine fever virus rapidly spread on other continents except African, and epidemic sources and possible transmission modes thereof can be traced back by identifying the genotypes, so that prevention and control measures can be timely taken.
The African swine fever virus OURT88/3 strain is a natural type I attenuated strain isolated from the inside of soft ticks of a pig farm. The research shows that: the OURT88/3 strain lacks 10L, 11L, 12L, 13L, 14L and MGF505/530 1R, 2R and 3R genes of the multigene family MGF360, so that the replication efficiency of viruses in macrophages or soft ticks is reduced, and simultaneously, the host is stimulated to generate a higher level IFN (Interferon), and the deletion strain can provide a certain virus attack protection capability for certain genotypes. In addition, OURT88/3 is frameshifted in the gene EP402R, EP R, which results in the inability to express the virulence protein CD2v, and therefore, does not cause clinical symptoms and viremia after infection of pigs.
The vaccine developed in China at present mainly takes African swine fever II type strain as a parent, and the constructed attenuated live vaccine with the gene deletion of MGF360-505R and the combined deletion of CD2V (EP 402R) and MGF360-505R has the main deleted virulence genes of multigene families MGF360-505 (MGF 360-9L, MGF360-10L, MGF360-11L, MGF360-12L, MGF360-13L, MGF360-14L, 505-1R, 505-2R and 505-3R) and EP402R, so that the vaccinated pigs have no obvious clinical symptoms and viremia compared with the infected type I attenuated strain, in addition, the current PCR detection kit designs probe primers by using vaccine-deleted gene fragments so as to identify wild strains and vaccine strains, however, the deletion fragments of the African swine fever type I natural attenuated strain and the vaccine strain deletion fragments have a large number of overlapping areas, and the method cannot identify the infection of the type I African swine fever natural attenuated strain, so that the method for rapidly, sensitively and accurately identifying the type I strain and the type II strain of the African swine fever is particularly important.
Disclosure of Invention
The invention aims to provide a double fluorescent probe primer combination for identifying African swine fever type I strain and type II strain, a kit and application thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a dual fluorescent probe primer combination for identifying a strain of african swine fever type i and a strain of type ii, comprising: a primer probe set for detecting the African swine fever virus I-type strain and a primer probe set for detecting the general P72 gene of the African swine fever virus;
the primer probe group for detecting the African swine fever virus I-type strain has the following sequence:
the upstream primer ASFV-I-F: as shown in SEQ ID NO.1
The downstream primer ASFV-I-R: as shown in SEQ ID NO.2
Fluorescent probe ASFV-I-P: as shown in SEQ ID NO. 3;
The primer probe group for detecting the African swine fever virus general P72 gene has the following sequence:
Upstream primer P72-F: as shown in SEQ ID NO.4
Downstream primer P72-R: as shown in 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, cy and ROX; the quenching group is selected from any one of BHQ1, MGB and BHQ 2.
Further, the 5' -end labeled fluorescent reporter groups of different specific probes are different.
A kit for identifying African swine fever type I strain and type II strain comprises the probe primer combination.
Further, the kit further comprises a positive control and a negative control. Further, the positive control is a recombinant plasmid containing African swine fever type I B117L gene fragment and P72 gene fragment; the negative control was double distilled water.
A method for identifying African swine fever virus type I strain and type II strain comprises the steps of performing real-time fluorescence quantitative PCR (polymerase chain reaction) by using the primer probe combination or the kit, collecting fluorescence signals, and determining whether the sample contains the African swine fever virus type I strain and/or type II strain.
The reaction conditions of the real-time fluorescent quantitative PCR are as follows: the circulation is carried out at 37 ℃ for 2min at 95 ℃ for 30s, and the circulation is carried out at 95 ℃ for 10s and 58 ℃ for 30s, and the total circulation is 45.
Compared with the prior art, the invention has the following advantages:
1) The genome of the type I strain and the genome of the type II strain are analyzed and compared in a conserved region of African swine fever virus, a gene B117L is finally screened out, a fragment with a large difference between the type I strain and the type II strain is selected, a probe primer is designed, and the specificity of the probe is improved by using a quenching group MGB, so that the gene sequences of the type I strain and the type II strain are distinguished.
2) The double fluorescent quantitative experiment is to amplify 2 target genes simultaneously in one reaction tube, so that the amplification of one target gene tends to affect the amplification of the other target gene, and the amplification efficiency is synchronous by the design of primers and the optimization of reaction conditions instead of simply mixing all the primers and templates in the same reaction tube. According to the invention, through the design of the primer and the probe, in a highly conserved sequence, a proper primer probe sequence is found, the amplification efficiency is close, the process is difficult, the result is not easy to control, multiple experiments are required, and meanwhile, the amplification efficiency of two virus genes in a sample is consistent with the sensitivity of each single reaction by adjusting the ratio of the primer to the fluorescent probe and optimizing the reaction condition.
3) The specificity of the invention is stronger, and the invention has no non-specific amplification curve for swine fever virus, porcine circovirus, porcine pseudorabies virus, porcine blue-ear virus, porcine parvovirus and porcine Japanese encephalitis virus, thereby ensuring the detection accuracy;
4) After one detection operation, the invention can identify the African swine fever virus type I infection, type II infection or common infection of two viruses in the sample, has the advantages of high sensitivity, strong specificity and good repeatability, and provides technical support for the 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 that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the results of gene sequence comparison of 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 graph of African swine fever virus type I.
FIG. 4 is a graph showing fluorescence amplification of the P72 gene of African swine fever virus.
FIG. 5 is a standard graph of the P72 gene of African swine fever virus.
FIG. 6 is a graph of fluorescence amplification curves from a specificity experiment.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
On one hand, aiming at the vaccine developed in China at present, mainly taking an African swine fever II type strain as a parent, the vaccinated pig has no obvious clinical symptoms and viremia with an infection I type attenuated strain, and great difficulty is brought to clinical diagnosis and epidemic prevention and control; on the other hand, the deletion fragments of the African swine fever type I natural attenuated strain and the vaccine strain deletion fragments have a large number of overlapping areas, and the method cannot identify the infection of the type I African swine fever natural attenuated strain. The invention analyzes and compares the genome in the conserved region of African swine fever virus, as shown in figure 1, finally screens out gene B117L, selects fragments with larger differences between I type strains and II type strains, designs probe primers, thereby distinguishing I type strains and II type strains, developing a kit convenient for diagnosis, and establishing a corresponding detection method.
The probe primer combination provided by the invention comprises the following components:
(1) A primer probe set for detecting a strain of african swine fever virus type i, the primer probe set comprising:
Upstream primer ASFV-I-F5'-TCTGCGACCACATCCC-3' (SEQ ID NO. 1)
The 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 sequence of interest:
AATAAAGCCCAAACCATTAAAAATATTTTTATCTGTTAGATTTAATTTAATAAATGGCTCATGGAATGTGTGGTGCGCCGCTGCATGAGGTGCGGCCACGGCCGCATGGGATGTGGTCGCATA(SEQ ID NO.7);
(2) A primer probe set for detecting the universal P72 gene of african swine fever virus, the primer probe set comprising:
upstream primer P72-F5'-CCACGGGAGGAATACCAA-3' (SEQ ID NO. 4)
Downstream primer P72-R:5'-GCAGATGCCGATACCACA-3' (SEQ ID NO. 5)
Fluorescent probe P72-P HEX-TCATATTAACGTATCCAGAGCAAGA-BHQ1 (SEQ ID NO. 6)
The sequence of interest:
GCAGATGCCGATACCACAAGATCAGCCGTAGTGATAGACCCCACGTAATCCGTGTCCCAACTAATATAAAATTCTCTTGCTCTGGATACGTTAATATGACCACTGGGTTGGTATTCCTCCCGTGG(SEQ ID NO.8).
the kit provided by the invention comprises the following reagents:
Positive control, negative control, primer premix, probe premix, PCR amplification solution;
Wherein:
The positive control is a recombinant plasmid containing African swine fever type I B117L gene fragment and a recombinant plasmid containing African swine fever type I P72 gene fragment;
The negative control is double distilled water;
The primer premix comprises: an ASFV-I-F, ASFV-I-R, P-F, P-R premix having an initial concentration of 10. Mu.M and a molar ratio of 1:1:1:1;
the probe premix comprises: a premix having an initial concentration of 10. Mu.M and an ASFV-I-P, P-P molar ratio of 1:1;
The PCR amplification solution comprises: fluorescence quantification 2× Animal Detection Probe Master Mix (nuuzan).
The double fluorescence PCR method for identifying the type I strain and the type II strain by using the probe primer or the kit comprises the following steps:
1) Extracting total DNA of a sample to be detected for standby;
2) Preparing a reaction system:
sample DNA 5. Mu.L, primer premix 2. Mu.L (400 nM), probe premix 1. Mu.L (200 nM), PCR amplification solution 12.5. Mu.L, ddH 2 O4.5. Mu.L;
3) Amplification procedure
2Min at 37 ℃,30 s at 95 ℃,10 s at 95 ℃ and 30s at 58 ℃ (collecting fluorescent signals) for 45 cycles in total; 2 fluorescence channels, namely a reporter group 'FAM', a quencher group 'MGB', a reporter group 'HEX' and a quencher group 'BHQ 1';
4) Result determination
The Ct value of the positive control 2 channels is less than 30, a specific S-shaped amplification curve appears, the negative control has no Ct value and no specific amplification curve, and the establishment of the Ct value and the specific amplification curve can judge that the experimental result is established.
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, and the detected sample is judged to be positive for virus nucleic acid; the Ct value of the detected sample is 35 < CT value less than or equal to 40, and a specific amplification curve appears, the suspicious sample is judged to be suspicious in virus nucleic acid, DNA needs to be extracted by resampling, the re-detection is carried out, the Ct value is less than or equal to 40, and the Ct value is judged to be positive, otherwise, the Ct value is judged to be negative. CT value > 40 or Ct value-free and specific amplification curve-free, and judging that the virus nucleic acid is negative.
The technical scheme of the invention is described in detail below. The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
Example 1
1. Preparation of positive standard
Positive standards of african swine fever type i B117L and P72 DNA was extracted using a known positive sample of african swine fever type i, total DNA extraction was performed according to DNA virus extraction kit of day root, target fragments of B117L and P72 were amplified respectively, using a system of 25 μl, a reaction system of 2×pcr Mix 12.5 μl, an upstream primer 1 μl, a downstream primer 1 μl L, DNA μl, TRUEscript Enzyme Mix 0.8.8 μl, and RNASE FREE H 2 O (nuclease free water) 7.7 μl. PCR amplification procedure: 94 ℃ for 5min; cycling at 94 ℃ for 30s,55 ℃ for 30s, and 72 ℃ for 30s, for 30 cycles; and then extending at 72 ℃ for 10min. After amplification was completed, all products were identified by 1% agarose gel electrophoresis. And (3) purifying and recovering the PCR product identified as positive by using a root gel recovery kit, connecting the PCR product to a pEASY-T1 vector and transforming the PCR product into DH5 alpha competent cells, picking up positive clones, carrying out shaking amplification by using LB culture solution, and then sending bacterial solution to Shanghai biological engineering Co.
2. Dual fluorescent quantitative PCR condition optimization
The plasmid template concentration is detected by a plasmid extraction kit of the day root, 10 times of gradient dilution is carried out on the two standard plasmids respectively, the concentration is 10 4 copise/mu L,1:1 is mixed and used as a template, the total system is 25 mu L, the upstream primer and the downstream primer are respectively mixed with the corresponding probe according to different probe final concentrations and primer final concentrations, 0.5 mu L and 0.25 mu L (200 nM:100 nM), 0.5 mu L and 0.5 mu L (200 nM:200 nM), 1 mu L and 0.5 mu L (400 nM:200 nM), 1 mu L and 1 nM (400 nM) are respectively added with the corresponding probes, the amplification program provided by the invention is 37 ℃ 2min,95 ℃ 30s, 58 ℃ 30s (collecting fluorescent signals) and 45 cycles are added in total, and the amplification is carried out, so that the optimal ratio of the lowest CT value and the fluorescence probe concentration at higher intensity is obtained.
The ratio of primer probe concentrations results are shown in the following table:
the best reaction system experiments are carried out by using different combination probes and primer concentrations, and the ASFV-I and P72 amplification efficiencies in the double qPCR reaction are closest and the CT value is lower when the final concentration of the probes is 200nM and the final concentration of the primers is 400 nM.
Respectively carrying out 10-time gradient dilution on two standard plasmids to obtain a template, taking the concentration of the template to be 107、106、105、104、103、102、101、100copise/μL for sensitivity measurement, and adopting a single qPCR reaction system: 5. Mu.L of DNA template, 1. Mu.L (400 nM) of upstream and downstream primers, 0.5. Mu.L (200 nM) of probe, 12.5. Mu.L of PCR amplification solution, 6. Mu.L of ddH 2 O, and 10. Mu.M of all primer probes were used. The amplification procedure was carried out at 37℃for 2min, at 95℃for 30s, at 95℃for 10s, and at 58℃for 30s (fluorescent signal was collected), for a total of 45 cycles, and PCR amplification was carried out and the standard curve thereof was analyzed. As shown in fig. 2-5, the results show that in the diluted current concentration range, the template amount and the corresponding Ct value have a better linear relationship, the correlation coefficient R 2 is 0.999 and 1.000 respectively, and the minimum detection amount of the fluorescent quantitative PCR is 10copise/μl, so that the fluorescent quantitative PCR established by the present invention has higher sensitivity.
3. Repeatability test
Positive standard substances diluted by 10 times of 8 concentrations are respectively used as templates, the final concentrations are 107、106、105、104、103、102、101、100copise/μL, respectively, fluorescent quantitative PCR is carried out according to a reaction system and a procedure for providing fluorescent quantification, 3 repeats are set for each gradient, and the repeatability of the method is verified. The results show that the variation coefficient (CV value) of the repeated experiments of the invention is below 0.5%, which shows that the invention has good repeatability.
4. Specificity test
The invention uses the positive samples of swine fever virus, porcine pseudorabies virus, porcine blue-ear virus, porcine parvovirus and porcine Japanese encephalitis virus as templates, and uses the primer and the probe of the invention to carry out fluorescent quantitative PCR amplification, as shown in figure 6, the detection results of different signal channels in the system are all negative, which shows that the method has strong specificity and has no cross reaction with other main infectious pathogens.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Sequence listing
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Claims (4)
1. A dual fluorescent probe primer combination for identifying African swine fever type I strain and type II strain is characterized in that: comprising the following steps: a primer probe set for detecting the African swine fever virus I-type strain and a primer probe set for detecting the general P72 gene of the African swine fever virus;
the primer probe group for detecting the African swine fever virus I-type strain has the following sequence:
the upstream primer ASFV-I-F: as shown in SEQ ID NO.1
The downstream primer ASFV-I-R: as shown in SEQ ID NO.2
Fluorescent probe ASFV-I-P: as shown in SEQ ID NO. 3;
5 '-end of fluorescent probe ASFV-I-P marks FAM and 3' -end marks MGB;
The primer probe group for detecting the African swine fever virus general P72 gene has the following sequence:
Upstream primer P72-F: as shown in SEQ ID NO.4
Downstream primer P72-R: as shown in SEQ ID NO.5
Fluorescent probe P72-P: as shown in SEQ ID NO. 6;
the fluorescent probe P72-P was labeled HEX at the 5 '-end and BHQ1 at the 3' -end.
2. A kit for identifying type i strain and type ii strain of african swine fever, characterized in that: a probe primer combination according to claim 1.
3. The kit of claim 2, wherein: the kit further comprises a positive control and a negative control.
4. A kit according to claim 3, wherein: the positive control is a recombinant plasmid containing African swine fever type I B117L gene fragment and P72 gene fragment; the negative control was double distilled water.
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| CN111876527A (en) * | 2020-08-13 | 2020-11-03 | 中国动物卫生与流行病学中心 | African swine fever virus wild strain and vaccine strain identification and detection kit |
| CN112831598A (en) * | 2019-11-22 | 2021-05-25 | 洛阳普泰生物技术有限公司 | Real-time fluorescent PCR amplification primer pair and probe primer for African swine fever virus identification and detection and prepared kit |
| CN113215109A (en) * | 2020-07-10 | 2021-08-06 | 中国农业科学院兰州兽医研究所 | Construction of African swine fever polygene combined deletion attenuated strain and application of attenuated strain as vaccine |
| CN113512534A (en) * | 2020-09-23 | 2021-10-19 | 杭州启函生物科技有限公司 | Compositions and methods for genetic modification and targeting |
| CN113543801A (en) * | 2019-03-27 | 2021-10-22 | 勃林格殷格翰动物保健有限公司 | Immunogenic composition and vaccine containing African swine fever virus peptide and protein and application thereof |
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| CN113543801A (en) * | 2019-03-27 | 2021-10-22 | 勃林格殷格翰动物保健有限公司 | Immunogenic composition and vaccine containing African swine fever virus peptide and protein and application thereof |
| CN112831598A (en) * | 2019-11-22 | 2021-05-25 | 洛阳普泰生物技术有限公司 | Real-time fluorescent PCR amplification primer pair and probe primer for African swine fever virus identification and detection and prepared kit |
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| CN113512534A (en) * | 2020-09-23 | 2021-10-19 | 杭州启函生物科技有限公司 | Compositions and methods for genetic modification and targeting |
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