CN112094948A - Application of target gene combination in African swine fever virus detection and kit - Google Patents
Application of target gene combination in African swine fever virus detection and kit Download PDFInfo
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Abstract
The invention discloses an application of a target gene combination in African swine fever virus detection and a kit. The target gene combination comprises B646L, B962L, C717R, D1133L and G1340L genes, and the primer sets respectively targeting the genes consist of nucleotide sequences shown in SEQ ID Nos. 1-30. The prepared African swine fever multiple nucleic acid detection kit establishes the whole detection process by using a honeycomb chip and an LAMP direct amplification method. Compared with the prior art, the invention designs primers aiming at a plurality of genes of ASFV, realizes simultaneous detection of a plurality of genes by combining a chip and can effectively avoid false negative results caused by single gene mutation, does not need a nucleic acid extraction step, can realize multiple visual detections, does not depend on precise instruments and professional operations, has the advantages of accuracy, rapidness, simplicity and convenience, and the like, is suitable for port inspection and quarantine and on-site instant detection, and has good application prospect.
Description
Technical Field
The invention relates to the technical field of inspection and quarantine, in particular to application of a target gene combination in African Swine Fever Virus (ASFV) detection and a kit, and particularly relates to an African swine fever virus multiple nucleic acid detection kit based on a honeycomb chip.
Background
African Swine Fever (ASF) is an acute, highly contagious and fatal disease in swine caused by African Swine Fever Virus (ASFV), and is characterized by short onset time, high mortality rate of 100%, and clinical manifestations of hyperpyrexia, diarrhea, extensive bleeding of skin and viscera, etc. (Normile, D.2018, Science,361,741; Ma, J.et al.,2020, Preventive Veterinary Medicine,175,104861; Galindo, I.A.,&alonso, C,2017, virues, 9(5), 103). ASF was first reported in the Kenya region of Africa in 1921 and then rapidly spread to over 60 countries including Europe, Lame and Asia, causing huge economic losses to the world (Montgomery, R.E.,1921, Journal of comparative research and therapeutics,34, 159-. In 2018, ASF erupts in Shenyang in Liaoning, China for the first time, and then rapidly spreads to other provinces. By 3/5 days in 2020, 165 outbreaks of African swine fever in China have been developed in 32 provinces, 1,193,000 live pigs are killed accumulatively, and great threat is brought to China and even the pig industry all over the world (http://www.fao.org/ag/againfo/programmes/en/empres/ASF/situation_ update.html). According to the 2019 version animal epidemic disease report directory issued by the world animal health Organization (OIE), the ASF is listed as the animal epidemic disease which needs to be reported.
ASFV is a dsDNA virus with icosahedral symmetry, is the only member of African swine fever virus of the African swine fever virus family, and is the only known arbovirus. The ASFV has a large genome with a length of about 170-190 kb and encodes 150-200 proteins (Alonso, C., et al, 2018, Journal of General Virology,99(5), 613: 614; Wang, N., et al, 2019, Science,366(6465), 640: 644). Due to the complex genetic composition and high variability of ASFV, the gene B646L encoding capsid protein Vp72, which has been classified into 24 genotypes according to the higher homology, is also classified into more subtypes in combination with other genes such as B602L (Quembo CJ., et al.,2018, transformed emery Dis,65(2): 420-431). At present, an effective treatment mode and a vaccine are not available for ASF, and killing and incineration are the most effective means for preventing epidemic spread, so that the ASFV is important to detect quickly and accurately.
The existing ASFV detection methods can be divided into two categories: (1) immunological detection methods include viral isolation (Rowlands, R.J., et al, 2008, Embedded in infectious diseases,14(12),1870), erythrocyte adsorption assay (Rodr I guez, J.M., et al, 1993, Journal of virology,67(9), 5312-. Although the virus isolation method (VI) and the erythrocyte adsorption assay (HAD) are very reliable and effective, they are time-consuming and cumbersome to operate, require high-quality tissue to culture cells, and are not suitable for rapid detection. Enzyme-linked immunosorbent assay (ELISA) and fluorescent antibody detection (FAT) are simple and convenient to operate, and can accurately identify ASFV antibodies, but the ASFV antibodies are limited by detection sensitivity and cannot be accurately detected at the early stage of infection, and pigs can be killed by virus infection before the antibodies are positive. Therefore, molecular biological detection is the mainstream detection method due to its simple and rapid operation, high sensitivity and high specificity. (2) The molecular biological detection method comprises fluorescent quantitative PCR (
M., et al,2003, Journal of clinical microbiology,41(9), 4431-. Fluorescent quantitative PCR is a gold standard for laboratory testing of ASFV as certified by the world animal health Organization (OIE), but relies on sophisticated thermocyclers and specialized operators and is not suitable for field testing in resource-limited areas. Isothermal amplification is simple and convenient to operate,The method has the advantages of no need of precise instruments, short time consumption and the like, and plays an increasingly important role in the field of molecular detection. In recent years, a plurality of researchers use LAMP or RPA in combination with CRISPR system and test strip for ASFV detection, which can improve sensitivity and realize on-site rapid detection (Wang X., ET al, 2020, ACS Nano,14(2): 2497-2508; Yuan, C.Q., ET AL.,2019, Anal Chem,92(5): 4029-4037; Wang, X., ET al, 2020, Communications Biology,3(1): 62; Bai, J., ET al, 2019, Frontiers in Microbiology,10:2830), but they need to enrich product by isothermal amplification and then transfer to the detection system, amplify and detect phase separation, uncovering easily causes aerosol pollution, causes false positive result, and increases operation steps. Furthermore, all the above mentioned methods target only a single gene of the ASFV, such as gene B646L encoding viral capsid protein VP72 (Wang, J., et al, 2017, Canadian Journal of viral Research,81(4),308-312), gene P1192R encoding topoisomerase II (James, H.E., et al, 2010, Journal of viral methods,164(1-2),68-74), gene K78R encoding DNA binding protein Vp10 (Wang, D., et al, 2020, Journal of viral methods,276:113775), which easily causes false negative results due to single gene mutations due to the large, complex and variable genomic characteristics of the ASFV.
The prior patent document CN110373500A discloses a double fluorescence PCR detection kit, which selects primers and probes for conserved segments of two genes (B646L and B438L) of african swine fever virus. In addition, patent document CN111020062A discloses a triple real-time fluorescent quantitative PCR kit for detecting wild strains and gene-deleted strains of african swine fever virus, which is directed against three genes of african swine fever virus CD2V, VP72 and MGF-36014L. But the disadvantages of the PCR are that the PCR is a temperature-variable reaction, a precise multi-channel fluorescent PCR instrument is required, the price is high, the volume is large, and the method is not suitable for the on-site instant detection of a pig farm. Meanwhile, the detection multiplicity is not easy to increase due to the competition and interference among multiple pairs of PCR primers in the same PCR tube, and the gene of the African swine fever serving as a virus is likely to be continuously mutated, so that the accuracy can be ensured by increasing the multiplicity.
In summary, there is an urgent need in the field of african swine fever detection to develop a multiple nucleic acid detection method targeting multiple genes of ASFV, which is simple and rapid, has high sensitivity, does not need to be uncapped, and is suitable for on-site detection.
Disclosure of Invention
Aiming at two main technical problems of the existing detection method: the invention provides an LAMP primer group and a kit for multiple gene detection of African Swine Fever Virus (ASFV), which only aim at ASFV single gene detection and depend on a complex and precise instrument. Aiming at the first problem, the LAMP primers are designed and screened for detection according to 5 genes of ASFV, so that false negative results caused by single gene mutation can be effectively avoided, and the positive detection rate is improved; aiming at the second problem, the invention combines the honeycomb chip to realize the simultaneous detection of a plurality of genes by one-time reaction, combines the LAMP direct amplification method to remove the step of nucleic acid extraction, can realize multiple visual detection, does not depend on precise instruments and professional operation, has the advantages of high sensitivity, strong specificity, simple, convenient and quick operation and the like, is suitable for port inspection and quarantine and on-site instant detection, and has good application prospect.
The purpose of the invention is realized by the following technical scheme:
the invention provides an application of a target gene combination in non-diagnostic African swine fever virus detection, wherein the target gene combination comprises B646L, B962L, C717R, D1133L and G1340L genes.
The invention also provides an LAMP primer group for multi-gene detection of African swine fever virus, which is characterized in that the primer group comprises 5 sets of LAMP primers which respectively target 5 genes of the African swine fever virus: B646L, B962L, C717R, D1133L and G1340L, which consist of nucleotide sequences shown as SEQ ID Nos. 1 to 30.
The invention also provides a multiple nucleic acid detection kit for the African swine fever virus, and the kit comprises the LAMP primer group.
Preferably, the kit further comprises a honeycomb chip, a sample adding joint, a sample lysate, a LAMP reaction solution, positive and negative controls, a sealing film and a reaction tube.
Preferably, the LAMP primer group is obtained by screening LAMP reaction layers in a PCR tube and on a chip, and is pre-fixed on corresponding positions of a honeycomb chip respectively.
Preferably, the honeycomb chip is a microarray integrated with a plurality of capillaries, is shaped like a "honeycomb", and thus is named as a "honeycomb chip", which is fixed in a reaction tube after hydrophobic modification and primer pre-fixing;
the sample adding joint can be tightly combined with the honeycomb chip and the reaction tube, so that simultaneous sample introduction of a plurality of capillaries is realized;
the main component of the sample lysate is NaOH, and the sample lysate is used for the pretreatment of a swine blood sample and releasing nucleic acid of African swine fever virus.
Preferably, the LAMP reaction solution contains 1 × ThermoPol buffer solution, 8.0mM MgSO41.4mM dNTPs, 0.8M betaine, 25. mu.M calcein, 0.5mM MnCl2,0.32UμL-1Bst DNA polymerase.
Preferably, the positive control is a plasmid without the nucleic acid sequence of African swine fever virus, and the corresponding LAMP primer is fixed in advance in a capillary labeled with "PC" on a honeycomb chip.
Preferably, the negative control is a control in which no primer is immobilized in the capillary labeled "NC" on the honeycomb chip.
The sealing film has optical permeability, can be used for sealing the reaction tube after sample adding is finished, avoids aerosol pollution, and cannot influence visual detection of results.
The invention also provides a method for multi-gene detection of African swine fever virus according to the kit of claim 3, which comprises the steps of collecting and pre-treating a swine blood sample, adding sample, incubating and detecting, wherein the total time consumption of the method is less than 70 min.
Preferably, the method comprises the following specific steps:
collecting a pig blood sample: collecting a pig blood sample and storing the pig blood sample in an anticoagulation tube;
pretreatment: fully mixing a pig blood sample stored in an anticoagulation tube with a sample lysate according to a volume ratio of 1:2, performing room temperature lysis for 3min, and then sucking the lysed mixed solution to mix with an LAMP reaction solution according to a volume ratio of 1: 24;
sample adding: sucking the mixed solution obtained from the pretreatment, inserting the mixed solution into a sample adding joint, moving the mixed solution to a reaction tube for sample adding, removing the sample adding joint, and sealing the reaction tube by using a sealing film;
incubation and detection: reacting at 63 ℃ for 1h, and performing visual detection by using ultraviolet irradiation.
Preferably, the ultraviolet irradiation is performed by a handheld ultraviolet irradiation device, and the temperature control and detection of the incubation can be integrated into an automatic device and automatically operated under the control of a software program.
The invention can detect single sample, or integrate 8 connecting pipes to detect multiple samples.
The ASFV multiple nucleic acid detection kit prepared by the LAMP primer group establishes the whole detection process by using a honeycomb chip and an LAMP direct amplification method.
Compared with the prior art, the invention has the following beneficial effects:
1. aiming at the characteristics of complexity and changeability of ASFV genome and numerous genotypes, the LAMP primers are designed and screened by selecting conserved sequences of a plurality of genes, so that false negative results caused by single gene mutation are effectively avoided, and the positive detection rate is improved;
2. and by combining the honeycomb chip, the quick sample adding, the sealed amplification and the multiple visual detection are realized.
3. The LAMP direct amplification method is combined, sample pretreatment is simplified, nucleic acid extraction is not needed, the detection efficiency is greatly improved, and the LAMP isothermal amplification mode is suitable for field detection in remote areas.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 shows the results of testing the effectiveness and specificity of primers in a PCR tube in example 1 of the present invention;
FIG. 2 shows the results of the specificity and sensitivity tests of the primers on the honeycomb chip in example 2 of the present invention;
FIG. 3 is a flowchart illustrating ASFV detection according to example 3 of the present invention;
FIG. 4 is a graph showing the results of the test of a swine blood sample in example 3;
FIG. 5 is a comparison of two pairs of PCR primers recommended by OIE (world animal health organization) in example 3 of the present invention with a target sequence in an existing ASFV strain;
FIG. 6 shows the results of the sensitivity test of the commercial kit of example 4 of the present invention as a performance comparison;
wherein, 1-5 in the chip pattern diagram respectively represent B646L, B962L, C717R, D1133L and G1340L, PC represents positive control, and NC represents negative control;
FIG. 7 shows the results of chip detection of the African swine fever virus genomic DNA sample in example 5 of the present invention;
FIG. 8 shows the results of the specificity test of the genomic DNAs of three porcine viruses other than the genomic DNAs of the porcine viruses in example 6 of the present invention.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.
Example 1: in-tube LAMP-based primer effectiveness and specificity test
According to the genome analysis result of the China/2018/AnhuiXCGQ virus strain (Bao, J., et al.,2019, Transboundary and observing diseases,66(3), 1167-.
The conserved sequence of about 1000bp selected in this example is only relatively conserved and eliminates the sequences after some mutant strains, because even though the PCR primers recommended by OIE are not completely conserved, strains still have mutations (FIG. 5), and therefore, we obtain 5 genes. The specific selection process and principle are as follows: (1) from genome comparisons of type II african swine fever virus strains (China/2018/AnhuiXCGQ) with other major type II strains (i.e. POL/2015/podraskie, Estonia 2014, Russia/odditiov _02/14/Boar and Georgia 2007/1) it was revealed that in all five type II african swine fever virus strains there are 118 identical open reading frames, including 16 structural proteins and proteins involved in morphogenesis, 25 proteins involved in nucleotide metabolism, transcription, replication and DNA repair, 3 proteins with other enzymatic activities, 4 proteins involved in host cell interactions, 25 proteins of the multigene family (MGF) and 47 proteins of unknown function (Bao, j., et al, 2019, transboundaundary and emergencies, 66 (1163), 7-; (2) since most of the variation between the genomes of African swine fever viruses is known to be caused by the addition and deletion of members of a multigene family, genes of this family are excluded; (3) the target gene should be selected from different gene function types as much as possible, so as to ensure the diversity of the gene types and avoid the mutation of a certain gene; (4) the length of the target gene is about 1000bp, so as to ensure that the target gene is subjected to multi-sequence alignment with known strains listed in a GenBank database to obtain a conserved sequence for primer design. Table 1 below is the gene information selected according to the above principle.
TABLE 1 genetic information contained in the target gene combinations for detection of African swine fever virus
TABLE 2 LAMP primer set for detection of African swine fever virus of 5 kinds selected
TABLE 3 unselected LAMP primer sets
The plasmid standard of 5 genes is used as a template, the effectiveness and specificity of the primer are tested by using the conventional LAMP containing calcein fluorescent dye, the specific reaction system is shown in Table 4, and the reaction product is detected by two modes of ultraviolet irradiation and agarose gel electrophoresis.
FIG. 1A shows the results of the primer validity test for 5 genes, the left panel of FIG. 1A shows the results of fluorescence detection after each gene was amplified with 4 sets of LAMP primers, and the right panel shows the results of gel electrophoresis of the amplification products in the corresponding tube on the left; p represents the addition of plasmid template, N represents the addition of ultrapure water as a control, and 1-4 represents 4 sets of primers. By comparing the fluorescence detection and gel electrophoresis results of the N group and the P group, the following results are found: the necessity of designing multiple sets of primers to screen for optimal primers is also demonstrated by the fact that 4 sets of primers of genes B646L, B962L, D1133L and G1340L can successfully detect the target gene, and only the primer set No. 2 of the gene C717R can successfully detect the target gene.
Based on the results of the primer validity tests, we selected 1 set of primers from each gene to perform specificity tests with each other, namely B646L-2, B962L-2, C717R-2, D1133L-3, and G1340L-1, and then used the 5 sets of primers to amplify each gene respectively to see whether non-specific amplification occurs, and the test results are shown in FIG. 1B. By comparing the fluorescence detection and gel electrophoresis results of the N group and the P group, no cross reaction exists between the 5 sets of primers and the template, and the product can be successfully detected only by adding the respective gene template and the corresponding primer into the same EP tube.
In conclusion, 5 primer sets capable of being used for ASFV detection are obtained by testing the effectiveness and specificity of the in-tube LAMP on the primers, and do not have cross reaction with each other.
TABLE 4 conventional LAMP reaction System
Example 2: specificity and sensitivity of LAMP test primer based on-chip
The specificity and sensitivity of primers were tested using a honeycomb chip, 5 LAMP primer sets and primer sets for positive control obtained in example 1 were pre-fixed at the corresponding capillary positions, 1 to 5 in the chip pattern diagram represent B646L, B962L, C717R, D1133L, and G1340L, respectively, PC represents positive control, NC represents negative control, and the specific reaction system is shown in Table 5, and the reaction product was detected by ultraviolet irradiation.
FIG. 2A shows the results of primer specificity tests of 5 genes, in which a plasmid template of a single gene and a positive control plasmid were sequentially mixed with a LAMP reaction solution, loaded on a chip, reacted at 63 ℃ for 1 hour, and visually detected using a hand-held ultraviolet irradiation device. The results showed that in 5 ASFV gene detection chips, fluorescence was successfully detected at the corresponding primer positions only when the respective gene templates were added, except for the positive control.
FIG. 2B shows the results of the primer sensitivity test of 5 genes, in which the plasmid templates of 5 genes and the positive control plasmid were diluted step by step, mixed with LAMP reaction solution and loaded on a chip, reacted at 63 ℃ for 1 hour, and visualized detection was performed with a hand-held ultraviolet irradiation device. The results show that when the template concentration is 880 copies/microliter, 88 copies/microliter and 30 copies/microliter, 5 gene targets can be successfully detected; whereas when the template concentration was 15 copies/microliter, only the two targets 3(C717R) and 4(D1133L) detected fluorescence, which is inconsistent with the expected signal, thus defining the lowest detection limit as 30 copies/microliter, i.e. 48 copies/capillary. The sensitivity of PCR primers recommended by the world animal health Organization (OIE) is about 600 copies/reaction (Wilkinson P.J. office International Des episomes 2000, pp.189-198; King DP, et al,2003, J Virol Methods,107(1): 53-61; Luo Y., et al, 2017, Arch Virol,162(1):191-199), further indicating that the sensitivity of the primers screened by us meets the actual detection requirements.
TABLE 5 chip LAMP reaction System
In conclusion, the 5 primer sets selected in example 1 were confirmed to have high specificity and overall sensitivity as low as 30 copies/microliter and individual targets as low as 15 copies/microliter by on-chip LAMP primer specificity and sensitivity tests.
Example 3: multiplex nucleic acid detection (containing single gene mutation detection node) for pig blood simulation sample based on honeycomb chip
Fruit)
The honeycomb chip is used for testing the swine blood simulation sample, the primer pre-fixing mode and the reaction system are the same as those in example 2, and the whole reaction flow is shown in figure 3.
Firstly, plasmid templates of 5 genes are mixed and then diluted step by step, and then 10 mu L of mixed plasmid is mixed with 90 mu L of anticoagulated pig blood to be used as a simulation sample; then 10. mu.L of the mock sample was mixed with 20. mu.L of the sample lysate, and lysed at room temperature for 3min, then 1. mu.L of the lysed sample and 1. mu.L of the positive control were mixed with 23. mu.L of the LAMP reaction solution, and loaded onto a chip, and reacted at 63 ℃ for 1h, followed by fluorescence detection, with the results shown in FIG. 4A. Two conclusions can be drawn from the chip result graph: (1) the LAMP direct amplification method does not affect the amplification and detection of ASFV genes, and the lowest detection limit of the swine blood simulation sample is 50 copies/microliter, namely 80 copies/capillary, which is equivalent to that of a pure plasmid sample, while the sensitivity of PCR primers recommended by the world animal health Organization (OIE) is about 600 copies/reaction (Wilkinson P.J. office International Des episomes 2000, pp.189-198; King DP, et al,2003, J Virol Methods,107(1): 53-61; Luo Y., et al, 2017, Arch Virol,162(1):191-199), which indicates that the sensitivity of the primers screened by the pig blood simulation sample meets the actual detection requirement; (2) the swine blood simulation sample only added with the positive control plasmid does not detect other fluorescent signals except the positive control hole, which shows that the complex background of the swine blood does not cause the non-specific amplification of the primer, and further verifies the specificity of the LAMP primer.
To further prove the necessity of ASFV multiplex detection, we performed sequence alignment on existing strains in GenBank database, and introduced the found point mutation into plasmid template for detection, and observed whether false negative result would occur. Taking the B646L gene encoding VP72 as an example, FIG. 4B shows the sequence alignment of 61 strains, and the base with dark labels indicates that although the conserved sequence is possibly cut out for primer design, point mutations still exist in some strains. Therefore, we introduced 5 mutation sites (C355A, C360T, C365G, C373A, A379G)) from 4 existing strains (GenBank: EF121429.1, MH025920.1, KM111295.1, MH025919.1) into the B646L plasmid according to the sequence alignment results, and performed chip detection, and the results are shown in FIG. 4C. In agreement with the expectation, capillary No.1 detected no positive signal against the B646L gene, with a false negative result; and the other 4 genes show positive signals, so that the problem of missed detection is avoided.
In addition, we also aligned P two pairs of PCR primers (OIE-F/OIE-R and PPA-1/PPA-2) recommended by OIE (world animal health organization) with the target sequence, and the results are shown in FIG. 5. The base marked in dark color indicates that a plurality of point mutations exist in the recognition positions of the primers OIE-F and PPA-1, and some mutations are positioned at the 3' end of the primers and probably cause false negative results, which further explains the necessity of ASFV multiplex detection.
Example 4: comparison of the detection Capacity of Honeycomb chips with commercial kits
We purchased an african swine fever detection PCR kit and a LAMP kit (guangzhou yuyo biotechnology limited) approved by the chinese animal epidemic prevention control center, and detected the samples using the honeycomb chip with the commercial kit at the same time, and the results are shown in fig. 6. In addition to the Positive Control (PC) and the Negative Control (NC), we set up 5 concentration gradients, 10 each5、104、103、10210 copies per reaction. The results show that the Ct value is 0 when the template content is 10 copies per reaction, indicating that the sensitivity of both commercial kits is 100 copies/reaction, lower than the sensitivity of 80 copies/reaction of the honeycomb chip, which further demonstrates the utility of our platform.
Example 5: honeycomb chip for detecting African swine fever virus genome DNA (actual sample)
To test the actual samples, we obtained 3 genomic DNA samples of African swine fever virus from the national African swine fever Virus regional laboratory institute of southern China university of agriculture and tested them with a honeycomb chip, the results of which are shown in FIG. 7. The visualization result of the chip is consistent with the fluorescence quantification result, and three samples can be successfully detected, so that the reliability of the actual detection of the platform is verified.
Example 6: specificity test (actual sample) for Honeycomb chip detection of genomic DNA of other porcine viruses
To further demonstrate the specificity between the LAMP primer sets screened and other common porcine viruses, 3 copies of each of genomic DNA samples of porcine pseudorabies virus (PRV), porcine circovirus type 2 (PCV2) and Porcine Parvovirus (PPV) were obtained from the university of agriculture in Huazhong and detected using a honeycomb chip, and the results are shown in FIG. 8. The visual result of the chip is consistent with the result of fluorescence quantification, and other capillaries have no amplification signals except for Positive Control (PC), so that the LAMP primer group has high specificity and has no cross reaction with other common porcine viruses.
The invention has many applications, and the above description is only a preferred embodiment of the invention. It should be noted that the above examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that various modifications can be made without departing from the principles of the invention and these modifications are to be considered within the scope of the invention.
Sequence listing
<110> Shanghai Zhengzhengzheng Biotech Co., Ltd
<120> application of target gene combination in African swine fever virus detection and kit
<130> DD10330
<160> 120
<170> SIPOSequenceListing 1.0
<210> 1
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ttagccccgt tacgtatccg tggaaactta tcgataagat tga 43
<210> 2
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tccgatatag atgaacatgc gtcttaacgc cattatgcag c 41
<210> 3
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aaacatttcc gtaactgctc atg 23
<210> 4
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
cctgaaagct tatctctgcg tg 22
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ggtatgtaag agctgcagaa c 21
<210> 6
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
<210> 7
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gattgcgcag gaaaatgagg gcggttaagg gatttttca 39
<210> 8
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
cctcactatc gatcggcaaa atgctgaata atgcaaatat ggac 44
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
<210> 10
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ttaacggcag cttttctttg g 21
<210> 11
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gaaacaatga cccttcttaa ag 22
<210> 12
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
aatacgcttc ctttcgatg 19
<210> 13
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ggaatttaag tgaacgtcca acgctccacg caacttattc tca 43
<210> 14
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
ttattgtgat ggagcggctt taccctagta agtccttatg ggc 43
<210> 15
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
aatgtccatt aaaagcgtcg c 21
<210> 16
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
cctcacataa aggatgccat agaa 24
<210> 17
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
<210> 19
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
cacatctcgg gattggcctt gccttgccga ataatgg 37
<210> 20
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
agcgacaaaa agctgccact ccagactggc ttctggaaaa c 41
<210> 21
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
tagcggaaaa tacactcaaa tgatg 25
<210> 22
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
<210> 23
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
<210> 24
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
<210> 25
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
ggattgcctt tctaccccac ccatggtttg ctccaccc 38
<210> 26
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
cataggacaa ggtatgacgc gatcggttct atattaacaa tacctgc 47
<210> 27
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
aaatacgata aaagagcccg acat 24
<210> 28
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
aagatcctcc aggtcttgtt ctaca 25
<210> 29
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
<210> 30
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
<210> 31
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
atattagttg ggacacggat tacgttgaac cgttctgaag aagaag 46
<210> 32
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
ggatacgtta atatgaccac tgggtggtgc gatgatgatt acctt 45
<210> 33
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
tgatcttgtg gtatcggcat ct 22
<210> 34
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gtattcctcc cgtggcttc 19
<210> 35
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
<210> 36
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
<210> 37
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
ggattaaaac ctacctggaa catctccatg tccgaacttg tgcc 44
<210> 38
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
caatgggcca tttaagagca gtccataaaa cgcaggtgac 40
<210> 39
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
<210> 40
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
ttcatcgtgg tggttattgt tg 22
<210> 41
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
<210> 42
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
<210> 43
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
tacacaacct ttttgtaaaa cgcgggttat tgttggtgtg ggtc 44
<210> 44
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
caggggttac aaacaggtta ttgatccatg gtttatccca ggag 44
<210> 45
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
<210> 46
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
ttattcgtga gcgagatttc atta 24
<210> 47
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
agacattagt ttttcatcgt gg 22
<210> 48
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
<210> 49
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
cttaaccgct aaagtggaaa aacccgtttc aatggtaagg cc 42
<210> 50
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
cacccacaga ttcttaattt ccgtttgccg atcgatagtg ag 42
<210> 51
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
<210> 52
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
ggtaggcctc attttcctgc 20
<210> 53
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
cgggatctat gacgtacttc 20
<210> 54
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
<210> 55
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
taacctccgc caccattgat aactgtcgtc cttccacca 39
<210> 56
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
attacaaagg gaatacgcag cgcatcgacc ttatgctcat gta 43
<210> 57
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
caacctattt tggtatcgga aaag 24
<210> 58
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
<210> 59
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
<210> 60
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
<210> 61
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
gaaaacattc atgatcgtcc cacaacatcg aaaggaagcg ta 42
<210> 62
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
tgagagccgt ttcgcaggcg acagtacggt gtgga 35
<210> 63
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
taattttcat gcccggtatg g 21
<210> 64
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
<210> 65
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
tccatatttg cattattcag c 21
<210> 66
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
<210> 67
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
ccaaatagtc ttcatggccc ttattggaaa ccattatcct gaacc 45
<210> 68
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
ctcaaaatct ttattgcgac gctgccgctc catcacaata a 41
<210> 69
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
caacgaagtc tagatctcgt gc 22
<210> 70
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
<210> 71
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
ccttcataga ggcttataat gc 22
<210> 72
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
ctatggcatc ctttatgtga g 21
<210> 73
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
cctctcgaat cgttagggga acggtactta attgcctttc gc 42
<210> 74
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
cgtatggcgg gaggtgttta tcacgtgtta aaaatgggga a 41
<210> 75
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
<210> 76
<211> 23
<212> DNA
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<400> 76
tgttccgact tagtcatcaa tgg 23
<210> 77
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
tgaagcccat aaggacttac t 21
<210> 78
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
tggttaatgc cttgcaaata c 21
<210> 79
<211> 43
<212> DNA
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<400> 79
gcgctatccc attgatgact aagcgattcg agaggtccta caa 43
<210> 80
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
ttaacacgtg gacgtatttg caaagctctc gggcaataaa atc 43
<210> 81
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
cctgcataat aaacacctcc cg 22
<210> 82
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
tgaaaacacg tctttgcagg a 21
<210> 83
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
tgaaaacacg tctttgcagg a 21
<210> 84
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
<210> 85
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
ggtttgaact ttcaggccgt gtgaataagc cggggaatat 40
<210> 86
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
cacaatcacc ttggacccga gctgtacgcg aacgaag 37
<210> 87
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
ggtacgagat gattatgtca ttgcc 25
<210> 88
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
<210> 89
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
caccaccctc ccaaata 17
<210> 90
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
<210> 91
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
tgtacgcgaa cgaagcctgg atgagaatac gcagctggt 39
<210> 92
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
ctctatttcg ctgtgcagaa tggattcgcg atgagcacac 40
<210> 93
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
ttaacgcctc gtccaacctg 20
<210> 94
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
cgtactggaa taaactgatg gtcg 24
<210> 95
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
<210> 96
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
<210> 97
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
aacaaacgga acccaaggct tactgtgctt gccatagaaa gg 42
<210> 98
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
caccatgtca taaatgctgt acgctgagta tcagcttgaa acgct 45
<210> 99
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
<210> 100
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
<210> 101
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
<210> 102
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
tacttacatt tcatcgaatg cc 22
<210> 103
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
tgggcacaaa gcggttctat atttgtgtat taagatcctc caggtc 46
<210> 104
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
ccttcgtctt ttggcagcac ctgactaccc tcatctaagc ct 42
<210> 105
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
<210> 106
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
<210> 107
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
tttccatagg acaaggtatg a 21
<210> 108
<211> 20
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<400> 108
<210> 109
<211> 40
<212> DNA
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<400> 109
cgtcatttac tcagaggccg acctccgtga aattccaata 40
<210> 110
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
cgtgggtgct ggcataggac cgacctgtat gacctg 36
<210> 111
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
<210> 112
<211> 18
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<213> Artificial Sequence (Artificial Sequence)
<400> 112
aaccgttgcc tgcgtgtc 18
<210> 113
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
<210> 114
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
<210> 115
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
ccggtgggaa acaatctacg gttaaactct tcctgggtaa tgt 43
<210> 116
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
gcatacgtgt tttggagcaa cgcgaacgga tgatcattat ttc 43
<210> 117
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
cgcaacattc gcatctacc 19
<210> 118
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
<210> 119
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
cacaaatagg taaacccaaa g 21
<210> 120
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
Claims (10)
1. The application of a target gene combination in the detection of African swine fever virus with non-diagnostic purpose is characterized in that the target gene combination comprises B646L, B962L, C717R, D1133L and G1340L genes.
2. An LAMP primer group for multi-gene detection of African swine fever virus, which is characterized in that the primer group comprises 5 sets of LAMP primers, and the LAMP primers respectively target 5 genes of the African swine fever virus: B646L, B962L, C717R, D1133L and G1340L, which consist of nucleotide sequences shown as SEQ ID Nos. 1 to 30.
3. An African swine fever virus multiple nucleic acid detection kit, which is characterized by comprising the LAMP primer group of claim 1.
4. The African swine fever virus multiple nucleic acid detection kit according to claim 3, wherein the kit further comprises a honeycomb chip, a sample adding joint, a sample lysate, a LAMP reaction solution, positive and negative controls, a sealing membrane and a reaction tube.
5. The African swine fever virus multiple nucleic acid detection kit according to claim 3 or 4, wherein the LAMP primer group is obtained by layer-by-layer screening of LAMP reaction in PCR tubes and on chips, and is pre-fixed at corresponding positions of a honeycomb chip.
6. The multiple nucleic acid detection kit for African swine fever virus according to claim 4, wherein the honeycomb chip is fixed in the reaction tube after hydrophobic modification and primer pre-fixation;
the sample adding joint can be tightly combined with the honeycomb chip and the reaction tube;
the main component of the sample lysate is NaOH;
the LAMP reaction solution contains 1 XThermoPol buffer solution and 8.0mM MgSO41.4mM dNTPs, 0.8M betaine, 25. mu.M calcein, 0.5mM MnCl2,0.32UμL-1Bst DNA polymerase.
7. The multiple nucleic acid detection kit for African swine fever virus according to claim 4, wherein the positive control is a plasmid without the nucleic acid sequence of African swine fever virus, and the corresponding LAMP primer is pre-fixed in a capillary labeled "PC" on a honeycomb chip.
8. The multiple nucleic acid detection kit according to claim 4, wherein the negative control is a control without any primer immobilized in a capillary labeled "NC" on a honeycomb chip.
9. A method for multiple gene detection of African swine fever virus according to the kit of claim 4, comprising the steps of collecting and pre-treating a swine blood sample, loading, incubating and detecting, wherein the total time consumption of the method is less than 70 min.
10. The method for detecting multiple genes of African swine fever virus according to claim 9, wherein the method comprises the following steps:
collecting a pig blood sample: collecting a pig blood sample and storing the pig blood sample in an anticoagulation tube;
pretreatment: fully mixing a pig blood sample stored in an anticoagulation tube with a sample lysate according to a volume ratio of 1:2, performing room temperature lysis for 3min, and then sucking the lysed mixed solution to mix with an LAMP reaction solution according to a volume ratio of 1: 24;
sample adding: sucking the mixed solution obtained from the pretreatment, inserting the mixed solution into a sample adding joint, moving the mixed solution to a reaction tube for sample adding, removing the sample adding joint, and sealing the reaction tube by using a sealing film;
incubation and detection: reacting at 63 ℃ for 1h, and performing visual detection by using ultraviolet irradiation.
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