CN112094948B - Application of target gene combination in African swine fever virus detection and kit - Google Patents
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
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Abstract
The invention discloses 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 groups respectively targeting the genes consist of nucleotide sequences shown in SEQ ID No. 1-30. The African swine fever multiplex nucleic acid detection kit prepared by the method establishes the whole detection flow by using a honeycomb chip and an LAMP direct amplification method. Compared with the prior art, the method has the advantages that primers are designed for a plurality of genes of ASFV, the genes are detected simultaneously by combining the chips, false negative results caused by single gene mutation can be effectively avoided, the nucleic acid extraction step is not needed, multiple visual detection can be realized, precise instruments and professional operation are not relied on, the method has the advantages of accuracy, rapidness, simplicity, convenience and the like, and the method 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 especially relates to a multiple nucleic acid detection kit for African swine fever virus based on a honeycomb chip.
Background
African swine fever (African swine fever, ASF) is an acute, highly infectious and fatal disease of pigs caused by African swine fever virus (African swine fever virus, ASFV), and is characterized by short onset time, mortality up to 100%, clinical manifestations of hyperthermia, diarrhea, extensive bleeding of skin and viscera, etc. (Normile, D., 2)018,Science,361,741；Ma,J.,et al.,2020,Preventive Veterinary Medicine,175,104861；Galindo,I.,&Alonso, C,2017, viruses,9 (5), 103). ASF was first reported in the Kenya of Africa in 1921, and then rapidly spread to more than 60 countries such as Europe, lamet and Asia, resulting in significant economic losses to the world (Montgomery, R.E.,1921,Journal of comparative pathology and therapeutics,34,159-191; costard, S., et al 2013,Virus research,173 (1), 191-197;Dei Giudici,S, 2019,Archives of virology,164 (3), 739-745). In 2018, 8 months, ASF first developed in the sun in the Chinese Liaoning, followed by rapid onset of other provinces. By 3 months and 5 days in 2020, 165 African swine fever epidemic situations are totally exploded in 32 provinces in China, 1,193,000 pigs are cumulatively killed, and huge threat is brought to pig industry in China and even worldwidehttp://www.fao.org/ag/againfo/programmes/en/empres/ASF/situation_ update.html). ASF is listed as an animal epidemic that must be reported according to the report list of animal epidemic of version 2019 issued by the world animal health Organization (OIE).
ASFV is a dsDNA virus with icosahedral symmetry, the only member of the genus African swine fever virus of the family African swine fever virus, and the only known arbovirus. ASFV has a large genome, about 170-190 kb in length, encoding 150-200 proteins altogether (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, genes B646L encoding capsid protein Vp72 have been classified into 24 genotypes with higher homology, and also into more subtypes in combination with other genes such as B602L (Quembo cj et al 2018,Transbound Emerg Dis,65 (2): 420-431). At present, no effective treatment mode and no effective vaccine exist for ASF, and the method for killing and burning is the most effective means for preventing epidemic situation from spreading, so that the rapid and accurate detection of ASFV is of great importance.
Existing ASFV detection methods can be divided into two categories: (1) Immunological detection methods, including viral isolation (Rowlan, R.J., et al, 2008,Emerging infectious diseases,14 (12), 1870), erythrocyte adsorption assays (Rodrii guez, J.M., et al, 1993,Journal of virolog)y,67 (9), 5312-5320), enzyme-linked immunosorbent assay (cube, c., et al, 2013,Virus research,173 (1), 159-167), fluorescent antibody detection (galrado, c., et al, 2015,Journal of clinical microbiology,53 (8), 2555-2565), and the like. Although the virus separation method (VI) and the red blood cell adsorption test (HAD) are very reliable and effective, they are time-consuming, cumbersome to handle, 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 the antibody of ASFV, but are limited by detection sensitivity, and cannot be accurately detected at the early stage of infection, and pigs may die due to virus infection before the antibody is positive. Therefore, molecular biological detection is the mainstream detection method because of its simple and rapid operation, high sensitivity and high specificity. (2) Molecular biological detection method including fluorescent quantitative PCRM., et al,2003,Journal of clinical microbiology,41 (9), 4431-4434), LAMP (James, h.e., et al, 2010,Journal of virological methods,164 (1-2), 68-74), RPA (Wang, j., et al, 2017,Canadian Journal of Veterinary Research,81 (4), 308-312), and the like. Fluorescent quantitative PCR is a gold standard for laboratory detection of ASFV as recognized by the world animal health Organization (OIE), but relies on sophisticated thermal cycling instrumentation and specialized operators and is not suitable for field detection in resource limited areas. Isothermal amplification plays an increasingly important role in the field of molecular detection due to the advantages of simple operation, no need of precise instruments, short time consumption and the like. In recent years, many researchers have used LAMP or RPA in combination with CRISPR systems and test strips for ASFV detection, both to increase sensitivity and to achieve rapid in-situ 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 have required enrichment of the product by isothermal amplification prior to transfer to the detection system, amplification and detection phase separation, uncapping is prone to aerosol contamination, false positive results, and increased procedures. In addition, toAll of the above-mentioned methods target only a single gene of ASFV, e.g., gene B646L encoding viral capsid protein VP72 (Wang, j., et al, 2017,Canadian Journal of Veterinary Research,81 (4), 308-312), gene P1192R encoding topoisomerase II (James, h.e., et al, 2010,Journal of virological methods,164 (1-2), 68-74), gene K78R encoding DNA binding protein VP10 (Wang, d., et al, 2020,Journal of virological methods,276:113775), easily cause false negative results from single gene mutations due to the large, complex and variable genomic characteristics of ASFV.
The prior patent document CN110373500A discloses a double fluorescence PCR detection kit, which selects primers and probes aiming at conserved fragments 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 African swine fever wild strain and gene deletion strain, and aims at three genes of African swine fever virus CD2V, VP and MGF-360 14L. However, they have the disadvantages that PCR is a temperature-changing reaction, a precise multichannel fluorescence PCR instrument is needed, the price is high, the volume is huge, and the method is not suitable for on-site instant detection in pig farms. Meanwhile, the PCR tube is limited by competition and interference among a plurality of pairs of PCR primers in the same PCR tube, the detection weight is not easy to increase, the African swine fever is taken as a virus, the gene can be continuously mutated, and the increase of the weight can ensure the accuracy.
In view of the above, in the field of african swine fever detection, there is an urgent need to develop a simple, rapid, highly sensitive multiplex nucleic acid detection method for targeting multiple genes of ASFV, which does not require uncapping, and which 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 multi-gene detection of African Swine Fever Virus (ASFV) only aiming at ASFV single-gene detection and relying on a complex and precise instrument. Aiming at the first problem, the invention designs and screens LAMP primers for detection according to 5 genes of ASFV, can effectively avoid false negative results caused by single gene mutation, and improves the positive detection rate; 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, and combines the LAMP direct amplification method to remove the nucleic acid extraction step, thereby not only realizing the multiple visual detection, but also not depending on precise instruments and professional operation, having the advantages of high sensitivity, strong specificity, simple and rapid operation and the like, being suitable for port inspection and quarantine and on-site instant detection and having good application prospect.
The invention aims at realizing the following technical scheme:
the invention provides an application of a target gene combination in African swine fever virus detection for non-diagnosis purposes, wherein the target gene combination comprises genes B646L, B962L, C717R, D1133L and G1340L.
The invention also provides an LAMP primer group for the polygene detection of African swine fever virus, which is characterized in that the primer group comprises 5 sets of LAMP primers for respectively targeting 5 genes of the African swine fever virus: B646L, B962L, C717R, D1133L, G1340L, consisting of the nucleotide sequences shown in SEQ ID Nos. 1 to 30.
The invention also provides a kit for detecting multiple nucleic acids of African swine fever virus, which comprises the LAMP primer group.
Preferably, the kit further comprises a honeycomb chip, a sample adding connector, a sample lysate, an LAMP reaction solution, positive and negative controls, a sealing film and a reaction tube.
Preferably, the LAMP primer group is obtained through LAMP reaction layer-by-layer screening in a PCR tube and on a chip, and is respectively pre-fixed at the corresponding position of the honeycomb chip.
Preferably, the honeycomb chip is a microarray integrated with a plurality of capillaries, and is shaped like a "honeycomb" and thus named as a "honeycomb chip", and the honeycomb chip is immobilized in a reaction tube after being hydrophobically modified and pre-immobilized with primers;
the sample adding joint can be tightly combined with the honeycomb chip and the reaction tube, so that simultaneous sample injection of a plurality of capillaries is realized;
the main component of the sample lysate is NaOH, which is used for pretreatment of pig blood samples and releases African swine fever virus nucleic acid.
Preferably, the LAMP reaction solution contains 1×Thermopol buffer, 8.0mM MgSO 4 1.4mM dNTPs,0.8M betaine, 25. Mu.M calcein, 0.5mM MnCl 2 ,0.32UμL -1 Bst DNA polymerase.
Preferably, the positive control is a plasmid without african swine fever virus nucleic acid sequence, and its corresponding LAMP primers are pre-immobilized in capillaries labeled "PC" on a honeycomb chip.
Preferably, the negative control is one in which no primer is immobilized within a capillary labeled "NC" on a honeycomb chip.
The sealing film has optical permeability, can be used for sealing the reaction tube after sample addition is completed, avoids aerosol pollution, and does not influence visual detection of results.
The invention also provides a method for carrying out the polygene detection of African swine fever virus by using the kit according to claim 3, which comprises the steps of collection and pretreatment, sample addition, incubation and detection of a swine blood sample, wherein the total time consumption of the method is less than 70 minutes.
Preferably, the specific steps of the method are as follows:
collecting a pig blood sample: the pig blood sample is required to be stored in an anticoagulation tube after being collected;
pretreatment: taking a pig blood sample stored in an anticoagulation tube, fully mixing the pig blood sample with a sample lysate according to a volume ratio of 1:2, performing room temperature pyrolysis for 3min, and then sucking the cracked mixed solution and mixing the LAMP reaction solution according to a volume ratio of 1:24;
sample adding: the mixed solution obtained by the pretreatment is absorbed and inserted into a sample adding joint, the sample adding joint is moved to a reaction tube for sample adding, then the sample adding joint is removed, and the reaction tube is sealed by a sealing film;
incubation and detection: the reaction was carried out at 63℃for 1 hour and visualized by ultraviolet irradiation.
Preferably, the ultraviolet irradiation adopts a handheld ultraviolet irradiation device, and the temperature control and detection of the incubation can be integrated into an automatic device, and the automatic operation of the automatic device is controlled by a software program.
The invention can detect single sample, and can integrate 8-connection tube to detect multiple samples.
The ASFV multiplex nucleic acid detection kit prepared by the LAMP primer group establishes the whole detection flow 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 complex and changeable ASFV genome and numerous genotypes, the LAMP primer is designed and screened by selecting the 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 chips, the rapid sample adding, sealing amplification and multiple visual detection are realized.
3. By combining the LAMP direct amplification method, the pretreatment of the sample is simplified, the 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 present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:
FIG. 1 shows the results of the validity and specificity test of the primer in the PCR tube in example 1 of the present invention;
FIG. 2 shows the results of the specificity and sensitivity test of primers on a honeycomb chip in example 2 of the present invention;
FIG. 3 is a flow chart of ASFV detection according to the present invention, which is exemplified in embodiment 3;
FIG. 4 shows the results of a simulated pig blood sample according to example 3 of the present invention;
FIG. 5 shows the result of the alignment of two pairs of PCR primers recommended by OIE (world animal health organization) in example 3 of the present invention with the target sequences in the existing ASFV strain;
FIG. 6 is the sensitivity test results of the commercial kit for performance comparison in example 4 of the present invention;
wherein 1-5 in the chip pattern diagram respectively represent B646L, B962L, C717R, D1133L, G1340L, PC represents positive control, and NC represents negative control;
FIG. 7 shows the results of the chip test of the African swine fever virus genomic DNA sample of example 5 of the present invention;
FIG. 8 shows the results of the specificity test of the genomic DNA of the porcine virus of example 6 according to 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 present invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications could be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention.
Example 1: effectiveness and specificity of primer based on in-tube LAMP test
According to the genome analysis results of the China/2018/AnhuiXCGQ virus strain (Bao, J., et al, 2019,Transboundary and emerging diseases,66 (3), 1167-1176), all single gene names of ASFV are input into the NCBI database one by one for searching, all the searched sequences are downloaded and subjected to sequence comparison, finally 5 genes with a conserved sequence of about 1000bp are selected, LAMP primer design is performed by PrimerExplorer V5 software, 4 sets of primers are designed for each gene, screening and verification are performed on all the primers by using the conventional LAMP reaction of calcein-containing dye, one set of LAMP primer sets which can be successfully detected is screened for each gene, 5 primer sets for ASFV detection are finally selected (as shown in Table 2), 5 genes B L, B962L, C717R, D1133L, G L are targeted respectively, and the unselected primer sets are shown in Table 3.
The conserved sequences of about 1000bp selected in this example were only relatively conserved, and sequences after some mutant strains were deleted, because even the OIE-recommended PCR primers were not completely conserved, there were still mutations in the strains (fig. 5), thus we obtained 5 genes. The specific selection process and principle are as follows: (1) Genomic comparison of the type II african swine fever virus strain (China/2018/AnhuiXCGQ) with other major type II virus strains (i.e. POL/2015/Podlaskie, estonia 2014, russia/odiotsov_02/14/Boar and Georgia 2007/1) revealed that in all five type II african swine fever virus strains there were 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,Transboundary and emerging diseases,66 (3), 1167-1176); (2) Since most of the variation between the African swine fever virus genomes is known to be caused by additions and deletions of members of the multigene family, genes of this family are excluded; (3) The target gene should be selected from different gene function types as much as possible in order to ensure the diversity of the gene types and avoid the mutation of a certain kind of genes; (4) The target gene is about 1000bp in length, and aims to ensure that multiple sequence alignment is carried out with known strains listed in GenBank database, and a conserved sequence for primer design is obtained. Table 1 below is the genetic information selected according to the above principles.
TABLE 1 Gene information contained in target Gene combinations for African swine fever Virus detection
TABLE 25 LAMP primer sets for African swine fever virus detection
TABLE 3 unselected LAMP primer sets
The effectiveness and specificity of the primers were tested by conventional LAMP using 5-gene plasmid standard as a template, and the specific reaction system is shown in Table 4, and the reaction products were detected by both ultraviolet irradiation and agarose gel electrophoresis.
FIG. 1A is a primer availability test result of 5 genes, the left graph of FIG. 1A shows fluorescence detection results after each gene is amplified by 4 sets of LAMP primers, respectively, and the right graph shows results of gel electrophoresis of amplified products in the corresponding tube on the left side; p represents addition of plasmid template, N represents 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 is found: the 4 sets of primers of gene B646L, B962L, D1133L, G1340L can successfully detect the target gene, while the gene C717R can only successfully detect the number 2 primer set, which also proves the necessity of designing multiple sets of primers to screen the optimal primers.
Based on the results of the primer validity test, we selected 1 set of primers from each gene to perform the specificity test between each other, namely B646L-2, B962L-2, C717R-2, D1133L-3, G1340L-1, and then amplified each gene with the 5 sets of primers to see if nonspecific amplification will occur, and the test results are shown in FIG. 1B. By comparing the fluorescence detection and gel electrophoresis results of the N groups and the P groups, the products can be successfully detected only if the respective gene templates and the corresponding primers are added into the same EP tube, and no cross reaction exists between the 5 sets of primers and the templates.
In summary, 5 primer sets capable of being used for ASFV detection were obtained through the effectiveness and specificity test of the in-tube LAMP on the primers, and no cross reaction was caused between them.
TABLE 4 conventional LAMP reaction System
Example 2: specificity and sensitivity of primers based on-chip LAMP test
The specificity and sensitivity test of the primers was performed using a honeycomb chip, the 5 LAMP primer sets obtained in example 1 and the positive control primer set were pre-immobilized at the corresponding capillary positions, 1-5 in the chip pattern diagram respectively represent B646L, B962L, C717R, D1133L, G L, PC represents the positive control, NC represents the negative control, and the specific reaction system is shown in Table 5, and the reaction products were detected by ultraviolet irradiation.
FIG. 2A is a primer specificity test result of 5 genes, a plasmid template of a single gene and a positive control plasmid were successively mixed with the LAMP reaction solution, then loaded onto a chip, reacted at 63℃for 1 hour, and visually detected by a hand-held ultraviolet irradiation device. The results showed that in the chip diagram of 5 ASFV gene assays, 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 primer sensitivity test results of 5 genes, wherein the plasmid templates of the 5 genes and the positive control plasmid are mixed and diluted step by step, then are mixed with LAMP reaction liquid and loaded on a chip for reaction for 1h at 63 ℃, and are visually detected by a handheld ultraviolet irradiation device. The results show that 5 gene targets can be successfully detected when the template concentration is 880 copies/microliter, 88 copies/microliter and 30 copies/microliter; whereas at a template concentration of 15 copies/microliter, only 3 (C717R) and 4 (D1133L) targets detected fluorescence, which is inconsistent with the expected signal, the lowest detection limit was defined as 30 copies/microliter, i.e., 48 copies/capillary. The sensitivity of PCR primers recommended by the world animal health Organization (OIE) was about 600 copies/reaction (Wilkinson P.J.office International Des Epizooties, 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 we screened met the practical detection requirements.
TABLE 5 chip LAMP reaction System
In summary, the specificity and sensitivity tests of the primers by on-chip LAMP confirm that the 5 primer sets selected in example 1 have high specificity and overall sensitivity as low as 30 copies/microliter, with individual targets below 15 copies/microliter.
Example 3: multiplex nucleic acid detection (detection junction containing single gene mutation) of pig blood simulation sample based on honeycomb chip
Fruit
The test of the pig blood simulation sample is carried out by using the honeycomb chip, 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, mixing plasmid templates of 5 genes, then diluting step by step, and then mixing 10 mu L of mixed plasmid with 90 mu L of anticoagulated pig blood to obtain a simulation sample; then, 10. Mu.L of the simulated sample was mixed with 20. Mu.L of the sample lysate, lysed at room temperature for 3min, then 1. Mu.L of the lysed sample and 1. Mu.L of the positive control were pipetted and mixed with 23. Mu.L of the LAMP reaction solution, loaded onto a chip, reacted at 63℃for 1 hour, and fluorescence was detected, and the result is shown in FIG. 4A. Two conclusions can be drawn from the chip result plot: (1) The LAMP direct amplification method does not affect the amplification and detection of ASFV genes, and the lowest detection limit of a pig blood simulated sample is 50 copies/microliter, namely 80 copies/capillary, which is equivalent to 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 Epizooties, 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 primers screened by us meets the actual detection requirements; (2) The pig blood simulation sample added with the positive control plasmid only, except the positive control hole, does not detect other fluorescent signals, which indicates that the complex background of the pig blood does not cause non-specific amplification of the primer, and further verifies the specificity of the LAMP primer.
To further demonstrate the necessity of multiple ASFV assays, we aligned the sequences of existing strains in the GenBank database and introduced the found point mutations into plasmid templates for detection, to see if false negative results occur. Taking the B646L gene encoding VP72 as an example, FIG. 4B shows the sequence alignment of 61 strains, and the darkly labeled bases indicate that point mutations are still present in some strains, although conserved sequences are cut as far as possible for primer design. Therefore, according to the sequence alignment, 5 mutation sites (C355A, C360T, C365G, C373A, A379G)) from 4 existing strains (GenBank: EF121429.1, MH025920.1, KM111295.1, MH 025919.1) were introduced into the B646L plasmid for chip detection, and the results are shown in FIG. 4C. Consistent with expectations, no positive signal was detected for capillary No.1 detected for the B646L gene, a false negative result occurred; the other 4 genes show positive signals, so that the problem of missed detection is avoided.
Furthermore, we also aligned the P two pairs of PCR primers (OIE-F/OIE-R and PPA-1/PPA-2) recommended by OIE (world animal health organization) with the targeting sequence, and the results are shown in FIG. 5. The darkly labeled bases indicate that there are many point mutations at the recognition sites of the primers OIE-F and PPA-1, and that some of the mutations are located at the 3' end of the primers, which are likely to cause false negative results, which also further illustrate the necessity of multiplex detection of ASFV.
Example 4: detection capability comparison of honeycomb chip and commercial kit
We purchased the African swine fever detection PCR kit and LAMP kit (Guangzhou Yuan Biotechnology Co., ltd.) approved by the Chinese animal epidemic prevention control center, and samples tested by using the honeycomb chip were tested by using the commercial kit at the same time, and the results are shown in FIG. 6. Except for yangIn addition to the sex control (PC) and the Negative Control (NC), we set 5 concentration gradients of 10 respectively 5 、10 4 、10 3 、10 2 10 copies per reaction. The results showed that when the template content was 10 copies per reaction, the Ct value was 0, indicating that the sensitivity of both commercial kits was 100 copies per reaction, which was lower than the sensitivity of 80 copies per reaction of the honeycomb chip, which further demonstrates the utility of our platform.
Example 5: detection of African swine fever Virus genomic DNA (actual sample) by honeycomb chip
For the detection of the actual samples, we obtained 3 samples of genomic DNA of African swine fever virus from the national African swine fever virus area laboratory institute of agricultural university of North China and used the honeycomb chip for the detection, and the results are shown in FIG. 7. The visual result of the chip is consistent with the result of fluorescence quantification, and three samples can be successfully detected, so that the reliability of actual detection of a platform is verified.
Example 6: specificity test (actual sample) of genomic DNA of Honeycomb chip for detecting other porcine viruses
To further demonstrate the specificity between the screened LAMP primer sets and other common porcine viruses, we obtained 3 copies of genomic DNA samples of porcine pseudorabies virus (PRV), porcine circovirus type 2 (PCV 2) and Porcine Parvovirus (PPV) from the university of Huazhong agriculture, and tested them with 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 except Positive Control (PC) have no amplification signal, so that the LAMP primer group has high specificity and has no cross reaction with other common porcine viruses.
There are many ways in which the invention may be practiced, and what has been described above is merely 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 modifications may be made without departing from the principles of the invention, and such modifications are intended to be within the scope of the invention.
Sequence listing
<110> Shanghai true testing Biotech Co., ltd
<120> application of target gene combination in African swine fever virus detection and kit
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tgaagcccat aaggacttac t 21
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tggttaatgc cttgcaaata c 21
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gcgctatccc attgatgact aagcgattcg agaggtccta caa 43
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ttaacacgtg gacgtatttg caaagctctc gggcaataaa atc 43
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cctgcataat aaacacctcc cg 22
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tgaaaacacg tctttgcagg a 21
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tgaaaacacg tctttgcagg a 21
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gcccttgata atttctttgg 20
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ggtttgaact ttcaggccgt gtgaataagc cggggaatat 40
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cacaatcacc ttggacccga gctgtacgcg aacgaag 37
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<400> 87
ggtacgagat gattatgtca ttgcc 25
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<400> 88
caggttggac gaggcgtt 18
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caccaccctc ccaaata 17
<210> 90
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attctgcaca gcgaaatag 19
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tgtacgcgaa cgaagcctgg atgagaatac gcagctggt 39
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ctctatttcg ctgtgcagaa tggattcgcg atgagcacac 40
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<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
tccacaatca ccttggac 18
<210> 96
<211> 15
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 96
gacccgctgc tccat 15
<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
gcctgtttaa ctcgacggaa 20
<210> 100
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 100
cgagatttta gggtcagggc 20
<210> 101
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 101
taccccgttt tccagaag 18
<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
cctcaccaag cacacgtttg 20
<210> 106
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 106
gacagtaatt tagggggtgg cg 22
<210> 107
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 107
tttccatagg acaaggtatg a 21
<210> 108
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 108
acctgcctac tatagctgcc 20
<210> 109
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<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
gccaagcgca aggagctag 19
<210> 112
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 112
aaccgttgcc tgcgtgtc 18
<210> 113
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 113
ggcagctata gtaggcaggt 20
<210> 114
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 114
tacgtgcgct tcaagaac 18
<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
attgatgtcc attttgcg 18
<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
gggatttatt cgtatgatcc 20
Claims (8)
1. The kit is characterized by comprising an LAMP primer group, wherein the primer group comprises 5 sets of LAMP primers for respectively targeting 5 genes of African swine fever virus: B646L, B962L, C717R, D1133L, G1340L, consisting of the nucleotide sequences shown in SEQ ID Nos. 1 to 30.
2. The african swine fever virus multiplex nucleic acid detection kit of claim 1, further comprising a honeycomb chip, a sample-adding adapter, a sample lysate, a LAMP reaction solution, positive and negative controls, a sealing film, a reaction tube.
3. The african swine fever virus multiplex nucleic acid detection kit of claim 1 or 2, wherein the LAMP primer sets are obtained by screening LAMP reaction layers in PCR tubes and on a chip, and are pre-fixed at corresponding positions of honeycomb chips, respectively.
4. The african swine fever virus multiplex nucleic acid detection kit of claim 2, wherein the honeycomb chip is immobilized in a reaction tube after hydrophobic modification and primer pre-immobilization;
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×Thermopol buffer, 8.0mM MgSO 4 1.4mM dNTPs,0.8M betaine, 25. Mu.M calcein, 0.5mM MnCl 2 ,0.32UμL -1 Bst DNA polymerase.
5. The african swine fever virus multiplex nucleic acid kit of claim 2, wherein the positive control is a plasmid containing no african swine fever virus nucleic acid sequence, and the corresponding LAMP primers are immobilized in advance in capillaries labeled "PC" on a honeycomb chip.
6. The african swine fever virus multiplex nucleic acid detection kit of claim 2, wherein the negative control is a control in which no primer is immobilized in a capillary labeled "NC" on a honeycomb chip.
7. The method for polygene detection of african swine fever virus non-disease diagnosis according to claim 2, characterized in that it comprises the steps of collection and pretreatment, sample addition, incubation and detection of swine blood samples, the total time of the method being less than 70min.
8. The method according to claim 7, characterized in that the specific steps of the method are as follows:
collecting a pig blood sample: the pig blood sample is required to be stored in an anticoagulation tube after being collected;
pretreatment: taking a pig blood sample stored in an anticoagulation tube, fully mixing the pig blood sample with a sample lysate according to a volume ratio of 1:2, performing room temperature pyrolysis for 3min, and then sucking the cracked mixed solution and mixing the LAMP reaction solution according to a volume ratio of 1:24;
sample adding: the mixed solution obtained by the pretreatment is absorbed and inserted into a sample adding joint, the sample adding joint is moved to a reaction tube for sample adding, then the sample adding joint is removed, and the reaction tube is sealed by a sealing film;
incubation and detection: the reaction was carried out at 63℃for 1 hour and visualized by ultraviolet irradiation.
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| CN113061650A (en) * | 2021-02-19 | 2021-07-02 | 中国科学院深圳先进技术研究院 | Instant detection system and method for pathogen nucleic acid |
| CN113073148A (en) * | 2021-04-08 | 2021-07-06 | 青岛农业大学 | On-site differential diagnosis kit for African swine fever virus and application thereof |
| CN113215311A (en) * | 2021-04-23 | 2021-08-06 | 华南农业大学 | Primer combination and kit for identifying African swine fever virus gene deletion strain and African swine fever epidemic strain by centrifugal microfluidic chip |
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| CN113186357A (en) * | 2021-06-02 | 2021-07-30 | 上海真测生物科技有限公司 | Novel RT-LAMP primer combination for multiple detection of coronavirus and kit thereof |
| CN114410587A (en) * | 2021-09-23 | 2022-04-29 | 中国农业科学院兰州兽医研究所 | Method for improving infection titer of African swine fever virus non-target cell MA-104 cell line |
| CN114410845A (en) * | 2022-02-09 | 2022-04-29 | 广东省农业科学院动物卫生研究所 | Locked nucleic acid modified one-step nested PCR (polymerase chain reaction) primer group and kit for detecting African swine fever virus |
| CN114774590B (en) * | 2022-05-21 | 2023-12-12 | 中国科学院青岛生物能源与过程研究所 | Double-target combination, primer combination, reagent, kit and method for detecting African swine fever virus and application |
| CN116875744B (en) * | 2023-09-08 | 2023-12-29 | 江西农业大学 | Kit for detecting African swine fever virus |
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