Primer group and method for detecting African swine fever virus

Abstract

The invention relates to the technical field of biology, in particular to a primer group and a method for detecting African swine fever virus. The primer group for detecting the African swine fever virus comprises the nucleotide sequences of primers as shown in SEQ ID NO. 1 to SEQ ID NO. 6; a pig saliva sample can be directly collected and added into an LAMP reaction reagent integrated in the micro-fluidic chip, the detection process is automatically completed by a full-automatic detection instrument, a spectrum is analyzed through a spectrum sensor, and a collected spectrum signal is comprehensively judged through an AI technology, so that full-automatic operation is realized, and the detection efficiency is improved. The detection time can be controlled within one hour, real-time diagnosis is realized, professionals and laboratory environments are not needed, the kit is portable and light, the kit can be applied to real-time detection in scenes such as pet hospitals on site, an innovative LAMP test method is adopted, nucleic acid extraction is not needed, a sample can be obtained, and the kit is low in price.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a primer set and method for detecting African swine fever virus. Background Art

[0002] African swine fever virus (ASFV) is the only species in the Asfarviridae family. It is a double-stranded nucleocytoplasmic large DNA virus (NCLDV). It is contagious and highly pathogenic. The clinical symptoms of acute cases are mainly characterized by high fever, short course of illness, high mortality, extensive bleeding in internal organs, and dysfunction of the respiratory and nervous systems. The 3D fine structure of the swine fever virus has been deciphered, but as of early 2020, there is still no specific vaccine or antiviral drug for ASFV that can effectively control the spread of the virus in a timely manner when an epidemic breaks out.

[0003] The incubation period of natural infection of African swine fever is 5 to 9 days, often shorter, and the incubation period of clinical experimental infection is 2 to 5 days. When the disease occurs, the body temperature rises to 41°C and lasts for about four days. It is characterized by the body temperature starting to drop forty-eight hours before death. At the same time, the clinical symptoms are not shown until the body temperature drops. Therefore, it is different from the symptoms of swine fever when the body temperature rises. During the first three to four days of fever, the pigs have no appetite and are extremely fragile. They lie in the corner of the house. If they are forced to get up and move around, they show extreme fatigue, especially in the hind legs. The pulse is Rapid pulse, cough, rapid breathing of about one-third, obvious difficulty breathing, serous or mucopurulent conjunctivitis, some strains can cause bloody diarrhea, vomiting, blood changes similar to swine fever, from three to five cases, showing a 50% decrease in white blood cell count, lymphocytes also decreased, leukocytic anemia occurred when the body temperature rose, the white blood cell count dropped to 40% on the fourth day and then stopped decreasing, an increase in the number of immature neutrophils can also be observed, often death on the seventh day after the fever, or death only one or two days after the onset of symptoms.

[0004] Depending on the virus strain, the number of bases in the genomic DNA is about 170,000 to 190,000. Like other NCLDVs, ASFV encodes many proteins that, in addition to structural proteins specifically used for virus assembly, are also involved in evading host defense mechanisms, such as type I interferon and apoptosis pathways, as well as biological processes such as DNA replication repair and regulation of gene expression. The functions of most ASFV genes are unknown and still need to be explored.

[0005] ASFV is a regular icosahedron with a diameter of about 200nm, and is composed of multiple layers of material: a protein coreshell containing a nucleoid in the center, and a lipid envelope and a protein capsid from the inside to the outside. The capsid is composed of 8280 major capsid proteins p72 and 60 pentaproteins. In addition, at least three proteins maintain the stability of the capsid structure by adhering to adjacent proteins.

[0006] African swine fever (ASF) is an acute, hemorrhagic, highly contagious disease caused by the African swine fever virus (ASFV) infecting domestic pigs and various wild boars (such as African wild boars, European wild boars, etc.). The World Organization for Animal Health (OIE) has listed it as a legally reportable animal disease, and the disease is also a type of animal epidemic that my country focuses on preventing. It is characterized by a short course of disease, with the most acute and acute infection mortality rate as high as 100%. The clinical manifestations are fever (up to 40-42°C), rapid heartbeat, difficulty breathing, some coughing, serous or mucopurulent secretions in the eyes and nose, cyanosis of the skin, and obvious bleeding in the lymph nodes, kidneys, and gastrointestinal mucosa. The clinical symptoms of African swine fever are similar to those of swine fever and can only be confirmed by laboratory monitoring.

[0007] Veterinarians can use molecular diagnostic technology - polymerase chain reaction (PCR) technology to diagnose ASFV in fecal samples. This method is currently a relatively effective method with a relatively high detection rate of ASFV. Primers targeting the conserved region of the viral genome are used for detection or primers designed using the viral VP-72 gene are used, and then the PCR method is used to detect African swine fever virus. The results are specific and sensitive. PCR technology is widely used due to its high detection sensitivity, wide range of applications, and simple operation. However, the detection technology is complex, requires sophisticated instruments, and takes a long time to detect, which is not conducive to on-site detection in non-laboratory environments and the promotion and application of grassroots laboratories. Summary of the invention

[0008] In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide a primer set and method for detecting African swine fever virus.

[0009] In one aspect, the present invention provides a primer set for detecting African swine fever virus, characterized in that the nucleotide sequence of the primers included in the primer set is as follows:

[0010] SEQ ID NO.1: 5'-TGCGTCCGTAATAGGAGTA-3';

[0011] SEQ ID NO.2: 5'-CAGGAGGTATCGGTGGAG-3';

[0012] SEQ ID NO.3:

[0013] 5'-CCATACCAACCCGAAATTCCTTTCTTTTTTGTTTACCTGCTGTTTGGAT-3';

[0014] SEQ ID NO.4:

[0015] 5'-GTTCGCTGCGTATCATTTTCATCTTTTCTCTCCTATGCAACATTCATG-3';

[0016] SEQ ID NO.5: 5'-CATTTTCCCGAGAACTCTCACAAT-3';

[0017] SEQ ID NO. 6: 5'-GTTTGCTTTGGTGCGGCTTGTG-3'.

[0018] Among them, the forward outer primer F3, its nucleotide sequence is shown in SEQ ID NO.1; the reverse outer primer B3, its nucleotide sequence is shown in SEQ ID NO.2; the forward inner primer FIP, its nucleotide sequence is shown in SEQ ID NO.3; the reverse inner primer BIP, its nucleotide sequence is shown in SEQ ID NO.4, the loop primer LB, its nucleotide sequence is shown in SEQ ID NO.5, and the loop primer LF, its nucleotide sequence is shown in SEQ ID NO.6.

[0019] Specifically, the present invention also provides the application of the above-mentioned primer set, which can be used in the preparation of an African swine fever virus kit, or the application of the primer set in detecting African swine fever virus, the application of the above-mentioned primer set in a microfluidic chip for detecting African swine fever virus, and especially the detection of African swine fever virus using a loop-mediated isothermal amplification-spectral sensor-artificial intelligence technology platform.

[0020] On the other hand, the present invention also provides a method for detecting African swine fever virus, comprising: performing loop-mediated isothermal amplification on the sample to be tested using LAMP detection technology in combination with the primer set as described above; and analyzing the amplification product.

[0021] Furthermore, the reaction temperature of the loop-mediated isothermal amplification is 65° C., and the reaction time is 35 minutes.

[0022] Furthermore, the loop-mediated isothermal amplification reaction is carried out in a microfluidic chip.

[0023] Furthermore, the method for analyzing the amplification product includes performing spectral analysis and / or colorimetric analysis on the LAMP reaction result.

[0024] The beneficial effect of the present invention is that the present invention innovatively develops a set of specific primer formulas for LAMP reaction directly using pig saliva samples without nucleic acid extraction for the conserved nucleic acid sequence corresponding to the ASFV-VP-72 protein, and collects data through a spectral sensor to perform a rapid nucleic acid detection method for African swine fever virus. The method can directly use pig saliva samples and add them to the LAMP reaction reagent integrated in the microfluidic chip. The detection process is automatically completed by a fully automatic detection instrument, and the spectrum is analyzed by a spectral sensor. The collected spectral signals are comprehensively judged by AI technology to achieve fully automatic operation, and the detection time is controlled within one hour to achieve instant diagnosis. It does not require professionals and laboratory environments, is portable and lightweight, and can be applied on-site to pet hospitals and other scenes for even-in-time detection. It adopts an innovative LAMP test method, does not require nucleic acid extraction, and achieves sample-in and result-out, and is low-priced.

[0025] Other features and advantages of the present invention will be described in the following description, and partly become apparent from the description, or understood by practicing the present invention. The purpose and other advantages of the present invention are realized and obtained by the structures particularly pointed out in the description and the drawings.

[0026] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, preferred embodiments are given below and described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 It is the screening result of LAMP reaction of 4 sets of primers designed for the genomic sequence of VP-72 protein of African swine fever virus;

[0029] Figure 2 It is the test result of the present invention for African swine fever virus positive / negative samples;

[0030] Figure 3 is the test result of the specific experiment of the present invention;

[0031] Figure 4 It is the detection result of the African swine fever virus detection method for saliva samples of the present invention. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] In the embodiments of the present invention, the standard positive sample of African swine fever virus (ASFV) is a synthetic ASFV-VP-72 protein gene sequence positive plasmid, Salmonella (Sal), feline calicivirus (FCV) and canine parvovirus (CPV) are all synthetic plasmid samples, and the primer sequences involved are all ordered from Sangon Biotech (Shanghai) Co., Ltd.

[0034] 1. Primer design and screening

[0035] By using a relatively high concentration of standard positive samples of African swine fever virus (ASFV), the LAMP primer set designed with the nucleic acid fragment corresponding to the S spike protein of the ASFV pathogen as a template was screened. The goal is to screen out a set of primers with the best sensitivity and specificity.

[0036] The primer design method is:

[0037] (1) Download the genomic sequence of African swine fever virus VP-72 protein from NCBI (Sequence ID: MN886925.1) as a template for primer design.

[0038] The conservative nucleic acid sequence of VP-72 protein is (SEQ ID NO.7):

[0039]

[0040] (2) Using PrimerExplorer V5

[0041] (http: / / primerexplorer.jp / lampv5e / index.html), using the African swine fever virus-VP-72 protein gene sequence as a template to design specific primers. LAMP reaction primers were designed based on 6 different regions of the African swine fever virus-VP-72 protein gene sequence, marked as F3, F2, F1, B1, B2, and B3 from the 5' end, where F1, F2, F3, B1, B2, and B3 are about 20 bp long sequence fragments on the target gene, and F1c and B1c are complementary regions of F1 and B1, respectively. The 4 primers include: upstream external primer F3 (Forward Outer Primer), downstream external primer B3 (Backward Outer Primer), upstream internal primer FIP (Forward Inner Primer) and downstream internal primer BIP (Backward Inner Primer). At the same time, use the free software PrimerExplorer V5 to design 1-2 loop primers LF (Loop Primer Forward) and LB (Loop Primer Backward) between F1c and F2c, or between B1c and B2c to speed up the LAMP reaction. m The melting temperature of DNA chain is 64℃~66℃, and the T m The value is 59℃~61℃, loop primer T m The value is 64℃~66℃. The free energy of the 3' end of F2 / B2, F3 / B3 and LF / LB and the 5' end of F1c and B1c should not be greater than -4kcal / mol. The free energy change (ΔG) is equal to the free energy of the product after the primer binds to the template minus the free energy of the initial reactant. The smaller the ΔG (the larger the absolute value of the negative number), the easier it is for the primer to bind to the template. And the GC content in the primer sequence should be controlled at 30%~65%. At the same time, ensure that each primer, especially the inner primer, is not easy to form a secondary structure to prevent the formation of primer dimers. There should be a certain appropriate spacing between the sequence fragments recognized by each primer. The spacing from the end of F2 to the end of B2 (LAMP amplification region) is 120 to 160 bases, the spacing from the 5' end of F2 to the 5' end of F1 (the region forming a loop structure) is 40 to 60 bases, and the spacing between F2 and F3 is 0 to 60 bases. All of the above conditions can be automatically calculated by the free software PrimerExplorer V5, and a list of designed primers is given;

[0042] (3) Select multiple sets of primers designed by the software PrimerExplorer V5 with different positions on the African swine fever virus-VP-72 protein gene sequence as templates, and enter the target product sequences of different primer sets into NCBI NucleotideBLAST

[0043] (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=Bl astSearch&LINK_LOC=blasthome) online tool was used to analyze the specificity of the primer set corresponding to the LAMP product to ensure that there was no non-specific amplification on the porcine genome and other pathogens.

[0044] Through the above method, 4 sets of LAMP primer sets were preliminarily designed for selection. The nucleotide sequences of each set of primers are shown in Table 1 below:

[0045] Table 1 Primer design

[0046]

[0047]

[0048] The above four primer sets were used to perform LAMP amplification experiments using the standard positive samples of African swine fever virus (ASFV) as positive samples and sterilized deionized water (ddH2O) as negative samples, and the color changes of the LAMP reaction system of each primer set were observed. Figure 1 As shown in the figure, the primer set of group 3 changed the color of the positive sample (+) in a short time (purple to bright yellow) and the negative sample (-) did not change color at 35 minutes, indicating that this primer set has higher sensitivity and specificity, while the other primer sets showed confusing positive and negative reactions and were not selected; in summary, group 3 is the primer set with the best sensitivity and specificity, therefore, the primer set of group 3 was selected as the primer set for detecting African swine fever virus.

[0049] 2. Reaction conditions

[0050] After several rounds of testing of different reaction conditions, the following conditions were finally selected as the final reagent ratio and reaction conditions:

[0051] (1) Preparation of 10× primer mixture:

[0052] Table 2 Primer set mixing concentration ratio

[0053] Working concentration (μM) 10× Primer Mix Concentration (μM) FIP 1.6 16 BIP 1.6 16 F3 0.2 2 B3 0.2 2 LB 0.4 4 LF 0.4 4

[0054] Table 3

[0055] Primer concentration (μM) Primer name Prepare 40 reactions (μL) 100 FIP 8 100 BIP 8 10 F3 10 10 B3 10 100 LB 2 100 LF 2 Sterile deionized water 10 Total volume (μL) 50

[0056] (2) Primers, 2×LAMP premix and positive / negative sample ratios in the LAMP reaction system:

[0057] Table 4

[0058] 1 reaction (μL) 2×LAMP premix 6.25 10× Primer Mix 1.25 Positive / Negative samples 5.0

[0059] The 2×LAMP premix solution is a well-known formula, which includes MgSO4 (MgSO4 concentration is 6mM to 12mM), buffer, dNTPs, KCl, (NH4)2SO4, Bst DNA polymerase, phenol red, sterile enzyme-free water, etc.

[0060] The positive sample can be a standard positive sample of African swine fever virus (ASFV), that is, a synthetic ASFV-VP-72 protein gene sequence positive plasmid.

[0061] The negative sample may be sterile deionized water (ddH2O).

[0062] (4) Reaction conditions

[0063] Under the condition of 65℃ temperature and constant temperature amplification for 35 minutes, better detection results were obtained.

[0064] Heating can be performed using a constant temperature heating device (water bath, metal bath, constant temperature oven, or PCR instrument, etc.).

[0065] 3. LAMP detection method for African swine fever virus

[0066] The present invention uses an African swine fever virus (ASFV) standard positive sample (synthesized ASFV-VP-72 protein gene sequence positive plasmid) to detect the ASFV pathogen according to the following steps:

[0067] Step 1: Preparation of positive / negative samples of African swine fever virus (ASFV)

[0068] The synthesized ASFV-VP-72 protein gene sequence positive plasmid was diluted with sterile deionized water, and the concentration of the African swine fever virus (ASFV) positive plasmid was diluted to 1000 copies / μL as a positive sample; sterile deionized water (ddH2O) was used as a negative sample.

[0069] Step 2: Prepare 10× primer mixture according to the method in Table 3 above.

[0070] Step 3: Take the African swine fever virus positive / negative samples prepared in the first step and prepare the LAMP reaction system for each reaction according to the method in Table 4 above.

[0071] Step 4: Heat at 65°C and react for 35 minutes.

[0072] The reaction results are as follows Figure 2 As shown, the LAMP reaction systems of 20 positive samples (rows 1 and 2) all turned yellow, and the LAMP reaction systems of 20 negative samples (rows 3 and 4) all turned purple-red. The positive accuracy rate and the negative accuracy rate of the reaction results were 100%, which shows that African swine fever virus can be clearly detected.

[0073] 4. Specificity experiments

[0074] In order to detect the specificity of the detection primer set for African swine fever virus, Salmonella (Sal), feline calicivirus (FCV) and canine parvovirus (CPV) plasmid solutions with a concentration of 1000 copies / μL were used as cross-detection samples.

[0075] Step 1: Preparation of positive / negative samples of African swine fever virus for cross-testing

[0076] African swine fever virus positive sample: ASFV positive plasmid diluted to 1000 copies / μL was used as a positive sample;

[0077] African swine fever virus negative samples: Salmonella (Sal), feline calicivirus (FCV) and canine parvovirus (CPV) plasmid solutions with a dilution concentration of 1000 copies / μL were used as negative samples.

[0078] Step 2: Prepare 10× primer mixture according to the method in Table 3 above.

[0079] The third step is to take the African swine fever virus-positive samples and the African swine fever virus-negative samples prepared in the first step, and prepare the LAMP reaction system for each reaction according to the method in Table 4 above.

[0080] Step 4: Heat at 65°C and react for 35 minutes.

[0081] The reaction results are as follows Figure 3 As shown, the LAMP reaction systems of 4 positive samples containing African swine fever virus (ASFV) plasmid solutions all turned yellow, while 4 negative samples containing Salmonella (Sal) plasmid solutions, 4 negative samples containing feline calicivirus (FCV) plasmid solutions, and 4 negative samples containing canine parvovirus (CPV) plasmid solutions did not change color, indicating that no false positive results occurred, demonstrating that the detection primer set of the present invention has good specificity.

[0082] according to Figure 2 and Figure 3The sensitivity and specificity of LAMP detection of African swine fever virus were preliminarily calculated based on the experimental results. Figure 2 and Figure 3 According to the experimental results, 24 of the 24 positive samples showed clear positive results, so the sensitivity was calculated as: As well as 32 of the negative samples of 12 other bacterial species and 20 sterile deionized water (ddH2O), all showed clear negative results, so the specificity was calculated as:

[0083] 5. LAMP detection method for African swine fever virus in saliva samples

[0084] Step 1: Preparation of positive / negative saliva samples

[0085] Use a cotton swab to dip different parts of the pig's mouth 3 to 5 times. When dipping, the head of the cotton swab should be completely immersed in the mouth, and try to cover all parts of the mouth. If there is a lot of saliva on the cotton swab, gently scrape off the excess saliva liquid; dip the cotton swab with the saliva sample completely into the pre-prepared 1mL sterile deionized water, shake the cotton swab dozens of times to dissolve the saliva sample in the water, and obtain a negative saliva sample;

[0086] A positive saliva sample can be obtained by adding a certain dilution ratio of the African swine fever virus (ASFV) standard positive sample to a portion of the negative saliva sample prepared above.

[0087] Step 2: Prepare 10× primer mixture according to the method in Table 3 above.

[0088] Step 3: Dilute the positive / negative saliva samples prepared in the first step by 1 / 50 with sterile deionized water, and make the concentration of the African swine fever virus (ASFV) positive plasmid in the positive saliva sample 1000 copies / μL; prepare the LAMP reaction system for each reaction according to the method in Table 4 above.

[0089] Step 4: Heat at 65°C and react for 35 minutes.

[0090] The reaction results are as follows Figure 4 As shown, the 10 negative saliva samples (below) all showed clear negative results, and the 10 positive saliva samples (above) all turned yellow, indicating that African swine fever virus (ASFV) in the saliva samples can be clearly detected.

[0091] The LAMP detection method for African swine fever virus in saliva samples is simple in collecting and processing saliva samples, does not require nucleic acid extraction, simplifies the detection steps, and shortens the detection time. It only takes about 1 hour from sample collection to test results.

[0092] This detection method can also detect African swine fever virus (ASFV) through the loop-mediated isothermal amplification-spectral sensor-artificial intelligence (LAMP-SpectralSensor-AI) technology platform. The diluted sample to be tested can be directly injected into the LAMP reaction reagent integrated in the microfluidic chip reaction chamber for amplification. The amplification product is analyzed by the spectral sensor, and the collected spectral signal is comprehensively judged by AI technology to quickly determine whether the sample contains African swine fever virus. The spectral detection method is combined with the AI ​​algorithm to make the detection accuracy reach the level of molecular detection. By limiting the sample to be tested to the microfluidic environment of the equipment, the risk of sample contamination is reduced, and the sample volume and reagents required for detection are minimized, thereby further reducing the overall cost of screening and detection. Moreover, this technology does not require any complex operations, only simple training, and can be completed on-site by medical staff in pet hospitals. It can realize fully automatic detection of samples in and results out, which is suitable for rapid and instant detection in scenarios such as pet hospitals.

[0093] Based on the above ideal embodiments of the present invention, the relevant staff can make various changes and modifications without departing from the technical concept of the present invention through the above description. The technical scope of the present invention is not limited to the contents of the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A primer set for detecting African swine fever virus, It is characterized in that The nucleotide sequences of the primers included in the primer set are as follows: SEQ ID NO.1: 5'-TGCGTCCGTAATAGGAGTA-3'; SEQ ID NO.2: 5'-CAGGAGGTATCGGTGGAG-3'; SEQ ID NO.3: 5'-CCATACCAACCCGAAATTCCTTTCTTTTTTGTTTACCTGCTGTTTGGAT-3'; SEQ ID NO.4: 5'-GTTCGCTGCGTATCATTTTCATCTTTTCTCTCCTATGCAACATTCATG-3'; SEQ ID NO.5: 5'-CATTTTCCCGAGAACTCTCACAAT-3'; SEQ ID NO. 6: 5'-GTTTGCTTTGGTGCGGCTTGTG-3'.

2. Use of the primer set according to claim 1 in preparing an African swine fever virus kit.

3. Use of the primer set according to claim 1 in detecting African swine fever virus.

4. Use of the primer set according to claim 1 in a microfluidic chip for detecting African swine fever virus.

5. A method for detecting African swine fever virus, It is characterized in that include: Using LAMP detection technology combined with the primer set as claimed in claim 1 to perform loop-mediated isothermal amplification on the sample to be tested; Analyze the results of the amplification products.

6. The method for detecting African swine fever virus according to claim 5, It is characterized in that The reaction temperature of the loop-mediated isothermal amplification was 65° C., and the reaction time was 35 minutes.

7. The method for detecting African swine fever virus according to claim 5, It is characterized in that The loop-mediated isothermal amplification reaction is carried out in a microfluidic chip.

8. The method for detecting African swine fever virus according to claim 5, It is characterized in that The method for analyzing the results of the amplification products includes performing spectral analysis and / or colorimetric analysis on the LAMP reaction results.

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