LAMP primer group sequence, kit containing primer group sequence, application of LAMP primer group sequence and kit, and ASFV detection method

Abstract

The invention relates to the technical field of virology and molecular biology, and discloses an LAMP primer group sequence, a kit containing the primer group sequence, application of the LAMP primer group sequence and the kit and a method for detecting ASFV. The primer group sequence comprises an external primer pair, an internal primer pair and a loop primer pair, the external primer pair comprises an external forward primer F3 and an external reverse primer B3, the internal primer pair comprises an internal forward primer FIP and an internal reverse primer BIP, the loop primer pair comprises a loop forward primer LF and a loop reverse primer LB, and the loop forward primer LF and the loop reverse primer LB are in one-to-one correspondence. The nucleotide sequences are shown as SEQ ID NO: 1-6 in sequence. According to the invention, the LAMP reaction is integrated and optimized through meticulous design, so that the problems that LAMP is easy to interfere and false positive is easy to appear during application are solved; the primer group sequence for detecting the ASFV has the advantages of high sensitivity and specificity, simplicity in operation, rapidness, economy and high efficiency; compared with the existing fluorescent quantitative PCR detection method for ASFV, the method has more advantages.

Description

Technical Field

[0001] The present invention relates to the technical field of virology and molecular biology, and in particular to a LAMP primer set sequence, a kit containing the primer set sequence, and their applications and a method for detecting ASFV. Background Art

[0002] African swine fever (ASF) is a highly contagious and lethal viral disease that currently poses a major threat to the global pig industry. ASF causes hemorrhagic fever in domestic and wild pigs, with clinical symptoms including high fever, loss of appetite, weakness, skin spots, diarrhea, dyspnea, abortion and sudden death, leading to death within 1 week of infection. The pathogen of ASF is African swine fever virus (ASF virus, referred to as ASFV), the only member of the Asfrisinidae family. The virus has a mortality rate of up to 100% and can be transmitted through contact with the body fluids of infected animals, ticks, and humans through contaminated vehicles or clothing. ASFV particles are 175-215nm icosahedral with a double-stranded DNA structure; its linear genome spans between 170-193K bases and contains 160-170 genes; ASFV can encode about 150-200 different proteins, of which the p72 structural protein accounts for most of the virus shell, and the amino acid sequence shows high conservation between different strains. Therefore, the p72 gene is a commonly used target gene for detecting ASFV.

[0003] ASF usually causes economic losses to farmers and farming-related enterprises. The increase in pork prices has an impact on consumers and enterprises that rely on pork products. ASF outbreaks also have macroeconomic impacts, resulting in reduced pork product exports, trade restrictions, and negative impacts on the tourism industry. To effectively control the outbreak of ASFV, early and sensitive detection and strict biosecurity measures are needed, which requires the detection of asymptomatic carriers and initial positive cases. The most relied upon detection methods are molecular diagnostic tests because they are the main method of identification. Different methods for diagnosing ASF include virus isolation, immunohistochemistry (IHC), serological tests such as enzyme linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and virus neutralization test (VNT). Although these detection methods have good accuracy, they require the use of specialized laboratories, skilled personnel, and a lot of time, and are less practical.

[0004] The currently widely accepted ASFV detection method is the molecular genomic detection method using polymerase chain reaction (PCR). Real-time quantitative polymerase chain reaction (qPCR) has become a widely used molecular technology due to its high sensitivity, real-time detection, quantification and rapid analysis of ASFV DNA. Although its analysis speed is very fast, its instruments are relatively expensive and require a high level of experimental operation environment and professional skills. Due to limited facility resources, equipment costs and professional technical knowledge in primary medical centers cannot meet the requirements of qPCR.

[0005] Loop-mediated isothermal amplification (LAMP) is a molecular biology detection technology that has been widely used in recent years. Compared with traditional PCR, LAMP detection does not require complex equipment, can be amplified at a constant temperature, is easy to operate, and produces rapid results. LAMP relies on Bst DNA polymerase to achieve self-circulating chain displacement of nucleic acids. It can significantly improve sensitivity and specificity by amplifying target DNA or RNA under isothermal conditions through specific LAMP primer set sequences. Developing LAMP technology for ASFV detection and achieving sensitivity comparable to qPCR will become a promising alternative and will be very beneficial to ASFV monitoring and control. However, the existing LAMP method for detecting ASFV has the defects of being easily interfered with and prone to false positives. Summary of the invention

[0006] The present invention aims to overcome the problem of easy interference and false positive during LAMP application by carefully designing and integrating and optimizing LAMP reaction; solve the problems that the commonly used fluorescence quantitative PCR detection of PRRSV in the market requires expensive precision instruments, high environmental requirements, complicated methods, and long time consumption; thereby providing a LAMP primer set sequence for detecting ASFV, a kit containing the primer set sequence, and their application and a method for detecting ASFV. The LAMP primer set sequence can realize a LAMP detection method with higher constant temperature reaction, higher sensitivity and specificity, faster and more efficient detection, and a wider clinical operation environment. Thus, the method for detecting ASFV provided by the present invention is a LAMP detection method that is efficient, reliable, accurate, flexible in detection, widely practical, simple and fast in operation, and convenient to transport and carry at room temperature, and can be used prospectively for the early diagnosis of ASFV.

[0007] In order to achieve the above object, the first aspect of the present invention provides a LAMP primer set sequence, the primer set sequence includes an external primer pair, an internal primer pair and a loop primer pair, the external primer pair includes an external forward primer F3 and an external reverse primer B3, the internal primer pair includes an internal forward primer FIP and an internal reverse primer BIP, and the loop primer pair includes a loop forward primer LF and a loop reverse primer LB;

[0008] Wherein, the external forward primer F3 has a nucleotide sequence as shown in SEQ ID NO: 1;

[0009] The external reverse primer B3 has a nucleotide sequence as shown in SEQ ID NO: 2;

[0010] The internal forward primer FIP has a nucleotide sequence as shown in SEQ ID NO: 3;

[0011] The internal reverse primer BIP has a nucleotide sequence as shown in SEQ ID NO:4;

[0012] The loop forward primer LF has a nucleotide sequence as shown in SEQ ID NO:5;

[0013] The loop reverse primer LB has a nucleotide sequence as shown in SEQ ID NO:6.

[0014] Preferably, the primer set sequence targets the p72 gene of ASFV.

[0015] The second aspect of the present invention provides a kit, which comprises the primer set sequence as described above.

[0016] Preferably, the kit further comprises a dye.

[0017] Preferably, the dye is a fluorescent dye or a colorimetric dye, more preferably a fluorescent dye.

[0018] Preferably, the fluorescent dye is selected from at least one of hydroxynaphthol blue, calcein, Sybr Green, EvaGreen and Syto.

[0019] The third aspect of the present invention provides the use of the primer set sequence as described above or the kit as described above in detecting ASFV.

[0020] A fourth aspect of the present invention provides a method for detecting ASFV, the method comprising the following steps:

[0021] 1) extracting DNA from a sample to be tested as the DNA to be tested;

[0022] 2) using the DNA to be tested as a template and using the primer set as described above to perform a LAMP reaction;

[0023] 3) Determine the result of the LAMP reaction product.

[0024] Preferably, in step 1), the sample to be tested is a pig secretion, preferably a pig nasopharyngeal swab.

[0025] Preferably, in step 2), the molar ratio of the external primer pair, the internal primer pair and the loop primer pair in the LAMP reaction is 1:3-5:1-3.

[0026] Preferably, in step 2), the conditions of the LAMP reaction include: temperature of 60-70° C. and time of 20-40 min.

[0027] Preferably, in step 3), the result is determined by fluorescence measurement.

[0028] Preferably, the fluorescence measurement process includes: adding a fluorescent dye to the LAMP reaction system, and testing the fluorescence value of the product of the LAMP reaction.

[0029] Through the above technical solution, the beneficial effects of the present invention are:

[0030] The primer set sequence provided by the present invention is applied to the LAMP reaction for detecting ASFV, has the advantages of higher constant temperature reaction, higher sensitivity and specificity, faster and more efficient detection, and low cost, can be used for the early diagnosis of ASFV, is not easily interfered with or has false positives, does not require expensive and complex instruments, is more convenient to operate, is easy to transport and carry at room temperature, and can quickly detect African swine fever virus at primary medical points;

[0031] This primer set sequence provides direction for the research and development of instant detection products, greatly shortens the product development cycle, and at the same time reduces the cost of product development; not only that, this primer set sequence can be used in laboratory detection of the pathogenesis of ASFV or clinical detection of sick pigs infected with ASFV. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a fluorescence curve result diagram of the LAMP primer set sequence Set-1 in Example 2 detecting 100 copies and 580 copies of the ASFV standard plasmid;

[0033] Figure 2 It is a fluorescence curve result diagram of the LAMP primer set sequence Set-2 in Example 2 detecting 100 copies and 580 copies of the ASFV standard plasmid;

[0034] Figure 3It is a fluorescence curve result diagram of the LAMP primer set sequence Set-3 in Example 2 detecting 100 copies and 580 copies of the ASFV standard plasmid;

[0035] Figure 4 It is a fluorescence curve result diagram of the LAMP primer set sequence Set-4 in Example 2 detecting 100 copies and 580 copies of the ASFV standard plasmid;

[0036] Figure 5 is a graph showing the detection capability of the LAMP reaction at 70, 80, and 90 plasmid copy numbers in Example 3;

[0037] Figure 6 is a graph showing the detection capability of the LAMP reaction at 100, 300, and 600 plasmid copy numbers in Example 3;

[0038] Figure 7 is a graph showing the detection capability of the LAMP reaction at 1200, 2500, and 5000 plasmid copy numbers in Example 3;

[0039] Figure 8 This is a result diagram of 20 replicate experiments of the LAMP reaction at 80 copies in Example 3;

[0040] Fig. 9 is a result graph of detecting 100, 200, 500 and 1000 plasmid copies using fluorescent quantitative PCR in Example 5;

[0041] Fig.10 is a graph showing the results of detecting 100, 200, 500 and 1000 plasmid copies using the LAMP method in Example 5;

[0042] Fig.11 It is a comparison chart of the peak time detected by the LAMP method for 100, 200, 500 and 1000 plasmid copies in Example 5 and the CT value detected by the qPCR method for the corresponding concentration of plasmids. DETAILED DESCRIPTION

[0043] The endpoints and any values ​​of the ranges described in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0044] The first aspect of the present invention provides a LAMP primer set sequence, the primer set sequence includes an external primer pair, an internal primer pair and a loop primer pair, the external primer pair includes an external forward primer F3 and an external reverse primer B3, the internal primer pair includes an internal forward primer FIP and an internal reverse primer BIP, and the loop primer pair includes a loop forward primer LF and a loop reverse primer LB;

[0045] Wherein, the external forward primer F3 has a nucleotide sequence as shown in SEQ ID NO: 1;

[0046] The external reverse primer B3 has a nucleotide sequence as shown in SEQ ID NO: 2;

[0047] The internal forward primer FIP has a nucleotide sequence as shown in SEQ ID NO: 3;

[0048] The internal reverse primer BIP has a nucleotide sequence as shown in SEQ ID NO:4;

[0049] The loop forward primer LF has a nucleotide sequence as shown in SEQ ID NO:5;

[0050] The loop reverse primer LB has a nucleotide sequence as shown in SEQ ID NO:6.

[0051] The nucleotide sequences shown in SEQ ID NO: 1-6 are as follows:

[0052]

[0053] Among them, Y represents C or T, R represents A or G, and V represents A or C or G.

[0054] According to the present invention, preferably, the primer set sequence targets the p72 gene of ASFV.

[0055] The inventors of the present invention found in the course of research that the LAMP detection method is a relatively new method that relies on Bst DNA polymerase to achieve self-circulating chain displacement of nucleic acids. Compared with traditional fluorescent quantitative PCR, LAMP not only has high sensitivity and specificity, short detection time and low cost, but also is more convenient because it does not require expensive and complex instruments. LAMP has great advantages in diagnosing pathogens such as viruses: it has extremely high sensitivity and specificity, and can accurately detect even a very small amount of viral genetic material; rapid results, versatility for various types of samples, ease of use and cost-effectiveness further enhance its diagnostic utility. It has been proven that LAMP is very valuable for virus monitoring and epidemic control, especially in the case of limited resources, it can achieve timely and reliable detection; it has been widely used to detect various pathogens such as influenza, classic swine fever and foot-and-mouth disease. In addition, LAMP amplicons can be visualized by agarose gel electrophoresis and ethidium bromide staining; signal detection requires professional equipment or the use of fluorescent dyes to observe color changes by naked eye; pH-sensitive neutral red chromogens are expected to enhance the contrast of color changes, providing potential for practical applications. However, the existing LAMP method for detecting ASFV has obvious disadvantages such as non-specificity, susceptibility to interference and false positives in the detection of domestic ASFV variants, and cannot meet the domestic needs of ASFV detection and early diagnosis.

[0056] Based on this, the inventors of the present invention carefully designed a primer set sequence with specific targeting by using the p72 gene (GenBank number: MN886926) of the ASFV conserved region stored in the National Center for Biotechnology Information (NCBI) of the United States as the target sequence, and obtained the LAMP primer set sequence provided by the present invention, which is used to detect ASFV, and can effectively cover possible variants of ASFV in China, and can detect all ASFV mutants currently in China, with wide coverage. In particular, the primer set sequence provided by the present invention has no reaction with pathogens other than ASFV, and will not cause false positives, and has higher specificity; in addition, the present invention integrates and optimizes the LAMP reaction, so that the detection limit of ASFV detection reaches or approaches the limit value of LAMP technology, and can be used for early diagnosis of ASF, and achieves the advantages of high efficiency, reliability and accuracy, flexible detection, wide practicality, no need for expensive and complex instruments, simple and fast operation, and convenient transportation and carrying at room temperature, and is suitable for various detection environments. The primer set sequence provided by the present invention can be used for clinical detection and has passed the test of positive and negative samples of clinically sick pigs, indicating that this set of LAMP primer set sequences can be used to develop ASFV clinical detection kits or rapid detection equipment, providing a very valuable primer design for the future development of ASFV instant detection products. The LAMP primer set sequence provided by the present invention will not only greatly shorten the research and development cycle of instant detection products, but also greatly reduce time, manpower and trial and error costs. Based on this, the second aspect of the present invention provides a kit, which comprises the primer set sequence as described above.

[0057] In the present invention, the kit may contain the above-mentioned primer set sequence. In addition, the kit may also contain a container for accommodating the primer set sequence, an enzyme for performing an amplification reaction, a buffer, a dye, and at least one of a specific probe. Preferably, the kit also includes a dye. Further preferably, the dye is a fluorescent dye or a colorimetric dye, more preferably a fluorescent dye.

[0058] According to the present invention, the fluorescent dye may be any fluorescent dye suitable for PCR. Preferably, the fluorescent dye is selected from at least one of hydroxynaphthol blue, calcein, Sybr Green, EvaGreen and Syto.

[0059] According to the present invention, preferably, by integrating and optimizing the LAMP reaction, the enzyme (complex enzyme preparation) and the fluorescent dye used for performing the LAMP reaction can be in the form of freeze-dried microspheres.

[0060] In the PCR reaction system, the purpose of the buffer is to provide an optimal enzymatic reaction condition for the enzyme preparation. It is understood that the buffer can be selected from but not limited to Tris-HCl buffer with a concentration of 15-60 mmol / L and a pH of 8.0-8.8.

[0061] The third aspect of the present invention provides the use of the primer set sequence as described above or the kit as described above in detecting ASFV toxin.

[0062] Specifically, the primer set sequence and the kit can be used to prepare reagents or rapid testing equipment for detecting ASFV.

[0063] The primer set sequence or kit provided by the present invention can be used for clinical detection. The testing of positive and negative samples of clinically sick pigs shows that the primer set sequence of the present invention can be used to develop ASFV clinical detection reagents or rapid detection equipment, which provides a very valuable primer design for the future development of ASFV instant detection products. The LAMP primer set sequence provided by the present invention will not only greatly shorten the research and development cycle of instant detection products, but also greatly reduce the time, manpower and trial and error costs.

[0064] A fourth aspect of the present invention provides a method for detecting ASFV, the method comprising the following steps:

[0065] 1) extracting DNA from a sample to be tested as the DNA to be tested;

[0066] 2) using the DNA to be tested as a template and using the primer set as described above to perform a LAMP reaction;

[0067] 3) Determine the result of the LAMP reaction product.

[0068] Based on the above-mentioned primer set sequence of the present invention, the method provided by the present invention can use the LAMP reaction to achieve a detection limit of ASFV that reaches or is close to the limit value of the LAMP technology, which illustrates that the primer set sequence of the present invention has the characteristic of high sensitivity.

[0069] In the present invention, those skilled in the art may use a commercial DNA extraction kit to extract DNA from a sample to be tested, or may use conventional techniques in the art to extract DNA from a sample to be tested.

[0070] According to the present invention, preferably, in step 1), the sample to be tested is a pig secretion, such as at least one of saliva, nasal mucus, urine and sweat, more preferably a pig nasopharyngeal swab.

[0071] According to the present invention, preferably, in step 2), the molar ratio of the external primer pair, the internal primer pair and the loop primer pair in the LAMP reaction is 1:3-5:1-3. It is understood that the forward primer and the reverse primer of each of the external primer pair, the internal primer pair and the loop primer pair are usually the same or approximately the same molar amount. Exemplarily, the molar ratio of the forward primer and the reverse primer of each of the external primer pair, the internal primer pair and the loop primer pair is 1:0.9-1.1.

[0072] According to the present invention, preferably, in step 2), the conditions of the LAMP reaction include: a temperature of 60-70°C, specifically 60°C, 62°C, 64°C, 66°C, 68°C, 70°C, or any value between the above two values; a time of 20-40min, specifically 20min, 25min, 30min, 35min, 40min, or any value between the above two values.

[0073] According to the present invention, preferably, in step 3), the result determination is performed by fluorescence measurement.

[0074] According to the present invention, preferably, the fluorescence measurement process includes: using the fluorescent dye in the freeze-dried microspheres of the LAMP reaction system to test the fluorescence value of the product of the LAMP reaction. Wherein, the fluorescent dye can be any fluorescent dye suitable for PCR, preferably, the fluorescent dye is selected from at least one of hydroxynaphthol blue, calcein, Sybr Green, EvaGreen and Syto.

[0075] Exemplarily, the LAMP reaction system can be established in the following manner: LAMP reaction freeze-dried microspheres (including complex enzyme preparations and fluorescent dyes), 0.5-5 μL of 10-fold concentration of LAMP premixed primers (including 2 μM each of external forward primer F3 and external reverse primer B3, 16 μM each of internal forward primer FIP and internal reverse primer BIP, 4 μM each of loop forward primer LF and loop reverse primer LB), 16-20 μL of viral DNA to be tested or lysate containing virus, or 16-20 μL of distilled water without DNase. The total volume of the LAMP reaction system is 20-25 μL.

[0076] The present invention will be described in detail below through examples.

[0077] In the following examples, unless otherwise specified, the reagents and raw materials used are conventional commercially available products.

[0078] Example 1 Primer Design

[0079] The p72 gene of the conserved region of ASFV stored in the National Center for Biotechnology Information (NCBI) (GenBank No.: MN886926) was used as the target sequence, and a LAMP primer set sequence (hereinafter referred to as Set-1) including an external primer pair, an internal primer pair and a loop primer pair was designed, wherein the external primer pair included an external forward primer F3 and an external reverse primer B3, the internal primer pair included an internal forward primer FIP and an internal reverse primer BIP, and the loop primer pair included a loop forward primer LF and a loop reverse primer LB;

[0080] The nucleotide sequence of the external forward primer F3 is shown in SEQ ID NO: 1;

[0081] The nucleotide sequence of the external reverse primer B3 is shown in SEQ ID NO: 2;

[0082] The nucleotide sequence of the internal forward primer FIP is shown in SEQ ID NO: 3;

[0083] The nucleotide sequence of the internal reverse primer BIP is shown in SEQ ID NO:4;

[0084] The nucleotide sequence of the loop forward primer LF is shown in SEQ ID NO:5;

[0085] The nucleotide sequence of the loop reverse primer LB is shown in SEQ ID NO:6.

[0086] The first set of comparative LAMP primer set sequences (hereinafter referred to as Set-2) was independently designed, and the LAMP primer set sequences of the external forward primer, external reverse primer, internal forward primer, internal reverse primer, loop forward primer and loop reverse primer are shown in SEQ ID NO: 7 to SEQ ID NO: 12.

[0087]

[0088] The second set of comparative LAMP primer set sequences (hereinafter referred to as Set-3) was independently designed, and the LAMP primer set sequences of the external forward primer, external reverse primer, internal forward primer, internal reverse primer, loop forward primer and loop reverse primer are shown in SEQ ID NO: 13 to SEQ ID NO: 18.

[0089]

[0090] The third set of LAMP primer set sequences designed independently (hereinafter referred to as Set-4) includes an external forward primer, an external reverse primer, an internal forward primer, an internal reverse primer, a loop forward primer and a loop reverse primer, and their LAMP primer set sequences are shown in SEQ ID NO: 19 to SEQ ID NO: 24.

[0091]

[0092]

[0093] The above primer set sequences were synthesized and purified by a primer synthesis company.

[0094] Example 2 Sensitivity Test

[0095] 1. The experimental steps of LAMP reaction are as follows:

[0096] (1) Before the experiment, prepare the following reagents:

[0097] Prepare a 10-fold primer mixture according to Table 1 and freeze at -20°C;

[0098] Table 1 Composition of 10-fold primer mixture and 1-fold primer working solution

[0099] Primers 10x primer mix 1x primer working solution F3 2μM 0.2μM B3 2μM 0.2μM FIP 16μM 1.6μM BIP 16μM 1.6μM LF 4μM 0.4μM LB 4μM 0.4μM

[0100] The LAMP reaction freeze-dried microspheres are from the DryMicro RT-LAMP Master Mix product of Genuin Biotechnologies LLC (a subsidiary of Hefei Shanben Biotechnology Co., Ltd.) in the United States (website: https: / / www.genuinbiotech.com / product-page / drymicro-rt-lamp-master-mix#product information), which contains the LAMP reaction complex enzyme preparation and the fluorescent dye SYBR GREEN I.

[0101] (2) Thaw the primer mixture at a concentration of 10 times on ice, mix well and centrifuge, and then prepare the LAMP reaction system according to Table 2, wherein the amount of LAMP reaction freeze-dried microspheres used in each 20 μL of the LAMP reaction system is 1.

[0102] Table 2 LAMP reaction system

[0103]

[0104] Mix the above reaction components thoroughly, centrifuge, adjust the program of the constant temperature PCR instrument or traditional fluorescence quantitative PCR instrument to 70°C, put the mixed PCR tube into the PCR instrument, react for 30 minutes, and the result of the PCR reaction can be read from the fluorescence curve displayed on the PCR instrument screen or the Ct value given by the software.

[0105] 2. The experimental steps of sensitivity test are as follows:

[0106] A pig nasopharyngeal swab sample (50 μL of the sample was drawn into 200 μL of virus lysis solution for lysis) (collected from a pig farm in Guangdong Province), an ASFV 100 copy standard plasmid (purchased from Beijing Tianzhitai Biotechnology Co., Ltd., product model GBW (E) 091034), and an ASFV 580 copy standard plasmid (purchased from Beijing Tianzhitai Biotechnology Co., Ltd., product model GBW (E) 091034) were mixed, and then added to the LAMP reaction system containing the LAMP primer set sequence Set-1, Set-2, Set-3, and Set-4 provided in Example 1 (as shown in Table 2), respectively, and the reaction temperature was kept constant at 70° C. for 30 min. At the same time, a fluorescence curve analysis (using a constant temperature nucleic acid amplification detector to read the fluorescence analysis results) was performed to detect the sensitivity of the four sets of LAMP primer set sequences. The specific results are shown in Figures 1 to 4 .

[0107] Wherein, the X-axis represents the detection period (time, in min), the Y-axis represents the fluorescence intensity of SYBR GREEN I, and Ct represents the time when the signal exceeds the threshold of the noise.

[0108] like Figures 1 to 4 As shown, the fluorescence LAMP results show that among all the synthesized primer set sequences, the ASFV DNA amplification of the primer set sequence Set-1 is the fastest, and the fluorescence can be detected for 10-11 minutes; the LAMP primer set sequence Set-1 designed by the present invention for detecting ASFV has a faster peak time in the LAMP reaction system than the other three independently designed primer set sequences, indicating that the sensitivity of the LAMP primer set sequence Set-1 is higher than that of the other three primer set sequences.

[0109] Example 3 Determination of limit of detection (LoD)

[0110] This example is to study the detection limit of the LAMP primer set sequence.

[0111] In order to investigate the detection capability of LAMP reaction at extremely low plasmid copy number, the target ASFV plasmid (5.8×10 3The concentration of the 5000, 2500, 1200, 600, 300, 100, 90, 80 and 70 copies was gradually diluted to 5000, 2500, 1200, 600, 300, 100, 90, 80 and 70 copies, and five independent experiments were repeated (five parallel experiments were conducted for each concentration group, and the positive detection rate was calculated as positive result / 5×100%) to ensure the reliability and consistency of the results; the specific results are shown in Figures 5 to 8 , Table 3.

[0112] Table 3

[0113] Copy number / reaction frequency Detection rate 70 4 / 5 80％ 80 5 / 5 100％ 90 5 / 5 100％ 100 5 / 5 100％ 300 5 / 5 100％ 600 5 / 5 100％ 1200 5 / 5 100％ 2500 5 / 5 100％

[0114] Figure 5 As shown in Table 3, when the target ASFV plasmid concentration was 70 copies, the positive detection rate was 80%; Figure 8 As shown, 20 repeated experiments were performed at 80 copies. If the repetition rate of 80 copies / reaction does not reach 95%, 80 copies / reaction cannot be used as the detection limit. The results show that the positive rate of 20 LAMP reactions at 80 copies is 100%, showing better stability and consistency, indicating that the definition of the detection limit of 80 copies / reaction is reliable. The LAMP primer sequence provided by the present invention can make the LoD of the LAMP reaction reach or approach the limit value of the LAMP technology, that is, 80 genome copies / reaction.

[0115] Example 4 Potential pathogenic microorganism interference experiment

[0116] The pathogenic microorganisms that may affect the results of LAMP detection of ASFV in clinical practice (see Table 1) were mixed with a lysis solution containing a pig nasopharyngeal swab sample (collected from a pig farm in Guangdong Province) (50 μL of the sample was aspirated into 200 μL of virus lysis solution for lysis), and then ASFV (purchased from Beijing Tianzhitai Biological Technology Co., Ltd.) at 1 times the detection limit concentration (1×LoD is 80 copies / μL), 2 times the detection limit concentration (2×LoD is 160 copies / μL) and 5 times the detection limit concentration (5×LoD is 400 copies / μL) were added. Technology Co., Ltd., product model GBW(E)091034) was used as the experimental group; at the same time, the pathogenic microorganisms shown in Table 1 were mixed with a lysate containing a pig nasopharyngeal swab sample (collected from a pig farm in Guangdong Province), and no ASFV was added as a negative control group (NTC). The experimental group and the control group were respectively added to the LAMP reaction system containing the LAMP primer set sequence Set-1 provided in Example 1 (as shown in Table 2), and the reaction was carried out at 70°C for 30 minutes. Each experiment was repeated 3 times. The specific results are shown in Table 4.

[0117] Table 4 List of pathogenic microorganisms that may potentially affect the results of LAMP detection of ASFV and the test results

[0118]

[0119] From the experimental results in Table 4, it can be seen that in the presence of pathogenic microorganisms but without ASFV, the ASFV detection results are all negative, indicating that the LAMP primer set sequence Set-1 designed by the present invention for detecting ASFV has high specificity. In the presence of pathogenic microorganisms and ASFV at the same time, the ASFV detection results are all positive, indicating that the LAMP primer set sequence Set-1 designed by the present invention for detecting ASFV is not interfered by other pathogenic microorganisms. The above experimental results show that the LAMP primer set sequence Set-1 designed by the present invention for detecting ASFV is not interfered by pathogenic microorganisms that may infect sick pigs.

[0120] Example 5

[0121] The LoD of the LAMP primer set sequence Set-1 for detecting ASFV provided in Example 1 in the LAMP reaction system was compared with the cycle threshold (cycle quantification, Cq) of the gold standard fluorescent quantitative PCR. The fluorescent quantitative PCR and LAMP methods were used to repeat the experiment 5 times for 100, 200, 500 and 1000 ASFV plasmid copies, respectively. Among them, the LAMP method adopted the LAMP primer set sequence Set-1 provided in Example 1 and the LAMP reaction system and steps described in Example 2, and the fluorescent quantitative PCR adopted the African swine fever virus fluorescent PCR detection kit (purchased from Beijing Tianzhitai Biotechnology Co., Ltd.); the specific results are shown in Figures 9 to 11 and Table 5.

[0122] The Cq value represents the cycle number at which the fluorescent signal from the PCR reaction crosses a predefined threshold (limit), which is usually set above the background noise, indicating that the target DNA or RNA has been amplified to the point where it can be detected reliably enough.

[0123] like Fig. 9 As shown, PCR exhibited high sensitivity and specificity at all copy number levels; Fig.10 As shown, although the Ct value of LAMP was unstable at low copy numbers (200 copies), at 500 and 1000 copies, LAMP was able to stably detect all positive samples, and its detection ability was comparable to that of PCR.

[0124] Table 5

[0125] Viral copies / reaction Cq±SEM 100 33.844±0.708 200 32.668±0.318 500 31.988±0.270 1000 30.732±0.179 NTC N / A

[0126] From Table 5 and Fig.11From the comparison results, compared with the Cq value (or Ct value) of the gold standard fluorescent quantitative PCR, the Cq value of 100 genome copies / reaction fluorescent quantitative PCR is 34 cycles, which is very close to the limit value Cq (or Ct) of 35 cycles of the gold standard fluorescent quantitative PCR, further illustrating that the LAMP primer set sequence Set-1 of the present invention is highly sensitive to ASFV detection.

[0127] Application Example 1: Clinical African Swine Fever Specimen Testing

[0128] Fluorescence quantitative PCR (qPCR) and LAMP detection were performed on 101 ASFV clinical positive samples and 50 negative samples (collected from a pig farm in Guangdong Province). qPCR detection: 200 μL of sample was extracted by nucleic acid to obtain 50 μL of nucleic acid, and finally 1 μL of nucleic acid was added to the qPCR system for reaction, and negative and positive results were counted. Fluorescence quantitative PCR used African swine fever virus fluorescence PCR detection kit (purchased from Beijing Tianzhitai Biotechnology Co., Ltd.); LAMP detection: 50 μL of sample was added to 200 μL of lysis solution for lysis, and 19 μL of the lysis product was reacted in the reaction system, and negative and positive results were counted. The LAMP method used the LAMP primer group sequence Set-1 provided in Example 1 and the LAMP reaction system and steps described in Example 2, and the results are shown in Table 6. In view of the fact that the samples used are retrospective clinical samples, the viral nucleic acids in the samples may have been degraded, so some negative results may be caused by the above reasons. As can be seen from Table 6, the LAMP primer group sequence Set-1 provided by the present invention has passed clinical testing and can be used in the development of IVD products.

[0129] Table 6 Comparison of LAMP and qPCR results for all clinical samples

[0130]

[0131]

[0132] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A LAMP primer set sequence, characterized in that: The primer set sequence includes an external primer pair, an internal primer pair and a loop primer pair, wherein the external primer pair includes an external forward primer F3 and an external reverse primer B3, the internal primer pair includes an internal forward primer FIP and an internal reverse primer BIP, and the loop primer pair includes a loop forward primer LF and a loop reverse primer LB; Wherein, the external forward primer F3 has a nucleotide sequence as shown in SEQ ID NO: 1; The external reverse primer B3 has a nucleotide sequence as shown in SEQ ID NO: 2; The internal forward primer FIP has a nucleotide sequence as shown in SEQ ID NO: 3; The internal reverse primer BIP has a nucleotide sequence as shown in SEQ ID NO:4; The loop forward primer LF has a nucleotide sequence as shown in SEQ ID NO:5; The loop reverse primer LB has a nucleotide sequence as shown in SEQ ID NO:

6.

2. The primer set sequence according to claim 1, characterized in that: The primer set sequence targets the p72 gene of ASFV.

3. A kit, characterized in that: The kit comprises the primer set sequence according to claim 1 or 2.

4. The kit according to claim 3, characterized in that The kit also includes a dye; Preferably, the dye is a fluorescent dye or a colorimetric dye, more preferably a fluorescent dye; Preferably, the fluorescent dye is selected from at least one of hydroxynaphthol blue, calcein, Sybr Green, EvaGreen and Syto.

5. Use of the primer set sequence described in claim 1 or 2 or the kit described in claim 3 or 4 in detecting ASFV.

6. A method for detecting ASFV, characterized in that The method comprises the following steps: 1) extracting DNA from a sample to be tested as the DNA to be tested; 2) using the DNA to be tested as a template and using the primer set sequence of claim 1 or 2 to perform a LAMP reaction; 3) Determine the result of the LAMP reaction product.

7. The method according to claim 6, characterized in that In step 1), the sample to be tested is a pig secretion, preferably a pig nasopharyngeal swab.

8. The method according to claim 6 or 7, characterized in that: In step 2), the molar ratio of the outer primer pair, the inner primer pair and the loop primer pair in the LAMP reaction is 1:3-5:1-3.

9. The method according to claim 6 or 7, characterized in that: In step 2), the conditions of the LAMP reaction include: temperature of 60-70° C. and time of 20-40 min.

10. The method according to claim 6 or 7, characterized in that: In step 3), the result is determined by fluorescence measurement; Preferably, the fluorescence measurement process includes: adding a fluorescent dye to the LAMP reaction system, and testing the fluorescence value of the product of the LAMP reaction.

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