Primers for distinguishing and detecting genotype I and II African swine fever viruses and their application

Abstract

The present invention discloses primers for distinguishing and detecting genotype I and type II African swine fever viruses and their applications, specifically a nucleic acid combination, including a first primer having a sequence as shown in SEQ ID NO.3, a second primer having a sequence as shown in SEQ ID NO.4, a third primer having a sequence as shown in SEQ ID NO.5, a first probe having a sequence as shown in SEQ ID NO.6, a Cy5 connected to the 5' end, and a BHQ2 connected to the 3' end, and a second probe having a FAM connected to the 5' end, and a TAMRA connected to the 3' end as shown in SEQ ID NO.7. The present invention also discloses a kit comprising the aforementioned nucleic acid combination, and the application of the aforementioned nucleic acid combination or the aforementioned kit in the preparation of a preparation or system for distinguishing and detecting genotype I and type II African swine fever viruses. The nucleic acid combination has good sensitivity and high specificity, and can effectively distinguish and detect nucleic acids of genotype I and type II African swine fever viruses by qPCR.

Description

Technical Field

[0001] The invention belongs to the field of veterinary diagnosis and relates to primers for distinguishing and detecting genotype I and II African swine fever viruses and applications thereof. Background Art

[0002] African swine fever (ASF) is an acute, severe, highly contagious disease caused by African swine fever virus (ASFV). Pigs of all breeds and ages are susceptible, and the morbidity and mortality rates can reach 100%. Its clinical features mainly include high fever, cyanosis of the skin, abortion, and congestion and swelling of internal organs and lymph nodes.

[0003] Currently, there is no effective drug or commercial vaccine to treat ASF. The key to ASF prevention and control is to ensure biosafety protection in all production links. Therefore, it is of great significance to strengthen the research on ASFV detection technology and develop rapid, sensitive and specific diagnostic reagents and methods.

[0004] The existing ASF laboratory detection methods are mainly divided into serological methods and pathogenic methods. The former mainly includes indirect fluorescent antibody test, indirect or blocking enzyme-linked immunosorbent (ELISA) test, etc.; the latter includes virus isolation (hemoglobin adsorption test, HA), direct immunofluorescence test (DIF), double antibody sandwich ELISA test and ASFV genomic DNA detection (PCR, qPCR), etc., among which the PCR and fluorescent PCR (qPCR) methods recommended by OIE are the most widely used. my country's "Technical Specifications for the Prevention and Control of African Swine Fever (Trial)" lists three pathogenic detection methods: double antibody sandwich ELISA test, PCR and qPCR.

[0005] ASFV can be divided into 24 genotypes based on its B646L gene (encoding p72 protein). Experiments have confirmed that the pathogenicity of the genotype I strain prevalent in my country is quite different from that of the genotype II strain, and their prevention and control strategies are also different. Therefore, it is extremely necessary to determine the ASF genotype prevalent in the farm through rapid differential diagnosis, and to distinguish the genotype I African swine fever virus and genotype II African swine fever virus that are mainly prevalent in my country, which is conducive to targeted prevention and control; at the same time, it is easy to grasp the distribution and epidemic trends of different ASFV genotypes in my country.

[0006] Conventional molecular diagnostic methods include ordinary PCR, fluorescent quantitative PCR, gene chips and constant temperature amplification detection technology. Ordinary PCR, gene chips and constant temperature amplification detection technology have disadvantages such as low sensitivity, high cost, difficult development technology, and easy to cause aerosol pollution. Fluorescent quantitative PCR has the advantages of high sensitivity, good specificity, high throughput and low pollution compared with other conventional molecular diagnostic technologies. It is widely used in animal disease diagnosis, genetic qualitative and quantitative detection, etc., and has become the main method for rapid detection of viral nucleic acids.

[0007] ASF is similar to viral diseases such as swine fever, highly pathogenic porcine blue ear disease and pseudorabies; bacterial diseases such as swine erysipelas, Streptococcus suis and Haemophilus parabies; and certain poisonings and sepsis in terms of clinical and pathological changes. Therefore, etiological detection and identification methods are of great significance. At present, commercial real-time fluorescence quantitative PCR detection kits for ASFV are all universal ASFV detection kits, which cannot achieve differential diagnosis and detection of genotype I and genotype II ASFV, and it is difficult to effectively respond to the current status and detection needs of ASFV infection in pig farms. Summary of the invention

[0008] In order to overcome the shortcomings of the prior art, the present invention discovered two SNPs on specific genomic fragments of genotype I African swine fever virus and genotype II African swine fever virus, and based on the principle of amplification refractory mutation system (ARMS), also known as allele-specific amplification (i.e., if the 3' end base of the primer is not complementary to the template base, it cannot be extended by a general heat-resistant DNA polymerase), combined it with the qPCR method and successfully developed a fluorescent quantitative PCR detection method for rapid differential diagnosis of ASFV genotype I and II, namely ARMS-qPCR, whose sensitivity is comparable to the qPCR detection method recommended by OIE.

[0009] The first aspect of the present invention provides a nucleic acid combination, wherein the nucleic acid combination is a first nucleic acid combination or a second nucleic acid combination;

[0010] The first nucleic acid combination includes a first primer, a second primer, a third primer, a first probe and a second probe;

[0011] The second nucleic acid combination includes a fourth primer, a fifth primer, a sixth primer, a third probe and a fourth probe;

[0012] in:

[0013] The specific nucleic acid sequence downstream of the first primer is shown in SEQ ID NO.3;

[0014] The specific nucleic acid sequence downstream of the second primer is shown in SEQ ID NO.4;

[0015] The specific nucleic acid sequence downstream of the third primer is shown in SEQ ID NO.5;

[0016] In the first probe, the nucleic acid sequence or its complementary sequence is as shown in SEQ ID NO.6, one end of which is connected to a first fluorescent group, and the other end of which is connected to a first quenching group;

[0017] In the second probe, the nucleic acid sequence or its complementary sequence is as shown in SEQ ID NO.7, one end of which is connected to a second fluorescent group, and the other end of which is connected to a second quenching group;

[0018] The first quenching group is capable of quenching the fluorescence emitted by the first fluorescent group;

[0019] The second quenching group is capable of quenching the fluorescence emitted by the second fluorescent group;

[0020] The first fluorescent group cannot quench the fluorescence emitted by the second fluorescent group or the second quenching group;

[0021] The first quenching group is not capable of quenching the second fluorescent group or the fluorescence emitted by the second quenching group;

[0022] The second fluorescent group cannot quench the fluorescence emitted by the first fluorescent group or the first quenching group;

[0023] The second quenching group cannot quench the first fluorescent group or the fluorescence emitted by the first quenching group;

[0024] The specific nucleic acid sequence downstream of the fourth primer is shown in SEQ ID NO.3;

[0025] The specific nucleic acid sequence downstream of the fifth primer is shown in SEQ ID NO.9;

[0026] The specific nucleic acid sequence downstream of the sixth primer is shown in SEQ ID NO.10;

[0027] In the third probe, the nucleic acid sequence or its complementary sequence is as shown in SEQ ID NO.6, one end of which is connected to a third fluorescent group, and the other end of which is connected to a third quenching group;

[0028] In the fourth probe, the nucleic acid sequence or its complementary sequence is as shown in SEQ ID NO.7, one end of which is connected to a fourth fluorescent group, and the other end of which is connected to a fourth quenching group;

[0029] The third quenching group is capable of quenching the fluorescence emitted by the third fluorescent group;

[0030] The fourth quenching group is capable of quenching the fluorescence emitted by the fourth fluorescent group;

[0031] The third fluorescent group cannot quench the fluorescence emitted by the fourth fluorescent group or the fourth quenching group;

[0032] The third quenching group cannot quench the fourth fluorescent group or the fluorescence emitted by the fourth quenching group;

[0033] The fourth fluorescent group cannot quench the fluorescence emitted by the third fluorescent group or the third quenching group;

[0034] The fourth quenching group cannot quench the third fluorescent group or the fluorescence emitted by the third quenching group.

[0035] In some embodiments, in the first probe, the 5' end is connected to a first fluorescent group, and the 3' end is connected to a first quenching group;

[0036] In the second probe, the 5' end is connected to a second fluorescent group, and the 3' end is connected to a second quenching group.

[0037] In some embodiments, the first fluorescent group is Cy5, the first quenching group is BHQ2, the second fluorescent group is FAM, and the second quenching group is TAMRA; or

[0038] The first fluorescent group is FAM, the first quenching group is TAMRA, the second fluorescent group is Cy5, and the second quenching group is BHQ2.

[0039] In some embodiments, in the third probe, the 5' end is connected to a third fluorescent group, and the 3' end is connected to a third quenching group;

[0040] In the fourth probe, the 5' end is connected to a fourth fluorescent group, and the 3' end is connected to a fourth quenching group.

[0041] In some embodiments, the third fluorescent group is Cy5, the third quenching group is BHQ2, the fourth fluorescent group is FAM, and the fourth quenching group is TAMRA; or

[0042] The third fluorescent group is FAM, the third quenching group is TAMRA, the fourth fluorescent group is Cy5, and the fourth quenching group is BHQ2.

[0043] In some embodiments, the nucleic acid sequence of the first primer is shown as SEQ ID NO.3;

[0044] The nucleic acid sequence of the second primer is shown in SEQ ID NO.4;

[0045] The nucleic acid sequence of the third primer is shown as SEQ ID NO.5.

[0046] In some embodiments, the nucleic acid sequence of the fourth primer is shown as SEQ ID NO.3;

[0047] The nucleic acid sequence of the fifth primer is shown in SEQ ID NO.9;

[0048] The nucleic acid sequence of the sixth primer is shown as SEQ ID NO.10.

[0049] The second aspect of the present invention provides a kit, wherein the kit contains the nucleic acid combination described in the first aspect of the present invention.

[0050] The third aspect of the present invention provides a method for identifying and detecting genotype I African swine fever virus and genotype II African swine fever virus for non-diagnostic purposes, the method comprising: using the nucleic acid combination described in the first aspect of the present invention to perform qPCR detection on the nucleic acid extracted from the target detection sample.

[0051] In some embodiments, the method comprises the steps of:

[0052] S1: Extract the genome template of the target test sample;

[0053] S2: mixing the genome template, the first nucleic acid combination or the second nucleic acid combination, and a qPCR reaction reagent to obtain a reaction system;

[0054] S3: thermally cycle the reaction system, and perform identification and detection on type I African swine fever virus and genotype II African swine fever virus through fluorescent signals.

[0055] In some embodiments, in the thermal cycle, the pretreatment is: 95°C, 30s, the temperature cycle program is: 95°C, 10s, 60°C, 30s, and the number of cycles is 38-42.

[0056] The fourth aspect of the present invention provides the use of the nucleic acid combination described in the first aspect of the present invention or the kit described in the second aspect of the present invention in the preparation of a preparation or system for identifying and detecting genotype I African swine fever virus and genotype II African swine fever virus.

[0057] The first, second, third, fourth, fifth, sixth and other sequential limitations of the present invention are for distinguishing scenarios and facilitating clear expression, and do not represent the order of importance or the order of use in operation, nor do they necessarily represent different structures / compositions.

[0058] The nucleic acid combination of the present invention has good sensitivity and high specificity, and can effectively identify and detect the nucleic acids of genotype I and type II African swine fever viruses through qPCR. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 The results of the sequence alignment of genotype I and genotype II ASFV genome fragments are shown.

[0060] Figure 2 The results of ARMS-qPCR detection of genotype I and type II ASFV positive cultures are shown.

[0061] Figure 3 The FAM channel reaction results of different concentrations of type I ASFV positive plasmid are shown.

[0062] Figure 4 The Cy5 channel reaction results of different concentrations of type I ASFV positive plasmid are shown.

[0063] Figure 5 The FAM channel reaction results of type II ASFV positive plasmids with different concentrations are shown.

[0064] Figure 6 The Cy5 channel reaction results of different concentrations of type II ASFV positive plasmid are shown.

[0065] Figure 7 Specific test results of qPCR diagnostic methods for genotype I and II are shown.

[0066] Figure 2-7 In the figure, the numbers on the horizontal axis represent the number of qPCR cycle reactions. DETAILED DESCRIPTION

[0067] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0068] The materials and instruments not described in the present invention are conventional materials and instruments in the art, and the operation details not described in the present invention are conventional operations in the art.

[0069] The nucleic acid sequences shown in the present invention are all written from left to right in the 5' to 3' direction.

[0070] The upstream in the present invention refers to the 5' end or the 5' end direction of the nucleic acid, and the downstream refers to the 3' end or the 3' end direction of the nucleic acid.

[0071] definition

[0072] The differential detection of African swine fever virus genotype 1 and African swine fever virus genotype 2 for non-diagnostic purposes refers to the differential detection of African swine fever virus genotype 1 and African swine fever virus genotype 2 for purposes or results other than diagnosis to obtain the infection status of pigs, evaluate the health status of pigs, or evaluate the treatment or prevention effects on pigs.

[0073] An example of an application of non-diagnostic operations: In order to select a site for a new pig farm, when conducting an environmental pathogen safety assessment and testing for a variety of potential pig pathogens (for example, African swine fever virus genotype I, African swine fever virus genotype II, classical swine fever virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine circovirus type 2, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine rotavirus, etc.), environmental samples are tested for potential African swine fever virus genotype I and African swine fever virus genotype II.

[0074] Material

[0075] (1) Cells and viruses

[0076] Porcine alveolar macrophages (PAM), type I ASFV strain: SD / DY-I / 21 strain (Genebank sequence number: MZ945537.1) and type II ASFV strain: Pig / CN / HLJ / 2018 strain (Genebank sequence number: MK333180.1) were all identified and preserved by the National African Swine Fever Professional Laboratory of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0077] (2) DNA preparation

[0078] Viral genomic DNA was extracted using the Viral Genomic DNA / RNA Extraction Kit (DP315) from Tiangen Biochemical Technology (Beijing) Co., Ltd. using cell cultures containing genotype I and genotype II ASFV, blood from infected pigs, oral and anal swab preservation fluid, spleen, tonsils, and submandibular lymph node tissues.

[0079] (3) Positive control

[0080] The positive plasmid contains the 89,416-91,356bp fragment of the genotype I ASFV genome with the GeneBank sequence number MZ945537.1 and the 103,618-105,558bp fragment of the genotype II ASFV genome with the GeneBank sequence number MK333180.1. The basic plasmid is: pCAGGS, which was constructed, identified and preserved by the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. It is used for susceptibility testing and genotype I and genotype II ASFV positive control samples in the detection reaction.

[0081] (4) Specificity verification samples

[0082] The nucleic acids of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine rotavirus (PoRV) were extracted from cell cultures of cells adapted to the respective viruses, and their titers were all greater than 10 6 TCID 50 / mL, take 0.2 ml, and extract and prepare using the nucleic acid extraction kit described in (2).

[0083] (5) Samples to be tested

[0084] First, genotype I ASFV strain (SD / DY-I / 21 strain) was diluted to 1×10 6 TCID 50 / ml and genotype II ASFV strain (Pig / CN / HLJ / 2018 strain) diluted to 1 × 10 2.5 HAD 50 / ml, then, 7-9 week old SPF pigs were infected by intramuscular injection at a dose of 1ml / head; and after infection, according to the clinical manifestations of the infected pigs (i.e., pigs infected with type I ASFV showed symptoms such as necrotic skin lesions and joint swelling; pigs infected with type II ASFV showed symptoms such as dying), autopsies were promptly performed, and blood, oral and anal swabs and tissue samples (spleen, tonsils, submandibular lymph nodes) were collected by conventional methods, and the nucleic acid extraction kit described in (2) was used to extract and prepare genome templates. The above alveolar macrophage infection test of genotype I and genotype II ASFV and the infection test of SPF pigs were all carried out in the P3 or P4 laboratory of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0085] Example 1: Discovery of SNPs and primer design

[0086] The whole genome sequences of several genotype I and II ASFV were downloaded from the Genebank database. The genome fragment sequences of 89,416-91,356 bp of the genotype I ASFV (Genebank sequence number: MZ945537.1) and 103,618-105,558 bp of the genotype II ASFV (Genebank sequence number: MK333180.1) were selected according to the sequence information. The Megalign program in DNAstar was used to align them with their respective homologous sequences. The results of partial sequence alignment are shown in Figure 2. Figure 1As shown, there are two SNPs between the compared genomic fragments of the specific fragment 89,579-89,727bp of the genome of genotype I ASFV (Genebank sequence number: MZ945537.1) (sequence see the following SEQ ID NO.1) and the specific fragment 103,781-103,929bp of the genome of genotype II ASFV (Genebank sequence number: MK333180.1) (sequence see the following SEQ ID NO.2), and the sequences of the homologous genomic fragments at other positions are consistent.

[0087] The bases at positions 89,647 (SNP1) and 89,701 (SNP2) of the genome of type I ASFV (Genebank sequence number: MZ945537.1) are T and C, respectively; the bases at positions 103,849 (SNP1) and 103,903 (SNP2) of the genome of type II ASFV (Genebank sequence number: MK333180.1) are C and T, respectively.

[0088] Type I ASFV-specific fragment (SEQ ID NO.1):

[0089]

[0090] Type II ASFV specific fragment (SEQ ID NO.2):

[0091]

[0092] It can be seen that these two SNPs can be used to distinguish and detect type I and type II ASFV.

[0093] Primers and probes were designed according to the two SNPs sites. Based on the principle of allele-specific amplification, conventional primer and probe design software Primer5 was used to screen candidate primers and probes, and then sequence comparison was performed in the Genebank database to ensure the specificity and amplification efficiency of primers and probes.

[0094] After screening and verification, the first set of primers and probes for differential diagnosis of type I and type II ASFV were finally obtained as shown in Table 1, all of which were synthesized by Jilin Kumei Biotechnology Co., Ltd.

[0095] Table 1: The first set of primers, probes and amplification sequence information

[0096]

[0097]

[0098] Note: “N” stands for no modification, and the boxes mark the bases corresponding to the SNP sites.

[0099] Based on the above information, a second set of primers and probes for differential diagnosis of type I and type II ASFV were designed, and the information is shown in Table 2.

[0100] Table 2: Second set of primers, probes and amplification sequence information

[0101]

[0102] Note: “N” stands for no modification, and the boxes mark the bases corresponding to the SNP sites.

[0103] The first set of primers and probes are used in Examples 2-4 of the present invention.

[0104] Example 2: Establishment of a fluorescence quantitative PCR method for differential diagnosis of type I and type II ASFV

[0105] (1) Sample processing

[0106] For genotype I and II ASFV cell cultures, tissue organs, blood, oral and anal swabs, genomic DNA was extracted in a P3 laboratory using the viral genomic DNA / RNA extraction kit (DP315) of Tiangen Biochemical Technology (Beijing) Co., Ltd., and 2 μl was used as an ARMS-qPCR template.

[0107] (2) Reaction system

[0108] The reaction system was prepared at 25 μl / tube, including 23 μl of qPCR reaction solution containing primers and probes and amplification reagent containing qPCR enzyme (amplification reagent is used according to BIO-RAD: CFX96 supporting reagents), and the template volume was 2 μl. The amplification reagent was prepared according to the proportion in Table 3 based on "number of samples to be tested (n) + type I and type II ASFV positive controls (2) + negative controls (1) + 1 = number of samples to be tested N", and then 2 μl of the extracted sample template (DNA template) was added, and negative and positive controls were set at the same time. After instant centrifugation, the sample was tested on the machine.

[0109] For the qPCR reaction solution containing primers and probes, each reaction system contains 1μl F (10pmol), 1μl IR (10pmol), 1μl II R (10pmol), 0.5μl Probe 1 (5pmol), 0.5μl Probe 2 (5pmol), 12.5μl 2×Mix, and 6μl ddH2O.

[0110] Table 3: Type I and Type II qPCR reaction system preparation

[0111]

[0112] (3) Reaction conditions and procedures

[0113] The program was set on a fluorescence quantitative PCR instrument (BIO-RAD: CFX96) according to Table 4; the fluorescence detection channels were selected: FAM and Cy5, and the specific detection channel settings were operated according to the instruction manual of each instrument.

[0114] Table 4: Programming parameters

[0115]

[0116] (4) Result determination

[0117] (a) Judgment principle: The reaction system contains F, IR, IIR, Probe 1 (including Cy5), and Probe 2 (including FAM). When the sample contains type I ASFV genome template, F, IR, Probe 1 (including Cy5), and Probe 2 (including FAM) participate in the reaction, and the FAM and Cy5 channels emit fluorescence. When the sample contains type II ASFV genome template, F, IIR, and Probe 2 (including FAM) participate in the reaction, and only the FAM channel emits fluorescence. It can be seen that according to the Ct values ​​in the two fluorescence channels of the qPCR reaction, it can be determined whether the sample contains genotype I African swine fever virus or genotype II African swine fever virus.

[0118] (b) Conditions for the test results to be valid: The positive control for type I ASFV has an obvious exponential amplification curve, and the Ct values ​​of both the FAM and Cy5 channels are ≤32; the positive control for type II ASFV has an obvious exponential amplification curve in the FAM channel, and the Ct value is ≤32, while the Cy5 channel result is no Ct value; the negative control test result is no Ct value. The test is considered valid only if all of the above are met, otherwise the test is invalid.

[0119] (c) Result judgment criteria: Under the condition that the test results are established, the result judgment is carried out according to the judgment criteria in Table 5; suspicious samples need to be tested a second time, and if the Ct value of the second test result is ≤40, it is judged as positive; otherwise, it is judged as negative.

[0120] Table 5: Judgment criteria

[0121]

[0122]

[0123] (5) Establishment of differential diagnosis methods

[0124] ARMS-qPCR was performed on DNA extracted from porcine alveolar macrophages infected with genotype I and II ASFV strains (SD / DY-I / 21 and Pig / CN / HLJ / 2018, respectively) and negative control samples (nuclease-free water) using the aforementioned method. The amplification curves are shown in Figure 2 The results showed that the Ct values ​​of the FAM and Cy5 fluorescence channels of the type I ASFV strain (SD / DY-I / 21 strain) sample were 21.34 and 19.44, respectively, and there was an obvious amplification curve; the Ct value of the FAM fluorescence channel of the type II ASFV strain (Pig / CN / HLJ / 2018 strain) sample was 20.37, and there was an obvious amplification curve, while the Ct value of the Cy5 fluorescence channel was No Ct, and there was no amplification curve; the Ct values ​​of the FAM and Cy5 fluorescence channels of the negative control sample were both No Ct, and there was no amplification curve.

[0125] It can be seen from this that: for type I ASFV samples: FAM Ct value ≤ 40 and Cy5 Ct value ≤ 40; type II ASFV samples: FAM Ct value ≤ 40 and Cy5 Ct value > 40; negative control samples: FAM Ct value > 40 and Cy5 Ct value > 40; this meets the judgment criteria for the differential diagnosis of type I and type II ASFV, and is fully capable of distinguishing type I ASFV from type II ASFV. The primers, probes and reaction conditions designed by the present invention can meet the needs of differential diagnosis of type I and type II ASFV.

[0126] Example 3: Sensitivity test

[0127] (1) Methods

[0128] According to the copy number calculation formula: copy number / ml = (6.02x10 23 The positive plasmids of type I and type II ASFV were diluted to 10 copies / mol x (concentration g / ml) / (MWg / mol) with nuclease-free water. 7 Then, ten-fold serial dilutions were performed, with each 2 μl containing 10 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 and 10 6 For the samples with different template copy numbers, three replicates were set for each dilution, and a negative control (nuclease-free water) was also set. The program was set in the PCR instrument according to the reaction conditions of the method established in Example 2, and ARMS-qPCR detection was performed.

[0129] (2) ASFV type I susceptibility test

[0130] ASFV type I positive plasmid (copy number 10 7 / 2μl), according to 1:10, 1:10 2 , 1:10 3 , 1:10 4 , 1:10 5 , 1:10 6 and 1:10 7 Serial dilutions were performed, with the copy number in each tube being 1×10 6 , 1×10 5 , 1 × 10 4 , 1×10 3 , 1×10 2 , 1×10 1 and 1×10 0 The results of ARMS-qPCR detection of FAM and Cy5 are as follows: Figure 3 and Figure 4 As shown: the Ct values ​​of the first 6 dilutions of each sample in the FAM and Cy5 fluorescence channels were ≤38, the results were all positive, and there was an obvious amplification curve, while 1:10 7 The Ct values ​​of the two fluorescence channels of FAM and Cy5 of the sample with a dilution of 1 copy / reaction were both No Ct, and there was no amplification curve; the Ct values ​​of the two fluorescence channels of FAM and Cy5 of the negative control sample were both No Ct, and there was no amplification curve. Therefore, the sensitivity test result of type I ASFV is 10 copies / reaction.

[0131] (3) Type II ASFV susceptibility testing

[0132] ASFV type II positive plasmid (copy number 10 7 / 2μl), according to 1:10, 1:10 2 , 1:10 3 , 1:10 4 , 1:10 5 , 1:10 6 and 1:10 7 Serial dilutions were performed, with the copy number in each tube being 1×10 6 , 1×10 5 , 1 × 10 4 , 1×10 3 , 1×10 2 , 1×10 1 and 1×10 0 The results of ARMS-qPCR detection of FAM and Cy5 are as follows: Figure 5 and Figure 6As shown in the figure: the Ct values ​​of the FAM fluorescence channel of each sample at 7 dilutions were all ≤40, the results were all positive, and there were obvious amplification curves, while the Ct values ​​of the Cy5 fluorescence channel were all No Ct, and there was no amplification curve; the Ct values ​​of the FAM and Cy5 fluorescence channels of the negative control samples were all No Ct, and there was no amplification curve. Therefore, the sensitivity test result of type II ASFV is 1 copy / reaction.

[0133] It can be seen that the primers, probes and reaction conditions designed in the present invention have a sensitivity of 1-10 copies / reaction for identifying and detecting type I and type II ASFV, which can meet actual detection needs.

[0134] Example 4: Specificity test

[0135] (1) Methods

[0136] The extracted nucleic acids of CSFV, PRRSV, PRV, PCV2, PEDV, TGEV and PoRV were used as template samples for specific detection. Similarly, 23 μl of amplification reagent was prepared for each sample in the reaction system, and 2 μl of template was added respectively after packaging. At the same time, a negative control (nuclease-free water), a type I ASFV positive control (type I ASFV positive plasmid) and a type II ASFV positive control (type II ASFV positive plasmid) were set. Finally, the program was set in the PCR instrument according to the primers, probes and reaction conditions shown in Example 2, and ARMS-qPCR detection was performed.

[0137] (2) Specificity test results

[0138] The nucleic acids of CSFV, PRRSV, PRV, PCV 2, PEDV, TGEV and PoRV were tested as samples for specificity verification. Figure 2 The Ct values ​​of the FAM and Cy5 fluorescence channels of the other samples were all No Ct, and there was no amplification curve, and the test results were all negative (see Figure 7 ). Therefore, the method of Example 2 of the present invention has good specificity for detecting type I and type II ASFV.

[0139] Example 5: Detection of test samples

[0140] Two samples of blood, oral swabs, anal swabs, spleen, tonsils and submandibular lymph nodes from pigs infected with ASFV strain SD / DY-I / 21; two samples of blood, oral swabs, anal swabs, spleen, tonsils and submandibular lymph nodes from pigs infected with ASFV strain Pig / CN / HLJ / 2018, and three samples of blood from pigs infected with type I and type II ASFV were collected and tested. The positive control was: type I ASFV positive plasmid and type II ASFV positive plasmid; the negative control was: nuclease-free water.

[0141] The above samples were tested in parallel using the OIE-qPCR method recommended by OIE. The primers designed for the conserved regions consistent with type I and type II by the OIE recommended method are shown in Table 6; the concentrations of primers OIE-F and OIE-R in each reaction were 50 pmol (1 μl), and the concentration of OIE-Probe was 5 pmol (1 μl); the reaction program was: 1.50°C, 2 min; 2.95°C, 10 min; 3.95°C, 15 s, 58°C (collect fluorescence in this step), 1 min, 40 cycles.

[0142] All test results are shown in Table 7.

[0143] Table 6: Primer and probe information recommended by OIE

[0144]

[0145]

[0146] Note: “N” stands for no modification.

[0147] Table 7: Test results of samples collected in experiments or on-site

[0148]

[0149]

[0150] Note: “N” stands for Not Applicable.

[0151] According to Table 7, for samples from pigs infected with type I ASFV strain SD / DY-I / 21, the Ct values ​​of the FAM and Cy5 fluorescence channels of all samples No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 were ≤38, which is consistent with the detection results of the fluorescence PCR method recommended by OIE.

[0152] For samples from pigs infected with type II ASFV strain Pig / CN / HLJ / 2018, the Ct values ​​of the FAM fluorescence channel of all samples 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 were ≤38, and the Ct values ​​of the Cy5 fluorescence channel were all No Ct, which is consistent with the detection results of the fluorescence PCR method recommended by OIE.

[0153] The Ct values ​​of the FAM and Cy5 fluorescence channels of samples 25, 27, and 29 were all ≤38; the Ct values ​​of the FAM fluorescence channel of samples 26, 28, and 30 were all ≤38, and the Ct values ​​of the Cy5 fluorescence channel were all No Ct, which were consistent with the detection results of the fluorescence PCR method recommended by OIE.

[0154] The above test results fully meet the judgment criteria and conditions for ASFV fluorescent quantitative PCR differential diagnosis, so this method can be used for the differential diagnosis of type I and type II ASFV.

[0155] The fluorescent quantitative PCR detection method of Example 2 of the present invention can sensitively, quickly, accurately and specifically differentiate and diagnose type I and type II ASFV genome fragments in samples such as blood, oral and anal swabs, and tissues.

[0156] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention. Sequence Listing <110> Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Harbin Branch of China Animal Health and Epidemiology Center) <120> Primers for distinguishing and detecting genotype I and II African swine fever viruses and their application <130> P1101CNCN220027 <160> 14 <170> SIPOSequenceListing 1.0 <210> 1 <211> 149 <212> DNA <213> African swine fever virus <400> 1 tggtattcct cccgtggctt caaagcaaag gtaatcatca tcgcacccgg atcatcgggg gttttaattg cattgcctcc gtagtggaag ggtatgtaag agctgcagaa ctttgatgga aacttatcga called accatgagc <210> 2 <211> 149 <212> DNA <213> Antimicrobial (African swine fever virus) <400> 2 tggtattcct cccgtggctt caaagcaaag gtaatcatca tcgcacccgg atcatcgggg gttttaatcg cattgcctcc gtagtggaag ggtatgtaag agctgcagaa ctttgatgga aatttatcga tagged accatgagc <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <400> 3 tggtattcct cccgtggc <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <400> 4 gctcatggta tcaatcttat cgataag <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <400> 5 cactacggag gcaatgcg 18 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <400> 6 ccatcaaagt tctgcagctc ttacatac 28 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <400> 7 ccgatgatcc gggtgcgat 19 <210> 8 <211> 86 <212> DNA <213> African swine fever virus <400> 8 tggtattcct cccgtggctt caaagcaaag gtaatcatca tcgcacccgg atcatcgggg 60 gttttaatcg cattgcctcc gtagtg 86 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <400> 9 cactacggag gcaatgca 18 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <400> 10 gctcatggta tcaatctttat cgataaa 27 <210> 11 <211> 86 <212> DNA <213> African swine fever virus <400> 11 tggtattcct cccgtggctt caaagcaaag gtaatcatca tcgcacccgg atcatcgggg 60 gttttaattg cattgcctcc gtagtg 86 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <400> 12 ctgctcatgg tatcaatctt atcga 25 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <400> 13 gataccacaa gatcagccgt 20 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <400> 14 ccacgggagg aatacccaacc cagtg 25

Claims

1. A nucleic acid combination, wherein the nucleic acid combination is a first nucleic acid combination or a second nucleic acid combination; The first nucleic acid combination includes a first primer, a second primer, a third primer, a first probe and a second probe; The second nucleic acid combination includes a fourth primer, a fifth primer, a sixth primer, a third probe and a fourth probe; in: The sequence of the first primer is shown in SEQ ID NO.3; The sequence of the second primer is shown in SEQ ID NO.4; The sequence of the third primer is shown in SEQ ID NO.5; In the first probe, the nucleic acid sequence or its complementary sequence is as shown in SEQ ID NO.6, one end of which is connected to a first fluorescent group, and the other end of which is connected to a first quenching group; In the second probe, the nucleic acid sequence or its complementary sequence is as shown in SEQ ID NO.7, one end of which is connected to a second fluorescent group, and the other end of which is connected to a second quenching group; The first quenching group is capable of quenching the fluorescence emitted by the first fluorescent group; The second quenching group is capable of quenching the fluorescence emitted by the second fluorescent group; The first fluorescent group cannot quench the fluorescence emitted by the second fluorescent group or the second quenching group; The first quenching group is not capable of quenching the second fluorescent group or the fluorescence emitted by the second quenching group; The second fluorescent group cannot quench the fluorescence emitted by the first fluorescent group or the first quenching group; The second quenching group cannot quench the first fluorescent group or the fluorescence emitted by the first quenching group; The sequence of the fourth primer is shown in SEQ ID NO.3; The sequence of the fifth primer is shown in SEQ ID NO.9; The sequence of the sixth primer is shown in SEQ ID NO.10; In the third probe, the nucleic acid sequence or its complementary sequence is as shown in SEQ ID NO.6, one end of which is connected to a third fluorescent group, and the other end of which is connected to a third quenching group; In the fourth probe, the nucleic acid sequence or its complementary sequence is as shown in SEQ ID NO.7, one end of which is connected to a fourth fluorescent group, and the other end of which is connected to a fourth quenching group; The third quenching group is capable of quenching the fluorescence emitted by the third fluorescent group; The fourth quenching group is capable of quenching the fluorescence emitted by the fourth fluorescent group; The third fluorescent group cannot quench the fluorescence emitted by the fourth fluorescent group or the fourth quenching group; The third quenching group cannot quench the fourth fluorescent group or the fluorescence emitted by the fourth quenching group; The fourth fluorescent group cannot quench the fluorescence emitted by the third fluorescent group or the third quenching group; The fourth quenching group cannot quench the third fluorescent group or the fluorescence emitted by the third quenching group.

2. The nucleic acid combination according to claim 1, characterized in that In the first probe, the 5' end is connected to a first fluorescent group, and the 3' end is connected to a first quenching group; In the second probe, the 5' end is connected to a second fluorescent group, and the 3' end is connected to a second quenching group.

3. The nucleic acid combination according to claim 1 or 2, characterized in that The first fluorescent group is Cy5, the first quenching group is BHQ2, the second fluorescent group is FAM, and the second quenching group is TAMRA; or The first fluorescent group is FAM, the first quenching group is TAMRA, the second fluorescent group is Cy5, and the second quenching group is BHQ2.

4. The nucleic acid combination according to claim 1, characterized in that In the third probe, the 5' end is connected to a third fluorescent group, and the 3' end is connected to a third quenching group; In the fourth probe, the 5' end is connected to a fourth fluorescent group, and the 3' end is connected to a fourth quenching group.

5. The nucleic acid combination according to claim 1 or 4, characterized in that The third fluorescent group is Cy5, the third quenching group is BHQ2, the fourth fluorescent group is FAM, and the fourth quenching group is TAMRA; or The third fluorescent group is FAM, the third quenching group is TAMRA, the fourth fluorescent group is Cy5, and the fourth quenching group is BHQ2.

6. A kit comprising the nucleic acid combination according to any one of claims 1 to 5.

7. A method for distinguishing and detecting genotype I African swine fever virus and genotype II African swine fever virus for non-disease diagnosis purposes, the method comprising: performing qPCR detection on nucleic acids extracted from a target detection sample using the nucleic acid combination described in any one of claims 1-5.

8. The method according to claim 7, comprising the steps of: S1: Extract the genome template of the target test sample; S2: mixing the genome template, the first nucleic acid combination or the second nucleic acid combination, and a qPCR reaction reagent to obtain a reaction system; S3: thermal cycling the reaction system, and performing identification and detection on type I African swine fever virus and genotype II African swine fever virus through fluorescence signals.

9. The method according to claim 8, wherein in the thermal cycle, the pretreatment is: 95°C, 30s, the temperature cycle program is: 95°C, 10s, 60°C, 30s, and the number of cycles is 38-42.

10. Use of the nucleic acid combination according to any one of claims 1 to 5 or the kit according to claim 6 in the preparation of a preparation for identifying and detecting genotype I African swine fever virus and genotype II African swine fever virus.

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