A rapid identification method for African swine fever virus genotype II and non-genotype II and its application

Abstract

A rapid identification method and its application for African swine fever virus genotype II and non-genotype II. In the present invention, the nucleotide at position 1500, a specific differential site of the B646L gene, is detected. For African swine fever virus genotype II, it is C, and for non-genotype II, it is T. By designing specific primers for the differential sites of different virus genotypes, African swine fever virus genotype II and non-genotype II can be effectively distinguished. The screened primers have a high amplification efficiency for African swine fever virus genotype II and a low amplification efficiency for African swine fever virus non-genotype II. Without sequencing, it can be determined whether the genotype of African swine fever virus is type II or non-type II, with a fast detection speed and low cost.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus detection, and particularly relates to a rapid identification method and application of African swine fever virus genotype II and non-genotype II. Background Art

[0002] China is the world's largest producer and consumer of pork, with pork production accounting for about half of the world's total. African swine fever is one of the most important diseases that currently cause significant harm and economic losses to the global pig industry and is recognized as the "number one killer" of the global pig industry. African swine fever is an acute, febrile, highly contagious animal infectious disease of pigs caused by African swine fever virus (ASFV). It is highly lethal to domestic pigs, and the mortality rate of highly pathogenic strains can reach 100%. ASFV has strong resistance in tissues and the environment. In addition, there are currently no effective vaccines and therapeutic drugs. Once it spreads widely, it will seriously affect China's people's livelihood and even international trade related to the pig industry.

[0003] According to the B646L gene, ASFV can be divided into 24 genotypes. Some genotype strains only appear in a certain country, while some genotype strains are spread in multiple countries (such as I, II, V, VIII, X, and XII). The ASFV prevalent in China belongs to genotype II. In order to monitor the distribution and epidemic trend of ASFV in China, trace the source of the epidemic, and take timely countermeasures, it is of great significance to carry out rapid differential diagnosis of the prevalent strains (genotype II) and other genotype strains (non-genotype II) in China. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a rapid identification method for African swine fever virus genotype II and non-genotype II, and it is found that their characteristic differential site is 1500C / T, where African swine fever virus genotype II is C and non-genotype II is T.

[0005] The purpose of the present invention is to provide a primer for rapidly distinguishing African swine fever virus genotype II and non-II;

[0006] The purpose of the present invention is to provide a probe for rapidly distinguishing African swine fever virus genotype II and non-II;

[0007] The purpose of the present invention is to provide a primer and a probe for African swine fever virus detection;

[0008] The purpose of the present invention is to provide a kit for rapidly distinguishing African swine fever virus genotype II and non-II;

[0009] The object of the present invention is to provide the application of the above primers, probes and their kits in rapidly differentiating African swine fever virus genotype II from non-genotype II.

[0010] The technical solution adopted by the present invention to solve its technical problems is as follows:

[0011] A rapid identification method for African swine fever virus genotype II and non-genotype II, the method is: detecting the nucleotide at position 1500 of the specific differential site of the B646L gene, where the African swine fever virus genotype II is C and the non-genotype II is T.

[0012] The method of the present invention can effectively differentiate African swine fever virus genotype II from non-genotype II by designing specific primers for different genotype differential sites of the virus.

[0013] A probe, the nucleotide sequence of which is shown in SEQ ID No.1, namely Probe: 5’-FAM-CAGATATAGATGAACATGCGTCTGGAAGAGC-BHQ1-3’.

[0014] Preferably, the fluorescent group FAM labeled at the 5’ end of the probe sequence is replaced by one of HEX, VIC, CY5, TET, and the quenching group BHQ1 labeled at the 3’ end of the probe sequence is replaced by a quenching group used in combination with the fluorescent group, and the quenching group is selected from BHQ-2, BHQ-3, TAMRA or DABCYL.

[0015] A primer composition, the primer composition includes a forward primer and a reverse primer, wherein, the type II forward primer is selected from one of the following three:

[0016] Type II primer F1: 5’-GTTGTTAACGCCATTATGCAGCAC-3’, the nucleotide sequence of which is shown in SEQ ID No.2,

[0017] or type II primer F2: 5’-GTTGTTAACGCCATTATGCAGCGC-3’, the nucleotide sequence of which is shown in SEQ ID No.3,

[0018] or type II primer F3: 5’-GTTGTTAACGCCATTATGCAGACC-3’, the nucleotide sequence of which is shown in SEQ ID No.4;

[0019] Reverse primer R: 5’-GTGGAAGGGTATGTAAGAGCTGCAGA-3’, the nucleotide sequence of which is shown in SEQ ID No.6.

[0020] A detection system for B646L gene mutation, including primers and probes. The primers are used to amplify nucleic acid fragments containing specific differential sites, including type II forward primers, universal forward primers and reverse primers. The reverse primer is used as a type II reverse primer and a universal reverse primer.

[0021] The type II forward primer, whose nucleotide sequence is selected from one of SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4.

[0022] Universal forward primer F: 5’-GACATGTTGTTAACGCCATTATGCAG-3’, and its nucleotide sequence is as shown in SEQ ID No.5.

[0023] Reverse primer R, and its nucleotide sequence is as shown in SEQ ID No.6.

[0024] The probe, and its nucleotide sequence is as shown in SEQ ID No.1.

[0025] A kit for quickly distinguishing between African swine fever virus genotype II and non-II, which contains the probe, or the primer composition, or the detection system for B646L gene mutation.

[0026] An application of the probe, or the primer composition, or the detection system for B646L gene mutation in quickly distinguishing between African swine fever virus genotype II and non-II.

[0027] A method for quickly identifying African swine fever virus genotype II and non-II, which includes the following steps:

[0028] S1. Extract or release viral DNA from the sample as a template.

[0029] S2. Amplify with the universal forward primer, reverse primer and probe to obtain Ct value 1; at the same time, amplify with the type II forward primer, reverse primer and probe to obtain Ct value 2.

[0030] S3. Calculate the difference between Ct value 1 and Ct value 2. |Ct value 2 - Ct value 1| ≤ 3 indicates that the detected African swine fever virus genotype is type II; |Ct value 2 - Ct value 1| > 3 indicates that the detected African swine fever virus genotype is non-II.

[0031] As is well known to those skilled in the art, adding or subtracting nucleotides at the 5' end of the primer can change the annealing temperature of the primer, but its amplification is initiated from the 3' end of the primer. Any addition or subtraction based on the above primer sequences is within the protection scope of the present invention.

[0032] The beneficial effects of the present invention are as follows:

[0033] 1. The primer pair screened by the present invention has a high amplification efficiency for African swine fever virus genotype II and a low amplification efficiency for non-genotype II of African swine fever virus. By comparing the Ct values amplified by the general primer and probe, the difference between the Ct value amplified by the typing primer and probe and the Ct value amplified by the general primer is ≤3, indicating that the genotype of African swine fever virus is genotype II; if the Ct value difference >3, it indicates that the detected genotype of African swine fever virus is non-genotype II.

[0034] 2. It is not necessary to sequence to determine whether the genotype of African swine fever virus is genotype II or non-genotype II, and the detection speed is fast and the cost is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a characteristic sequence comparison diagram of "C / T" at the 1500th site of the B646L gene for ASFV genotype II and non-genotype II, where genotype II is C and non-genotype II is T. DETAILED IMPLEMENTATION METHODS

[0036] The following are specific examples to further illustrate the technical solutions of the present invention. It should be understood that the implementation of the present invention is not limited to the following examples, and any form of variation and / or change made to the present invention will fall within the protection scope of the present invention.

[0037] In the present invention, unless otherwise specified, all parts and percentages are in weight units, and the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following examples are conventional methods in the art unless otherwise specified.

[0038] The reagents used in the following examples can be obtained from a conventional biochemical reagent store unless otherwise specified.

[0039] Example Characteristic Differential Sequences of African Swine Fever Virus Genotype II and Non-genotype II

[0040] By performing ClustalW multiple sequence alignment on the B646L gene sequences of 24 genotypes of African swine fever virus and the epidemic strains in China, the alignment results are shown in Figure 1 , Figure 1 showing that there is a characteristic sequence of "C / T" at the 1500th site of the B646L gene for ASFV genotype II and non-genotype II, where ASFV genotype II is C and non-genotype II is T.

[0041] Example Rapid Identification Method for African Swine Fever Virus Genotype II and Non-genotype II

[0042] Synthesize partial sequences of the B646L gene of African swine fever virus genotype II and partial sequences of the B646L gene of African swine fever virus non-genotype II, ligate the sequences into a vector (such as pUC57) respectively, extract the plasmid, measure the concentration and dilute it to the same concentration as the PCR template.

[0043] The partial sequence of the B646L gene of African swine fever virus genotype II has a nucleotide sequence as shown in SEQ ID No.7. The partial sequence of the B646L gene of African swine fever virus non-genotype II has a nucleotide sequence as shown in SEQ ID No.8.

[0044] Synthesize 17 primers described in Table 1, synthesize the reverse primer R with a nucleotide sequence as shown in SEQ ID No.6, and synthesize the probe with a nucleotide sequence as shown in SEQ ID No.1.

[0045] Table 1

[0046]

[0047]

[0048] The Taqman fluorescence quantitative PCR reaction system is as follows: 5 μL of Taqman qPCR Mix, 0.4 μL each of the genotype II-specific F and the universal reverse primer R, 0.2 μL of the probe, 0.4 μL of the genotype II or non-genotype II plasmid template, and make up to 10 μL with water. The same template is amplified with the universal primer at the same time. Except that the forward primer uses the universal forward primer (universal type) F primer, the rest are the same as the genotype II amplification system.

[0049] The Taqman fluorescence quantitative PCR amplification reaction conditions are: 95°C for 30 s; 95°C for 5 s, 65°C for 1 min, 40 cycles.

[0050] Table 2 Amplification results of different primers for African swine fever virus genotype II and non-genotype II templates

[0051]

[0052]

[0053] The experimental results show that the genotype II F1 and the reverse primer R pair in Table 2 can effectively amplify both genotype II and non-genotype II templates, indicating that it is difficult to distinguish African swine fever virus genotype II and non-genotype II with these primers.

[0054] The present invention designed and screened different F primers, hoping to screen primers that can efficiently amplify genotype II of the gene, but have a relatively low amplification efficiency for non-genotype II of the gene. The results in Table 2 show that there are very few primers that can meet the above requirements. For example, when amplifying genotype II templates, the primers with amplification efficiency comparable to that of the universal primers are only F1, F2, F3, F4, F5, F9, and F13. However, when amplifying non-genotype II templates, the amplification efficiencies of F1, F9, and F13 are also relatively high, which does not meet the expectations. Overall, F2, F4, and F5 better meet the above conditions and can be used for the detection of African swine fever virus genotype II. These three screened primers are respectively named forward primer F1 for genotype II, forward primer F2 for genotype II, and forward primer F3 for genotype II. Moreover, the amplification efficiencies of these 3 primers are comparable to those of the universal primers, that is, these primer pairs have a high amplification efficiency for genotype II of the gene and can also be used as primers for the detection of African swine fever virus genotype II.

[0055] Application Example 1

[0056] A kit for rapidly differentiating genotype II and non-genotype II of African swine fever virus, the kit contains:

[0057] Forward primer F1 for genotype II: 5’-GTTGTTAACGCCATTATGCAGCAC-3’, and its nucleotide sequence is shown as SEQ ID No.2,

[0058] Universal forward primer F: 5’-GACATGTTGTTAACGCCATTATGCAG-3’, and its nucleotide sequence is shown as SEQ ID No.5;

[0059] Reverse primer: R: 5’-GTGGAAGGGTATGTAAGAGCTGCAGA-3’, and its nucleotide sequence is shown as SEQ ID No.6.

[0060] Probe, the nucleotide sequence is shown as SEQ ID No.1, that is, Probe: 5’-FAM-CAGATATAGATGAACATGCGTCTGGAAGAGC-BHQ1-3’.

[0061] The kit also contains PCR reaction solution, positive control, negative control and blank control.

[0062] The usage method of the kit is as follows:

[0063] S1. Extract DNA from the sample.

[0064] S2. PCR reaction. Detect the specific differential site 1500th nucleotide of the B646L gene. For African swine fever virus genotype II, it is C, and for non-genotype II, it is T.

[0065] Perform amplification using the described universal forward primer, reverse primer, and probe to obtain a Ct value of 1; simultaneously, perform amplification using the type II forward primer, reverse primer, and probe to obtain a Ct value of 2;

[0066] S3. Calculate the difference between Ct value 1 and Ct value 2,

[0067] |Ct value 2 - Ct value 1| ≤ 3 indicates that the genotype of the detected African swine fever virus is type II;

[0068] |Ct value 2 - Ct value 1| > 3 indicates that the genotype of the detected African swine fever virus is non-type II.

[0069] Application Example 2

[0070] A kit for rapidly differentiating between genotype II and non-genotype II of African swine fever virus, the kit contains:

[0071] Type II forward primer F2: 5’-GTTGTTAACGCCATTATGCAGCGC-3’, and its nucleotide sequence is as shown in SEQ ID No. 3,

[0072] The rest is the same as Application Example 1.

[0073] Application Example 3

[0074] A kit for rapidly differentiating between genotype II and non-genotype II of African swine fever virus, the kit contains:

[0075] Type II forward primer F3: 5’-GTTGTTAACGCCATTATGCAGACC-3’, and its nucleotide sequence is as shown in SEQ ID No. 4;

[0076] The rest is the same as Application Example 1.

[0077] Application Example 4

[0078] A rapid identification method for genotype II and non-genotype II of African swine fever virus, which detects the nucleotide at position 1500 of the specific difference site of the B646L gene. For African swine fever virus genotype II, it is C, and for non-genotype II, it is T. Specifically, a detection system for B646L gene mutation is used, including primers and probes. The primers are used to amplify nucleic acid fragments containing specific difference sites, including type II forward primers, universal forward primers, and reverse primers. The reverse primer is used as a type II reverse primer and a non-type II reverse primer,

[0079] The type II forward primer, its nucleotide sequence is selected from one of SEQ ID No. 2, SEQ ID No. 3, or SEQ ID No. 4,

[0080] Universal forward primer F: 5'-GACATGTTGTTAACGCCATTATGCAG-3', and its nucleotide sequence is shown in SEQ ID No. 5;

[0081] Reverse primer R: 5'-GTGGAAGGGTATGTAAGAGCTGCAGA-3', and its nucleotide sequence is shown in SEQ ID No. 6;

[0082] For the said probe, its nucleotide sequence is shown in SEQ ID No. 1.

[0083] The rapid discrimination method for African swine fever virus genotype II and non-II type is specifically as follows:

[0084] S1. Extract or release viral DNA from the sample as a template;

[0085] S2. Amplify using the said universal forward primer, reverse primer and probe to obtain Ct value 1; at the same time, amplify using the type II forward primer, reverse primer and probe to obtain Ct value 2;

[0086] S3. Calculate the difference between Ct value 1 and Ct value 2, |Ct value 2 - Ct value 1| ≤ 3 indicates that the detected African swine fever virus genotype is type II; |Ct value 2 - Ct value 1| > 3 indicates that the detected African swine fever virus genotype is non-II type.

[0087] In this specification, each embodiment is described in a progressive manner. Each embodiment focuses on the differences from other embodiments. For the same or similar parts between each embodiment, reference can be made to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple. For the relevant parts, reference can be made to the description in the method part.

[0088] The above has introduced in detail a rapid discrimination method for African swine fever virus genotype II and non-II type and its application. In this article, specific examples are used to elaborate on the principle and implementation manner of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and modifications can still be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention. Sequence Listing <110> Zhejiang University <120> A rapid discrimination method for African swine fever virus genotype II and non-II type and its application <130> ZJDX - 001 <160> 21 <170> SIPO Sequence Listing 1.0 <210> 1 <211> 31 <212> DNA <213> Synthetic (Probe) <400> 1 cagatataga tgaacatgcg tctggaagag c 31 <210> 2 <211> 24 <212> DNA <213> Synthetic (Type II Primer F1) <400> 2 gttgttaacg ccattatgca gcac 24 <210> 3 <211> 24 <212> DNA <213> Synthetic (Type II Primer F2) <400> 3 gttgttaacg ccattatgca gcgc 24 <210> 4 <211> 24 <212> DNA <213> Synthetic (Type II Primer F3) <400> 4 gttgttaacg ccattatgca gacc 24 <210> 5 <211> 26 <212> DNA <213> Synthetic (Universal Forward Primer F) <400> 5 gacatgttgt taacgccatt atgcag 26 <210> 6 <211> 26 <212> DNA <213> Synthetic (Reverse Primer R) <400> 6 gtggaagggt atgtaagagc tgcaga 26 <210> 7 <211> 466 <212> DNA <213> African swine fever virus (type II B646L) <400> 7 cacaagttcg gacatgttgt taacgccatt atgcagccca ctcaccacgc agagataagc 60 tttcaggata gagatacagc tcttccagac gcatgttcat ctatatctga tattagcccc 120 gttacgtatc cgatcacatt acctattatt aaaaacattt ccgtaactgc tcatggtatc 180 aatcttatcg ataaatttcc atcaaagttc tgcagctctt acataccctt ccactacgga 240 ggcaatgcga ttaaaacccc cgatgatccg ggtgcgatga tgattacctt tgctttgaag 300 ccacgggagg aataccaacc cagtggtcat attaacgtat ccagagcaag agaattttat 360 attagttggg acacggatta cgtggggtct atcactacgg ctgatcttgt ggtatcggca 420 tctgctatta actttcttct tcttcagaac ggttcagctg tgctgc 466 <210> 8 <211> 466 <212> DNA <213> African swine fever virus (non-type II B646L) <400> 8 cacaagttcg gacatgttgt taacgccatt atgcagccta ctcaccacgc agagataagc 60 tttcaggata gagatacagc tcttccagac gcatgttcat ctatatctga tattagcccc 120 gttacgtatc cgatcacatt acctattatt aaaaacattt ccgtaactgc tcatggtatc 180 aatcttatcg ataaatttcc atcaaagttc tgcagctctt acataccctt ccactacgga 240 ggcaatgcga ttaaaacccc cgatgatccg ggtgcgatga tgattacctt tgctttgaag 300 ccacgggagg aataccaacc cagtggtcat attaacgtat ccagagcaag agaattttat 360 attagttggg acacggatta cgtggggtct atcactacgg ctgatcttgt ggtatcggca 420 tctgctatta actttcttct tcttcagaac ggttcagctg tgctgc 466 <210> 9 <211> 24 <212> DNA <213> Synthetic (Primer II-F1) <400> 9 gttgttaacg ccattatgca gccc 24 <210> 10 <211> 24 <212> DNA <213> Synthetic (Primer II-F3) <400> 10 gttgttaacg ccattatgca gctc 24 <210> 11 <211> 24 <212> DNA <213> Synthetic (Primer II-F6) <400> 11 gttgttaacg ccattatgca gaac 24 <210> 12 <211> 24 <212> DNA <213> Synthetic (Primer II-F7) <400> 12 gttgttaacg ccattatgca gatc 24 <210> 13 <211> 24 <212> DNA <213> Synthetic (Primer II-F8) <400> 13 gttgttaacg ccattatgca gagc 24 <210> 14 <211> 24 <212> DNA <213> Synthetic (Primer II-F9) <400> 14 gttgttaacg ccattatgca gtcc 24 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence (Primer II-F10) <400> 15 gttgttaacg ccattatgca gtac 24 <210> 16 <211> 24 <212> DNA <213> Synthetic (Primer II-F11) <400> 16 gttgttaacg ccattatgca gttc 24 <210> 17 <211> 24 <212> DNA <213> Synthetic (Primer II-F12) <400> 17 gttgttaacg ccattatgca gtgc 24 <210> 18 <211> 24 <212> DNA <213> Synthetic (Primer II-F13) <400> 18 gttgttaacg ccattatgca ggcc 24 <210> 19 <211> 24 <212> DNA <213> Synthetic (Primer II-F14) <400> 19 gttgttaacg ccattatgca ggac 24 <210> 20 <211> 24 <212> DNA <213> Synthetic (Primer II-F15) <400> 20 gttgttaacg ccattatgca ggtc 24 <210> 21 <211> 24 <212> DNA <213> Synthetic (Primer II-F16) <400> 21 gttgttaacg ccattatgca gggc 24

Claims

1. A primer composition, characterized in that The primer composition includes a type II forward primer and a reverse primer. The type II forward primer is used for amplifying the B646L gene of African swine fever virus type II. Among them, the type II forward primer is selected from one of the following three: Type II primer F1: 5’-GTTGTTAACGCCATTATGCAGCAC-3’, and its nucleotide sequence is shown as SEQ ID No.2, or Type II primer F2: 5’-GTTGTTAACGCCATTATGCAGCGC-3’, and its nucleotide sequence is shown as SEQ ID No.3, or Type II primer F3: 5’-GTTGTTAACGCCATTATGCAGACC-3’, and its nucleotide sequence is shown as SEQ ID No.4; Reverse primer R: 5’-GTGGAAGGGTATGTAAGAGCTGCAGA-3’, and its nucleotide sequence is shown as SEQ ID No.

6.

2. A detection system for B646L gene mutation, comprising primers and a probe, characterized in that: the primers are used for amplifying a nucleic acid fragment containing specific differential sites, including a type II forward primer, a universal forward primer and a reverse primer, the type II forward primer is used for amplifying the B646L gene of African swine fever virus type II, and its nucleotide sequence is selected from one of SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4, Universal forward primer F: 5’-GACATGTTGTTAACGCCATTATGCAG-3’, and its nucleotide sequence is shown as SEQ ID No.5; Reverse primer R, and its nucleotide sequence is shown as SEQ ID No.6; the nucleotide sequence of the probe is shown as SEQ ID No.1, that is, Probe: 5’-FAM-CAGATATAGATGAACATGCGTCTGGAAGAGC-BHQ1-3’.

3. The detection system for the B646L gene mutation according to claim 2, wherein: The fluorescent group FAM labeled at the 5’ end of the probe sequence is replaced by one of HEX, VIC, CY5, TET, and the quenching group BHQ1 labeled at the 3’ end of the probe sequence is replaced by a quenching group used in combination with the fluorescent group. The quenching group is selected from BHQ-2, BHQ-3, TAMRA or DABCYL.

4. A kit for rapidly distinguishing African swine fever virus gene type II from non-type II, which contains the primer composition described in claim 1, or the detection system for B646L gene mutation described in claim 2.

5. A rapid identification method for African swine fever virus genotype II and non-genotype II, characterized in that The method is as follows: Using the detection system for B646L gene mutation described in claim 2, detect the nucleotide at position 1500 of the specific differential site of the B646L gene. For African swine fever virus gene type II, it is C, and for non-type II gene, it is T.

6. The rapid identification method according to claim 5, characterized in that The method includes the following steps: S1. Extract or release viral DNA from the sample as a template; S2. Amplify using the universal forward primer, reverse primer and probe described in claim 2 to obtain Ct value 1; Simultaneously perform amplification using the type II forward primer, reverse primer, and probe described in claim 2 to obtain a Ct value of 2; S3. Calculate the difference between Ct value 1 and Ct value 2. If |Ct value 2 - Ct value 1| ≤ 3, it indicates that the detected genotype of African swine fever virus is type II; if |Ct value 2 - Ct value 1| > 3, it indicates that the detected genotype of African swine fever virus is non-type II.

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