Kit for detecting African swine fever virus and triple digital PCR detection method

Abstract

The invention provides a kit for detecting African swine fever viruses and a triple digital PCR detection method, the African swine fever viruses comprise ASFV I + R type strains, ASFV II type strains and ASFV recombinant strains, and the kit comprises a forward primer, a reverse primer and a probe for detecting the African swine fever viruses; the sequences of a forward primer and a reverse primer of the ASFV I + R type strain are respectively as shown in SEQ ID NO.1 and SEQ ID NO.2, and the sequence of the probe is as shown in SEQ ID NO.3; the sequences of a forward primer and a reverse primer of the ASFV II type strain are respectively as shown in SEQ ID NO.4 and SEQ ID NO.5, and the sequence of the probe is as shown in SEQ ID NO.6; the sequences of a forward primer and a reverse primer of the ASFV recombinant strain are respectively shown as SEQ ID NO.7 and SEQ ID NO.8, and the sequence of the probe is shown as SEQ ID NO.9. The triple digital PCR detection method provided by the invention can accurately identify the ASFV type I, the ASFV type II and the recombinant strains, has no cross reaction with PCV2, PRV, PRRSV, PEDV, PDCoV and other common swine disease pathogens, and provides a high-sensitivity and high-accuracy detection means for rapid identification of the ASFV type I, the ASFV type II and the recombinant strains.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus detection, and specifically relates to a kit for detecting African swine fever virus and a triple digital PCR detection method. Background Art

[0002] African swine fever (ASF) is a highly contagious disease common to domestic and wild pigs and caused by the African swine fever virus (ASFV, a member of the Assurviridae family). Due to its high mortality rate and lack of an effective vaccine, ASFV poses a significant threat to the global swine industry. Since its first report in Kenya in 1921, the virus has spread globally, causing significant economic losses to the swine industry. ASFV exhibits high genetic diversity, with 24 genotypes identified based on sequence differences within the 3′ end of the B646L gene. Recently, natural recombinant strains of type I and type II have been discovered in China and Vietnam. These recombinants are highly pathogenic and lethal in domestic pigs. Currently, co-circulation of type I, type II, and type I / II recombinant strains occurs in China, and cases of mixed infection with type I and type II have been reported. Type II has been the primary strain responsible for recent outbreaks in Europe, Africa, the Caribbean, and Asia.

[0003] Current clinical studies have found that ASFV undergoes genetic recombination. Genetic recombination between different genotypes may change the virulence and transmission characteristics of the virus, posing new challenges to clinical diagnosis, prevention, purification and prevention and control. Traditional PCR and real-time fluorescence quantitative PCR (qPCR) technologies have limitations in the joint detection of multiple genotypes and recombinant strains of ASFV, while the third-generation digital PCR (dPCR) technology is particularly suitable for multiple nucleic acid detection due to its ultra-high sensitivity and precise quantitative advantages. The present invention designs and verifies a triple dPCR detection method that can simultaneously identify ASFV type I, type II and recombinant strains, providing a highly sensitive and highly accurate detection method for the rapid identification of ASFV type I, type II and recombinant strains. The application of this technology will enhance diagnostic efficiency and provide a new tool for global ASFV prevention and control. Summary of the Invention

[0004] The present invention provides a kit for detecting African swine fever virus and a triple digital PCR detection method. The detection limit (LOD) of the present invention for all target genes is extremely low. Recombinant strains, type I+R, and type II only require 1.740, 1.810, and 0.840 copies / reaction, respectively. Compared with the reported dual qPCR method, this method not only increases the sensitivity by nearly 12 times (LOD 0.84 vs 10 copies / reaction), but also adds the function of detecting recombinant strains without cross-reaction. The unique ability of this method to simultaneously identify type I, type II, and recombinant strains is of great value in monitoring the evolution and spread of ASFV. Rapid and accurate diagnosis helps to take quarantine and culling measures in a timely manner, reducing economic losses in the pig farming industry.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] In the first aspect, the present invention provides a kit for detecting African swine fever virus, wherein the African swine fever virus includes ASFV I+R type strain, ASFV II type strain and ASFV recombinant strain, and the kit includes a forward primer, a reverse primer and a probe for detecting African swine fever virus.

[0007] Preferably, the forward primer and reverse primer sequences of the ASFV I+R type strain are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively, and the probe sequence is shown as SEQ ID NO.3; the forward primer and reverse primer sequences of the ASFV type II strain are shown as SEQ ID NO.4 and SEQ ID NO.5, respectively, and the probe sequence is shown as SEQ ID NO.6; the forward primer and reverse primer sequences of the ASFV recombinant strain are shown as SEQ ID NO.7 and SEQ ID NO.8, respectively, and the probe sequence is shown as SEQ ID NO.9.

[0008] Preferably, the probes for the ASFV I+R type strain, the probes for the ASFV type II strain, and the probes for the ASFV recombinant strain are all modified with fluorescent groups, and the fluorescent groups are at least one of FAM, MGB, HEX, BHQ2, and ROX.

[0009] Preferably, the kit further comprises a recombinant standard plasmid.

[0010] Preferably, the recombinant standard plasmid includes a 159bp fragment of the NP1450L gene of the ASFV recombinant strain, a 147bp fragment of the P72 gene of the ASFV I+R type strain, and a 137bp fragment of the P72 gene of the ASFV II type strain.

[0011] Preferably, the kit further comprises 10×dPCR Taq Master Mix and template DNA.

[0012] In a second aspect, the present invention provides the use of the above-mentioned kit in detecting African swine fever virus.

[0013] In a third aspect, the present invention provides a PCR reaction system for detecting African swine fever virus, wherein the reaction system comprises the above-mentioned forward primer, reverse primer and probe for detecting African swine fever virus, as well as the above-mentioned 10×dPCR Taq MasterMix and template DNA.

[0014] In a fourth aspect, the present invention provides a triple digital PCR method for detecting African swine fever virus, comprising the following steps:

[0015] S1: Collect African swine fever virus nucleic acid samples;

[0016] S2: preparing the PCR reaction system according to claim 8;

[0017] S3: After droplets are generated using a droplet generator, PCR amplification is performed and the data is analyzed.

[0018] Preferably, the PCR amplification conditions are: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds; annealing and extension at 52°C for 45 seconds; 40 cycles; and permanent storage at 4°C.

[0019] Compared with the prior art, the at least one technical solution adopted in the embodiments of this specification can achieve the following beneficial effects:

[0020] (1) The triple dPCR method of the present invention achieved extremely low limits of detection (LOD) for all target genes: only 1.740, 1.810, and 0.840 copies / reaction were required for recombinant strains, type I+R, and type II, respectively. Compared with the reported duplex qPCR method, this method not only increased the sensitivity by nearly 12 times (LOD 0.84 vs 10 copies / reaction), but also added the ability to detect recombinant strains without cross-reaction.

[0021] (2) The droplet segmentation technology of the present invention effectively overcomes the interference of common PCR inhibitors (such as hemoglobin) in clinical samples. This advantage solves the technical bottleneck of conventional qPCR.

[0022] (3) The method of the present invention has significant sensitivity, with detection limits (LODs) of 1.740, 1.810, and 0.840 copies / reaction for the NP1450L gene (recombinant strain), P72 gene (I+R type), and P72 gene (type II), respectively. It has excellent repeatability, with an intra-batch coefficient of variation (CV) of 0.16%-2.03% and an inter-batch CV of 0.82%-4.96%. It has good specificity and can accurately identify ASFV type I, type II, and recombinant strains. It has no cross-reaction with common swine disease pathogens such as PCV2, PRV, PRRSV, PEDV, and PDCoV, which is crucial for on-site diagnosis of mixed infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on the drawings without creative work.

[0024] Figure 1 This is a multiple sequence alignment analysis of the NP1450L and P72 gene regions of different ASFV strains;

[0025] Figure 2 This is the result diagram of digital PCR single set of primers and probes verification;

[0026] Figure 3 Graphs showing primer and probe concentration optimization experiments, including (A) ASFV recombinant strain (NP1450L), (B) ASFV-I+R type (P72), and (C) ASFV-II type (P72). Each panel shows the amplification results for 12 different probe and primer concentration combinations.

[0027] Figure 4 This is an analysis diagram of the annealing temperature optimization experiment results, where (A) is the ASFV recombinant strain; (B) is the ASFV-I+R type; (C) is the ASFV-II type; the panel shows the amplification results at different annealing temperatures; NC is the negative control;

[0028] Figure 5 For different final concentrations (1.0×10 4 to 1.0×10 0 The positive droplet detection results of the plasmid constructs at 400 copies / μL are shown, where (A) is the ASFV recombinant strain, (B) is the ASFV-I+R type, (C) is the ASFV-II type, and (D) is the standard curve;

[0029] Figure 6The results of triple dPCR specificity analysis are shown, where (A) is the ASFV recombinant strain, (B) is the ASFV-I+R type, (C) is the ASFV-II type, and NC is the negative control;

[0030] Figure 7 This is a triple digital PCR sensitivity analysis chart, where (A) is the ASFV recombinant strain, (B) is the ASFV-I+R type, (C) is the ASFV-II type, and NC is the negative control. DETAILED DESCRIPTION

[0031] The embodiments of the present application are described in detail below with reference to the accompanying drawings.

[0032] The following describes the embodiments of the present application through specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the contents disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The present application can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, in the absence of conflict, the features in the following embodiments and embodiments can be combined with each other. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of this application.

[0033] In the present invention, African swine fever virus (ASFV) nucleic acid samples were provided by the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Pseudorabies virus (PRV), porcine circovirus type 2 (PCV-2), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), porcine reproductive and respiratory syndrome virus type 1 (PRRSV-1) and type 2 (PRRSV-2) were isolated and preserved by this laboratory. All clinical samples were extracted with the Vazyme VAMNE virus DNA / RNA extraction kit version 3.0, and then reverse transcribed using the Thermo Fisher Scientific RevertAid first-strand cDNA synthesis kit. The extracted DNA was used for ASFV, PCV2 and PRV detection, and cDNA was used for PEDV, PDCoV, PRRSV-1 and PRRSV-2 detection. DNA quality and concentration were detected by the Thermo Fisher Scientific NanoDrop-2000 spectrophotometer.

[0034] Example 1 Establishment of triple digital PCR detection method

[0035] 1. Primer and Probe Design

[0036] (1) Download the corresponding pathogen genome sequence from the public database NCBI, and find the most conserved sequence after multiple sequence alignment analysis. Figure 1 As shown, the conserved region of the ASFV p72 gene was selected for ASFV type I+R strains and type II strains, and the conserved region of the NP1450L gene was selected for ASFV recombinant strains; the G3048A mutation site of the NP1450L gene was stably present in all recombinant ASFV isolates and could be used as a characteristic marker to distinguish recombinant strains from type I and type II parental strains; the T1597C and A1048G mutation sites of the P72 gene were stably present in recombinant ASFV and type I strains, and were key molecular markers for distinguishing them from type II strains;

[0037] (2) Designing specific primers and probes for conserved regions, where the probes are labeled with different fluorescent groups to achieve multiplex detection;

[0038] (3) Perform primer dimer analysis on all primers and probes to ensure that there is no obvious dimer structure between all primers and probes;

[0039] (4) The primers and probes were analyzed for specificity using the NCBI-prime Blast online tool (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). Finally, the primers and probes were screened using the IDT Analyzer online tool. The primer and probe sequences involved are shown in Table 1. The primers and probes were synthesized by Shanghai Jierui Bioengineering Co., Ltd. and stored at -20°C for future use.

[0040] Table 1. Primer sequences and probe sequences

[0041]

[0042] 2. Preparation of recombinant standard plasmid

[0043] The gene sequences of the NP1450L gene (1219bp-1378bp) of the ASFV recombinant strain (Genbank No.: OQ504954.1), the P72 gene (226bp-373bp) of the ASFV type I strain (Genbank No.: MZ945536.1), and the P72 gene (804bp-941bp) of the ASFV type II strain (Genbank No.: MK333180.1) were synthesized into the pUC-19 vector by Qingke Biotechnology Co., Ltd. (Shanghai Branch). The sequence information of the NP1450 gene (1219bp-1378bp) of the ASFV recombinant strain is shown in SEQ ID NO.10, the sequence information of the P72 gene (226bp-373bp) of the ASFV type I strain is shown in SEQ ID NO.11, and the sequence information of the ASFV type II strain is shown in SEQ ID NO.12. The sequence information of the P72 gene (804bp-941bp) of the type II strain is shown in SEQ ID NO. 11, as shown in Table 2. Recombinant standard plasmids containing the following fragments were obtained: a 159bp fragment of the NP1450L gene of the recombinant ASFV strain, a 147bp fragment of the P72 gene of the ASFV I+R type, and a 137bp fragment of the P72 gene of the ASFV type II, and the concentrations of the three plasmids were all 1.0×10 7 copies / μL, stored at -20℃ for future use.

[0044] Table 2. Sequence information of genes related to ASFV recombinant strains, type I strains and type II strains

[0045]

[0046] 3. Validation of Single and Triple Primers and Probes

[0047] In order to verify whether the triple detection primers and probes designed by the present invention can effectively amplify the target gene of ASFV-I+R type, type II and recombinant strain, the corresponding positive plasmid standards (1×10 6 The single- and triple-plex qPCR amplifications were performed with 100 copies / μL concentration. The qPCR test results (Table 3) showed that both the single template and the triple-mixed template showed good amplification effects. The cycle threshold (Ct) values ​​of the triple-mixed template amplification were highly similar, and there was no significant difference in the amplification efficiency of each target. It can be used for subsequent digital PCR detection ( Figure 2 ).

[0048] Table 3. Validation results of single set of qPCR primers and probes

[0049]

[0050]

[0051] 1 The value represents the cycle threshold (Ct value) of the qPCR amplification curve, and a negative sign indicates that no amplification curve was detected in this reaction.

[0052] 2 The mixed plasmid was prepared by mixing the three plasmids containing the above targets at a rate of 1.0×10 7 The final concentration of copies / μL was prepared by mixing in equal proportions.

[0053] 4. Optimization of Primer and Probe Concentrations for Triple dPCR

[0054] Three standard plasmid mixtures (each plasmid concentration was 5.0×10 2 The probe and primer concentrations were optimized using 100 copies / μL. The annealing temperature was fixed at 59°C, and the permutations and combinations of primers and probes at different concentrations were analyzed using GraphPad Prism software (GraphPad Software, Boston, USA). Figure 3 The optimal concentrations of primers and probes are shown in Table 4.

[0055] Table 4. Triple dPCR reaction system

[0056]

[0057] 5. Optimization of Triple dPCR Annealing Temperature

[0058] The annealing temperature of triple dPCR was optimized using ASFV recombinant strain (NP1450L), ASFV-I+R type (P72) and ASFV-II type (P72) standard plasmids. The plasmids were diluted 10-fold and 5.0×10 2 The mixture of 100 copies / μL was used as a template and triple dPCR detection was performed in the annealing temperature range of 50-62℃. The results showed that 52℃ was the optimal annealing temperature, which could produce the largest number of total droplets and positive droplets ( Figure 4 ).

[0059] 6. Establishment of Triple dPCR Standard Curve

[0060] The final concentration was 1.0 × 10 4 to 1.0×10 0 The standard plasmid constructs with 100 copies / μL were used as templates: ASFV recombinant strain (NP1450L), ASFV-I+R type (P72) and ASFV-II type (P72). The results showed the slope and R of the standard curve of P72 gene and NP1450L gene. 2 The values ​​were: 1.0051 and 0.9992 (P72 gene); 1.023 and 0.9969 (P72 gene);

[0061] 1.0234 and 0.9983 (NP1450L gene) (see Figure 5 ).

[0062] 7. Establishment of Digital PCR Detection Method

[0063] Triplex detection was performed using the AccuONE-200 Droplet Digital PCR System (Shanghai, China). Each 20 μL reaction system contained: 2 μL 10× dPCR Taq Master Mix (with Rox, China), 1 μL each of forward and reverse primers, 0.4–0.6 μL each of primer / probe sets, and 1 μL template DNA, which was made up to 20 μL with ddH₂O. Droplets were generated using the AccuONE-200 Droplet Generator, and PCR amplification was performed using optimized cycling conditions: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 52°C annealing and extension for 45 s, 40 cycles, and storage at 4°C. Data were analyzed using AccuONE-200 Analysis Software (China, China), with thresholds set based on the negative control (ddH₂O).

[0064] Example 2 Method Specificity and Sensitivity Analysis

[0065] The following viral nucleic acid samples were used for triple dPCR specificity validation: ASFV recombinant strain; ASFV type I; ASF type VII; PCV2; PRV; PEDV; PDCoV; PRRSV-1 and PRRSV-2. The established method was used for detection, and ddH2O was used as a negative control. The results showed that only ASFV type I and II and recombinant strains could detect positive droplets, and there was no cross-reaction with other porcine viruses ( Figure 6 ).

[0066] To evaluate the limit of detection (LOD) of triplex dPCR, 1.0 × 10 7 The three plasmids were mixed into a template with a final concentration of 1.0×10-1 copies / μL: ASFV recombinant strain (NP1450L); ASFV-I+R type (P72); ASFV-II type (P72), and a negative control was set to determine the minimum detection limit of the established method. The results showed that the number of positive droplets decreased with the decrease of template concentration. The LOD values ​​calculated based on Poisson distribution were: ASFV recombinant strain (NP1450L): 1.740 copies / reaction; ASFV-I+R type (P72): 1.810 copies / reaction; ASFV-II type (P72): 0.840 copies / reaction (see Figure 7 ).

[0067] Three plasmids: ASFV recombinant strain (NP1450L), I+R type (P72) and type II (P72) were serially diluted (450 to 0.45 copies / reaction) to determine the positive droplet rate and detection rate. The results are shown in Table 5.

[0068] Table 5. Number of positive samples and detection rate of serial dilutions of plasmid constructs

[0069]

[0070] Example 3 Repeatability Analysis

[0071] The final concentration was 5×10 4 , 5×10 3 and 5×10 2 The reproducibility of this method was analyzed using a mixture of the three plasmid constructs at 50 copies / μL as a template. The coefficient of variation (CV) was calculated to determine the stability of the established method. The intra-group coefficient of variation (CV) ranged from 0.16% to 2.03%, and the inter-group coefficient of variation ranged from 0.39% to 4.96, indicating good reproducibility of the method (Table 6).

[0072] Table 6. Repeatability analysis of triplex dPCR

[0073]

[0074]

[0075] Example 4 Clinical Verification

[0076] The triple digital PCR method developed in this paper was used to test 45 African swine fever virus nucleic acid samples of known genotypes provided by the Harbin Veterinary Research Institute. The results showed that among the 45 ASFV-positive samples, 10 were type I positive, 15 were type II positive, and 20 were recombinant positive (Table 7).

[0077] Table 7. Clinical sample triple digital PCR test results

[0078] genotype total ASFV-Ⅰ+R ASFV-Ⅱ ASFV-Recombinant Positive rate (%) I 10 10 - - 100 II 15 - 15 - 100 recombinant 20 20 - 20 100 total 45 30 15 20 100

[0079] The present invention designs three pairs of specific primers and probes for the P72 gene and NP1450L gene of ASFV, which can detect type I, type II and recombinant strains respectively. By optimizing the multiple reaction conditions, a triple digital PCR (dPCR) detection method was established. The method has significant sensitivity, and the detection limits (LOD) of the NP1450L gene (recombinant strain), P72 gene (I+R type) and P72 gene (type II) are 1.740, 1.810 and 0.840 copies / reaction respectively; the intra-batch coefficient of variation (CV) is 0.16%-2.03%, and the inter-batch CV is 0.82%-4.96%, with excellent repeatability; the standard curve R 2 The CV value was ≥0.997, indicating a good linear relationship between template amount and the number of positive droplets. This low CV value confirms the suitability of this technique for routine diagnosis. It also exhibits excellent specificity, detecting only ASFV types I and II, and recombinant strains, with no cross-reactivity with porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus types I / II (PRRSV-I / II), porcine epidemic diarrhea virus (PEDV), and porcine deltacoronavirus (PDCoV). Clinical validation was performed using 45 ASFV nucleic acid samples with known genotypes, with a 100% detection rate for each type and an overall concordance rate exceeding 98.98% with the reference method, using triplex qPCR, demonstrating its potential to become a gold standard diagnostic technique.

[0080] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made based on the essence of the present invention are intended to be covered by the scope of protection of the present invention.

Claims

1. A kit for detecting African swine fever virus, characterized in that: The African swine fever virus includes ASFVI+R type strain, ASFVII type strain and ASFV recombinant strain, and the kit includes forward primers, reverse primers and probes for detecting African swine fever virus.

2. The kit according to claim 1, wherein The forward primer and reverse primer sequences of the ASFV I+R type strain are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and the probe sequence is shown in SEQ ID NO.3; the forward primer and reverse primer sequences of the ASFVII type strain are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively, and the probe sequence is shown in SEQ ID NO.6; the forward primer and reverse primer sequences of the ASFV recombinant strain are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively, and the probe sequence is shown in SEQ ID NO.

9.

3. The kit according to claim 2, wherein The probes for the ASFV I+R type strain, the probes for the ASFV II type strain, and the probes for the ASFV recombinant strain are all modified with fluorescent groups, and the fluorescent groups are at least one of FAM, MGB, HEX, BHQ2, and ROX.

4. The kit according to claim 1, wherein The kit also includes a recombination standard plasmid.

5. The kit according to claim 4, wherein The recombinant standard plasmid includes a 159bp fragment of the ASFV recombinant strain NP1450L gene, a 147bp fragment of the ASFV I+R type strain P72 gene, and a 137bp fragment of the ASFV type II strain P72 gene.

6. The kit according to claim 1, wherein The kit also includes 10× dPCR Taq MasterMix and template DNA.

7. Use of the kit according to any one of claims 1 to 6 in detecting African swine fever virus.

8. A PCR reaction system for detecting African swine fever virus, characterized in that: The reaction system includes the forward primer, reverse primer and probe for detecting African swine fever virus according to claim 1 and the 10×dPCRTaq Master Mix and template DNA according to claim 6.

9. A triple digital PCR method for detecting African swine fever virus, characterized in that: The following steps are involved: S1: Collect African swine fever virus nucleic acid samples; S2: preparing the PCR reaction system according to claim 8; S3: After droplets are generated using a droplet generator, PCR amplification is performed and the data is analyzed.

10. The triple digital PCR method according to claim 9, wherein: The PCR amplification conditions are as follows: pre-denaturation at 95° C. for 5 min; denaturation at 95° C. for 30 s; annealing and extension at 52° C. for 45 s; 40 cycles; and permanent storage at 4° C.

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