The Development of a Multiplex Real-Time Quantitative PCR Assay for the Differential Detection of the Wild-Type Strain and the MGF505-2R, EP402R and I177L Gene-Deleted Strain of the African Swine Fever Virus

Simple Summary African swine fever (ASF) was first reported in August 2018 in China, and the naturally gene-deleted ASFV strain was first identified in 2020 in this country. The vaccine candidates that deleted some virulent genes from the virulent parental strains have also been reported in many countries. To differentiate the wild-type and gene-deleted ASFV strains, four pairs of specific primers and TaqMan probes targeting the ASFV B646L (p72), I177L, MGF505-2R and EP402R (CD2v) genes were designed. After optimizing the reaction conditions, a multiplex real-time qPCR assay for the differential detection of the wild-type and gene-deleted ASFV strains was developed. The assay was further used to test 4239 clinical samples, and 534 samples were positive for ASFV, of which 30 samples lacked B646L, I177L, MGF505-2R and/or EP402R genes. The assay showed high specificity, sensitivity and repeatability, and it provided a reliable method for evaluating ASFV in clinical samples. Abstract African swine fever virus (ASFV) causes African swine fever (ASF), a devastating hemorrhagic disease of domestic pigs and wild boars. Currently, the MGF505R, EP402R (CD2v) and I177L gene-deleted ASFV strains were confirmed to be the ideal vaccine candidate strains. To develop an assay for differentiating the wild-type and gene-deleted ASFV strains, four pairs of specific primers and TaqMan probes targeting the ASFV B646L (p72), I177L, MGF505-2R and EP402R (CD2v) genes were designed. A multiplex real-time qPCR assay for the differential detection of the wild-type and gene-deleted ASFV strains was developed after optimizing the reaction conditions, including the annealing temperature, primer concentration and probe concentration. The results showed that the multiplex real-time qPCR assay can specifically test the ASFV B646L (p72), I177L, MGF505-2R and EP402R (CD2v) genes with a limit of detection (LOD) of 32.1 copies/μL for the B646L (p72) gene, and 3.21 copies/μL for the I177L, MGF505-2R and EP402R (CD2v) genes. However, the assay cannot test for the classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), porcine circovirus type 2 (PCV2), PCV3 and pseudorabies virus (PRV). The assay demonstrated good repeatability and reproducibility with coefficients of variation (CV) less than 1.56% for both the intra- and inter-assay. The assay was used to test 4239 clinical samples, and the results showed that 12.60% (534/4239) samples were positive for ASFV, of which 10 samples lacked the EP402R gene, 6 samples lacked the MGF505-2R gene and 14 samples lacked the EP402R and MGF505-2R genes. The results indicated that the multiplex real-time qPCR developed in this study can provide a rapid, sensitive and specific diagnostic tool for the differential detection of the ASFV B646L, I177L, MGF505-2R and EP402R genes.


Clinical Samples
From January 2021 to December 2021, a total of 4239 clinical samples were collected from different pig farms, slaughterhouses, farmers' markets and harmless disposal sites in Guangxi province, southern China. The samples included environmental swabs, nasopharyngeal swabs, whole blood and tissue samples (kidneys, spleens and lymph nodes). The samples were transported to our laboratory at conditions of 0-4 • C immediately after collection and were stored at −70 • C until they were used.

DNA Extraction
The homogenized material from the tissue samples, including kidneys, spleens and lymph nodes, was obtained using a blender (TIANLONG Scientific, Xi'an, China), freezethawed 3 times and centrifuged at 10,000× g for 10 min at 4 • C. The supernatants were used for total nucleic acids extraction.
Each environmental sample (swab) and nasopharyngeal swab was diluted in a tube with 1.0 mL PBS (pH 7.2), vortexed for 10 s and centrifuged at 10,000× g for 5 min at 4 • C. The supernatants were used for total nucleic acids extraction.
The anticoagulant whole blood samples with EDTA-Na 2 (15 g/L) were vortexed for 10 s and centrifuged at 10,000× g for 5 min at 4 • C. The supernatants were used for total nucleic acids extraction.
The total nucleic acids were extracted from 200 µL of the supernatants from the clinical samples using the GeneRotex 96 Automatic Nucleic Acid Extractor (TIANLONG Scientific, Xi'an, China) with Viral DNA/RNA Isolation Kit 4.0 (TIANLONG Scientific, Xi'an, China) according to the manufacturer's instructions. The extracted nucleic acids were tested for ASFV by the developed multiplex real-time qPCR using the QuantStudio 6 qPCR system (ABI, Carlsbad, CA, USA).

Primers and Probes
Four pairs of specific primers and corresponding TaqMan probes were designed using the Primer Express Software v3.0 (ABI, Los Angeles, CA, USA), which targeted the conserved regions of the ASFV B646L gene, MGF505-2R gene, EP402R gene and I177L gene. The sequences of the primers and probes are shown in Table 1.

Construction of Standard Plasmids
Using ASFV DNA as a template, the targeted fragments of the B646L gene, MGF505-2R gene, EP402R gene and I177L gene were amplified by PCR, purified, ligated to the pMD18-T vector (TaKaRa, Beijing, China) and then transformed into E. coli DH5α competent cells. The positive clones were selected and grown at 37 • C for about 20 h. The recombinant plasmids were extracted, named as pASFV-B646L, pASFV-MGF505-2R, pASFV-EP402R and pASFV-I177L, respectively, and used as standard plasmids for developing the multiplex real-time qPCR. A NanoDrop spectrophotometer (Thermo Fisher, Waltham, MA, USA) was used to quantify the ultraviolet absorbance of the standard plasmids at 260 nm and 280 nm. The following formula was used to determine their concentrations: plasmid copy number (copies/µL) = (plasmid concentration × 10 −9 × 6.02 × 10 23 )/(660 Dalton/bases × DNA length).

Optimization of the Reaction Conditions of the Multiplex Real-Time qPCR
The multiplex real-time qPCR was performed using the QuantStudio 6 qPCR system (ABI, Carlsbad, CA, USA). The annealing temperature and the concentrations of the primers and probes were determined by the following amplification parameters: 95 • C for 2 min, 40 cycles of 95 • C for 5 s and 56 • C for 30 s. To determine the optimal reaction conditions for the multiplex real-time qPCR, the 20 µL of the fundamental systems was made as follows: 10 µL Premix Ex Taq (Probe qPCR) (TaKaRa, Beijing, China), 2 µL mixtures of the four standard plasmids (each with final concentration 10 8 copies/µL), 4 pairs of the primers and probes (each with different final concentration) and distilled water. The recombinant plasmids pASFV-B646L, pASFV-MGF505-2R, pASFV-EP402R and pASFV-I177L were used as templates. The different annealing temperatures (52 • C, 54 • C, 56 • C, 58 • C, 60 • C and 62 • C), primer concentrations (0.1 pmol/µL, 0.2 pmol/µL, 0.3 pmol/µL, 0.4 pmol/µL and 0.5 pmol/µL) and probe concentrations (0.1 pmol/µL, 0.2 pmol/µL, 0.3 pmol/µL, 0.4 pmol/µL and 0.5 pmol/µL) were used for amplification by an arrangement and combination test to obtain the optimal reaction conditions. At the end of each cycle, the fluorescent signals were determined.

Specificity Analysis of the Multiplex Real-Time qPCR
The DNA or cDNA of the ASFV, PRRSV, CSFV, PCV2, PCV3, PEDV, PDCoV and PRV, as well as the recombinant gene-deleted plasmids pASFV-∆MGF505-2R, pASFV-∆EP402R and pASFV-∆I177L, were used to evaluate the specificity of the developed assay. The sterilized distilled water was used as the negative control.

Repeatability Analysis of the Multiplex Real-Time qPCR
The standard plasmids pASFV-B646L, pASFV-MGF505-2R, pASFV-EP402R and pASFV-I177L were mixed and 10-fold serially diluted. Three final concentrations of 3.21 × 10 8 , 3.21 × 10 6 and 3.21 × 10 4 copies/µL for each plasmid were used to run the intra-and inter-assay tests. The coefficients of variation (CVs) were used to evaluate the repeatability and the reproducibility of the assay.

Test of Clinical Samples by the Multiplex Real-Time qPCR
The developed multiplex real-time qPCR assay was used to test 4239 clinical samples collected in Guangxi province, southern China, from January 2021 to December 2021.
At the same time, the above samples were tested for ASFV by the OIE-recommended real-time qPCR for ASFV [38] with a total volume of 25 µL, as shown in Table 2. The amplification was performed at 50 • C for 2 min and 95 • C for 10 min and then with 40 cycles of 95 • C for 15 s and 58 • C for 60 s, and the fluorescent signals at the end of each cycle were determined. The coincidence rate between the developed multiplex real-time qPCR and the OIErecommended real-time qPCR was evaluated.

Statistical Analysis
The statistical analyses were conducted by regression analysis, the Pearson's correlation coefficient and an analysis of variance (ANOVA) using the GraphPad Prism version 7.04 software (GraphPad Software, Santiago, CA, USA) for linear regression and for the tested results of clinical samples.

Optimal Reaction Conditions of the Multiplex Real-Time qPCR
The standard plasmids were used to optimize the multiplex real-time qPCR reaction parameters, including the annealing temperature and the primer and probe concentrations using an arrangement and combination test.

Standard Curves of the Multiplex Real-Time qPCR
The multiplex real-time qPCR standard curves were generated using 10-fold serially diluted standard plasmids from 3.21 × 10 8 to 3.21 × 10 2 copies/µL (final concentration). The results showed that the corresponding slope of the equation, correlation coefficient (R 2 ) and amplification efficiency (E) were as follows: −3.211, 0.999 and 104.9% for the B646L gene; −3.159, 0.999 and 107.3% for the I177L gene; −3.152, 0.999 and 107.6% for the EP402R gene; and −3.115, 0.998 and 109.4% for the MGF505-2R gene ( Figure 1). tion coefficient and an analysis of variance (ANOVA) using the GraphPad Prism version 7.04 software (GraphPad Software, Santiago, CA, USA) for linear regression and for the tested results of clinical samples.

Optimal Reaction Conditions of the Multiplex Real-Time qPCR
The standard plasmids were used to optimize the multiplex real-time qPCR reaction parameters, including the annealing temperature and the primer and probe concentrations using an arrangement and combination test.
The  Table 2). The amplification was performed at 95 °C for 10 s and then with 40 cycles of 95 °C for 5 s, 56 °C for 30 s, and the fluorescent signals at the end of each cycle were determined. A sample with a Ct value of ≤35 cycles was judged as positive, whereas a sample with a Ct value of >35 cycles was judged as negative.

Standard Curves of the Multiplex Real-Time qPCR
The multiplex real-time qPCR standard curves were generated using 10-fold serially diluted standard plasmids from 3.21 × 10 8 to 3.21 × 10 2 copies/μL (final concentration). The results showed that the corresponding slope of the equation, correlation coefficient (R 2 ) and amplification efficiency (E) were as follows:

Specificity of the Multiplex Real-Time qPCR
The multiplex real-time qPCR assay was validated for specificity using DNA/cDNA from the ASFV, PRRSV, CSFV, PCV2, PCV3, PEDV, PDCoV and PRV and using recombinant plasmids pASFV-ΔMGF505-2R, pASFV-ΔEP402R and pASFV-ΔI177L. The results showed that only the ASFV generated positive amplification curves for the four fluorescence channels of FAM, VIC, Cy5 and Texas Red corresponding to the B646L gene, MGF505-2R gene, EP402R gene and I177L gene, respectively, whereas other viruses and plasmids did not generate amplification curves, indicating that the developed multiplex real-time qPCR could specifically test the B646L gene, MGF505-2R gene, EP402R gene and I177L gene of the ASFV (Figure 2).

Specificity of the Multiplex Real-Time qPCR
The multiplex real-time qPCR assay was validated for specificity using DNA/cDNA from the ASFV, PRRSV, CSFV, PCV2, PCV3, PEDV, PDCoV and PRV and using recombinant plasmids pASFV-∆MGF505-2R, pASFV-∆EP402R and pASFV-∆I177L. The results showed that only the ASFV generated positive amplification curves for the four fluorescence channels of FAM, VIC, Cy5 and Texas Red corresponding to the B646L gene, MGF505-2R gene, EP402R gene and I177L gene, respectively, whereas other viruses and plasmids did not generate amplification curves, indicating that the developed multiplex real-time qPCR could specifically test the B646L gene, MGF505-2R gene, EP402R gene and I177L gene of the ASFV (Figure 2).

Specificity of the Multiplex Real-Time qPCR
The multiplex real-time qPCR assay was validated for specificity using DNA/cDN from the ASFV, PRRSV, CSFV, PCV2, PCV3, PEDV, PDCoV and PRV and using recomb nant plasmids pASFV-ΔMGF505-2R, pASFV-ΔEP402R and pASFV-ΔI177L. The resul showed that only the ASFV generated positive amplification curves for the four fluore cence channels of FAM, VIC, Cy5 and Texas Red corresponding to the B646L gen MGF505-2R gene, EP402R gene and I177L gene, respectively, whereas other viruses an plasmids did not generate amplification curves, indicating that the developed multiple real-time qPCR could specifically test the B646L gene, MGF505-2R gene, EP402R gene an I177L gene of the ASFV (Figure 2).

Repeatability of the Multiplex Real-Time qPCR
The repeatability and reproducibility of the developed multiplex real-time qPCR was evaluated using three concentrations of the standard plasmids: 3.21 × 10 8 , 3.21 × 10 6 and 3.21 × 10 4 copies/μL (final concentration). The results showed that the coefficients of variation (CVs) of the intra-and inter-assay were from 0.26% to 0.92% and from 0.53% to 1.56%, respectively (Table 3).

Repeatability of the Multiplex Real-Time qPCR
The repeatability and reproducibility of the developed multiplex real-time qPCR was evaluated using three concentrations of the standard plasmids: 3.21 × 10 8 , 3.21 × 10 6 and 3.21 × 10 4 copies/µL (final concentration). The results showed that the coefficients of variation (CVs) of the intra-and inter-assay were from 0.26% to 0.92% and from 0.53% to 1.56%, respectively (Table 3).

Evaluation of Clinical Samples
A total of 4239 clinical samples were collected from January 2021 to December 2021 in Guangxi province, southern China, and were tested by the developed multiplex real-time qPCR. The results showed that 12.60% (534/4239) of samples were positive for the ASFV ( Table 4)

Evaluation of Clinical Samples
A total of 4239 clinical samples were collected from January 2021 to December 2021 in Guangxi province, southern China, and were tested by the developed multiplex realtime qPCR. The results showed that 12.60% (534/4239) of samples were positive for the ASFV ( Table 4). Out of the different sample types, the harmless disposal sites showed the highest positive rate of 28.34% (121/427), followed by the slaughterhouses with 17.30% (330/1980), the farmers' markets with 10.70% (67/626) and the breeding farms with the lowest positive rate of 1.25% (15/1278). Additionally, the results showed that, of the 534 positive samples, 10 samples lacked the EP402R gene, 6 samples lacked the MGF505-2R gene, 14 samples lacked the EP402R and MGF505-2R genes and no sample lacked the I177L gene. Three different kinds of gene-deleted clinical samples are shown in Figure 4. The data in the same column which are marked with different small letters indicate significant differences (p < 0.05). The data in the last line which are marked with different capital letters indicate significant differences (p < 0.05). The harmless disposal site is the place where dead pigs are concentrated from one or more different pig farms for harmless treatment.   The clinical samples were also tested by the OIE-recommended real-time qPCR targeting the ASFV B646L gene [38]. The results of the developed multiplex real-time qPCR were consistent with the results of the OIE-recommended real-time qPCR. The coincidence rate of these two assays was 100%, suggesting good consistency between these two methods while using the B646L gene as a target.

Discussion
Currently, ASF is still an epidemic in Africa, the trans-Caucasus region, eastern Europe, the Russian Federation and Asia, which poses a great challenge to the pig industry and port trade all over the world [9,10]. At present, there is no completely effective way to prevent and control ASF, and strict biosafety and culling of the infected pigs are common measures to reduce the losses of this disease. The development of safe and effective vaccines is one of the key strategies. In order to generate attenuated ASF vaccines, virulent ASFV strains have had several virulence-associated genes deleted, such as I177L, 9GL, UK, EP402R (CD2v), DP148R, A238L, MGF505, MGF360, etc., and have shown decreased virulence and high efficacy to protect pigs infected with their parental strains [24,39,40]. Scientists from China and the United Kingdom have reported that the ASFV strains, which were deleted the MGF505 and MGF360 genes or the MGF505, MGF360 and EP402R (CD2v) genes, greatly attenuated their virulence and protected pigs from challenges with their parental trains [24,41]. Scientists from the USA and Vietnam have reported that the ASFV strain with the I177L gene deletion can also provide homology protection [20][21][22]. Furthermore, the naturally gene-deleted ASFV strains of MGF360-505R and EP402R genes, which show decreased virulence with lower morbidity and mortality, were identified in several provinces in China [27]. Therefore, to differentiate the wild-type strains and the gene-deleted strains, including the naturally gene-deleted strains and the man-made gene-deleted vaccine candidate strains, a rapid, sensitive and specific assay is necessary to differentially detect these ASFV strains.
The qRT-PCR/PCR is a rapid, specific, sensitive and accurate method for the detection of viral nucleic acids and can be conveniently used for the detection and quantification of viral swine pathogens [42,43]. In this study, a quadruplex real-time qPCR based on four pairs of specific primers and corresponding probes was developed, and it can be used for the differential detection of the wild-type and gene-deleted ASFV strains in one reaction. The assay showed high sensitivity with an LOD from 3.21 copies/µL to 32.1 copies/µL for different templates and can specifically test the ASFV B646L, I177L, MGF505-2R and EP402R genes without cross-reaction with other porcine viruses. Using this multiplex real-time qPCR assay, the ASFV strain with the B646L gene can be tested regardless of whether it is the wild-type or gene-deleted ASFV. The wild-type ASFV strain shows the simultaneous presence of these four genes, and the gene-deleted ASFV strain shows the deletion of one or more than one gene of the I177L, MGF505-2R and EP402R genes. The assay was verified for its practicality and usefulness by testing 4239 clinical samples in this study.
Since ASF was first reported in China in August 2018, this disease has spread rapidly throughout all provinces in mainland China in a short time [6,44]. In this study, a total of 4239 clinical samples form Guangxi province, southern China, were tested by the developed multiplex real-time qPCR. The results showed that 12.60% of samples were positive for ASFV, of which the positive rates of the harmless disposal sites, slaughterhouses, farmers' markets and breeding farms were 28.34%, 17.30%, 10.70% and 1.25%, respectively, indicating that the ASFV was still a serious epidemic in Guangxi province, southern China. Of the 534 positive samples, 285 (53.37%) were environmental samples, which came from the harmless disposal sites, slaughterhouses, farmers' markets and breeding farms. Since ASFV can survive for a long period in the environment [45], the viruses easily spread from these places through moving humans and vehicles. Therefore, it is very important to clean and disinfect the environments of these places, especially harmless disposal sites, to cut off the transmission of ASFV.
It is noteworthy that the gene-deleted ASFV strains are circulating in the field. Among the 534 ASFV-positive samples, 10 samples lacked the EP402R gene, 6 samples lacked the MGF505-2R gene, 14 samples lacked the EP402R and MGF505-2R genes and no sample lacked the I177L gene. This indicates that the wild-type and gene-deleted ASFV strains were simultaneously circulating in the field in Guangxi province, which further verified the existence of different gene-deleted ASFV strains in China [27]. They may be natural variations, or they may come from man-made illegal vaccines, which need to be further investigated and verified. At present, no I177L gene-deleted ASFV strain, which was verified to be an ideal vaccine candidate and has been approved officially to be used in the field as an attenuated vaccine since June 2022 in Vietnam [20], was found in the field in China. However, since the border between China and Vietnam is more than 1300 km, and since personnel and goods exchanges are frequent, the illegal trade of live pigs and pork products between these two countries is inevitable. Therefore, the I177L gene-deleted ASFV strain needs to be continuously monitored at present in the case of the transmission of this strain from Vietnam to China.

Conclusions
A multiplex real-time qPCR assay was successfully developed for the differential detection of the wild-type and gene-deleted ASFV strains in this study. The assay showed high specificity, sensitivity and repeatability, and it provided a reliable method for evaluating ASFV in clinical samples. The tested results of the clinical samples showed that harmless disposal sites, slaughterhouses and farmers' markets in Guangxi province, southern China, are severely polluted by ASFV at present, which are important risk points for the prevention and control of ASF.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ani12141754/s1, Table S1: The sequences of recombinant plasmids pASFV-∆MGF505-2R, pASFV-∆EP402R and pASFV-∆I177L.  Informed Consent Statement: Written informed consent was obtained to use the clinical samples in this study from the owners of the animals.

Data Availability Statement:
The data used in the analysis can be obtained from the authors on request.