Development of a multiplex qRT-PCR assay for detection of classical swine fever virus, African swine fever virus, and Erysipelothrix rhusiopathiae

Classical swine fever virus (CSFV), African swine fever virus (ASFV), and Erysipelothrix rhusiopathiae (E. rhusiopathiae) remain endemic in many parts of China. Co-infections make distinguishing their clinical symptoms and pathological changes difficult. This study developed a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) that can simultaneously detect CSFV, ASFV, and E. rhusiopathiae. Three sets of primers and probes were designed to target the CSFV 5΄ untranslated region, ASFV p72 gene, and E. rhusiopathiae 16sRNA gene. Multiplex qRT-PCR for simultaneous differential detection of these three pathogens was developed after optimizing reaction parameters such as annealing temperature, primer and probe concentrations, amplification cycles, etc. The multiplex qRT–PCR could detect CSFV, ASFV, and E. rhusiopathiae simultaneously but could not amplify other porcine pathogens. The assay’s limit of detection (LOD) was 2.89 × 102 copies/μL for CSFV, ASFV, and E. rhusiopathiae. All correlation coefficients (R2) at higher than 0.99, and the amplification efficiency was 98, 90, and 84%, respectively. All correlation coefficients (R2) were higher than 0.99, and the efficacy of amplification was 84%. In a repeatability test utilizing standard recombinant plasmids, the intra- and inter-assay coefficients of variation (CVs) were less than 2.27 and 3.79 percent, respectively. Lastly, 150 clinical samples were used to evaluate the assay’s applicability in the field. The positive rates of CSFV, ASFV, and E. rhusiopathiae were 1.33%, 0, and 3.33%, respectively. And no co-infection among the three pathogens was found. The concordance rate between the multiplex qRT-PCR and single-plex commercial PCR kits reached 100%. This study’s multiplex qRT-PCR could provide a rapid, sensitive, and specific method for the simultaneous and differential detection of CSFV, ASFV, and E. rhusiopathiae.


Introduction
Classical swine fever virus (CSFV), a single-stranded, positive-sense RNA virus in the Pestivirus genus of the Flaviviridae family (1), is the causative agent of classical swine fever (CSF), which frequently manifests as fever, organ hemorrhage, and immunosuppression (2). The African swine fever virus (ASFV), an enveloped double-stranded DNA virus, is currently the sole member of the Asfivirus genus within the Asfarviridae family (3). This virus can cause African swine fever (ASF), typified by high fever, pulmonary edema, severe hemorrhage, and extensive necrosis of lymphoid tissue, with close to 100% higher morbidity and mortality (4). Erysipelothrix rhusiopathiae (E. rhusiopathiae), a nonsporulating, gram-positive, rod-shaped bacterium, pertains to the Erysipelothrix genus. E. rhusiopathiae can cause swine erysipelas, displaying the primary clinical manifestation with fever, lameness, diamond-shaped lesions on the skin, and sudden mortality in growing and adult swine (5,6). CSFV, ASFV, and E. rhusiopathiae are still prevalent in many countries, resulting in significant economic losses for the global swine industry. Similar clinical signs and pathological alterations make it difficult to distinguish between CSF, ASF, and swine erysipelas in the field. To diagnose these diseases accurately and quickly, developing a specific, sensitive, and rapid test method to detect these pathogens simultaneously and discriminately is essential.
Multiplex real-time quantitative polymerase chain reaction (multiplex qRT-PCR) offers excellent performance in clinical testing, which is based on constant measurements of the change of fluorescent signals during the amplification reaction (7). It provides greater detection capacity, faster speed, and lower labor costs (8). Most multiplex qRT-PCR assays are based on target-specific TaqMan probes, which have higher specificity than conventional RT-PCR, and it is widely used for pathogen diagnosis and monitoring (9). Currently, several qRT-PCR and qPCR assays have been established for the detection of CSFV (10,11), ASFV (12, 13), E. rhusiopathiae (14, 15), and CSFV+ASFV (16, 17). However, a multiplex qRT-PCR assay that can simultaneously and differentially detect CSFV, ASFV, and E. rhusiopathiae has not been reported. Hence, developing a rapid and specific method for differentiating between these three pathogens is essential. This study aimed to establish multiplex qRT-PCR methodbased TaqMan probes for simultaneously and differentially detecting CSFV, ASFV, and E. rhusiopathiae.

Design of primers and probes
The study involved the design of specific primers and corresponding TaqMan probes targeted toward the 5′ untranslated region (UTR) of CSFV, the p72 gene of ASFV, and the 16sRNA gene of E. rhusiopathiae. Then the primers and probes were synthesized by Sangon Biotech Co. Ltd. (Shanghai, China). Table 1 lists detailed data relating to the primers and probes.

Nucleic acid extraction
Major swine pathogens and clinical samples nucleic acids were extracted using GeneRotex96 automatic nucleic acid extractor with Tianlong virus DNA / RNA extraction kit and bacterial genomic DNA extraction kit (Xi'an TianLong Science and Technology Co., Ltd., Xi'an, China) according to the manufacturer's instructions and stored at −80°C until use.

Optimization of the reaction conditions of the multiplex qRT-PCR
Optimizing multiplex qRT-PCR conditions, encompassing annealing temperature, primer and probe concentrations, amplification cycles, and other relevant parameters, was conducted using Analytik Jena qTOWER3/G (Jena, Germany). The optimal reaction conditions for the multiplex qRT-PCR were determined using the following 20 μL basic systems:10 μL of One Step PrimeScript III RT-qPCR (2×) (Takara, Dalian, China), 2.0 μL of standard plasmid (containing 2.89 × 104 copies/μL), and three pairs of primers and probes with varying final concentrations, along with distilled water. The amplification parameters were as follows: reverse transcription at 52°C for 5 min, predenaturation at 95°C for 10 s, 40 cycles of 95°C for 5 s, varying annealing temperature for 30 s, and scanning for the The specificity assay, sensitivity assay, reproducibility assay, and detection of clinical samples in the following study were conducted under the optimized reaction conditions.

Analytical specificity assay
The specificity of the established multiplex qRT-PCR was estimated by utilizing standard DNAs or RNAs of prominent swine pathogens, such as CSFV, ASFV, E. rhusiopathiae, PEDV, PDCoV, TGEV, SADS-CoV, FMDV-O, FMDV-A, PRRSV, PRV, PCV2, swine Escherichia coli, and swine Pasteurella multocida as templates for amplification. Nuclease-free water was utilized as a negative control, while standard plasmids were employed as a positive control.

Repeatability assay
The repeatability assay included intra-and inter-assay measurements. The DNA templates used in the repeatability test were diluted to 2.89 × 10 3 , 2.89 × 10 4 , and 2.89 × 10 5 copies/μL of pUC57-CSFV-ASFV-ER. Intra-assay was conducted by performing one repeat of the assay on each of the three dilutions, with nine replicates for each dilution. Inter-assays were conducted by performing the assay three times on each of the three dilutions on three separate occasions.

Detection of clinical samples
A total of 150 clinical samples were extracted and tested with the optimized multiplex real-time PCR assay. Positive (RT-qPCR system with pUC57-CSFV-ASFV-ER plasmid as template) and negative (RT-qPCR system with nuclease-free water as template) controls were included in each run.

Comparison of multiplex qRT-PCR with single-plex commercial PCR kits
Two CSFV-positive and five E. rhusiopathiae-positive nucleic acids from the clinical samples, along with eight ASFV-positive nucleic acids, were confirmed through Sanger sequencing. These nucleic acids, including 20 negative clinical samples, were subsequently amplified using both the established multiplex qRT-PCR assay and the single-plex commercial PCR kits. The ASFV commercial PCR kit was produced by Beijing SCENK biotechnology development Co., Ltd. (Beijing, China), while the CSFV and E. rhusiopathiae commercial PCR kits were produced by Aodong Inspection & Testing Co., Ltd. (Shenzhen, China). The diagnostic sensitivity and specificity of the assay were evaluated using commercial PCR kits as the standard.

Optimal reaction conditions of the multiplex qRT-PCR
The multiplex qRT-PCR was performed using probes concentrations ranging from 50 to 125 nM and primers with concentrations ranging from 100 to 250 nM. The fluorescence intensity and Ct values of all possible combinations were compared. After  Figure 1). The developed multiplex qRT-PCR with a total volume of 20 μL, containing 10 μL 2× One-Step PrimerScript III RT-PCR (TaKaRa), 0.50 μL forward primer (10 μM), 0.50 μL reverse primer (10 μM), 0.25 μL probe (10 μM), 4.25 μL RNase-free ddH 2 O, and 2.0 μL template. The amplification process was conducted with the following parameters: reverse transcription at 52°C for 5 min, predenaturation at 95°C for 10 s, followed by 45 cycles of denaturation at 95°C for 5 s, and annealing and extension at 59°C for 30 s. Fluorescent signals were collected at the end of each cycle.

Specificity of the multiplex qRT-PCR
The DNA or RNA from various porcine pathogens were used as templates for the multiplex qRT-PCR. The amplification curves were observed only for CSFV, ASFV, and E. rhusiopathiae. Other viruses or bacterium, including PEDV, PDCoV, TGEV, SADS-CoV, FMDV-O, FMDV-A, PRRSV, PRV, PCV2, swine Escherichia coli, swine Pasteurella multocida, and nuclease-free water exhibited no fluorescent signals or amplification curves, indicating the assay's high specificity (Figure 3).

Repeatability of the multiplex qRT-PCR
To assess the repeatability of the multiplex qRT-PCR, three different concentrations of standard plasmids (2.89 × 10 3 , Optimal reaction conditions of the multiplex qRT-PCR. (A-C) Amplification curves of CSFV, ASFV, and E. rhusiopathiae detected by multiplex real-time PCR with different probe and primer concentrations. Plasmid standards with a concentration of 2.89 × 10 4 copies/μL were chosen as reaction templates. The red lines are the amplification curves of three fluorescence of the most suitable reaction tube.
Frontiers in Veterinary Science 05 frontiersin.org 2.89 × 10 4 , and 2.89 × 10 5 copies/μL) were utilized as templates for both intra-and inter-assay comparisons. The results reveal that the coefficients of variation (CVs) for both intra-and inter-assay of the Ct values were less than 5% (Table 3), suggesting the assay's excellent repeatability.

Detection of the clinical samples
A total of 150 clinical samples were gathered from Guangdong Province, Southern China, during the period spanning from February 2021 to December 2022. These samples were subsequently analyzed using the established multiplex qRT-PCR method to verify its suitability. As a result, the positive rates of CSFV, ASFV, and E. rhusiopathiae were 1.33% (2/150), 0 (0/75), and 3.33% (5/150), respectively (Supplementary Table 1). And no co-infection among the three pathogens was found.

Comparison of multiplex qRT-PCR with single-plex commercial PCR kits
Upon comparing the results obtained from the multiplex qRT-PCR and single-plex commercial PCR, the analysis revealed a  Frontiers in Veterinary Science 06 frontiersin.org 100% concordance rate for both positive and negative samples (Table 4). Additionally, the diagnostic sensitivity and specificity were both 100%, indicating the high clinical value of the assay.

Discussion
CSFV, ASFV, and E. rhusiopathiae are significant pathogens severely damaging the global swine industry. In certain pig herds, there may be cases of co-infection between CSFV and ASFV, resulting in clinical manifestations and pathological changes that are difficult to differentiate in the field. Similar observations also exist between CSFV and E. rhusiopathiae (18,19). The clinical symptoms and pathological changes exhibited by these pathogens can be similar, posing a challenge in accurately identifying the causative agent just through clinical indicators. Hence, it is essential to differentially detect these three pathogens through laboratory procedures to diagnose these diseases accurately. Multiplex qPCR is one of the best options among the different methods for diagnosing because it can rapidly, precisely, sensitively, and accurately identify several pathogenic nucleic acids in a single reaction. It is possible to achieve simultaneous rapid detection of multiple pathogens, which is significant for rapid diagnosis, prevention, and epidemiological study of mixed infectious diseases. It is expected to be more useful in some sudden outbreak areas (20). In this study, we used three sets of specific primers and corresponding probes to develop a multiplex TaqMan probe-based qRT-PCR system that can detect CSFV, ASFV, and E. rhusiopathiae simultaneously and differentially. With a detection limit of 289 copies per reaction, The assay demonstrated the ability to precisely detect CSFV, ASFV, and E. rhusiopathiae, while exhibiting no cross-reactivity with other porcine pathogens. Moreover, the intra-and inter-assay CVs were all less than 3.79%. Thus demonstrating a high level of specificity, sensitivity, and repeatability. Furthermore, the overall concordance rate between the multiplex qRT-PCR and single-plex commercial PCR kits was 100%. Lastly, the positive rates of CSFV, ASFV, and E. rhusiopathiae in 150 clinical samples were 1.33%, 0, and 3.33%, respectively, indicating that CSFV and E. rhusiopathiae were still sporadically prevalent in pig herds in southern China. Even without co-infection among the three pathogens found, significant efforts are necessary to prevent and control these pathogens.
In summary, using the genomic sequences of CSFV, ASFV, and E. rhusiopathiae, specific primers and probes were designed. After optimizing the reaction conditions, a TaqMan-probe-based multiplex qRT-PCR capable of simultaneous and differential detection of CSFV, ASFV, and E. rhusiopathiae was developed with high specificity, sensitivity, repeatability, and clinical value.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

Author contributions
LZ, X-HW, and QZ designed the primers and probes, optimized the reaction conditions, and wrote the manuscript. C-LJ, X-RZ, and S-JL tested the diagnostic method. D-HL and QZ designed the experiments and revised the manuscript. All authors contributed to the article and approved the submitted version.