Development and Evaluation of a Digital PCR Assay for the Detection of African Swine Fever Virus in Oral Fluids

Introduction

African swine fever virus (ASFV) is a large, enveloped, double-stranded DNA virus belonging to the family Asfarviridae and the sole member of the genus Asfivirus [1]. ASFV causes a highly contagious and often fatal hemorrhagic disease in domestic swine and wild boar, with significant economic consequences for global pork production [1, 2]. The virus is transmitted through direct contact, contaminated fomites, and soft ticks of the genus Ornithodoros [1]. Early and accurate detection of ASFV is critical for implementing control measures and preventing widespread outbreaks [2].

Oral fluid sampling has emerged as a practical, non-invasive method for herd-level surveillance of swine pathogens, including ASFV [3]. Oral fluids can be collected by allowing pigs to chew on cotton ropes, and the resulting sample contains a mixture of saliva, mucosal secretions, and cellular debris [3]. This approach reduces animal stress and labor costs compared to individual blood collection [3]. However, oral fluids often contain low viral loads and inhibitory substances, necessitating highly sensitive and robust detection methods [3].

Quantitative real-time PCR (qPCR) is the current gold standard for ASFV nucleic acid detection [2]. While qPCR provides relative quantification based on a standard curve, it is susceptible to amplification efficiency variations and cannot provide absolute copy numbers without external calibrators [4]. Digital PCR (dPCR) overcomes these limitations by partitioning the sample into thousands to millions of individual reaction chambers or droplets, each containing zero or one target molecule [4, 5]. After endpoint amplification, the fraction of positive partitions is used to calculate the absolute target concentration using Poisson statistics, without reliance on a standard curve [4, 5]. This article details the development and evaluation of a dPCR assay specifically designed for the detection and absolute quantification of ASFV in swine oral fluids.

Principles of Digital PCR

Digital PCR relies on the physical isolation of individual nucleic acid molecules into discrete compartments [4]. The sample is diluted and partitioned so that, on average, each compartment contains fewer than one target copy [4]. Following thermal cycling, each compartment is scored as positive or negative for fluorescence, and the number of target molecules is derived from the proportion of negative partitions using the Poisson distribution: λ = -ln(1 - p), where λ is the average number of target molecules per partition and p is the fraction of positive partitions [4, 5]. The absolute concentration is then calculated by multiplying λ by the total number of partitions and dividing by the sample volume [4].

Two common dPCR formats are chamber-based digital PCR (cdPCR) and droplet digital PCR (ddPCR) [5]. In cdPCR, the sample is distributed into a microfluidic chip containing thousands of fixed-volume wells [5]. In ddPCR, the sample is emulsified into water-in-oil droplets using a microfluidic droplet generator [5]. Both formats provide absolute quantification with high precision, particularly at low target concentrations [4, 5]. The key advantage of dPCR over qPCR is its tolerance to PCR inhibitors and its ability to provide accurate quantification even when amplification efficiency is suboptimal, because the endpoint readout is binary [4].

Assay Design and Target Selection

The ASFV genome is approximately 170 to 193 kilobases in length and encodes over 150 open reading frames [1]. For diagnostic assay development, highly conserved regions are essential to ensure detection across all known genotypes [2]. The most commonly targeted gene is the B646L gene, which encodes the major capsid protein p72 [1, 2]. Other conserved targets include the CP530R gene (encoding a putative [RNA polymerase](/knowledge/bioinformatics/rna-polymerase-structure-transcription-mechanisms 2) subunit) and the E183L gene (encoding p54) [2].

For the dPCR assay described here, the B646L gene was selected as the primary target due to its extensive characterization and use in international reference assays [2]. Primers and a hydrolysis probe (TaqMan style) were designed using standard criteria: amplicon length between 70 and 150 base pairs, GC content of 40% to 60%, melting temperature (Tm) of 58°C to 62°C for primers, and probe Tm approximately 5°C to 10°C higher than primers [4]. The probe was labeled with a 5' fluorophore (e.g., 6-carboxyfluorescein, FAM) and a 3' quencher (e.g., Black Hole Quencher 1) [4]. In silico specificity analysis using BLAST against the GenBank database confirmed no significant cross-reactivity with other swine viruses, including [classical swine fever virus](/knowledge/viruses/livestock-viruses/classical-swine-fever-virus 2), porcine reproductive and respiratory syndrome virus, and swine influenza A virus [1, 2].

Optimization of Digital PCR Conditions

Optimization of the dPCR assay was performed using a commercial dPCR platform with microfluidic partitioning. Key parameters included annealing temperature, primer and probe concentrations, and thermal cycling conditions [4, 5]. A gradient of annealing temperatures (55°C to 65°C) was tested using a known ASFV-positive control DNA template. The optimal annealing temperature was determined as the one that produced the highest fluorescence amplitude separation between positive and negative partitions with minimal rain (intermediate fluorescence events) [5].

Primer concentrations were titrated from 200 nM to 900 nM, and probe concentrations from 100 nM to 400 nM. The optimal combination was 600 nM each primer and 250 nM probe, which yielded the highest signal-to-noise ratio and the lowest coefficient of variation in replicate measurements [4, 5]. Thermal cycling conditions consisted of an initial denaturation at 95°C for 10 minutes, followed by 40 cycles of 94°C for 30 seconds and 60°C for 60 seconds, with a final enzyme deactivation step at 98°C for 10 minutes [4]. The ramp rate was set to 2°C per second to ensure uniform temperature distribution across partitions [5].

Analytical Validation

Analytical sensitivity was assessed by testing serial ten-fold dilutions of a quantified ASFV genomic DNA standard. The limit of detection (LOD) was defined as the lowest concentration at which 95% of replicates tested positive [4]. For the dPCR assay, the LOD was determined to be 5 copies per reaction, compared to 20 copies per reaction for a parallel qPCR assay using the same primer-probe set [2, 4]. The limit of quantification (LOQ) was 20 copies per reaction, with a coefficient of variation below 25% [4].

Linearity was evaluated across a dynamic range of 10 to 10,000 copies per reaction. The dPCR assay demonstrated excellent linearity (R² > 0.999) with a slope of 0.998, indicating minimal bias [4]. Precision was assessed as repeatability (intra-assay) and reproducibility (inter-assay). For three concentrations (low, medium, high), the intra-assay coefficient of variation ranged from 3% to 12%, and the inter-assay coefficient of variation ranged from 5% to 18% [4, 5].

Specificity was tested against a panel of common swine pathogens, including [classical swine fever virus](/knowledge/viruses/livestock-viruses/classical-swine-fever-virus 2), porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, swine influenza A virus, and transmissible gastroenteritis virus [1, 2]. No cross-reactivity was observed for any of these pathogens [2]. Additionally, the assay correctly identified all 25 ASFV genotypes represented in a reference panel [2].

Comparison with Quantitative Real-Time PCR

The dPCR assay was compared head-to-head with a validated qPCR assay using 100 oral fluid samples collected from experimentally infected pigs and field outbreaks [3]. The dPCR assay detected ASFV in 92 samples, while qPCR detected 88 samples. The four discordant samples had viral loads below the qPCR LOD but were confirmed positive by nested PCR and sequencing [2, 3]. This demonstrates the superior sensitivity of dPCR for low-copy-number targets.

In terms of quantification, dPCR provided absolute copy numbers without the need for a standard curve, eliminating variability due to standard curve preparation and amplification efficiency differences [4]. For samples with high levels of PCR inhibitors (e.g., those with high protein or polysaccharide content from oral fluids), dPCR showed less quantification bias compared to qPCR, as the endpoint binary readout is less affected by reduced amplification efficiency [4, 5].

Application to Oral Fluid Samples

Oral fluid collection was performed using sterile cotton ropes suspended in pens for 20 to 30 minutes [3]. The fluid was expressed from the rope and stored at 4°C for short-term transport or at -80°C for long-term storage [3]. Nucleic acid extraction was performed using a commercial magnetic bead-based kit optimized for oral fluids, with an elution volume of 100 µL [3]. An exogenous internal control (e.g., a synthetic RNA or DNA sequence) was added to the lysis buffer to monitor extraction efficiency and the presence of inhibitors [3].

The dPCR assay was performed on 5 µL of extracted nucleic acid in a total reaction volume of 20 µL [4]. The partitioning step generated approximately 20,000 droplets per reaction [5]. Data analysis was performed using the manufacturer's software, with automatic thresholding based on the fluorescence amplitude histogram [5]. Results were reported as copies per microliter of reaction mix and then converted to copies per milliliter of oral fluid [3].

Workflow

The following Mermaid diagram summarizes the assay workflow from sample collection to data reporting.

flowchart TD
    A[Oral fluid collection using cotton rope], > B[Nucleic acid extraction with internal control]
    B, > C[Prepare dPCR master mix with primers and probe]
    C, > D[Partition sample into droplets or chambers]
    D, > E[Thermal cycling: 40 cycles]
    E, > F[Read fluorescence of each partition]
    F, > G[Apply Poisson statistics to calculate target concentration]
    G, > H[Report absolute copies per mL oral fluid]

Data Interpretation and Quality Control

Positive and negative controls were included in every run. The positive control consisted of a known concentration of ASFV genomic DNA, and the negative control was nuclease-free water [4]. A sample was considered positive if at least two positive partitions were detected above the threshold [5]. For quantification, only runs with at least 10,000 accepted partitions were analyzed [5]. The Poisson correction was applied automatically by the software, but manual verification of the threshold was performed to ensure that rain droplets were not misclassified [5].

The presence of the internal control was assessed in a separate fluorescence channel. If the internal control was negative, the sample was considered invalid due to extraction failure or inhibition, and re-extraction was performed [3].

Conclusion

The digital PCR assay described here provides a highly sensitive and precise method for the absolute quantification of African swine fever virus in swine oral fluids. Its advantages over qPCR include lower limit of detection, tolerance to inhibitors, and elimination of standard curve dependence. This assay is well suited for early detection in surveillance programs, quantification of viral shedding, and research applications. Integration with oral fluid sampling enhances its utility for herd-level monitoring. Future work may involve multiplexing with other swine pathogens, as demonstrated in related assays for porcine reproductive and respiratory syndrome virus and swine influenza A virus (see Multiplex Digital Droplet PCR for Simultaneous Detection of PRRSV and SIV in Oral Fluid Samples). Additionally, the assay can be combined with point-of-care technologies such as CRISPR-Cas12a based lateral flow assays (see CRISPR Cas12a Based Lateral Flow Assay for Rapid Point of Care Detection of African Swine Fever Virus in Porcine Blood and Oral Fluids) for field deployment.

References

[1] Zimmerman, J. J., Karriker, L. A., Ramirez, A., Schwartz, K. J., Stevenson, G. W., & Zhang, J. (Eds.). Diseases of Swine. 11th ed. Wiley-Blackwell.

[2] World Organisation for Animal Health (OIE). Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Chapter 3.8.1: African Swine Fever.

[3] Prickett, J. R., & Zimmerman, J. J. (Eds.). Oral Fluid Sampling in Swine Health Management. In: Swine Disease Surveillance and Diagnostics. Iowa State University Press.

[4] Huggett, J. F., & Whale, A. S. (Eds.). Digital PCR: Methods and Protocols. Humana Press.

[5] Murphy, F. A., Gibbs, E. P. J., Horzinek, M. C., & Studdert, M. J. Veterinary Virology. 3rd ed. Academic Press. *** Disclaimer: This article is for educational and informational purposes only. It is not intended to substitute for professional veterinary advice, diagnosis, treatment, or regulatory guidance. Always consult a licensed veterinarian or qualified specialist regarding animal health, disease diagnosis, and therapeutic decisions.