Development and Validation of a Quantitative Synthetic Molecular Standard for African Swine Fever Virus

African swine fever virus (ASFV) is a double-stranded DNA virus within the Asfarviridae family. ASFV infects both wild and domestic pigs, resulting in the highly contagious and deadly disease African swine fever. The virus can be transmitted either directly from sick to healthy pigs or indirectly through contact with contaminated products or the bite of infected Ornithodoros ticks, which are natural hosts and reservoirs of the virus. The mortality rate is close to 100% for infected domestic pigs and clinical symptoms include sudden death, fever, reddening of skin, vomiting, diarrhea, abortion in pregnant sows, and malaise. There is currently no treatment or vaccine for ASFV, and prevention and control include culling of infected individuals and disinfection of the infected zone and surrounding area.

Diagnosis of infected individuals is necessary for infection management and preventing further spread of the virus. Disease presentation of ASFV is similar to classical swine fever and bacterial septicemia; therefore, laboratory testing is required for accurate diagnosis. While a culture-based approach can be used for detection, viral growth and purification can be difficult, time-consuming, and costly. PCR-based methods provide a sensitive and rapid alternative approach; however, these molecular-based methods are dependent on the availability of high-quality reference materials. To address this need, ATCC has designed and developed a quantitative synthetic molecular standard for ASFV. We used a proprietary strategy that incorporated key target regions from the genome as well as conserved regions used for viral detection and identification in various published assays. 

The synthetic standard was validated through next-generation sequencing, and precise DNA copy number (copies/µL) was quantified using droplet digital™ PCR (Bio-Rad). The standard was then tested using an independent, publicly available qPCR assay, which verified the efficacy of the design against a relevant assay. Ten-fold dilutions were used to create a standard curve, with DNA concentrations ranging from approximately 10² to 10⁶ copies/µL. The ASFV standard had an R² value of 0.997, indicating a high degree of linearity. This standard displayed high efficiency and amplification, with a slope of M = -3.342. Overall, these data demonstrate the applicability of the ASFV synthetic molecular standard as a control in the development and validation of molecular-based detection and quantification assays.