Development and validation of a fast quantitative real-time PCR assay for the detection of African swine fever virus

Abstract

African swine fever virus (ASFV) is a double-stranded DNA virus that causes African swine fever (ASF), a lethal hemorrhagic fever that is highly contagious among domestic pigs and wild boars. Due to the high mortality rates and highly contagious nature of the ASF, it is important to develop a fast detection method for ASFV with high sensitivity and specificity to take an immediate action to stop wide spread of the virulent disease. Therefore, a fast and quantitative molecular detection method of ASFV is presented in this study. A total of 24 genotypes of ASFV have been identified based on nucleic acid sequences of the major capsid protein p72. The primers and probe of the present assay was designed to detect all of the p72-based genotypes of ASFV. The turnaround time for PCR detection was within 50 min which is at least about two-times faster compared to other PCR assays. Limit of detection (LoD) was 6.91 genomic copies/reaction for the most virulent genotype II. LoD values for other genotypes were within 10–20 copies/reaction. Cross-reactivity of the assay was validated using a panel of pathogens related to swine disease, and no cross-reactivity was observed. Positive and negative clinical samples (50 samples each) obtained from sick and healthy animals, were used to validate the assay. The results showed that 100% agreement for both positive and negative samples. In summary, the assay described in this study offers the advantage of rapid detection of all genotypes of ASFV with high sensitivity and specificity. The assay is a valuable tool both in clinical and laboratory uses for sensitive and fast detection of ASFV.

Development of a triplex real-time quantitative PCR for detection and differentiation of genotypes I and II African swine fever virus

“…The real-time quantitative PCR (qPCR) is a convenient, efficient, sensitive, accurate and high throughput technology, and has been widely used to detect viral nucleic acids (20,21). To date, the qPCR has been developed for detection and diagnosis of ASFV (22)(23)(24)(25)(26). Several multiplex qPCR based on the E296R, B646L, or E183L genes of ASFV have been reported for the detection of genotypes I and II ASFV in 2022 (27)(28)(29).…”

Novel triplex nucleic acid lateral flow immunoassay for rapid detection of Nipah virus, Middle East respiratory syndrome coronavirus and Reston ebolavirus

“…This reduced efficiency of high multiplexing in endpoint LFA readouts may be overcome by adopting a different strategy for coating capture proteins on the membrane (Anfossi et al 2018). Amplification multiplexing of more than three targets has been successfully deployed in realtime PCR using specific labeled probes; however, conventional PCR multiplexing beyond four targets is not as efficient (Shaopeng et al 2023). These points may be held accountable for triplexed DNA amplification-based LFA end-point detection assays that have been developed without IC in previous studies (Crannell et al 2016).…”

Advanced Strategies for Developing Vaccines and Diagnostic Tools for African Swine Fever

“…Furthermore, these highly sensitive assays can lead to false positives due to cross-contamination and false negatives due to the presence of inhibitors or damaged nucleic acids. Therefore, novel PCR assays, which were developed by redesigning the primer sequence or forming a multiplex assay to improve efficacy and reduce processing time, showed higher sensitivity, specificity, and efficiency than those previously used globally [109][110][111][112][113][114]. Wang et al added a new set of primers and probes, which cover the 5 end region of p72, leading to potent diagnostic sensitivity and specificity; the limit of detection (LOD) was six copies of positive standard plasmids per reaction, or about 0.1-1 TCID 50 of ASFV isolates per reaction, and ASFV was well distinguished from nontargeted common swine virus [109].…”