Development of a TaqMan-Probe-Based Multiplex Real-Time PCR for the Simultaneous Detection of African Swine Fever Virus, Porcine Circovirus 2, and Pseudorabies Virus in East China from 2020 to 2022

Abstract

African swine fever virus (ASFV), porcine circovirus 2 (PCV2), and pseudorabies virus (PRV) are important DNA viruses that cause reproductive disorders in sows, which result in huge losses in pig husbandry, especially in China. The multiplex qPCR assay could be utilized as a simultaneous diagnostic tool for field-based surveillance and the control of ASFV, PCV2, and PRV. Based on the conserved regions on the p72 gene of ASFV, the Cap gene of PCV2, the gE gene of PRV, and the porcine endogenous β-Actin gene, the appropriate primers and probes for a multiplex TaqMan real-time PCR test effective at concurrently detecting three DNA viruses were developed. The approach demonstrated high specificity and no cross-reactivity with major pathogens related to swine reproductive diseases. In addition, its sensitivity was great, with a detection limit of 101 copies/L of each pathogen, and its repeatability was excellent, with intra- and inter-group variability coefficients of <2%. Applying this assay to detect 383 field specimens collected from 2020 to 2022, the survey data displayed that the ASFV, PCV2, and PRV single infection rates were 22.45%, 28.46%, and 2.87%, respectively. The mixed infection rates of ASFV+ PCV2, ASFV + PRV, PCV2 + PRV, and ASFV + PCV2 + PRV were 5.22%, 0.26%, 1.83%, and 0.26%, respectively. Overall, the assay established in this study provides an effective tool for quickly distinguishing the viruses causing sow reproductive disorders, suggesting its huge clinical application value in the diagnosis of swine diseases.

Saccharomyces cerevisiae oral immunization in mice using multi-antigen of the African swine fever virus elicits a robust immune response

“…Cellular immune responses may be nonspecific but enhance host resistance, confirming the excellent applicability of SC as a probiotic. The invasion and proliferation of pathogenic bacteria contribute to host death after ASFV infection ( 70 , 71 ). Therefore, enhancing host immune resistance can reduce ASFV invasion.…”

Development of rapid nucleic acid testing techniques for common respiratory infectious diseases in the Chinese population

“…Compared with the single detection methods used in other studies, the multiplex PCR method showed higher specificity and sensitivity, allowing for the simultaneous detection of multiple pathogens in a single reaction. This approach not only saves time and resources, but also facilitates the rapid identification of various pathogens in complex samples ( Liu et al, 2023 ). Our multiplex fluorescent quantitative PCR assays demonstrated high specificity, sensitivity, and efficiency in detecting respiratory pathogens, such as SARS-CoV-2, FluA, PIV, and RSV.…”

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].…”