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

Abstract

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.

A duplex fluorescent quantitative PCR assay to distinguish the genotype I, II and I/II recombinant strains of African swine fever virus in China

“…The pUC57-ASFV standard plasmid was generated through the synthesis and cloning of gene sequences amplified from the MGF_110-1L gene of ASFV genotype I isolate (MZ945537) and the O61R gene of genotype II isolate (MK333180) in GenBank into the pUC57 vector. Quantification of the standard plasmid was performed using a UV–visible spectrophotometer, and copy numbers were determined using a specific formula ( 15 ). Subsequently, a 10-fold serial dilution was conducted, resulting in concentrations ranging from 2.95 × 10 9 to 2.95 × 10 −1 copies/μL, which were then stored at −20°C for further use.…”

Enhancing Point-of-Care Diagnosis of African Swine Fever Virus (ASFV) DNA with the CRISPR-Cas12a-Assisted Triplex Amplified Assay

“…Compared to the above methods, the detection of ASFV DNA is widely adopted due to its ease of sampling, high sensitivity, and low contamination rate. Quantitative real-time polymerase chain reaction (qRT-PCR) is the most reliable method for the laboratory diagnosis of ASFV DNA. − Despite its efficacy in detecting nucleic acids, PCR techniques were not suitable for rapid diagnostic and onsite monitoring at the POC in endemic areas due to its intricate procedures, equipment-intensive demands, and lengthy analysis time. Moreover, professional laboratories and equipment are usually not available in pig farms, and even a tiny amount of the virus could potentially cause an outbreak of ASF.…”