Establishment of a Dual Real-Time PCR Assay for the Identification of African Swine Fever Virus Genotypes I and II in China

Abstract

Since the first outbreak of ASFV genotype II in China in 2018, ASF has posed a significant threat to the swine industry. After the emergence of genotype I in China in 2020, the epidemic prevention and control have become more difficult. No effective commercial vaccine is currently available, and the disease is difficult to eradicate; therefore, the identification of the ASFV genotype is critical to establish biosafety control measures. In this study, a dual real-time PCR detection method based on B646L and E183L genes was developed to distinguish between ASFV genotypes I and II by specifically amplifying the genotype I E183L gene. The method is strongly specific, detects B646L and E183L genes simultaneously, and does not cross-react with PEDV, PCV, PRRSV, PRV, and CSFV. The double real-time PCR detection of ASFV genotypes I and II showed a B646L amplification curve, and only genotype I showed an E183L amplification curve, consistent with our expectations. The method has high sensitivity and the lowest copy numbers detected for recombinant plasmids B646L and E183L were 1.07 × 102 and 3.13 × 104 copies/μL, respectively. The method is reproducible, and the coefficient of variation for detecting the coefficient of variation (CV) values of the two recombinant plasmids was <2%. Seven samples were positive and 277 were negative, and the results of the two methods were consistent. The dual real-time PCR presented in this study provides a rapid detection method for the identification of ASFV genotypes I and II, which may lead to improving efficient prevention and control measures for ASF in China.

Intradermal needle-free injection prevents African Swine Fever transmission, while intramuscular needle injection does not

“…Genotype II is currently the dominant genotype circulating in several European and Asian countries 1 . Although the main risk factors for the transmission of the ASF virus (ASFV) between herds are live animal transportation and poor compliance with biosecurity measures 8 , the abundance of small farms and the recent emergence of diversified ASFV strains including less virulent ones 9 represent new threats to the global swine industry. Currently, there are no effective and safe ASFV vaccines.…”

Development of a Competitive Enzyme-Linked Immunosorbent Assay Targeting the-p30 Protein for Detection of Antibodies against African Swine Fever Virus

“…We hypothesized that mAb-2B4 may recognize an immunodominant epitope on the p30 protein, and mAb-2B4 had an excellent competitive effect with the p30 antibody in the ASFV-positive serum. Since the isolation of the ASFV genotype I in China, multiple studies have developed various multiplex PCRs which can simultaneously detect genotype I and II ASFVs [ 29 , 30 , 31 , 32 , 33 ]. However, there were few cases reported of ASFV genotype I strains when the aforementioned PCRs were introduced to analyze the filed samples [ 30 , 31 , 33 ].…”

“…Since the isolation of the ASFV genotype I in China, multiple studies have developed various multiplex PCRs which can simultaneously detect genotype I and II ASFVs [ 29 , 30 , 31 , 32 , 33 ]. However, there were few cases reported of ASFV genotype I strains when the aforementioned PCRs were introduced to analyze the filed samples [ 30 , 31 , 33 ]. Animal challenge testing proved the efficient transmissibility of genotype I ASFV strain SD/DY-1/21 in pigs, and infected pigs developed low-level viremia, which makes early diagnosis more difficult than attenuated genotype II strains in the field.…”

On-site detection and differentiation of African swine fever virus variants using an orthogonal CRISPR-Cas12b/Cas13a-based assay

“… 29 , 32 Later, several groups also developed PCR-based assays to differentiate low virulent genotype I from wild-type genotype II strains. 4 , 24 , 33 , 34 , 35 However, PCR-based methods are usually conducted in central laboratories by highly skilled operators and require expensive instruments, making them less suitable for field applications. 36 Isothermal nucleic acid amplification (INA), such as recombinase polymerase amplification (RPA) 37 , 38 and loop-mediated isothermal amplification (LAMP), 39 circumvents the use of thermal cyclers and is usually faster than real-time PCR, but it has the disadvantages of nonspecific amplification and low sensitivity.…”