Dataset Information

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.

SUBMITTER: Gao Q

PROVIDER: S-EPMC9198542 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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

Gao Qi Q Feng Yongzhi Y Yang Yunlong Y Luo Yizhuo Y Gong Ting T Wang Heng H Gong Lang L Zhang Guihong G Zheng Zezhong Z

Frontiers in veterinary science 20220601

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 o ...[more]

PMID: 35720851

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Project description:African swine fever virus (ASFV) was first identified in 1921 and is extensively prevalent around the world nowadays, which has a significant negative impact on the swine industry. In China, genotype II ASFV was first discovered in 2018, and has spread quickly to different provinces in a very short time; genotype I ASFV was first found in 2020, and has been reported in several provinces since then. To establish an accurate method for detection and differentiation of genotypes I and II ASFV, three primers and probes were designed targeting the ASFV B646L gene for different genotypes, the F1055L gene for genotype I, and the E183L gene for genotype II, and a triplex real-time quantitative PCR (qPCR) for differential detection of genotypes I and II ASFV was developed after optimizing the reaction conditions. The assay showed high sensitivity, and the limits of detection (LOD) of the B646L, F1055L, and E183L genes were 399.647 copies/reaction, 374.409 copies/reaction, and 355.083 copies/reaction, respectively; the coefficients of variation (CVs) of the intra-assay and the inter-assay were 0.22-1.88% and 0.16-1.68%, respectively, showing that this method had good repeatability; the assay could detect only ASFV, without cross-reactivity with other swine viruses including PRRSV, PEDV, PDCoV, CSFV, PRV, and PCV2, showing excellent specificity of this method. A total of 3,519 clinical samples from Guangxi province, southern China, were tested by the developed assay, and 8.16% (287/3,519) samples were found to be positive for ASFV, of which 0.17% (6/3,519) samples were positive for genotype I, 7.19% (253/3,519) samples for genotype II, and 0.80% (28/3,519) samples for genotypes I and II. At the same time, these clinical samples were also tested by a previously reported multiplex qPCR, and the agreement between these two methods was more than 99.94%. In summary, the developed triplex qPCR provided a fast, specific and accurate method for detection and differentiation of genotypes I and II ASFV.

Dataset Information

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

Publications

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

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