The Development of a Multiplex Real-Time Quantitative PCR Assay for the Differential Detection of the Wild-Type Strain and the MGF505-2R, EP402R and I177L Gene-Deleted Strain of the African Swine Fever Virus

Zhao, Kang; Shi, Kaichuang; Zhou, Qingan; Xiong, Chenyong; Mo, Shenglan; Zhou, Haifang; Long, Feng; Wei, Haina; Hu, Liping; Mo, Meilan

doi:10.3390/ani12141754

Cited by 17 publications

(20 citation statements)

References 43 publications

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“…The spread of ASFV across Europe and into Asia has been rapid, vast, and in the face of no effective vaccine, it will probably keep expanding its range into new countries globally [ 48 ]. While spreading across Eurasia, the clonal populations of the virus accrue mutations that can be common in a region and used for evaluating ASFV radiation events [ 24 , 30 , 41 ].…”

Section: Discussionmentioning

confidence: 99%

“…Fluorescent probe-based multiplex real-time PCR technology has been determined and applied to the field of molecular diagnostics. Recently, a multiplex real-time quantitative PCR assay was outlined for the detection of ASFV and simultaneous discrimination between virulent and multi-gene deleted candidate vaccine strains [ 48 ]. Real-time PCR can be applied as an alternative technique to SNP-based differentiation between isolates.…”

Section: Discussionmentioning

confidence: 99%

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A Guide to Molecular Characterization of Genotype II African Swine Fever Virus: Essential and Alternative Genome Markers

et al. 2023

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African swine fever is a contagious viral disease that has been spreading through Europe and Asia since its initial report from Georgia in 2007. Due to the large genome size of the causative agent, the African swine fever virus (ASFV), the molecular epidemiology, and virus evolution are analyzed by employing different markers. Most of these markers originate from single nucleotide polymorphisms or disparities in the copy number of tandem repeat sequences observed during the comparisons of full genome sequences produced from ASFVs isolated during different outbreaks. Therefore, consistent complete genome sequencing and comparative analysis of the sequence data are important to add innovative genomic markers that contribute to the delineation of ASFV phylogeny and molecular epidemiology during active circulation in the field. In this study, the molecular markers currently employed to assess the genotype II ASFVs circulating in Europe and Asia have been outlined. The application of each of these markers to differentiate between ASFVs from related outbreaks is described to implement a guideline to their suitability for analyzing new outbreaks. These markers do not signify the complete repertoire of genomic differences between ASFVs, but will be beneficial when analyzing the first outbreaks in a new region or a large number of samples. Furthermore, new markers must be determined via complete genome sequence analyses for enabling in-depth insights into the molecular epidemiology of ASFV.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A Guide to Molecular Characterization of Genotype II African Swine Fever Virus: Essential and Alternative Genome Markers

et al. 2023

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“…Using OIE-recommended quantitative PCR and ELISA methods, researchers can accurately judge whether pigs are infected with wild-type ASFV. Recently, multiplex real-time qPCR was developed to provide a diagnostic tool for the differential detection of B646L , I177L , MGF505-2R and EP402R genes ( 28 ). For the early diagnosis and efficient prevention of circulating ASFV, antigen detection is very limited because of the marked decline in viral copy number.…”

Section: Discussionmentioning

confidence: 99%

A highly efficient indirect ELISA and monoclonal antibody established against African swine fever virus pK205R

Qiao

Liu

et al. 2023

Front. Immunol.

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African swine fever (ASF) is a contagious infectious disease with high lethality which continuously threatens the global pig industry causing huge economic losses. Currently, there are no commercially available vaccines or antiviral drugs that can effectively control ASF. The pathogen of ASF, ASF virus (ASFV) is a double-stranded DNA virus with a genome ranging from 170 to 193 kb and 151 to 167 open reading frames in various strains, which encodes 150–200 proteins. An effective method of monitoring ASFV antibodies, and specific antibodies against ASFV to promote the development of prevention techniques are urgently needed. In the present study, pK205R of ASFV was successfully expressed in mammalian cells using a suspension culture system. An indirect enzyme-linked immunosorbent assay (ELISA) based on the purified pK205R was established and optimized. The monoclonal antibody (mAb) against pK205R recognized a conservative linear epitope (2VEPREQFFQDLLSAV16) and exhibited specific reactivity, which was conducive to the identification of the recombinant porcine reproductive and respiratory syndrome virus (PRRSV) expressing pK205R. The ELISA method efficiently detected clinical ASFV infection and revealed good application prospects in monitoring the antibody level in vivo for recombinant PRRSV live vector virus expressing the ASFV antigen protein. The determination of the conserved linear epitope of pK205R would contribute to further research on the structural biology and function of pK205R. The indirect ELISA method and mAb against ASFV pK205R revealed efficient detection and promising application prospects, making them ideal for epidemiological surveillance and vaccine research on ASF.

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“…An earlier report showed that a qPCR assay was established to detect DNA at 1.0 × 10 1 copies/μL using the special primers and probe for the conservative region of the ASFV p72 gene. Recently, the wild-type and gene-deleted ASFV strains were detected simultaneously using a multiplex real-time qPCR assay, which tested for ASFV with a detection limit of 32.1 copies/L for the p72 gene and 3.21 copies/L for the I177L, MGF505-2R, and CD2v genes [ 40 ]. In addition, multiple reports have shown qPCR assays being used for PRV detection.…”

Section: Discussionmentioning

confidence: 99%

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

Liu

Zou

Liu

et al. 2023

Veterinary Sciences

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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.

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The Development of a Multiplex Real-Time Quantitative PCR Assay for the Differential Detection of the Wild-Type Strain and the MGF505-2R, EP402R and I177L Gene-Deleted Strain of the African Swine Fever Virus

Cited by 17 publications

References 43 publications

A Guide to Molecular Characterization of Genotype II African Swine Fever Virus: Essential and Alternative Genome Markers

A Guide to Molecular Characterization of Genotype II African Swine Fever Virus: Essential and Alternative Genome Markers

A highly efficient indirect ELISA and monoclonal antibody established against African swine fever virus pK205R

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

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