Rapid and highly sensitive portable detection of African swine fever virus

Daigle, Jade; Onyilagha, Chukwunonso; Truong, Thang; Le, Van Phan; Nga, Bui Thi To; Nguyễn, Thị Lan; Clavijo, Alfonso; Ambagala, Aruna

doi:10.1111/tbed.13770

Cited by 15 publications

(16 citation statements)

References 19 publications

(34 reference statements)

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“…The most sensitive PCR was obtained from the IndiField ASFV PCR, which was amplified on the IndiField thermocycler. The study of Daigle et al (2020) has ascertained the functionality of the IndiField thermocycler [ 19 ]. The slightly increased analytical sensitivity of the IndiField ASFV PCR compared with the other tested PCR assays can be most likely explained by the high template volume, possibly due to the lyophilized format of the kit.…”

Section: Discussionmentioning

confidence: 99%

Swift and Reliable “Easy Lab” Methods for the Sensitive Molecular Detection of African Swine Fever Virus

Elnagar

Pikalo

Beer

et al. 2021

IJMS

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African swine fever (ASF) is a contagious viral hemorrhagic disease of domestic pigs and wild boars. The disease is notifiable to the World Organisation for Animal Health (OIE) and is responsible for high mortality and serious economic losses. PCR and real-time PCR (qPCR) are the OIE-recommended standard methods for the direct detection of African swine fever virus (ASFV) DNA. The aim of our work was the simplification and standardization of the molecular diagnostic workflow in the lab. For validation of this “easy lab” workflow, different sample materials from animal trials were collected and analyzed (EDTA blood, serum, oral swabs, chewing ropes, and tissue samples) to identify the optimal sample material for diagnostics in live animals. Based on our data, the EDTA blood samples or bloody tissue samples represent the best specimens for ASFV detection in the early and late phases of infection. The application of prefilled ready-to-use reagents for nucleic acid extraction or the use of a Tissue Lysis Reagent (TLR) delivers simple and reliable alternatives for the release of the ASFV nucleic acids. For the qPCR detection of ASFV, different published and commercial kits were compared. Here, a lyophilized commercial kit shows the best results mainly based on the increased template input. The good results of the “easy lab” strategy could be confirmed by the ASFV detection in field samples from wild boars collected from the 2020 ASFV outbreak in Germany. Appropriate internal control systems for extraction and PCR are key features of the “easy lab” concept and reduce the risk of false-negative and false-positive results. In addition, the use of easy-to-handle machines and software reduces training efforts and the misinterpretation of results. The PCR diagnostics based on the “easy lab” strategy can realize a high sensitivity and specificity comparable to the standard PCR methods and should be especially usable for labs with limited experiences and resources.

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Section: Discussionmentioning

confidence: 99%

Swift and Reliable “Easy Lab” Methods for the Sensitive Molecular Detection of African Swine Fever Virus

Elnagar

Pikalo

Beer

et al. 2021

IJMS

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“…The gold standard detection method is real-time PCR, which takes up to several hours to deliver results. Two modified PCR assays have been developed to speed up the testing and simplifying the extraction method [ 31 , 32 ]. The total run time was two hours for nine samples implementing a cartridge-based kit, which is easily deployable but required various hands-on steps per sample [ 32 ].…”

Section: Discussionmentioning

confidence: 99%

“…Two modified PCR assays have been developed to speed up the testing and simplifying the extraction method [ 31 , 32 ]. The total run time was two hours for nine samples implementing a cartridge-based kit, which is easily deployable but required various hands-on steps per sample [ 32 ]. When using a magnetic-bead-based extraction protocol, an automated expensive device was required [ 31 ].…”

Section: Discussionmentioning

confidence: 99%

Rapid Extraction and Detection of African Swine Fever Virus DNA Based on Isothermal Recombinase Polymerase Amplification Assay

Ceruti

Kobialka

Ssekitoleko

et al. 2021

Viruses

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African swine fever virus (ASFV) is the causative agent of a deadly disease in pigs and is spread rapidly across borders. Samples collected from suspected cases must be sent to the reference laboratory for diagnosis using polymerase chain reaction (PCR). In this study, we aimed to develop a simple DNA isolation step and real-time recombinase polymerase amplification (RPA) assay for rapid detection of ASFV. RPA assay based on the p72 encoding B646L gene of ASFV was established. The assays limit of detection and cross-reactivity were investigated. Diagnostic performance was examined using 73 blood and serum samples. Two extraction approaches were tested: silica-column-based extraction method and simple non-purification DNA isolation (lysis buffer and heating, 70 °C for 20 min). All results were compared with well-established real-time PCR. In a field deployment during a disease outbreak event in Uganda, 20 whole blood samples were tested. The assay’s analytical sensitivity was 3.5 DNA copies of molecular standard per µL as determined by probit analysis on eight independent assay runs. The ASFV RPA assay only detected ASFV genotypes. Compared to real-time PCR, RPA diagnostic sensitivity and specificity were 100%. Using the heating/lysis buffer extraction procedure, ASFV-RPA revealed better tolerance to inhibitors than real-time PCR (97% and 38% positivity rate, respectively). In Uganda, infected animals were identified before the appearance of fever. The ASFV-RPA assay is shown to be as sensitive and specific as real-time PCR. Moreover, the combination of the simple extraction protocol allows its use at the point of need to improve control measures.

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“…A number of molecular assays including laboratory based, portable conventional and real‐time loop‐mediated isothermal amplification (LAMP) assays have been developed and validated for ASFV genome detection. Real‐time PCR is the preferred assay used in many diagnostic laboratories since it is quantitative and faster compared to conventional PCR (Daigle et al., 2020 ; King et al., 2003 ; Tignon et al., 2011 ; Wang, Jia et al., 2020 ; Wang, Xu et al., 2020 ). Most of these assays are based on B646L (p72) gene.…”

Section: Discussionmentioning

confidence: 99%

Development of a novel real‐time PCR assay targeting p54 gene for rapid detection of African swine fever virus (ASFV) strains circulating in Vietnam

Trinh

Truong

Nguyen

et al. 2021

Veterinary Medicine & Sci

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African swine fever (ASF) continues to cause outbreaks throughout regions of Africa, Europe and Asia. The disease can cause severe morbidity and mortality resulting in serious economic losses. Since there is no vaccine available to control ASF, early detection is critical to contain and control the disease. The aim of this study was to develop a novel real-time PCR assay based on highly conserved ASFV gene E183L (p54). The limit of detection of the assay, VNUA-p54 real-time PCR, was 2.63 copies/reaction and 2 Log 10 HAD 50 /ml. The VNUA-p54 real-time PCR was able to detect fifteen different ASFV reference strains representing p72 genotypes I, II and V. The assay was specific and did not amplify other swine viruses including CSFV, FMDV, PRRSV and PEDV. The diagnostic sensitivity of the real-time PCR assay was evaluated using 200 field clinical specimens collected from swine farms located in different provinces in Vietnam. The VNUA-p54 real-time PCR assay is an additional tool for ASF diagnostics and can be used in combination with other p72 based ASFV real-time PCR assays as a rapid confirmatory assay.

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Rapid and highly sensitive portable detection of African swine fever virus

Cited by 15 publications

References 19 publications

Swift and Reliable “Easy Lab” Methods for the Sensitive Molecular Detection of African Swine Fever Virus

Swift and Reliable “Easy Lab” Methods for the Sensitive Molecular Detection of African Swine Fever Virus

Rapid Extraction and Detection of African Swine Fever Virus DNA Based on Isothermal Recombinase Polymerase Amplification Assay

Development of a novel real‐time PCR assay targeting p54 gene for rapid detection of African swine fever virus (ASFV) strains circulating in Vietnam

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