Development of a Rapid and Sensitive Method for Detection of African Swine Fever Virus Using Loop-Mediated Isothermal Amplification

Wu, Xulong; Lu, Xiao; Wang, Yin; Yang, Zexiao; Yao, Xueping; Peng, Bin

doi:10.1590/1678-4324-2016160500

Cited by 8 publications

(11 citation statements)

References 19 publications

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“…Previously, James et al succeeded to utilize LAMP to rapidly identify ASFV by targeting the topoisomerase II gene and showed the LOD value of approximately 330 genome copies (James et al., 2010). Wu's group used LAMP to detect ASFV successfully with the LOD of approximately 6 copies of the targeted gene per µl (Wu et al., 2016). Atuhaire and colleagues extensively evaluated the performance of LAMP and compared with the conventional OIE‐recommended diagnostic PCR for detecting ASFV (Atuhaire et al., 2014).…”

Section: Discussionmentioning

confidence: 99%

“…Loop‐mediated isothermal amplification (LAMP) has begun to gain huge attention since its first introduction (Notomi et al., 2000). Accordingly, the LAMP assays were previously established for the detection of ASFV (Atuhaire et al., 2014; Bredtmann, 2014; James et al., 2010; Wang et al., 2020; Wu et al., 2016). However, due to the lack of evaluation of the assay performance using directly crude samples or simple visualization of reaction outcome, earlier developed ASF LAMP assays were still less feasible for field applications with restricted resources.…”

Section: Introductionmentioning

confidence: 99%

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Direct colorimetric LAMP assay for rapid detection of African swine fever virus: A validation study during an outbreak in Vietnam

Tran

et al. 2020

Transbound Emerg Dis

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African Swine Fever (ASF) is a highly infectious viral disease with high mortality. The most recent ASF outbreak in Vietnam occurred in 2019, posing a threat to spread to the neighboring Asian countries. Without a commercial vaccine or efficient chemotherapeutics successfully developed, rapid diagnosis and necessary biosecurity procedures are required to control the disease. While the diagnosis method of ASF recommended by the World Organization of Animal Health is real-time PCR, it is not . CC-BY-NC-ND 4.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Direct colorimetric LAMP assay for rapid detection of African swine fever virus: A validation study during an outbreak in Vietnam

Tran

et al. 2020

Transbound Emerg Dis

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“…Several LAMP assays exist for the detection of ASFV, with preliminary validation of these assays performed on extracted nucleic acid [ 36 , 37 , 38 ]. The LAMP assay targeting the topoisomerase II (TPII) gene developed at the Institute for Animal Health, United Kingdom, was selected for further in-field verification as part of our study [ 36 ].…”

Section: Introductionmentioning

confidence: 99%

Field Verification of an African Swine Fever Virus Loop-Mediated Isothermal Amplification (LAMP) Assay during an Outbreak in Timor-Leste

Mee

Wong

O’Riley

et al. 2020

Viruses

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Recent outbreaks of African swine fever virus (ASFV) have seen the movement of this virus into multiple new regions with devastating impact. Many of these outbreaks are occurring in remote, or resource-limited areas, that do not have access to molecular laboratories. Loop-mediated isothermal amplification (LAMP) is a rapid point of care test that can overcome a range of inhibitors. We outline further development of a real-time ASFV LAMP, including field verification during an outbreak in Timor-Leste. To increase field applicability, the extraction step was removed and an internal amplification control (IAC) was implemented. Assay performance was assessed in six different sample matrices and verified for a range of clinical samples. A LAMP detection limit of 400 copies/rxn was determined based on synthetic positive control spikes. A colourmetric LAMP assay was also assessed on serum samples. Comparison of the LAMP assay to a quantitative polymerase chain reaction (qPCR) was performed on clinical ASFV samples, using both serum and oral/rectal swabs, with a substantial level of agreement observed. The further verification of the ASFV LAMP assay, removal of extraction step, implementation of an IAC and the assessment of a range of sample matrix, further support the use of this assay for rapid in-field detection of ASFV.

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“…Generally, the amplification time of LAMP varies from 60-120 min (Francois et al, 2011). The previous LAMP studies for ASFV detection revealed that the optimal incubation time is 60 min (Atuhaire et al, 2014;Wu et al, 2016). For this novel LAMP, the amplification was observed at a temperature of 60 °C as early as 50 min.…”

Section: Optimal Novel Lamp Reaction and Visualizationmentioning

confidence: 80%

“…Recently, a study of LAMP based on K205R gene of ASFV established more sensitive results than conventional PCR. However, the products of LAMP were observed as the fluorescent dye changed color under a UV illuminator or by agarose gel electrophoresis (Wu et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

Development of a loop-mediated isothermal amplification assay for rapid detection of African swine fever

Dokphut¹,

Boonpornprasert²,

Songkasupa³

et al. 2020

VIS

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Since the first African swine fever (ASF) outbreak was reported in China in 2018, the disease has spread rapidly to several countries in Asia. The early detection of this disease is essential for the ASF control strategy to be effective. Loop-mediated isothermal amplification (LAMP) is a nucleic acid detection assay that is rapid, simple, cost-effective and field-friendly. In this study, we have developed a colorimetric assay of LAMP to detect ASF virus (ASFV). A set of LAMP primers was designed to target the conserved region of the VP72 gene. The conditions of LAMP were optimized. The amplification products were easily detected by the naked eye using hydroxynaphthol blue (HNB). The positive LAMP reaction generated a violet to sky blue color change. The sensitivity and specificity of LAMP assay were demonstrated in comparison with the OIE-recommended real-time PCR. A total of 211 samples including 121 confiscated pork products and 90 spiked clinical specimens were tested. The optimal amplification of ASFV DNA by LAMP was incubation at 60 °C for 90 min. The analytical sensitivity of ASFV LAMP assay was at least 368 plasmid DNA copies/µL without cross-reactivity with other swine pathogens. The diagnostic sensitivity and specificity of LAMP were 88% and 100%, respectively. There was almost perfect agreement between LAMP and real-time PCR assays (Kappa value=0.84). This novel LAMP assay is deemed to be a rapid, simple, sensitive, specific diagnostic tool and suitable for early detection of ASF to minimize the likelihood of ASF spread nationwide.

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Development of a Rapid and Sensitive Method for Detection of African Swine Fever Virus Using Loop-Mediated Isothermal Amplification

Abstract: A loop-mediated isothermal amplification (LAMP)

Cited by 8 publications

References 19 publications

Direct colorimetric LAMP assay for rapid detection of African swine fever virus: A validation study during an outbreak in Vietnam

Direct colorimetric LAMP assay for rapid detection of African swine fever virus: A validation study during an outbreak in Vietnam

Field Verification of an African Swine Fever Virus Loop-Mediated Isothermal Amplification (LAMP) Assay during an Outbreak in Timor-Leste

Development of a loop-mediated isothermal amplification assay for rapid detection of African swine fever

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