Direct colorimetric LAMP assay for in-field detection of African swine fever virus: a validation study during an outbreak in Vietnam

Tran, Diem Hong; Tran, Hau Thi; Le, Uyen Phuong; Vu, Xuan Dang; Trinh, Thi Bich Ngoc; Khoa, Hoang Dang; Than, Van Thai; Bui, Le Minh; Vu, Van V.; Nguyễn, Thị Lan; Phung, Huong Thi Thu; Le, Van Phan

doi:10.1101/2020.06.03.132944

2020

DOI: 10.1101/2020.06.03.132944

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Direct colorimetric LAMP assay for in-field detection of African swine fever virus: a validation study during an outbreak in Vietnam

Diem Hong Tran

Hau Thi Tran

Uyen Phuong Le

et al.

Abstract: 1 3

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2021

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Development of a loop-mediated isothermal amplification (LAMP) assay based on the C962R gene for african swine fever virus detection

2021

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Aim. The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) assay for African swine fever virus (ASFV) detection. Methods. Primer design was performed using publicly available full genome sequences of ASFV. A panel of heterologous DNA samples and reference ASFV DNA samples were used for the assay specificity testing. The limit of detection (LOD) was assessed using purified and quantified serial dilution of the amplified target sequence. LAMP product detection was performed via gel-electrophoresis and via ethidium bromide fluorescence under UV after adding the ethidium bromide directly to the tube with the LAMP product. Results. Three primer sets amplifying different regions of ASFV gene C962R were developed, of which the set № 2 providing the most intense product synthesis with the most vivid and clear pattern was selected for further studies. The optimal concentration of reaction mix components for the most effective primer set was established. In the final protocol the LAMP reaction was carried out at 60 °C for 40 min. The limit of detection (LOD) of the assay was 50 copies of the target sequence per reaction. In a preliminary testing the assay proved specific, using 10 reference and 4 heterologous viral and two bacterial DNA samples. Our LAMP assay detected ASFV genotypes I and II that are currently spread in Europe, Asia, and the Pacific and IX, occurring in Africa. Conclusion. A LAMP assay was developed based on the C962R gene that proved in preliminary validation to be specific and sensitive and was able to detect down to 50 copies per reaction of purified target gene within 40 minutes. Classical gel electrophoresis and direct staining using ethidium bromide were used for product visualisation in this study. Colorimetric approaches or the use of lateral flow devices in the visuali- sation step could make the assay less equipment dependent. Further validation of the assay, determining analytical specificity, selectivity and reproducibility performance characteristics also using clinical samples under field condi- tions and inclusion of an internal control would possibly enable its use as a test of choice at point-of-care and at low resource laboratories.

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Development of a loop-mediated isothermal amplification (LAMP) assay based on the C962R gene for african swine fever virus detection

2021

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Development of Diagnostic Tests Provides Technical Support for the Control of African Swine Fever

Qiu

Yan

et al. 2021

Vaccines

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African swine fever is a highly contagious global disease caused by the African swine fever virus. Since African swine fever (ASF) was introduced to Georgia in 2007, it has spread to many Eurasian countries at an extremely fast speed. It has recently spread to China and other major pig-producing countries in southeast Asia, threatening global pork production and food security. As there is no available vaccine at present, prevention and control must be carried out based on early detection and strict biosecurity measures. Early detection should be based on the rapid identification of the disease on the spot, followed by laboratory diagnosis, which is essential for disease control. In this review, we introduced the prevalence, transmission routes, eradication control strategies, and diagnostic methods of ASF. We reviewed the various methods of diagnosing ASF, focusing on their technical characteristics and clinical test results. Finally, we give some prospects for improving the diagnosis strategy in the future.

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Direct colorimetric LAMP assay for in-field detection of African swine fever virus: a validation study during an outbreak in Vietnam

Abstract: 1 3

Cited by 2 publications

References 39 publications

Development of a loop-mediated isothermal amplification (LAMP) assay based on the C962R gene for african swine fever virus detection

Development of a loop-mediated isothermal amplification (LAMP) assay based on the C962R gene for african swine fever virus detection

Development of Diagnostic Tests Provides Technical Support for the Control of African Swine Fever

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