Direct colorimetric LAMP assay for rapid 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:10.1111/tbed.13879

Cited by 15 publications

(19 citation statements)

References 10 publications

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“…Analytical sensitivity of the LAMP assay using purifi ed PCR product (ASF of genotype II, strain ASFV "Estonia") of target sequence for LAMP primer set № 2, detecting ASFV gene C962R. 10 10 -1 -analyte concentration (copies/μl), NTCnegative template control (deionized water) Tran DH et al, 2020;James HE et al, 2010). The assay we developed detected ASFV DNA extracted from three different sources, spleen, liver and macrophage cell cultures.…”

Section: Resultsmentioning

confidence: 99%

“…Several LAMP assays for ASFV detection have been developed to date. Genes coding structural proteins p72 (Dokphut A et al, 2021, Tran DH et al, 2020, Zhu YS et al, 2020), p22 (Elsukova AA et al, 2019, p10 (Wang D et al, 2020), uncharacterised protein K205R (Wu X et al, 2016), C717R (Zhu YS et al, 2020, enzymes topoisomerase II (James HE et al, 2010), helicase (Zhu YS et al, 2020) have been used as target sequences. However, there is a need for other molecular markers for molecular diagnostics.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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“…The RT-LAMP assays developed in this study could be further adapted for point of use application; however, some future work is required and should consider either the removal of nucleic extraction steps [39] or the use of field-suitable extraction procedures [41]. To help reduce the cost of in-field diagnostics, the development of an alternative visualization strategy, such as a direct colorimetric LAMP assay [42], could be considered. Ideally, to implement these improvements, the RT-LAMP assay validation should be performed directly in the field during outbreak investigations.…”

Section: Discussionmentioning

confidence: 99%

Development of a Novel Loop Mediated Isothermal Amplification Assay (LAMP) for the Rapid Detection of Epizootic Haemorrhagic Disease Virus

Rajko-Nenow

Howson

Clark

et al. 2021

Viruses

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Epizootic haemorragic disease (EHD) is an important disease of white-tailed deer and can cause a bluetongue-like illness in cattle. A definitive diagnosis of EHD relies on molecular assays such as real-time RT-qPCR or conventional PCR. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a cost-effective, specific, and sensitive technique that provides an alternative to RT-qPCR. We designed two sets of specific primers targeting segment-9 of the EHD virus genome to enable the detection of western and eastern topotypes, and evaluated their performance in singleplex and multiplex formats using cell culture isolates (n = 43), field specimens (n = 20), and a proficiency panel (n = 10). The limit of detection of the eastern and western RT-LAMP assays was estimated as ~24.36 CT and as ~29.37 CT in relation to real-time RT-qPCR, respectively, indicating a greater sensitivity of the western topotype singleplex RT-LAMP. The sensitivity of the western topotype RT-LAMP assay, relative to the RT-qPCR assay, was 72.2%, indicating that it could be theoretically used to detect viraemic cervines and bovines. For the first time, an RT-LAMP assay was developed for the rapid detection of the EHD virus that could be used as either a field test or high throughput screening tool in established laboratories to control the spread of EHD.

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“…In order to better cope with the conditions on the scene, researchers continue to improve the detection methods, trying to express the results of experiments through visible changes. For example, Tran et al established the real-time LAMP assay and the visual LAMP assay using pH-sensitive dyes to develop color, which directly detected ASFV from serum samples [ 111 ]. Yu et al combined image processing and a hue saturation value (HSV) color model with LAMP assay to display the detection results in the form of images [ 112 ].…”

Section: Diagnosis Of Asfmentioning

confidence: 99%

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 rapid detection of African swine fever virus: A validation study during an outbreak in Vietnam

Cited by 15 publications

References 10 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 a Novel Loop Mediated Isothermal Amplification Assay (LAMP) for the Rapid Detection of Epizootic Haemorrhagic Disease Virus

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

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