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

Abstract

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.

Use of Field Based Loop Mediated Isothermal Amplification (LAMP) Technology for a Prevalence Survey and Proof of Freedom Survey for African Swine Fever in Timor-Leste in 2019

“…The use of LAMP technology in Timor-Leste has provided a fast, simply performed and robust test suitable for use in a basic laboratory or field situations. Test specificity and sensitivity appear very high (>99.9%) based on previous research (15) and preliminary validation work in Australia with serum samples from known ASF free animals and seeded manufactured DNA samples (unpublished data). Serum samples were used in this prevalence survey to maximize the familiarity of local field staff experience and the availability of blood tube supplies.…”

“…Serum samples were used in this prevalence survey to maximize the familiarity of local field staff experience and the availability of blood tube supplies. Acknowledging that serum generally contains less viral genome than whole blood, preliminary LAMP testing was also conducted on whole blood samples in a variety of types of anticoagulants and is reported in a separate publication ( 15 ). The use of liquid reagents for the field tests undertaken with the LAMP machine created minor issues when the reagents were exposed to high ambient temperatures associated with the tropical climate in Timor-Leste.…”

“…Epitools online calculator ( 14 ) was used to calculate the true disease prevalence at a village level. Assumptions for the calculation were the LAMP test sensitivity (Se) 0.98 and test specificity (Sp) 0.999 ( 15 ) and a confidence level of 0.95. The calculation tool recommends the use of Blaker's interval for confidence levels for general use.…”

“…Serum was separated from blood by centrifuging for 20 min at 3,000 g. Serum was removed from the tubes and tested straight away or stored at −20°C until testing. Samples were tested for ASFV using an ASFV LAMP assay ( 15 ). Briefly, serum was diluted 1 in 10 in nuclease-free water before 2 μL of each sample was heat treated at 95°C for 2 min.…”

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

“…The LAMP assay fi ts this gap, since the reaction can be carried out under fi eld conditions using such simple devices as a water bath or thermoblock. Different simple product visualisation techniques are also compatible with LAMP, which makes it a good tool for rapid molecular diagnostics (Wong Y-P et al, 2017;Mee PT et al, 2020).…”