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.

Keywords: ASFV; LAMP; Timor-Leste; colourmetric LAMP; qPCR.

Figure 1

Limit of detection of the ASFV LAMP performed with the ASFV gBlock in three different sample matrices. The sample matrices of serum and fluoride oxalate blood were diluted 1 in 10 in nuclease-free water before being spiked with the ASFV gBlock. All runs were performed in triplicates with error bars representing the standard deviation.

Figure 2

Repeatability of the ASFV LAMP assay was assessed by spiking a low (400 copies/rxn) and medium (40,000 copies/rxn) concentration of the ASFV gBlock into two sample matrices, serum and whole blood collected in fluoride oxalate tubes. All samples were tested in triplicates with error bars representing standard deviation.

Figure 3

The effect of sample matrix on the detection of a low (400 copies/rxn) ASFV gBlock spike (a) and a medium (40,000 copies/rxn) ASFV gBlock spike (b), with the sample matrix tested at neat (black), 1 in 10 (dark grey) and 1 in 100 (light grey) dilution in nuclease-free water. All runs were performed in triplicates. No signal was detected for the ASFV gBlock when spiked into neat blood.

Figure 4

Comparison of dry and fresh blood swabs on the detection sensitivity of the ASFV LAMP assay, using porcine blood spiked with a low concentration (200 copies/µL) and medium concentration (20,000 copies/µL) of the ASFV gBlock.