Genie 3

The RtF-RT-LAMP reaction was conducted in a final reaction volume of 25 μl consisting of 2 μl RNA template, FIP and BIP primers (1.6 μM each), Loop F and Loop B primers (0.8 μM each), F3 and B3 primers (0.2 μM each), Isothermal Mastermixes (OptiGene, UK), 0.15 u of AMV reverse transcriptase (15 u/μl; Invitrogen, USA). Amplification reactions were performed at 63 °C for 40 min (with fluorescence detection followed by melt curve analysis from 90 to 70 °C at 0.05 °C/s), and then heated at a start temperature of 98 °C and end temperature of 80 °C for 10 min with a ramp rate of 0.05 °C/sec to terminate the reactions using Genie® III (OptiGene, UK). The fluorescence of the reaction was measured in real time, verifying the start of the amplification.

The LAMP amplification results were detected with three different methods in a laboratory setting: 1. on 1% TBE agarose gel stained with GelGreen^® Nucleic Acid Stain (Biotium, Fremont, CA) for visual observation; 2. with SYBR^™ Green 1 nucleic acid gel stain (Invitrogen, Carlsbad, CA) for observation under UV light; and 3. using Genie^® III (OptiGene, Horsham, WS, UK) instrument by obtaining the amplification curves and analyzing data using Genie Explorer software (OptiGene, Horsham, WS, UK). All reactions were repeated at least three times.

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 (link)). 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. LAMP reactions were setup with 15 μL of Isothermal Mastermix ISO-DR004-DT (OptiGene Ltd., Horsham UK), 2.5 μL of primer mix targeting the topoisomerase II gene (19 (link)) with a final primer concentration of F3/B3 0.2 μM, FIP/BIP 1.6 μM and loop primers at 0.8 μM and reaction made up to 25 μL with nuclease-free water. Reactions were run on a Genie III (OptiGene, Horsham, UK), instrument with run conditions of 65°C for 25 min. Each run included a synthetic positive control as well as a no template control. A sample was classified positive if the time to positivity (T_p) < 20 min and had an annealing temperature (T_a) of 87.42°C (± 0.56°C).

Analytical sensitivity of the RT-LAMP assays was determined with a dilution series of HIV-1 MN RNA isolated as described above. Master mix (OptiGene ISO-001) was combined with 0.2 Units of avian myeloblastosis virus reverse transcriptase (AMV-RT) and 3 pairs of primers [18 (link)] targeting the HIV-1 p24 gene (Table 1) in a single tube with a final volume of 25 µl including 3–4 µl RNA at 65 °C. SYBR Green I interchelating dye, included in the OptiGene Mastermix formulation and allows following real-time fluorescence in a Genie III (OptiGene, Horsham, UK) portable isothermal amplification device. In a manner similar to melting curve analyses, annealing curves were obtained immediately following LAMP to allow post- amplification display of products by increasing the temperature to 92 °C and gradually cooling to 85°C.

Two independent laboratories (A and B) participated in evaluating the LAMP assays. Laboratory A conducted the tests in field portable Genie^® II or Genie^® III instruments (OptiGene) and laboratory B in a real-time thermocycler CFX96 (Biorad). Both laboratories used the same fast isothermal master mix (ISO-004, Optigene) and primer concentrations. LAMP reactions consisted of 15 μL of the fast isothermal master mix (ISO-004, OptiGene), 0.2 µM of each primer F3 and B3, 1.0 µM of each LoopF and LoopB, 2.0 µM of each FIP and BIP, and either plasmid standards or test DNA (2–5 µL), to a final volume of 25 μL. Tests were run over 30 min at the optimised temperature. Real-time monitoring of the isothermal amplification and the annealing temperature of the amplicon was measured as the change of fluorescence in the FAM channel. The reaction time (measured in minutes:seconds) was the time when fluorescence changed for the Genie instruments (time of positivity “Tp”) or the time to reach 1000 (t1000) relative fluorescence units (RFU) for the CFX96 thermocycler.

LAMP reactions were carried out in 25 μL volumes using 15 μL OptiGene GspSSD2.0 Isothermal Mastermix (ISO-DR004), 5 μL primer mix (final concentrations of 1.6 μM FIP and BIP, 0.2 μM F3, B3 and LF) (Bioneer), and 5 μL template. Template included MilliQ nuclease free water as no template control, and control isolate A198 and C305 gDNA. Assays were run on both the Genie II and Genie III (Optigene) real-time fluorometer, with results reported as time to positive (Tp) (minutes.seconds) and anneal derivative melting temperature (Tm) (°C), given when the sample florescence crossed the pre-set threshold of the Genie machines. Primer concentration ratios of 1:10, 1:8, 1:6 and 1:4 (F3/B3:FIP/BIP) were assessed and 1:8 chosen to optimise the discrimination of aprV2 fluorescent signals from aprB2 fluorescent signals based on Tp. Temperature gradients from 60°C—67°C were performed and assessed for speed and discrimination. LAMP assays were run with a pre-heating step of 40°C for 1 minute, followed by 20 minutes at 65°C before an annealing step from 94°C to 84°C, at a rate of 0.5°C/second, with these conditions used throughout.