100 bp ladder

Using the RNA isolated from tissues, as described above, cDNA was made using the iScript cDNA Synthesis Kit (Bio-rad) using RNAse-free water (Qiagen) under sterile conditions to prevent contamination. A previously published primer (Supplementary Table 1) was used to generate cDNA of the negative-strand. The JEV NS5 region of the negative-strand was subsequently amplified using Platinum™ Hot Start PCR Master Mix (Thermo Fisher, Waltham, MA) using previously published primers^{70 (link)} that are also provided in Supplementary Table 1. The reaction conditions used for PCR were 95 °C for 15 min, 40 cycles of 95 °C for 15 s, 55 °C for 30 s, and 72 °C for 10 s. The PCR products were subsequently run on a 1% agarose gel to visualize the product bands specific to the primers. A 100-bp ladder (Promega, Madison, WI) was utilized to identify the 250bp PCR product. An uncut gel image is provided in Supplementary Data 1.

Primer3Plus [20 (link)] was utilized to generate specific 16S rRNA gene primers for L. plantarum. The primers’ efficacy and specificity were examined utilizing Insilico PCR online tool [21 (link)]. A 50 µL combined volume consisted of 5 µL 10X PCR buffer, 5 µL 2X dNTPs mixture (0.4 mM each), 1 µL 10 pmol of each primer (16S rRNA, Table 1), 0.2 µL Taq DNA polymerase (Premix TaqTM, TaKaRa TaqTM Version 2.0, Code No. R004A), 1 µL 50 ng DNA template, and 37.8 µL free RNase water to complete the reaction volume that underwent thermal cycling in a Thermocycler (Labcycler Gradient 96 block, SensoQuest, Hannah-Vogt-Str. 1, 37085, Göttingen, Germany). The amplification conditions included 4 min at 94 °C before commencing thirty-five cycles at a 1 min denaturation at 94 °C followed by sixty-second annealing at 60 °C, and elongation lasted for another minute at 72 °C and concluded with an additional 10 min extension step running again at 72 °C. Upon completion, the amplicon was stained with an EtBr in 1X TAE buffer after being run on a gel containing 1.5% agarose and was monitored via a Gel Doc XR + System afterward. As a marker, a 100 bp ladder (Promega, UK) was used, as described by Mahmoud et al. [19 (link)].

The integrity of the E2 gene was assessed using overlapping primers that spanned the full length of the HPV16 E2 gene using primers shown in Supplementary Table S1. For the outer PCR reaction, 8 μl of cervical DNA was amplified in a gradient thermocycler (Eppendorf) using 12.5 μl of 2X PCR master mix (Promega) and 10 pmol of outer PCR primers under the cycle conditions; 95 °C for 15 min, followed by 60 cycles of 95 °C for 30 s, 54 °C for 45 s and 72 °C for 1 min. For the inner PCR reaction, 5 μl of product was amplified in a gradient thermocycler (Eppendorf) using 12.5 μl of 2X PCR master mix (Promega) and 10 pmol of inner PCR primers under the cycle conditions; 95 °C for 15 min, followed by 40 cycles of 95 °C for 30 s, 55 °C for 45 s and 72 °C for 1 min. PCR products were electrophoreised on a 2% agarose gel alongside 1 μg of 100 bp ladder (promega). DNA extracted from PHK containing episomal HPV16 and SiHa cells which contains integrated HPV16 were used as controls for intact and disrupted E2 repectively. The water control amplified in the first PCR reaction was carried through to the second PCR reaction to control for PCR product contamination.

The complementary DNA (cDNA) synthesis kit (PrimeScript First Strand cDNA Synthesis Kit, Takara) was used according to the manufacturer’s instructions. Briefly, cDNA was synthesized using random hexamers, and samples were eluted in a total volume of 20 μl. PCR experiments were performed using cDNA corresponding to 5 ng of the reverse transcripted RNA and 400 nM primers. Each primer was designed using the website Primer3Plus [67 (link)]; the primers are listed in Additional file 1: Table S1. Amplification was performed with 1.25 units of GoTaq DNA Polymerase (Promega) in a thermocycler (peqSTAR) at 95 °C, 30 s, 58–62 °C, 30 s and 72 °C, 30 sec for 35 cycles. Amplification products were visualized with ethidium bromide on a 3% electrophoresis gel. Negative controls were performed omitting the reverse transcription step or with H₂O. A 100-bp ladder (Promega) was used to verify the size of the amplification products.

PCR was performed at least on three independent biological replicates for each sample using specific primers and reaction conditions. The PCR was performed with HotStarTaq Master Mix Kit (Qiagen GmbH, Hilden, Germany) using 5 ng of cDNA for each amplification and including a positive reaction control (Table 1). Amplicon fragments were separated on a 2% agarose gel, stained with ethidium bromide and with a 100 bp ladder as a marker (Promega).

To assess efficacy of RPA reactions, fluorometry and electrophoretic analyses were used. Fluorometry was performed on a Qubit 2.0 with a dsDNA HS Assay Kit (ThermoFisher, Dartford, UK), which was powered by the 18 V, 2 Ah Osci-lyser battery via a USB adapter and plug, which adjusted voltage from 5 V to 9 V, as shown in Figure 8C,D.
All gel electrophoresis was performed using 1% agarose gel at 110 V using a 100 bp ladder (Promega). This could have been conducted in the field using the Bento lab; however, all electrophoresis testing in the study was undertaken using laboratory equipment.
An Agilent Tapestation 4150 using a D1000 screen tape was used to confirm the sizes and quantities of the RPA products obtained.