Process of Extraction of Nucleic Material

Kibera is the largest informal settlement in Nairobi, Kenya and one of the largest in Africa and the world. It is is located 7km southwest of Nairobi and houses approximately one million people and several animal species. Conditions in Kibera are extremely poor and heavily polluted by human and animal refuse and garbage.

Stool specimens were collected in sterile cups and maintained at 4oC until tested. All study stool samples were stored at -80oC upon reception at the KEMRI laboratories in Nairobi.

Extraction of Nucleic Material

180 – 220 mg or 200ul of liquid stool samples was extracted using a modified QIAamp Fast Stool Mini Kit procedure where they underwent a lysate preparation process that included a mechanical disruption step (bead beating), removal of inhibitors and elution of nucleic material using spin columns. The lysate was transferred into the spin columns and centrifuged. The columns were washed twice following the manufacturer’s instructions; centrifuging at 14,000Xg for 1 minute and eluted with 100µl total nucleic acid (TNA) (Liu et al., 2013).

RT-PCR

Samples were tested for HEV by RT-PCR. Briefly, 25µl reaction mixtures were made containing: 12.5µL of 2x Taq man one-step RT-PCR master mix buffer, 1µl 40x Multi Scribe and RNase inhibitor mixture, 1µM forward primer, 1µM reverse primer,5.75µl molecular grade water and 5µL of purified RNA. (The reactions were incubated at 50°C for 30 min followed by 94°C for 3 min. Thermo cycling was performed for 45 cycles at 94°C for 30 s, 42°C for 30 s and 72°C for 30 s in a 7500F model thermocycler (Applied Biosystems). Cycle threshold CT values of ≤30 were considered for sequencing.

cDNA Synthesis and Amplification

A standardized in-house protocol one step cDNA synthesis and amplification assay was used to obtain amplicons for sequencing as follows:

A master mix containing 2.0 μL RNase free water; 16 μL of 2X reaction mix; 1 μL of 20 pmoles/µL Forward and reverse primers (292 and 222 Table 1); 1 μL of SuperScript III RT/Platinum Taq mix and 4 μL of the RNA template. The reaction mix was incubated at 50°C for 45 min; 94°C for 3minutes then thermocycling was performed for 40 cycles of 94°C for 30s; 42°C for 1 min and 60°C for 2 min in a model 9700 thermocycler (Applied Biosystems, Foster City, CA) (Oberste et al., 2006). Thermocycling was followed by a final extension at 60°C for 10 min. The reaction products were analyzed by electrophoresis in a 1% agarose gel and staining with 0.5 mg/ml ethidium bromide.