Fig 1.
Schematic depicting protocol for preparation and use of evaporated cellular reagents.
Fig 2.
Comparison of lyophilized and evaporated Taq DNA polymerase cellular reagents prepared with or without solid support.
Activities of cellular reagents were compared by performing 20 cycle PCR amplification of 10 ng CT16S plasmid templates using either 3 μL of rehydrated evaporated or lyophilized cellular reagents prepared without solid support (‘Liquid’) or a single 3 mm paper disc containing evaporated cellular reagents added into the PCR reaction mix (PTR5 and GF800: two types of glass fiber conjugate pad paper; 691, 693, 161: three types of glass fiber filter paper). Agarose gel electrophoretic analysis of resulting PCR amplicons is depicted. Expected product size is indicated by an asterisk. Data shown are representative of eight biological replicates.
Fig 3.
Activity of evaporated KlenTaq cellular reagents.
KlenTaq DNA polymerase cellular reagents evaporated at the indicated temperature with (GF800) or without (None) solid support were tested after 2 months of preparation by performing qPCR reactions with or without CT16S plasmid templates. Quantitative PCR reactions performed using commercially obtained pure KlenTaq enzyme served as control. Amplification curves observed in real-time by measuring increase in fluorescence of intercalating EvaGreen dye are depicted in panel A. Melting temperature analysis of resulting PCR amplicons is shown in panel B. Data shown are representative of three biological replicates.
Fig 4.
Activity of Bst-LF evaporated cellular reagents.
Bst-LF DNA polymerase cellular reagents evaporated at the indicated temperature with (GF800) or without (None) solid support were tested after 2 months of preparation by performing LAMP-OSD reactions with or without human gapd plasmid DNA templates. LAMP-OSD reactions performed using commercially obtained pure Bst 2.0 enzyme or with lyophilized Bst-LF cellular reagents (Lyo CR) served as controls. Amplification curves observed in real-time by measuring increase in fluorescence of OSD probes are depicted. Data shown are representative of three biological replicates.
Fig 5.
Activity and stability assays of evaporated OpenVent cellular reagents produced in Ghana and Cameroon.
(A) Left panel: OpenVent evaporated cellular reagents were used to amplify the synthetic beta-globin human gene. OpenVent is functional down to 25% of the standard concentration (2μL of reagent in 20μL PCR reaction). Right panel: OpenVent evaporated cellular reagents are stable for at least 7 months stored at 4°C in an airtight container in the presence of silica desiccant beads. (B) OpenVent evaporated cellular reagents amplify DNA fragments up to 7.5 kb from lambda genome. The shorter amplicons seen along with the 7.5 kb PCR product is due to unintended mispriming of lambda DNA. No amplicons were observed in the negative controls lacking lambda DNA templates. MW: molecular weight (A: 100bp DNA ladder, Newmarket Scientific, B: 1Kb plus DNA ladder, Beneficial Bio; C-: negative control).
Fig 6.
Detection of SARS-CoV-2 using Br512 polymerase evaporated cellular reagents and multiplex RT-LAMP-OSD assays.
Multiplex RT-LAMP-OSD assays for viral N and ORF1AB genes were operated using only Br512 polymerase cellular reagents and indicated amounts of inactivated SARS-CoV-2 virions. Images of endpoint OSD fluorescence representative of five biological replicates (A) and colorimetric readout using fluorescein/biotin lateral flow dipsticks representative of three biological replicates (B) are depicted.
Fig 7.
DNA assembly using evaporated cellular reagents.
Pure enzymes (NEB) or evaporated cellular reagents (CR) for BsaI, T7 DNA ligase, and T4 DNA ligase were used to assemble a PCR product comprising the coding sequence of pink chromoprotein FP595 flanked by two BsaI restriction sites into a chloramphenicol resistant plasmid bearing two BsaI restriction sites downstream of a constitutive lac promoter. Cellphone image of pellets of individual FP595-expressing (pink) and non-expressing (white) transformants grown overnight in chloramphenicol-containing growth media is depicted. Data are representative of four biological replicates.
Fig 8.
PCR-based molecular testing kit using Taq DNA polymerase evaporated cellular reagents.
(A) Schematic depicting design of probe-based colorimetric detection of PCR using fluorescein/biotin-specific lateral flow dipsticks. (B) HF183 PCR performed using evaporated Taq cellular reagents and analyzed by agarose gel electrophoresis. M: 100 bp molecular weight ladder; (-): no template; (+): with 10 ng plasmid template. (C) Indicated amounts of HF183 plasmid templates were PCR amplified using pure Taq DNA polymerase or Taq DNA polymerase evaporated cellular reagents and analyzed using lateral flow dipsticks. Cellphone images of the dipsticks taken after development of red colored control and test lines are depicted. Data are representative of three biological replicates.
Fig 9.
LAMP-OSD based molecular testing kit using Bst-LF evaporated cellular reagents.
80,000 (+) or 0 (—) copies of HF183 plasmid templates were amplified using HF183 LAMP-OSD assays operated with either Bst-LF cellular reagents (CR) or with pure Bst 2.0 enzymes. Endpoint image of OSD fluorescence (A) and colorimetric readout on lateral flow devices (B) are depicted. Data are representative of three biological replicates.