DIFFUSION OF MICROBIAL DNA ON PAPER USING DIFFERENT EXTRACTION METHODS.

SAID ADAMO, MARÍA DEL MILAGRO; REYES, SARITA ISABEL; MAIDANA KULESZA, MARÍA NOEL; POMA, RAMIRO HUGO; RAJAL, VERÓNICA BEATRIZ; CRISTÓBAL, HÉCTOR ANTONIO

Congreso; LVII SAIB Meeting - XVI SAMIGE Meeting; 2021

Paper is an extremely versatile material with many desirable properties that makes it an excellent substrate for manufacturingdevices for medical diagnostics, food quality control, and environmental monitoring due to its compatibility with biologicalsystems and biomolecules. There is a progressive tendency to develop applications based on the identification andquantification of specific DNA sequences of bacterial cells in miniaturized laboratories, lab-on-a-chip (LOC), that are easy touse, low cost, portable, reproducible, sensitive and that allow obtaining results quickly. The transport of nucleic acid moleculesacross paper is a key process in several operations implemented in the development of analytical devices. Integration andoptimization of DNA extraction method in a same device remain a challenge for in situ detection. The objective of this studywas to evaluate the diffusion of DNA obtained after the application of different extraction methods on paper; so that it wouldbe possible to choose the method that best suit the development of LOC devices based on paper. Pure cultures of SalmonellaTyphimurium ATCC 14028 were used. Five DNA extraction methods were employed: two commercial kits (PURO Virus RNA PB-L and QIAmp Viral RNA QIAGEN), thermal shock, phenol-chloroform and a lysis solution with Proteinase K and lysozyme. Chromatography paper (Whatman 3 mm, CHR) was cut into strips (80 x 3 mm) as support for assays. A volume (50 µL) of DNA extracts dilutions (1/100) from each method was applied on one end of the strips. After, diffusion time was standardized at 3 min from its application. Fragments of paper from the beginning, middle and end of the strips were eluted using TE buffer (Tris-EDTA). Subsequently, they were quantified by Real Time PCR (qPCR) and by spectrophotometry (NanoDrop). This procedure allowed us evaluating the distance diffused throughout the strips. In all cases, DNA were detected by qPCR and the best performance of DNA diffusion, in terms of cycle threshold (Ct) value, was observed in beginning and middle fragments of strips with constant values. The averages (Ct ± S.D.) of both fragments were: 24,1±0,1 (lysis solution), 24,2±0,04 (thermal shock), 24,8±0,01 (PURO Virus RNA), 28,3±0,7 (QIAmp Viral RNA), 33,8±0,7 (phenol-chloroform protocol). The end fragments showed some Ct (±S.D.) differences: 31,7±0,1 (lysis solution), 25,6±0,1 (thermal shock), 34,7±0,6 (phenol-chloroform protocol), 24,5±0,04 (PURO Virus RNA), 28,2±0,1 (QIAmp Viral RNA). Using ratio of absorbance at 260 nm and 280 nm, it was possible to infer the purity of DNA eluted of strips. The purest DNA was obtained by both commercial kits, followed by phenol-chloroform protocol, thermal shock and lysis solution. Thermal shock and lysis solution could be good candidates to molecular detection LOC devices based on paper according to optimal requirements, bringing alternative solutions that improve device autonomy.