Comparison of nucleic acid extraction methods to improve the gDNA recovery in drinking water samples

CORIMAYO, SN; RAJAL, VB; CRUZ, MC

Los Cocos, Córdoba

Congreso; Congreso Argentino de Microbiología General; 2022

SAMIGE

Drinking water quality is essential for human health; therefore, effective health surveillance is needed. According to the current legislation, this quality is evaluated by culture-dependent methods of indicators microorganisms, representing a late stage for the application of corrective measurements and not representative of the total microorganisms present. Currently, molecular techniques like shotgun sequencing and real time PCR (qPCR) allow to determine most of the member present in the drinking water microbial community and to rapidly detect pathogens and/or opportunists microorganisms. Thus, improving the monitoring design conducting to the mitigation of risk for public health. However, these methods are hindered because of the low microbial biomass of drinking water distribution system, which leads to deficient nucleic acid extraction. The aim of this work was to compare extraction methods to determine the protocol with the highest recovery of DNA in drinking water samples. Ten liters of water samples were collected and concentrated using a membrane filtration method. Four DNA extraction methods were evaluated: i) commercial kit of national production (PURO Soil, Productos Bio-lógicos), ii) imported commercial kit (Fast DNA, MP Biomedicals), iii) Guanidine thiocyanate agent based protocol, commercially available (TRIZol), and iv) traditional method that used chloroform. The gDNA concentration was quantified by fluorometry with QubitTM dsDNA HS Assays Kit (Invitrogen). Furthermore, qPCR was used to amplify a bacterial 16S fragment gene from the extracts recovered. The reactions were carried out with StepOnePlus™ Real-Time PCR System (Applied Biosystems). The SYBR Green intercalant agent and primers previously validated were used. PURO Soil method recovered the highest DNA concentration (102.00 ± 13.31 ng/mL), followed by the traditional method (51.00 ± 15.26 ng/mL), then the Trizol protocol (18.33 ± 3.97 ng/mL) and finally the Fast DNA that was under the detection limit by fluorometry (< 5.00 ng/mL). The Trizol, PURO Soil and the traditional method showed statistically significant differences (p-valor < 0.001). Only the samples extracted with the PURO Soil method were amplified by qPCR. It is important to note during the Fast DNA procedure, the FastPrep® homogenizer recommended by the manufacturer was not used. Considering the cost/benefit of the compared methods, PURO Soil was found to be the most effective method due to its highest yield, low cost, and performance time that improve the genetic material extraction for the monitoring of the drinking water quality carried out in laboratories with limited resources.