Experimental design
Different experiments were designed to assess DNA sterilization methods such as decontamination using 10% bleach solution, ethanol solution, DNA-ExitusPlus IF (PanReac AppliChem, Germany) in two different DNA laboratory settings, i.e., low template DNA lab and reference DNA lab (abbreviated as CW and DB respectively). In this study, we tested various approaches to remove DNA from hard laboratory surfaces and instruments. We applied gDNA of ~ 20 ng/ul in clean surfaces. The DNA was dried and left for 15 minutes before any treatment. The collection of cells was done using cotton swabs (SceneSafe, UK). All research was performed in accordance with relevant guidelines/regulations.
- Applying 85% ethanol solution to disinfect the working area surfaces and instruments
Applying 85% ethanol solution to sterilize surface of widely used instruments and working areas such as thermomixers (Eppendorf, Germany), drawers and pipettors in both CW and DB labs. Each instrument was first pre-swabbed, then applied the 85% ethanol solution.
- Applying 85% ethanol solution and DNA-ExitusPlus IF to disinfect working area surfaces and instruments
Applying 85% ethanol solution and DNA-ExitusPlus IF (PanReac AppliChem, Germany) to sanitize surfaces of working areas such as DNA extraction benches and PCR cabinets in both CW and DB labs. Each instrument was first pre-swabbed then applied the 85% ethanol solution and DNA-ExitusPlus IF then swabbed again.
- Applying different exposure time of UV light to disinfect PCR cabinets
By applying different exposure time of UV light to decontaminate the PCR cabinets using the following time intervals: 5 min, 10 min, 15 min, 20 min and 25 min.
- Applying different exposure time of DNA-ExitusPlus IF to disinfect working area
By applying different exposure time of DNA-ExitusPlus IF (PanReac AppliChem, Germany) to decontaminate the working area using the following time intervals: 10 min and 15 min. To ensure proper decontamination, we have applied induced DNA on the tested surfaces, sprayed the solution then waited for the studied time then swabbed again to check for efficiency.
- Applying different exposure time of bleach to disinfect working area
By applying different exposure time of 10% bleach solution (commercially available) to decontaminate the working area using the following time intervals: 10 min, 15 min, 20 min, 25 min, 30 min and 35 min. To ensure proper decontamination, we have applied induced DNA on the tested surfaces, sprayed the solution then waited for the studied time then swabbed again to check for efficiency.
- Applying different concentrations of ethanol solution to disinfect working area
By applying different concentrations of ethanol solution to decontaminate the working area using the following concentrations: 70%, 75%, 80% and 85%. To ensure proper decontamination, we have applied induced DNA on the tested surfaces, sprayed the ethanol solution using the abovementioned concentrations and waited for 10 minutes then swabbed again to check for efficiency.
- DNA testing of gloves during work
Random swabbing was done during DNA testing for different DNA experts.
- Talking in the presence of an open tube.
We have talked and coughed inside DNA test tubes prior to proceed for pre and post PCR amplification to study the effect of DNA contamination from unprocessed DNA such as saliva.
- Presence of DNA in the air
Random swabbing was done in the air to check for the presence of DNA, i.e., working areas, PCR cabinets and offices for CW and DB labs.
DNA processing
Genomic DNAs (gDNA) were extracted from the collected cotton swabs samples (SceneSafe, UK) using AutoMate Express DNA Extraction System (Thermo fisher Scientific, Inc., Waltham, MA, USA) following magnetic beads principle [11].
Subsequently the extracted DNAs were quantified using Quantifiler HP DNA Quantification Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) in the 7500 Real-Time PCR System (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to manufacturer’s recommendation [12]. About 1.2 ng of the extracted DNA was amplified using GlobalFiler PCR Amplification Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to manufacturer’s recommendation [13].
A total of 24 loci were amplified, including 21 autosomal STR loci and three gender determination loci in 29 cycles via MicroAmp Optical 96-Well Reaction Plate (Thermo Fisher Scientific Company, Carlsbad, USA) along with the Previously genotyped male control (provided with the kit) and low TE buffer as a negative control using 96-Veriti thermal cycler (Thermo Fisher Scientific Company, Carlsbad, USA). The PCR products (1µl) were separated by capillary electrophoresis in an ABI 3500xl Genetic Analyzer (Thermo Fisher Scientific Company, Carlsbad, USA) with reference to the LIZ600 size standard v2 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) in total of 10 µl master mix consisting of LIZ600 size standard and Hi-Di formamide (Thermo Fisher Scientific, Inc., Waltham, MA, USA). GeneMapper ID-X Software v1.4 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used for genotype assignment [13]. DNA typing and assignment of nomenclature were based on the ISFG recommendations.
Analysis
The results from the 7500 Real-Time PCR System (Thermo Fisher Scientific, Inc., Waltham, MA, USA) were detected using the HID Real-Time PCR Analysis Software v1.2. All the results were input in a table format. Additionally, the STR profiles were analyzed and interpreted using GeneMapper ID-X Software v1.4 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) by direct counting the number of loci/ peaks found in the STR profiles and inserted in the table format.
RFU for reference samples were done using in house validation for the GlobalFiler Amplification Kit to differentiate between the stochastic threshold and possible allele drop out [14].