Glycerol-Driven Contamination in PCR Labs: Implications for Diagnostic Accuracy and Laboratory Safety

BackgroundA key component of diagnostics in low- and middle-income (LMIC) nations is polymerase chain reaction (PCR). Despite its high sensitivity, PCR is vulnerable to laboratory contamination, particularly from aerosolized DNA. Glycerol, a common additive that enhances enzyme activity, may also promote aerosol generation--an issue not yet thoroughly investigated. ObjectiveTo determine whether PCR master mixes containing glycerol lead to increased aerosol-based contamination and false-positive results. MethodsUsing glycerol-free and 30% glycerol master mixes, SARS-CoV-2 N1,N2 and human RNaseP gene templates were pipetted 30 times in biosafety cabinets in two labs. Template-free tubes were exposed to ambient air for 1, 2, or 5 minutes and subjected to 40-cycle real-time PCR. Aerosol particle counts (<0.3 {micro}m) were measured at each time point. Positive PCR counts and Ct values were compared between circumstances. Analysis was also done on retrospective contamination data comparing 10% and 30% glycerol-mixes. FindingsNegative controls exposed to 30% glycerol containing master-mixes resulted in significantly earlier amplification (lower Ct values) and more frequent positivity compared to glycerol-free and 10% glycerol-mixes (P< 0.05 across all targets and time points). Aerosol particle counts were upto 3-fold higher in the 30% glycerol group than the 10% group (P< 0.001). Retrospective analysis confirmed significantly greater contamination in 30% glycerol incidents, with lower Ct values for all targets (P= 0.0001). ConclusionThis work is the first to show that aerosol-mediated contamination is increased when glycerol is added to PCR master mixes. These findings emphasize strengthening biosafety measures and improving diagnostic accuracy in molecular laboratories, particularly in LMICs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/25341814v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@6231edorg.highwire.dtl.DTLVardef@bbfa4corg.highwire.dtl.DTLVardef@12dc21dorg.highwire.dtl.DTLVardef@12334e4_HPS_FORMAT_FIGEXP M_FIG C_FIG