Presentation Title

An Optimization Method for Live/Dead Differentiation by PMA-qPCR Targeting HF183 in Environmental Water Matrices

Faculty Mentor

Dr. Phillip Gedalanga

Start Date

23-11-2019 10:00 AM

End Date

23-11-2019 10:45 AM

Location

51

Session

poster 3

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

HF183 is a genetic marker within the 16s rRNA gene in Bacteroides dorei, an indicator of human fecal pollution in environmental waters. However, limitations of risk assessments include difficulty in differentiating the source of DNA from viable or non-viable cells. The objective of this study is to optimize a quantitative polymerase chain reaction (qPCR) method targeting HF183 for live and dead differentiation using propidium monoazide (PMA). PMA permeates damaged cell membranes and binds to double-stranded DNA, preventing DNA amplification during qPCR. The samples analyzed included B. dorei, Escherichia coli, and secondary clarifier effluent (SCE) wastewaters. To produce the non-viable condition, samples were killed at different times at 72ºC, 85ºC or 100ºC, or by autoclave at 121ºC and 15 psi for 15 min. Samples were directly treated on isopore filters or resuspended biomass at varying concentrations of PMA (10μM - 500μM), followed by LED or halogen light inactivation. Bacterial growth on R2A or sheep blood agar plates confirmed heat-kills. Heat kill was effective for B. dorei at 85ºC, but not for SCE, as bacterial growth would occur. Autoclaving successfully killed cells, but destroyed DNA and led to inaccurate qPCR results. PMA treatment on SCE samples was more effective on filters than resuspension due to a greater decrease in cell abundance in the non-viable condition, a 28.2-fold difference compared to an 8.2-fold difference, respectively. LED light had a 50-fold difference in abundance with the addition of PMA in non-viable cells of E. coli (halogen light was 17.6-fold). Lastly, efficient PMA treatments utilized less than 100 μM PMA. This study supports PMA-qPCR targeting HF183 as a plausible method for detecting human fecal pollution in environmental waters. Additionally, organizations could utilize this method to improve wastewater treatment. On-going research will explore alternative methods to destroy cells without denaturing DNA in SCE samples.

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Nov 23rd, 10:00 AM Nov 23rd, 10:45 AM

An Optimization Method for Live/Dead Differentiation by PMA-qPCR Targeting HF183 in Environmental Water Matrices

51

HF183 is a genetic marker within the 16s rRNA gene in Bacteroides dorei, an indicator of human fecal pollution in environmental waters. However, limitations of risk assessments include difficulty in differentiating the source of DNA from viable or non-viable cells. The objective of this study is to optimize a quantitative polymerase chain reaction (qPCR) method targeting HF183 for live and dead differentiation using propidium monoazide (PMA). PMA permeates damaged cell membranes and binds to double-stranded DNA, preventing DNA amplification during qPCR. The samples analyzed included B. dorei, Escherichia coli, and secondary clarifier effluent (SCE) wastewaters. To produce the non-viable condition, samples were killed at different times at 72ºC, 85ºC or 100ºC, or by autoclave at 121ºC and 15 psi for 15 min. Samples were directly treated on isopore filters or resuspended biomass at varying concentrations of PMA (10μM - 500μM), followed by LED or halogen light inactivation. Bacterial growth on R2A or sheep blood agar plates confirmed heat-kills. Heat kill was effective for B. dorei at 85ºC, but not for SCE, as bacterial growth would occur. Autoclaving successfully killed cells, but destroyed DNA and led to inaccurate qPCR results. PMA treatment on SCE samples was more effective on filters than resuspension due to a greater decrease in cell abundance in the non-viable condition, a 28.2-fold difference compared to an 8.2-fold difference, respectively. LED light had a 50-fold difference in abundance with the addition of PMA in non-viable cells of E. coli (halogen light was 17.6-fold). Lastly, efficient PMA treatments utilized less than 100 μM PMA. This study supports PMA-qPCR targeting HF183 as a plausible method for detecting human fecal pollution in environmental waters. Additionally, organizations could utilize this method to improve wastewater treatment. On-going research will explore alternative methods to destroy cells without denaturing DNA in SCE samples.