A Propidium Monoazide (PMAxx)-Droplet Digital PCR (ddPCR) for the Detection of Viable <i>Burkholderia cepacia</i> Complex in Nuclease-Free Water and Antiseptics
Pharmaceutical products contaminated with <i>Burkholderia cepacia</i> complex (BCC) strains constitute a serious health issue for susceptible individuals. New detection methods to distinguish DNA from viable cells are required to ensure pharmaceutical product quality and safety. In this...
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MDPI AG
2022-04-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/10/5/943 |
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| author | Soumana Daddy Gaoh Ohgew Kweon Yong-Jin Lee David Hussong Bernard Marasa Youngbeom Ahn |
| author_facet | Soumana Daddy Gaoh Ohgew Kweon Yong-Jin Lee David Hussong Bernard Marasa Youngbeom Ahn |
| author_sort | Soumana Daddy Gaoh |
| collection | DOAJ |
| description | Pharmaceutical products contaminated with <i>Burkholderia cepacia</i> complex (BCC) strains constitute a serious health issue for susceptible individuals. New detection methods to distinguish DNA from viable cells are required to ensure pharmaceutical product quality and safety. In this study, we have assessed a droplet digital PCR (ddPCR) with a variant propidium monoazide (PMAxx) for selective detection of live/dead BCC cells in autoclaved nuclease-free water after 365 days, in 0.001% chlorhexidine gluconate (CHX), and in 0.005% benzalkonium chloride (BZK) solutions after 184 days. Using 10 μM PMAxx and 5 min light exposure, a proportion of dead BCC was quantified by ddPCR. The detection limit of culture-based method was 10<sup>4</sup> CFU/mL, equivalent to 9.7 pg/μL for <i>B. cenocepacia</i> J2315, while that of ddPCR was 9.7 fg/μL. The true positive rate from nuclease-free water and CHX using PMAxx-ddPCR assay was 60.0% and 38.3%, respectively, compared to 85.0% and 74.6% without PMAxx (<i>p</i> < 0.05), respectively. However, in BZK-treated cells, no difference in the detection rate was observed between the ddPCR assay on samples treated with PMAxx (67.1%) and without PMAxx (63.3%). This study shows that the PMAxx-ddPCR assay provides a better tool for selective detection of live BCC cells in non-sterile pharmaceutical products. |
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| last_indexed | 2024-03-10T03:22:42Z |
| publishDate | 2022-04-01 |
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| series | Microorganisms |
| spelling | doaj.art-08a2a05323c54cd089ee44a5fe0689ff2023-11-23T12:15:22ZengMDPI AGMicroorganisms2076-26072022-04-0110594310.3390/microorganisms10050943A Propidium Monoazide (PMAxx)-Droplet Digital PCR (ddPCR) for the Detection of Viable <i>Burkholderia cepacia</i> Complex in Nuclease-Free Water and AntisepticsSoumana Daddy Gaoh0Ohgew Kweon1Yong-Jin Lee2David Hussong3Bernard Marasa4Youngbeom Ahn5Division of Microbiology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079, USADivision of Microbiology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079, USADepartment of Natural Sciences, Albany State University, Albany, GA 31705, USAOffice of Pharmaceutical Science, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Beltsville, MD 20993, USAOffice of Pharmaceutical Quality, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD 20993, USADivision of Microbiology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079, USAPharmaceutical products contaminated with <i>Burkholderia cepacia</i> complex (BCC) strains constitute a serious health issue for susceptible individuals. New detection methods to distinguish DNA from viable cells are required to ensure pharmaceutical product quality and safety. In this study, we have assessed a droplet digital PCR (ddPCR) with a variant propidium monoazide (PMAxx) for selective detection of live/dead BCC cells in autoclaved nuclease-free water after 365 days, in 0.001% chlorhexidine gluconate (CHX), and in 0.005% benzalkonium chloride (BZK) solutions after 184 days. Using 10 μM PMAxx and 5 min light exposure, a proportion of dead BCC was quantified by ddPCR. The detection limit of culture-based method was 10<sup>4</sup> CFU/mL, equivalent to 9.7 pg/μL for <i>B. cenocepacia</i> J2315, while that of ddPCR was 9.7 fg/μL. The true positive rate from nuclease-free water and CHX using PMAxx-ddPCR assay was 60.0% and 38.3%, respectively, compared to 85.0% and 74.6% without PMAxx (<i>p</i> < 0.05), respectively. However, in BZK-treated cells, no difference in the detection rate was observed between the ddPCR assay on samples treated with PMAxx (67.1%) and without PMAxx (63.3%). This study shows that the PMAxx-ddPCR assay provides a better tool for selective detection of live BCC cells in non-sterile pharmaceutical products.https://www.mdpi.com/2076-2607/10/5/943<i>Burkholderia cepacia</i> complexpropidium monoazide (PMAxx)droplet digital polymerase chain reaction (ddPCR)nuclease-free waterantiseptics |
| spellingShingle | Soumana Daddy Gaoh Ohgew Kweon Yong-Jin Lee David Hussong Bernard Marasa Youngbeom Ahn A Propidium Monoazide (PMAxx)-Droplet Digital PCR (ddPCR) for the Detection of Viable <i>Burkholderia cepacia</i> Complex in Nuclease-Free Water and Antiseptics Microorganisms <i>Burkholderia cepacia</i> complex propidium monoazide (PMAxx) droplet digital polymerase chain reaction (ddPCR) nuclease-free water antiseptics |
| title | A Propidium Monoazide (PMAxx)-Droplet Digital PCR (ddPCR) for the Detection of Viable <i>Burkholderia cepacia</i> Complex in Nuclease-Free Water and Antiseptics |
| title_full | A Propidium Monoazide (PMAxx)-Droplet Digital PCR (ddPCR) for the Detection of Viable <i>Burkholderia cepacia</i> Complex in Nuclease-Free Water and Antiseptics |
| title_fullStr | A Propidium Monoazide (PMAxx)-Droplet Digital PCR (ddPCR) for the Detection of Viable <i>Burkholderia cepacia</i> Complex in Nuclease-Free Water and Antiseptics |
| title_full_unstemmed | A Propidium Monoazide (PMAxx)-Droplet Digital PCR (ddPCR) for the Detection of Viable <i>Burkholderia cepacia</i> Complex in Nuclease-Free Water and Antiseptics |
| title_short | A Propidium Monoazide (PMAxx)-Droplet Digital PCR (ddPCR) for the Detection of Viable <i>Burkholderia cepacia</i> Complex in Nuclease-Free Water and Antiseptics |
| title_sort | propidium monoazide pmaxx droplet digital pcr ddpcr for the detection of viable i burkholderia cepacia i complex in nuclease free water and antiseptics |
| topic | <i>Burkholderia cepacia</i> complex propidium monoazide (PMAxx) droplet digital polymerase chain reaction (ddPCR) nuclease-free water antiseptics |
| url | https://www.mdpi.com/2076-2607/10/5/943 |
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