Propidium Monoazide (PMAxx)-Recombinase Polymerase Amplification Exo (RPA Exo) Assay for Rapid Detection of Burkholderia cepacia Complex in Chlorhexidine Gluconate (CHX) and Benzalkonium Chloride (BZK) Solutions
Abstract
1. Introduction
2. Materials and Methods
2.1. Primer and Probe Design
2.2. 10 μM PMAxx-RPA Exo Assay
2.3. Optimization of Temperature, Reaction Time and Concentration of Magnesium Acetate for PMAxx-RPA Exo Assay
2.4. True-Negative Rate (Specificity) and Limit of Detection (LOD)
2.5. Effect of PMAxx-RPA Exo Assay in Presence of Antiseptics and Cell Lysates
2.5.1. Evaluation of Various Concentrations of CHX and BZK
2.5.2. Comparing DNA Extraction Methods Using the Boiling Method and a Commercial Kit
2.6. Detection of Live/Dead B. multivorans HI2229 in CHX and BZK Solutions
3. Results
3.1. Optimization of the PMAxx-RPA Exo Assay
3.2. Evaluation of Live/Dead Cells with PMAxx Treatment
3.3. Specificity and LOD of PMAxx-RPA Exo Assay
3.4. Assessment of PMAxx-RPA Exo Conditions
3.4.1. Effect of CHX and BZK on the PMAxx-RPA Exo Assay
3.4.2. Comparing Pure DNA and Cell Lysates on the PMAxx-RPA Exo Assay
3.5. Assessing Live/Dead Cells in CHX and BZK Solutions
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primer/Probe | Sequence (5′–3′) | Primer Length (nt) |
---|---|---|
gbpT-F gbpT-R | ACGCTGTCGTCGACGATCATCAGCCTCGTGCT ACCATCGACAGCGCCATCATGATCGTCTGGTT | 32 32 |
Probe | TCGGCCGCGTGCCGGGGATCCTGTCGACGG[FAM-dT]G[THF]-[BHQ1-dT]CTTCGCGATGCCGCC | 49 |
Group | Species | Strain | Results |
---|---|---|---|
BCC | Burkholderia cepacia | PC783 | + |
AU24442 | + | ||
Burkholderia stabilis | AU23340 | + | |
Burkholderia ambifaria | HI2468 | + | |
Burkholderia anthina | HI2738 | + | |
Burkholderia metallica | AU0553 | + | |
AU16697 | + | ||
Burkholderia contaminans | HI3429 | + | |
AU24637 | + | ||
Burkholderia diffusa | AU1075 | + | |
Burkholderia arboris | ES0263a | + | |
AU22095 | + | ||
Burkholderia lata | HI4002 | + | |
Burkholderia multivorans | HI2229 | + | |
AU24571 | + | ||
Burkholderia vietnamiensis | HI2212 | + | |
AU24694 | + | ||
Burkholderia cenocepacia | AU1054 | + | |
AU0222 | + | ||
AU19236 | + | ||
HI2976 | + | ||
HI2485 | + | ||
J2315 | + | ||
Non-BCC | Burkholderia glumae | AU6208 | + |
AU12450 | + | ||
Burkholderia gladioli | AU26454 | + | |
AU29541 | + | ||
AU30473 | + | ||
AU16341 | + | ||
Burkholderia concitans | AU12121 | – | |
Burkholderia oklahomensis | ES0634 | + | |
Burkholderia plantarii | AU9801 | + | |
AU37486 | + | ||
Burkholderia thailandensis | AU13555 | + | |
AU36262 | + | ||
Burkholderia tropica | AU15822 | – | |
AU19944 | – | ||
Burkholderia fungorum | AU18377 | – | |
AU35949 | – | ||
Non-Burkholderia | Caballeronia zhejiangensis | AU10475 | – |
AU12096 | – | ||
Enterococcus faecalis | ATCC29212 | – | |
Enterococcus durans | ATCC6056 | – | |
Proteus mirabilis | ATCC7002 | – | |
Enterococcus faecium | ATCC35667 | – | |
ATCC49624 | – | ||
Bacillus subtilis | ATCC6051 | – | |
Citrobacter freundii | ATCC8090 | – | |
Pseudomonas aeruginosa | PAO1 | – | |
ATCC27853 | – | ||
Yersinia enterocolitica subsp. entrocolitica | ATCC27729 | – | |
Shigella sonnei | ATCC9290 | – | |
Lactobacillus salivarius subsp. salivarius | ATCC11741 | – | |
Enterobacter aerogenes | ATCC13048 | – | |
Klebsiella pneumoniae | ATCC13883 | – | |
Candida albicans (Robin) Berkhout | ATCC10231 | – | |
Salmonella enterica | isolates | – | |
Paenibacillus lautus | isolates | – | |
Brevibacillus laterosporus | isolates | – |
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Daddy Gaoh, S.; Kweon, O.; Ahn, Y. Propidium Monoazide (PMAxx)-Recombinase Polymerase Amplification Exo (RPA Exo) Assay for Rapid Detection of Burkholderia cepacia Complex in Chlorhexidine Gluconate (CHX) and Benzalkonium Chloride (BZK) Solutions. Microorganisms 2023, 11, 1401. https://doi.org/10.3390/microorganisms11061401
Daddy Gaoh S, Kweon O, Ahn Y. Propidium Monoazide (PMAxx)-Recombinase Polymerase Amplification Exo (RPA Exo) Assay for Rapid Detection of Burkholderia cepacia Complex in Chlorhexidine Gluconate (CHX) and Benzalkonium Chloride (BZK) Solutions. Microorganisms. 2023; 11(6):1401. https://doi.org/10.3390/microorganisms11061401
Chicago/Turabian StyleDaddy Gaoh, Soumana, Ohgew Kweon, and Youngbeom Ahn. 2023. "Propidium Monoazide (PMAxx)-Recombinase Polymerase Amplification Exo (RPA Exo) Assay for Rapid Detection of Burkholderia cepacia Complex in Chlorhexidine Gluconate (CHX) and Benzalkonium Chloride (BZK) Solutions" Microorganisms 11, no. 6: 1401. https://doi.org/10.3390/microorganisms11061401
APA StyleDaddy Gaoh, S., Kweon, O., & Ahn, Y. (2023). Propidium Monoazide (PMAxx)-Recombinase Polymerase Amplification Exo (RPA Exo) Assay for Rapid Detection of Burkholderia cepacia Complex in Chlorhexidine Gluconate (CHX) and Benzalkonium Chloride (BZK) Solutions. Microorganisms, 11(6), 1401. https://doi.org/10.3390/microorganisms11061401