Loop-Mediated Isothermal Amplification (LAMP) Assay for Detecting Burkholderia cepacia Complex in Non-Sterile Pharmaceutical Products
Abstract
:1. Introduction
2. Materials and Methods
2.1. LAMP Primer Design
2.2. Bacterial Strains and DNA Preparation
2.3. LAMP and qPCR Assay
2.4. Optimization of LAMP
2.5. True-Positive Rate (Sensitivity) and True-Negative rate (Specificity) in Analysis Using LAMP
2.6. Limit of Detection (LOD) and Limit of Quantification (LOQ) of LAMP
2.7. Evaluation of LAMP in Autoclaved Nuclease-Free Water and Antiseptic Solutions
2.7.1. Autoclaved Nuclease-Free Water
2.7.2. Chlorhexidine Gluconate (CHX) and Benzalkonium Chloride (BZK)
3. Results
3.1. Primers Designed for LAMP
3.2. Optimal Conditions of LAMP
3.3. Specificity Analysis of LAMP
3.4. Limit of Quantification (LOQ) and Limit of Detection (LOD) of the LAMP Assay
3.5. Testing BCC Detection from Autoclaved Nuclease-Free Water, and Antiseptic Solutions with the LAMP Assay
3.5.1. Nuclease-Free Water
3.5.2. CHX
3.5.3. BZK
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Disclaimer
References
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Primers | Sequences (5′-3′) | Length (bp) | ||
---|---|---|---|---|
qPCR | LAMP | |||
RibB5 | RibB5-F | RibB5 F3 | GGCCGGATGGTGATCCT | 17 |
RibB5-R | RibB5 B3 | GTCATCAGCGGCAGGTG | 17 | |
RibB5 FIP | TCCGGTGTGACGAATTCGGC- CGAAGAAGACCGCGAAAACG | 40 | ||
RibB5 BIP | AACTTCATGGCCAAGTACGGCC-AAGCTGCTTGCAGCGTT | 39 | ||
RibB5 LF | GATCACGAGGTCGCCCT | 17 | ||
RibB5 LB | CGGCCTGGTTTGTCTGA | 17 | ||
RibB67 | RibB67-F | RibB67 F3 | GCGAYACGAAGGAACAYCTG | 20 |
RibB67-R | RibB67 B3 | CGTAGCCGGACATGCTG | 17 | |
RibB67 FIP | TGAAATCGACCGGCCGGC-TCTTCAAGGCGTTCGACGAG | 38 | ||
RibB67 BIP | AGATCCTGCGCGATGTCGG-CCAGCTTGCGCGGATTC | 36 | ||
RibB67 LF | TGAGCGCAGCGGCCTTTTCTT | 21 | ||
RibB67 LB | TCGGCAAGATGCAGGTGC | 18 | ||
RibB16 | RibB16-F | RibB16 F3 | ARGGCGACCTCGTGATC | 17 |
RibB16-R | RibB16 B3 | CGAGATGCCGGTCGTCAC | 18 | |
RibB16 FIP | TCAGACAAAYCAGGCCGCG-CGTCACACCGGAAGCGAT | 37 | ||
RibB16 BIP | ACGCTGCAAGCAGCTTCACC-CGATGCTGACCGTGAACG | 38 | ||
RibB16 LF | CCGTACTTGGCCATGAAGTT | 20 | ||
RibB16 LB | GCTGATGACCTACCGCAAC | 19 |
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Daddy Gaoh, S.; Kweon, O.; Lee, Y.-J.; LiPuma, J.J.; Hussong, D.; Marasa, B.; Ahn, Y. Loop-Mediated Isothermal Amplification (LAMP) Assay for Detecting Burkholderia cepacia Complex in Non-Sterile Pharmaceutical Products. Pathogens 2021, 10, 1071. https://doi.org/10.3390/pathogens10091071
Daddy Gaoh S, Kweon O, Lee Y-J, LiPuma JJ, Hussong D, Marasa B, Ahn Y. Loop-Mediated Isothermal Amplification (LAMP) Assay for Detecting Burkholderia cepacia Complex in Non-Sterile Pharmaceutical Products. Pathogens. 2021; 10(9):1071. https://doi.org/10.3390/pathogens10091071
Chicago/Turabian StyleDaddy Gaoh, Soumana, Ohgew Kweon, Yong-Jin Lee, John J. LiPuma, David Hussong, Bernard Marasa, and Youngbeom Ahn. 2021. "Loop-Mediated Isothermal Amplification (LAMP) Assay for Detecting Burkholderia cepacia Complex in Non-Sterile Pharmaceutical Products" Pathogens 10, no. 9: 1071. https://doi.org/10.3390/pathogens10091071
APA StyleDaddy Gaoh, S., Kweon, O., Lee, Y.-J., LiPuma, J. J., Hussong, D., Marasa, B., & Ahn, Y. (2021). Loop-Mediated Isothermal Amplification (LAMP) Assay for Detecting Burkholderia cepacia Complex in Non-Sterile Pharmaceutical Products. Pathogens, 10(9), 1071. https://doi.org/10.3390/pathogens10091071