Mar 27, 2024
Version 2
The culture-independent Bcc NAD method for the rapid detection and quantification of Bcc in water V.2
- Huong Thu Duong1,
- Shannon Fullbrook1,
- Kate Reddington1,
- Elizabeth Minogue1,
- Thomas Barry1
- 1University of Ireland Galway, Ireland
Protocol Citation: Huong Thu Duong, Shannon Fullbrook, Kate Reddington, Elizabeth Minogue, Thomas Barry 2024. The culture-independent Bcc NAD method for the rapid detection and quantification of Bcc in water. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzxox8gx1/v2Version created by Huong Duong
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 27, 2024
Last Modified: March 27, 2024
Protocol Integer ID: 97442
Keywords: Burkholderia cepacia complex, water testing, culture-independent, nucleic acid diagnostics, qPCR, bcc microbial culture diagnostic methodology, negative legionella species in water, negative legionella species, independent bcc nad method, quantification of bcc, bcc species, microbial culture, bcc, water sample, valuable diagnostic tool, water risk management strategy in the pharmaceutical sector, quality of these water system, independent nad method, independent bcc nad method for the rapid detection
Abstract
Here we present a rapid (<4 hours from sample in to result out) culture-independent Bcc NAD method, incorporating a quantitative real-time Polymerase Chain Reaction (qPCR) assay. This method can be used to detect and simultaneously identify the Bcc species directly from water samples. This culture-independent Bcc NAD method is validated to the testing method equivalent of the ISO/TS 12869:2019 standard (a widely used rapid culture-independent NAD method for detecting Gram-negative Legionella species in water) in terms of specificity, sensitivity, accuracy and turnaround time to result compared to Bcc microbial culture diagnostic methodologies.
We propose that the rapid culture-independent Bcc NAD method presented could gain widespread acceptance and be applied as a valuable diagnostic tool supporting a water risk management strategy in the pharmaceutical sector to assure the safety and quality of these water systems and water used for manufacturing.
The development and validation of this culture-independent Bcc NAD method has been published in:
Citation
LINK
Guidelines
- The laboratory workspace and equipment were sterilized by UV-light, DNase solution, and 70% ethanol.
- Due to the sensitivity of the assays, extra care must be paid to avoid sample contamination during analysis, and as such Good Laboratory Practice must be followed. Wear laboratory coats and gloves at all times.
- Filter pipet tips were used in all steps of the laboratory work.
- The material to be used (tubes, tips, water, etc.) must be free of nucleases.
Materials
- Filtration apparatus (mounted either on an air pump or on a vacuum flask), suitable for sterilization by steam (autoclave)
- Filter funnel has visible graduations that are appropriate to the volumes of the sample typically analysed (should be sterilized by autoclaving before use)
- A vacuum pump provides a pressure which permits the suction of filtration through the membrane filter
- A vessel to collect the filtered water (e.g. 1 litre bottle)
- Sterile forceps for handling membrane filters
- 0.4 µm hydrophilic polycarbonate membrane filters of 47 mm diameter (Isopore membrane filters, Merck Millipore) with a low absorption capacity for extracellular nucleic acids using a vacuum pump. The membrane filters shall be sterilized.
Citation
LINK
- Quick-gDNA‱ MiniPrep Kit (Zymo Research)
WATER FILTRATION
Water preparation:
Water is collected in a sterile container containing sterile 0.1% v/v sodium thiosulfate pentahydrate solution to inactivate any residual disinfectant chlorine (if needed) and immediately used for experimental analysis upon arrival in the laboratory (within approximately 2 hours of collection).
Ethanol-flame sterilise the forceps and, with them, remove the sterile membrane filter from its packaging.
Note
To sterilise forceps, dip forceps in alcohol and flame in an alcohol burner. Cool
before use.
Place the sterile hydrophilic polycarbonate membrane filter on the sterile membrane filter base of the filtration apparatus, taking care that only the outer edge of the membrane filter is grasped with sterile forceps.
Fit the sterile funnel securely on the filter base of the apparatus.
Note
Do not touch the inside of the funnel.
Flame the pouring lip of the sample container and pour the desired volume of test water sample to be studied (e.g. 100 ml) into the funnel.
Turn on the vacuum pump (pressure differential of 30-50 kPa (kiloPascals)) and allow the complete separation of filtrate through the membrane filter.
Note
It may be advisable to rinse the funnel's interior with one to three 10 ml to 30 ml portions of sterile water while the filter is still in place to remove organisms adhering to the funnel.
Power off the vacuum pump and remove the funnel.
With ethanol-flame sterile forceps, carefully remove the membrane filter and aseptically place the membrane filter into a prepared sterile 20-ml tube.
The funnel may be re-used without disinfection for the same sample so that the smallest volumes and/or the most diluted samples are filtered first.
To filter another sample, a separate sterile apparatus shall be used, or where appropriate, the funnel can be disinfected by rinsing the funnel with at least 100 ml of sterile water and then with alcohol to prevent cross-contamination.
Note
In every batch of experiments, always include a processing control sterile water confirmed to be free of Bcc, to be used as a negative control to indicate any contamination during the process.
BACTERIAL gDNA EXTRACTION AND PURIFICATION
Add 2 ml of Genomic Lysis Buffer of the Quick-gDNA MiniPrep Kit to each filter in the 20-ml tube and incubate at room temperature on a rolling platform for 30 mins.
Each tube is vortexed for 1 minute.
The remainder of the DNA extraction and purification protocol follows steps 2-4 of the section "Cell Suspensions and Proteinase K Digested Samples" from the Quick-gDNA MiniPrep Kit (Zymo Research) manufacturer’s instructions to extract total gDNA.
https://files.zymoresearch.com/protocols/_d3024_d3025_quick-dna_miniprep_kit.pdf
The column is transferred to a new tube and centrifuged again at maximum speed for 1 minute to ensure the complete removal of remnants of the washing solution from the column before DNA elution.
Transfer the spin column to a clean microcentrifuge tube. Add 25 µl nuclease-free water to the spin column. Incubate 5 minutes at room temperature and then centrifuge at top speed for 30 seconds to elute the DNA.
Note
- It is advisable to incubate the column with resuspension solution for 5 minutes to increase DNA yield. Centrifuge again; the eluted DNA in solution is collected in the tube, and the column is discarded.
- The eluted DNA can be used immediately for molecular-based applications or stored at ≤-20ºC for future use.
THE Bcc qPCR NAD ASSAY
The eluted DNA samples are used as templates in the previously described Bcc qPCR NAD assay.
Link:
Protocol
CREATED BY
Huong Duong
Protocol references
1. Duong HT, Fullbrook S, Reddington K, Minogue E, Barry T. Design, Development and Validation of a Culture-Independent Nucleic Acid Diagnostics Method for the Rapid Detection and Quantification of the Burkholderia cepacia Complex in Water with an Equivalence to ISO/TS 12869:2019. PDA Journal of Pharmaceutical Science and Technology. 2023. doi:10.5731/pdajpst.2021.012728
2. ISO 12869:2019 Water quality — Detection and quantification of Legionella spp. and/or Legionella pneumophila by concentration and genic amplification by quantitative polymerase chain reaction (qPCR). 2019.
3. Barry, T.; Reddington, K.; Minogue, E. A Method for the Detection of Legionella. WO 2018/065497 Al, April 12, 2018.