TITLE: The culture-independent Bcc NAD method for the rapid detection and quantification of Bcc in water
AUTHORS: Huong Thu Duong, Shannon Fullbrook,  Kate Reddington, Elizabeth Minogue, Thomas Barry

[DESCRIPTION]
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: 
 


[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.

[STEPS]
SECTION: WATER FILTRATION
1. 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).
SECTION: WATER FILTRATION
2. Ethanol-flame sterilise the forceps and, with them, remove the sterile membrane filter from its packaging.

 

SECTION: WATER FILTRATION
3. 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.

SECTION: WATER FILTRATION
4. Fit the sterile funnel securely on the filter base of the apparatus.

 

SECTION: WATER FILTRATION
5. 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.
SECTION: WATER FILTRATION
6. Turn on the vacuum pump (pressure differential of 30-50 kPa (kiloPascals)) and allow the complete separation of filtrate through the membrane filter.
 

SECTION: WATER FILTRATION
7. Power off the vacuum pump and remove the funnel. 
SECTION: WATER FILTRATION
9. 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.  

 


SECTION: BACTERIAL gDNA EXTRACTION AND PURIFICATION
10. 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. 
SECTION: BACTERIAL gDNA EXTRACTION AND PURIFICATION
11. Each tube is vortexed for 1 minute. 
SECTION: WATER FILTRATION
8. With ethanol-flame sterile forceps, carefully remove the membrane filter and aseptically place the membrane filter into a prepared sterile 20-ml tube.
SECTION: BACTERIAL gDNA EXTRACTION AND PURIFICATION
12. 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
SECTION: BACTERIAL gDNA EXTRACTION AND PURIFICATION
14. 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.

 

SECTION: BACTERIAL gDNA EXTRACTION AND PURIFICATION
13. 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. 
SECTION: THE Bcc qPCR NAD ASSAY
15. The eluted DNA samples are used as templates in the previously described Bcc qPCR NAD assay. 
Link: