naica® Droplet Chip Digital PCR System for Absolute Quantification of Dominant Bacteria Related to Water Quality Environment
In modern aquaculture, the water quality of aquaculture systems directly affects the health and production of fish. Microorganisms play a vital role in the removal of organic matter and nitrogen cycle, the production of toxic hydrogen sulfide (H2S), but if the microorganisms are pathogenic or have probiotic properties to fish, it will directly affect the health of fish.
Recently, Stilla of France and SINTEF Ocean of Norway cooperated to publish an article titled "Absolute quantification of priority bacteria in aquaculture using digital PCR" in the Journal of Microbiological Methods, aiming to detect relevant dominant bacteria in aquaculture.
In this article, the authors focused on three different bacteria associated with salmon production:
The first is fish pathogens, Moritella viscosa, which is associated with ulcerative diseases in fish, Yersinia ruckeri, which causes enteric redmouth disease, and Flavobacterium psychrophilum, which is associated with bacterial coldwater disease in fish.
The second is a human pathogen that can be transferred from seafood to consumers, Listeria monocytogenes.
The third is bacteria that threaten fish health by damaging the breeding environment. Sulfate-reducing bacteria (SRB) usually affect fish health by converting sulfate (SO42-) into toxic hydrogen sulfide (H2S) under anaerobic conditions. SRB detection can be performed using Desulfovibrio desulfuricans as a reference strain.
The researchers used the naica® Droplet Chip Digital PCR System in singleplex and multiplex detection methods to absolutely quantify the above dominant species. The results showed that the detection limit of Moritella viscosa, Yersinia ruckeri, and Flavobacterium psychrophilum was around 20 fg, and the detection content of Listeria monocytogenes and Desulfovibrio desulfuricans DNA could be as low as 2 fg, and they all had a high linear dynamic range (Figure 1). The multiplex cdPCR detection results were very consistent with the target gene concentrations detected in the corresponding single-plex analysis (Figure 2, Figure 3). This experiment fully demonstrated that the naica® droplet chip digital PCR system can accurately quantify multiple dominant strains in complex water quality samples at the same time.
▲Figure 1: Linear regression diagram of 5 dominant bacterial species quantified by naica® microdroplet chip digital PCR system, with corresponding equations and regression coefficients given respectively
▲Figure 2: Comparison of single and double analysis results for Yersinia ruckeri (A) and Flavobacterium psychrophilum (B).
Yersinia ruckeri and Flavobacterium psychrophilum gDNA were added to the MMC-DNA background (1 ng/μl) and gene copy number was quantified after 10-fold dilution.
▲Figure 3: Target gene copy concentrations of single (circle) and triple (triangle) assays are plotted against a 1 ng/μl MMC-DNA background.
The identity line indicates the position where the x- and y-coordinates of each point are equal.
The article uses the naica® droplet chip digital PCR system to complete quantitative detection of multiple dominant strains.
naica®Microdroplet chip digital PCR system
The naica® microdroplet chip digital PCR system from Stilla Technologies in France has unique advantages in nucleic acid testing. The system uses the cutting-edge microfluidic innovative chip - Sapphire chip (or high-throughput Opal chip) as a consumable for the digital PCR process. The sample enters the 2D chip in the form of 30,000 microdroplets through the capillary channel grid. The 3-color fluorescence detection instrument only takes two and a half hours for the entire process, and data quality control and result traceability analysis can be performed, and the data obtained is true and reliable.
naica®Six-channel droplet chip digital PCR system
The naica® six-channel droplet chip digital PCR system from Stilla Technologies of France is based on the Crystal droplet chip digital PCR technology. It automates droplet generation and amplification. Each sample well can detect 6 fluorescent channels, intelligently identify droplets and perform quality control. The absolute copy number concentration of at least 6 target genes can be obtained within 3 hours.
