Abstract
In this research, the detection method for absolute quantification of total coliforms was established based on Droplet Digital Polymerase Chain Reaction (DDPCR) technology using lacZ as the target gene for coliform group detection. The experimental conditions (e.g. primer and probe concentrations, annealing temperatures, etc) were well optimized. Besides, the linear range, precision and limit of quantification (LOQ) of this method were investigated and evaluated. The results illustrated that the optimal primer concentration was 0.2 μmol/L, whereas the optimal probe concentration was 0.5 μmol/L. The optimal annealing temperature was 56°C. The linear relationship between the total coliform genome DNA concentrations derived from DDPCR and DNA fluorometer was quite good (R2 = 0.999). The linear range was 3.95 ~ 7.80 × 104 copies/20 μL DDPCR reaction system. The LOQ for total coliforms was single copy per reaction system. Practical applications using real water samples collected from water supply system in Macao illustrated that this innovative method possessed high efficiencies and capabilities. This is probably the first research using DDPCR technology to absolutely qualify and quantify total coliforms and successfully applied it in Macao water supply system. The achievements from this research could provide with significant values for setting-up the emergency mechanism of water pollution in early stage.
Highlights
Rapid economic development and its corresponding population explosion are putting incredible strains on our environment
The Droplet digital PCR (DDPCR) detection method for absolute quantification of total coliforms in Macao water supply system was established by selection of lacZ as the target gene for coliform group detection
The positive fluorescent amplitude increased as the probe concentration increased, more lacZ genome DNA was amplified by DDPCR method
Summary
Rapid economic development and its corresponding population explosion are putting incredible strains on our environment. Recognized total coliforms detection methods are mainly membrane filtration [7, 8], multiple-tube fermentation [9, 10], immunomagnetic separation [11, 12], etc Even though these traditional methods have been regarded as the “Golden Standards” worldwide, they pose disadvantages such as longer detection period (several days or a dozen of days), tedious operation procedures (requirement for verification experiments), failure to detect damaged microbes, lower sensitivity and potential biohazards, etc [13,14,15,16]. It has been the innovation “hotspot” among water industries [17,18,19,20]
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