Date Published: April 2, 2018
Publisher: Springer Berlin Heidelberg
Author(s): Zhuoran Han, Jingfeng Sun, Aijun Lv, YeongYik Sung, Xueliang Sun, Hongyue Shi, Xiucai Hu, Anli Wang, Kezhi Xing.
A modified genomic DNA extraction method named the combination of lysozyme and ultrasonic lysis (CLU) method was used to analyze the fish intestinal microflora. In this method, the physical disruption and chemical lysis steps were combined, and some parameters in the key steps were adjusted. In addition, the results obtained by this method were compared with the results obtained by the Zirmil-beating cell disruption method and the QIAamp Fast DNA Stool Mini Kit. The OD260/OD280 ratio and concentration of the DNA extracted using the CLU method were 2.02 and 282.8 µg/µL, respectively; when the incubation temperatures for lysozyme and RNase were adjusted to 37 °C, those values were 2.08 and 309.8 µg/µL, respectively. On the agarose gel, a major high-intensity, discrete band of more than 10 kb was found for the CLU method. However, the smearing intensity of degraded DNA was lower when the incubation temperatures were 60 °C for lysozyme and 30 °C for RNase than when incubation temperatures of 37 °C for lysozyme and 37 °C for RNase were used. The V3 variable region of the prokaryotic 16S rDNA was amplified, and an approximately 600-bp fragment was observed when the DNA extracted using the CLU method was used as a template. The CLU method is simple and cost effective, and it yields high-quality, unsheared, high-molecular-weight DNA, which is comparable to that obtained with a commercially available kit. The extracted DNA has potential for applications in critical molecular biology techniques.
The gut microbiota is associated with many key functions of the host, such as resistance to infectious diseases and the decomposition of nutrients, and it provides the host with physiologically active materials, such as enzymes, amino acids and vitamins (Sugita et al. 1997). An altered microbiota in the intestine can lead to altered host immune function, as well as an increased risk of disease (Brown et al. 2012; Morgan et al. 2012). For fish, the gut microbiota also plays important roles in health and physiology (Ganguly and Prasad 2011). Over the last decade, investigations of the intestinal microbiota of fish have aimed to study its significant biological functions and make use of probiotic bacteria (Gatesoupe 1999; Narrowe et al. 2015; Xia et al. 2014). In addition, these studies were expected to contribute in a meaningful way to enhancing immunity and reducing mortality in cultured fish.
Several protocols for extracting DNA from the fish intestinal microflora, including physical and chemical methods, have been described. Generally, common physical disruption methods have been employed, such as freezing–thawing (Fan et al. 2014), sonication (Yang et al. 2006) and bead beating (Carrigg et al. 2008). In addition, a variety of chemical lysis approaches, including cetyl trimethyl ammonium bromide (Chapela et al. 2007) and Triton X-100 (Wang et al. 2012), have been used to obtain higher purity DNA. Many methods for DNA extraction employ lysozyme and proteinase K to quicken the process and increase the DNA yield. However, the incubation temperatures in the key steps are vital for the extraction process due to their influence on the activity of the enzymes used.