Background

Colony PCR is commonly used to verify the expected genome DNA in Escherichia coli, but it fails in Gram-positive bacteria such as Bacillus subtilis even when treated with chemical reagents (Packeiser et al., 2013; Azevedo et al., 2017). The reason colony PCR fails in these cases is the thick cell wall that exists in most Gram-positive bacteria, which impedes cell lysis and subsequent DNA release. Enzymes such as lysozyme, proteinase K, and other chemical reagents are used to lyse the Gram-positive bacterial cell wall (Mertens et al., 2014); however, these methods are costly, time consuming, and not eco-friendly for the DNA extraction of a huge quantity of samples. For example, it is very difficult to achieve a fast screen towards thousands of mutants generated in random mutagenesis. It is therefore necessary to set up a convenient and economical method.

Sonication is a common physical method employed to disrupt cells by transmitting ultrasonic energy to the liquid region (Taylor et al., 2001). Sonication-based DNA extraction is considered as an efficient and cost-effective method. In the intestinal microflora and compost microbes of fish, the ultrasonic lysis method combined with lysozyme was used to extract DNA (Yang et al., 2006; Han et al., 2018). Moreover, direct extraction of DNA from sputum clinical sample (containing diverse microbes) for real-time PCR proved to be convenient and suitable (Bello et al., 2020). In this study, a sonication-based DNA extraction method was developed, which could screen large samples in just one day. Despite not being as pure as the commercial kit, this method can be used for high-throughput screening purposes, which requires extracting great amounts of DNA, in Gram-positive bacteria such as Bacillus cereus, Bacillus thuringiensis, Bacillus subtilis, and Listeria monocytogenes.