A universal PCR method and its application in sequence-based identification of microorganisms in dairy

Abstract

To resolve problems with dairy product quality related to microbial contamination, it is necessary to be able to identify microorganisms in those dairy products quickly and accurately. With traditional PCR-based methods for microbial identification, specific primers need to be designed for the different microorganisms. In this study, we developed a universal PCR method with two semi-random primers to amplify microbial genomic DNA without considering the gene sequence of the test microorganism. The sequence obtained using this method was specific and could be used to identify the microorganism. The DNA sequences of known microorganisms related to dairy were amplified and identified to the species level, confirming the validity of this method. The DNA sequences of unknown common microorganisms were then amplified directly without culturing, and these microorganisms could also be identified to the species level. Compared with the common rRNA sequencing methods, this new method has high universality and resolution.

Introduction

Dairy products can encounter quality problems, frequently related to microbial contamination; in such cases, microbiological identification is required. The main microorganisms in dairy products are prokaryotic and eukaryotic microorganisms. However, the sequences of rRNA are different, and the identification methods are also different. Moreover, many of the microorganisms in dairy products are difficult to purify and culture; therefore, a quick sequencing method without culturing is highly advantageous. The common non-culturing methods for identification include sequencing of rRNA or other conserved genes, sequencing by clone library, metagenomic sequencing, and so on. Although all of these methods have been successfully applied, there are still some shortcomings.

Sequencing of rRNA or other conserved genes has been widely applied in microbial identification. Prokaryotic microorganisms mainly include 16S, 23S and 16S23S rRNA, whereas eukaryotic microorganisms mainly include 5S, 18S, 28S rRNA (26S for yeasts) and internal transcribed spacer region (ITS) (Han, Schepartz, Pellegrini, & Dervan, 1994). Since the rRNA sequences of eukaryotic and prokaryotic microorganisms are different (Huys et al., 2008, Kuhn et al., 2011, Tremblay et al., 2015), different primers need to be selected.

A major limitation of the rRNA sequencing method is its inability to discriminate some species among all microbiological taxa. For example, Bacillus cereus and Bacillus anthracis have identical 16S rDNA sequences and cannot be separated reliably, nor can Escherichia coli and Shigella flexneri or Bacillus subtilis and Bacillus tequilensis (Petti, 2007). Other conserved genes such as rpoB, gyrA, gyrB, sodA (Kawamura et al., 2005, Madhusudan et al., 1994, Randall et al., 2005), tuf (Huang, Chang, Huang, & Chu, 2014), are also used to identify microorganisms. All these methods require selection of specific primers for different microorganisms, and they have a lower universality (Huys et al., 2008, Kuhn et al., 2011, Tremblay et al., 2015); sometimes it is difficult to distinguish similar species and genus.

Sequencing by clone library has a high universality, but it is associated with a heavier workload, a higher cost, and a lower specificity (Brady, 2007, Courtois et al., 2003). It requires construction of plasmids and digestion of the restrictive endonucleases. Due to the poor specificity of the sequences, more sequences must be measured to identify the microbe.

In recent years, metagenomic sequencing has been increasingly used for research on dairy products. The composition (Almeida et al., 2014, Walsh et al., 2017) and dynamic changes of microorganisms (Doyle et al., 2016, Walsh et al., 2016) in dairy products, the effects of dairy products on intestinal bacteria (Berni Canani et al., 2017, Veiga et al., 2014) were studied. The commonly used methods were 16S rRNA and shotgun. The method of 16S rRNA is easy to operate, but it is difficult to distinguish the similar species; the primers are not universal, and deviation might occur for some bacteria when the primers are amplified. The method of shotgun metagenomics is universal and accurate (Dunne et al., 2012, Faure and Joly, 2015, Lim et al., 2014, Pitta et al., 2016), but time-consuming and complicated because the DNAs of the sample need to be cut and ligated to the oligonucleotide adapters for sequencing. Additionally, a long time can be required to gather a certain number of samples before experimentation and specialised software packages are also necessary to assemble and analyse the sequences. It can, however, be applied to the analysis of microbial flora and the species and abundance of the microorganisms can be determined.

Therefore, a convenient and universal method of sequencing is required. Common PCR methods normally require the design of two primers according to the target sequence, although walking PCR only needs one primer to be designed according to the target sequence (Dominguez and Lopez-Larrea, 1994, Parker et al., 1991, Siebert et al., 1995). In this study we developed a universal PCR method based on the previous walking PCR method (Zhang et al., 2014). All microbial DNAs could be amplified with just two universal primers, regardless of the sequences of the target gene. The sequences we obtained had a good specificity, and most of the microorganisms in dairy could be identified to the species level.

Dairy product bacteria are Gram+ve and Gram-ve; eukaryotic microorganisms include fungi and yeasts. Because of the complex microbial composition, the methods of DNA extraction are different according to the different structures of cell walls (Cheng and Jiang, 2006, Parrish and Greenberg, 1995, Stirling, 2003, Tapia-Tussell et al., 2006). Sequencing identification needs to prejudge the species of microbes in the sample. Therefore, it was also necessary to find a universal microbial DNA extraction method to establish a universal sequencing identification method.