A universal PCR method and its application in sequence-based identification of microorganisms in dairy
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.
Section snippets
Primers
The primers used in this experiment are shown in Table 1. EU27F and 1492R (Frank et al., 2008, Magray et al., 2011) were the primers of 16S rRNA for prokaryotic microorganisms; NL-1F and NL-4R were the primers of rRNA for eukaryotic microorganisms (Kurtzman & Robnett, 1998). Semi-random primers (AP1 and AP2) consisted of 3 components (5’–3’): DQW or 360 primer sequences, 6 bases of degenerate sequence and 7 fixed bases. DQW and 360 primer sequences were not microbial sequences.
Sample collection and DNA extraction
The known strains
Identification of the known strains
All the 36 selected known strains of common microorganisms in dairy belong to 25 species, including 22 prokaryotic and 3 eukaryotic microorganisms. Primers AP1 and AP2 were used to obtain the gene sequences of different strains. At the same time, primers EU27F and 1492R were used to obtain the 16S rRNA gene sequences of prokaryotic microorganisms, and NL-1F and NL-4R were used to obtain the 26S rRNA D1/D2 sequences of eukaryotic microorganisms. The gel electrophoresis results of the universal
Discussion
Compared with the ordinary PCR methods, the universal PCR has a higher and stronger specificity. The primers AP1 and AP2 consist of three parts (5′–3′): (1) sequencing primer, which can increase the pairing length between the primers and the target sequence, and is helpful to form a panhandle structure between the same primers (AP1-AP1 or AP2-AP2) to avoid the nonspecific amplification; (2) six degenerate bases, which can add more complementary sites to increase annealing temperature (about
Acknowledgements
This work was supported by the National Key R&D Program of China (2017YFC1600404), the Program of Shanghai Subject Chief Scientist (15XD1520300) and Minhang District Science and Technology Development Funds (2016MH237).
References (39)
- et al.
Utilization of elongation factor Tu gene (tuf) sequencing and species-specific PCR (SS-PCR) for the molecular identification of Acetobacter species complex
Molecular and Cellular Probes
(2014) - et al.
Metagenomic assessment of the functional potential of the rumen microbiome in Holstein dairy cows
Anaerobe
(2016) - et al.
Construction of a dairy microbial genome catalog opens new perspectives for the metagenomic analysis of dairy fermented products
BMC Genomics
(2014) - et al.
Specific signatures of the gut microbiota and increased levels of butyrate in children treated with fermented cow's milk containing heat-killed Lactobacillus paracasei CBA L74
Applied and Environmental Microbiology
(2017) Construction of soil environmental DNA cosmid libraries and screening for clones that produce biologically active small molecules
Nature Protocols
(2007)- et al.
Extremely rapid extraction of DNA from bacteria and yeasts
Biotechnology Letters
(2006) - et al.
Recombinant environmental libraries provide access to microbial diversity for drug discovery from natural products
Applied and Environmental Microbiology
(2003) - et al.
Gene walking by unpredictably primed PCR
Nucleic Acids Research
(1994) - et al.
Impacts of seasonal housing and teat preparation on raw milk microbiota: A high-throughput sequencing study
Applied and Environmental Microbiology
(2016) - et al.
Next-generation and whole-genome sequencing in the diagnostic clinical microbiology laboratory
European Journal of Clinical Microbiology & Infectious Diseases
(2012)
Next-generation sequencing as a powerful motor for advances in the biological and environmental sciences
Genetica
Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes
Applied and Environmental Microbiology
Systematic improvement of amplicon marker gene methods for increased accuracy in microbiome studies
Nature Biotechnology
Mapping RNA regions in eukaryotic ribosomes that are accessible to methidiumpropyl-EDTA.Fe(II) and EDTA.Fe(II)
Biochemistry
Coamplification of eukaryotic DNA with 16S rRNA gene-based PCR primers: Possible consequences for population fingerprinting of complex microbial communities
Current Microbiology
High genetic similarity of Streptococcus agalactiae and Streptococcus difficilis: S. difficilis Eldar et al. 1995 is a later synonym of S. agalactiae Lehmann and Neumann 1896 (Approved Lists 1980)
International Journal of Systematic and Evolutionary Microbiology
Evaluation of commercial universal rRNA gene PCR plus sequencing tests for identification of bacteria and fungi associated with infectious endocarditis
Journal of Clinical Microbiology
Irradiation in combination with higher storage temperatures maintains chip-making quality of potato
Journal of Food Science
Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences
Antonie Van Leeuwenhoek
Cited by (3)
Three phylogenetically distinct and culturable diazotrophs are perennial symbionts of leaf-cutting ants
2021, Ecology and EvolutionExtracellular and intracellular DNA for bacterial profiling of long-ripened cheeses
2020, FEMS Microbiology LettersMicrobial contamination level and microbial diversity of occupational environmentin commercial and traditional dairy plants
2019, Annals of Agricultural and Environmental Medicine