Introduction to Microbial Identification
Microbiology research revolves around the detection and identification of microorganisms. It can be used in a variety of fields, including the environment, industry, and medicine. Microbial detection and identification techniques have evolved over time, from conventional methods that are based heavily on culture, morphology, physiology, pathology, and biochemical testing, which are time-consuming and labor-intensive, to advanced mass spectrometry and genetic technologies, which have resulted to the recent surge in microbiology studies.
Whole-Genome Sequencing
Whole-genome sequencing offers a structured knowledge of the overall genome, which not only defines the likelihood that genetic material comes from an individual or group, but also implies additional information on genetic relationships, origin, and disease susceptibility. Alignment, variant calling, and annotation are the main steps in a whole-genome sequencing workflow and calculating related metrics.
Ribotyping
Ribotyping is a phylogenetic analysis based on rRNA for bacterial identification and classification. Because rRNA genes (such as 16S rRNA) are highly conserved within bacterial species, classifying 16S rRNA gene polymorphisms reflects the bacterial species’ evolutionary lineage and can show light on bacterial classification, taxonomy, and epidemiological investigation, and population biology.
DNA Fingerprinting
RAPD-PCR: Short primers (8-12 nucleotides long) with arbitrary sequences bind nonspecifically to template bacterial DNA, resulting in amplification of random, repetitive areas of template DNA, resulting in a unique profile for bacterial identification.
RFLP: Restriction fragment length polymorphism (RFLP) is a technique for assessing bacterial strains that depend on the existence of variants in homologous DNA sequences to create unique fingerprints. Restriction enzymes, which can identify and cut amplified DNA (PCR product) into DNA fragments of various lengths, are used in this PCR-based method. Individuals, populations, and species are distinguished by electrophoresis of enzyme digests, which can also be used to identify the sites of genes within a sequence.
AFLP: Amplified fragment length polymorphism (AFLP) fragments genomic DNA with restriction enzymes (usually a pair) before amplifying a subset of restriction fragments with ligated adaptors. Primers that are complementary to the adaptor sequences but also have some unique nucleotides are used to accomplish this amplification. As a result, only a small proportion of restriction fragments are amplified selectively. After that, gel electrophoresis is used to separate the AFLP fingerprints, resulting in a set of distinct DNA fragments from a single bacterial genomic DNA sample with high specificity and discriminatory potential in the absence of any prior genome sequence understanding.
Real-Time PCR
Real-time polymerase chain reaction (real-time PCR), also known as quantitative polymerase chain reaction (qPCR), is a common technique used in biology labs to amplify and supervise targeted DNA molecules while simultaneously quantifying or semi-quantifying them.
Metagenomics
Metagenomics is a culture-independent technology that can reveal information about a microbial community’s collective genome, revealing microbial diversity and ecology in specific environments. Next-generation sequencing, which reads billions of DNA base pairs in a single run, is commonly used in metagenomics. Microbial metagenomics provides a solid foundation for the layout and analysis of omics studies of microbiome characteristics because it contains a wealth of genetic data.
References:
1. Patil-Joshi A, Rangaswamy BE, Apte-Deshpande A. based PCR method development, validation and application for microbial detection. Journal of Genetic Engineering and Biotechnology. 2021 Dec;19(1).
2. Ribarits A, Eckerstorfer M, Simon S, Stepanek W. Genome-edited plants: Opportunities and challenges for an anticipatory detection and identification framework. Foods. 2021 Feb;10(2).
