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Sample Term Paper on Molecular Methods for Identifying Microbes

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Sample Term Paper on Molecular Methods for Identifying Microbes

There has been an explosion in the advancement and application of molecular methods for identifying microbes as well as their activities in the recent years. These methods are gradually more applied in lactic acid bacteria (LAB) strains comprising of those applied in fermentation and those marketed as probiotics for recognition and study of their activities. Most of these methods are built on 16S rDNA successions and exploits of PCR hybridization techniques. In addition, partial or complete genomes of some bifidobacteria and LAB have been established and offer omics based approaches to study the activity of the microbes given that the mechanism of their actions are known. Lastly, fluorescent probes joined to flow cytometry are applied to examine the physiological capacity of microbe cells in situ. These methods can be applied in selection as well as screening of the LAB, evaluating their function in taste and fermentation formation in fermented products. Other probiotic LAB features comprise of their vigor and feasibility during processing and study of their performance, persistence as well as presence in the gastrointestinal tract. An outline of these methods is presented as well as definite examples of their purpose in lactic cultures are provided (Borneman, Skroch, & Jansen, 2012).

Most of the current molecular methods are rooted on 165S ribosomal DNA series, partial or full genomes, or particular fluorescent probes, which supervise the physiological microbial cells activity. These high throughput techniques are largely used in strains of LAB and bifidobacteria, which provide benefits on health and are marketed as probiotic bacteria. The main intention for these approaches is to present better strain recognition as needed for good and legal manufacturing practices. Additionally, these identification methods can be applied in tracing and tracking LAB comprising of probiotics in the development phase and foodstuff products and after eating in the intestinal tract. Likewise, most of these identification methods can be influential in the selection of other species or strains of LAB or the bifidobacteria as appetizers, for taste developments or the probiotics that develop from them. There are also sequences of functional techniques being formed and validated which can further be applied in controlling quality. They are either built on general microbial properties or purposely address the technique by which probiotics and LAB can exert their beneficial or functional effects. In this case, the general systems are used to supervise the vitality, stress or viability reaction of LAB in bioreactors, food products or during fermentation, while the exact ones have the capability to establish the probiotic microbe performance in every of these system as well as in the consumer’s tract of the intestine (Fredrick, Tina & Marrazzo, 2005).

The methods that are available for indentifying microbes as well as studying their activities can be categorized into those found on nucleic acids as well as other macromolecules and the approaches aimed at studying the activity of entire cells. These nucleic acid based methods are frequently used than the rest because of their high throughput capability provided by the use of PCR magnification or rather in situ hybridization with RNA, DNA or peptide nucleic acid probes. Normally, these comprise of 16S rDNA series that may be used to keep diagnostics into a phylogenic structure and can be connected to database that provide about 100,000 sequences. The 16S rDNA based methods are usually superior and robust than traditional approaches based on phenotypic methods that are regularly undependable and do not have the resolving ability to study the bacterial composition as well as activity of microbial populations. Additionally, panoply methods that are built on DNA series rather than rDNAs, has been used repeatedly to probiotic microbe (Hermie, Gerwin & Jacques, 2000).   These have been revealed to be useful especially for strain identification as discussed bellow.

Recently, techniques based on partial or complete genomes have been initiated in the food production. These comprise of DNA arrays which can be employed in a relative genomics or genome wide expression profiling. These omics techniques have currently become viable for probiotic microbes because of the latest understanding of the whole human genome series segregates of Lactobacillus plantarum and the Bifidobacterium longum. There are other functional techniques based on other macromolecules properties like proteins. Surprisingly, the relation with the partial or whole genomes provides the root for further growth of proteomics as well as other omics related techniques to discover, identify and study the functionality of bifidobacteria and LAB. Additionally, to study the collective group of microbes or their individual group in a LAB strain, the entire cells can be targeted too. This provides the possibility of studying the physiological properties of undamaged cells in situ by use of fluorescently labeled substrates or probes together with high throughput techniques, for instance, flow cytometry. These approaches are especially useful in providing information regarding stresses and feasibility in lactic groups (Schaad, Jones & Chun, 2001).

Over the past years, hybridizations with ribosomal RNA targeted analysis have offered a distinctive insight towards the spatiotemporal dynamics and structure of complex microbial species. Nucleic acid analysis may be planned to particularly aim at taxonomic sets at diverse levels of specificity, by the virtue of valuable evolutionary preservation of the ribosomal RNA molecules. Nucleic acid analysis is built on two main approaches, that is, the entire cell in situ hybridization as well as dot-blot hybridization. The latter is an earlier situ approach in which a whole RNA is extracted from the sample and is immobilized on a membrane with combination of RNAs series of reference trains. Consequently, the membrane gets hybridized with a radioactively marked probe and after rigorous washing, the amount of targeted ribosomal RNA is quantified. Since cellular ribosomal RNA content is depends on the cells physiological activity, there is no direct evaluation of the cell counts that can be obtained (Borneman, Skroch, & Jansen, 2012).

Genotyping is a molecular typing technique that has been developed to identify and classify bacteria at the strain level. DNA fingerprinting technique is the most powerful generic based molecular approaches, which have been used widely for the LAB genotyping and bifidobacteria as well as infraspecific identification from human gastrointestinal tract and fermented food products. Mainly, these methods depend on the DNA polymorphism detection between strains and species and vary in their dynamic range of taxonomic inequitable power, simplicity of interpretation, reproducibility and standardization (Fredrick, Tina & Marrazzo, 2005).

Pulse field gel electrophoresis (PFGE) is another method for identifying microbes. Restriction fragment length polymorphism (RFLP) study of microbial DNA involves genomic DNA digestion with rare cutting enzymes to give way few relatively larger fragments. These restriction fragments are later size fractioned by use of PFGE that facilitates separation of big genomic fragments. The fingerprint of DNA produced relies on the specificity of the applied control enzyme and the series of microbial genome, which is attribute of a specific strain or group of microbe. The fingerprint stands for the whole genome, hence can detect certain changes such as DNA deletion, rearrangements or insertions, within a certain strain over time (Weston-Hafer, 2006).

Ribotyping is the other molecular technique used to indentify microbes. It represents the conventional RFLP analysis variation. It unites the DNA fingerprints’ Southern hybridization formed from the analysis of electrophoretic of the genomic DNA digest within ribosomal DNA targeted probing. This method uses probes that differ from partial series of the intergenic spacer sections or the ribosomal DNA genes to the entire ribosomal DNA operon. Ribotyping method has been applied to differentiate between the bifidobacterium and the Lactobacillus strains from human being fecal samples and from the business products. Nevertheless, ribotyping method provides a higher inequitable influence at the species and subspecies stage as compared to the strain stage (Gozalez, 2005).

Another method is known as arbitrarily amplified polymorphic DNA and is also referred to as RAPD. It has been extensively reported as a sensitive, fast and reasonably priced method for hereditary typing of diverse strains of bifidobacteria and LAB. This approach uses random primers that are capable of binding under low rigidity to a number of perfectly or partially complementary series of unknown site in the genome of a creature. When binding locations take place in orientation and spacing that facilitate enlargement of DNA sections, fingerprints samples are created that are unique to every strain. RAPD profiling is being used to differentiate between the strains of Lb. acidophilus and Bifidobacterium as well as other related strains. There are several factors that have been discovered to influence the inequitable and reproducibility power of the RAPD fingerprints, that is, annealing temperature, primer combinations and DNA template concentration. The analysis revealed that the use of five single primer reactions in an optimized state improved the accuracy and resolution of the RAPD approach for the classification of daily related bifidobacteria that include B. adolescentis, B. bifidum, B. animalis, B. breve, B. longum and B. infantis (Weston-Hafer, 2006).

 

References

Borneman, P., Skroch, M. & Jansen, J. (2012).Molecular microbial diversity of an agricultural soil in Wisconsin. 

Fredrick, N., Tina, L. & Marrazzo, D. (2005). Molecular Identification of Bacteria Associated with Bacterial Vaginosis. 

Gozalez, A. (2005). Application of molecular techniques for identification and enumeration of acetic acid bacteria.

Hermie, J., Gerwin, C. & Jacques, G. (2000). Analysis of Intestinal Flora Development in Breast-Fed and Formula-Fed Infants by Using Molecular Identification and Detection Methods. 

Schaad, N. Jones, B. & Chun, W. (2001). Laboratory Guide for Identification of Plant Pathogenic Bacteria, Third Edition.

Weston-Hafer, K. (2006). Identifying Unknown Bacteria Using Biochemical and Molecular Methods. 

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