"Molecular Methods Detection and Identification of Micro-Organisms" is an interesting example of the applied research. Molecular techniques and methods have provided a significant tool in the evaluation of micro-organisms. There have been various techniques that have been developed over a long period of time in the evaluations of food as well as other micro-organisms. The techniques that have been developed provide solutions to a large variety of agents with the application of just one test. Additionally, more complex and accurate techniques that can be relied on are believed to be coming up as the days go.
Various sectors such as the water processing units, analytical laboratories as well as food industries are rigorous on the development of the molecular techniques. On the other hand, various molecular techniques will be used with respect to their characteristic traits. Some of the techniques are specially designed to meet certain conditions while others do not. On the contrary, some of the techniques are easy to use while others are complex. This write up will report some of the molecular detection techniques that are applicable in the identification of micro-organisms. PCR (Polymerase chain reaction) The polymerase chain reaction has been a powerful tool in the detection as well as the identification of micro-organisms.
The method identifies the micro-organism at the gene level. A scientist is known as Kary Mullis initially developed the technique in the year 1987. The technique has had a great shift in the manner of identification of microorganisms in diverse biological applications (Sivaganensan et. Al 2014). The technique involves the amplification of a single or many copies of the piece of DNA. The DNA in question can be copied transversely numerous orders of magnitudes.
The technique has been applied in DNA cloning that needs to be used in sequencing, phylogenetic studies based on DNA, genes functionalities, diagnostic of diseases, DNA fingerprint, and identification of microorganisms. The PCR technique functionality relies on thermal cycling. This consists of a series of cycles of heating and cooling that are in a continuous repetitive manner. The molecular technique has been very appropriate in the detection of micro-organisms. In fact, Campylobacter, which is the most common causative agent of bacterial gastroenteritis in the various parts of the world, was identified from meat using the PCR technique.
Additionally, the powerful technique has been used in the identification of the lactobacillus curvatures well as the Lactobacillus Sake has been identified by the Polymerase chain reaction (Sivaganensan et. Al 2014). Some of the techniques that are used at the species level, especially for a defined function are like in the hybridization techniques. These hybridization techniques can be utilized with DNA probes and RNA probes that are specific to the specie that is being determined. The techniques involve the identification of a particular region of the organism’ s genome.
These methods have always been used in the proof of identity of wine lactic acid bacteria. The techniques have also been used in the identification of the specific roles of the micro-organisms. Some of the methods are like; Northern blotting technique This is a molecular technique that is used in the identification of micro-organisms from the gene expression level. The method mainly utilizes the detection of the Ribose Nucleic acids (RNA) in a given sample of questions. The technique allows for the analysis of the cellular control of the structure as well as the functions of the specified gene, especially at the expression levels.
The technique further allows for the investigation of the expression levels of the gene of an organism through the determination of the mRNA; the messenger RNA in an organism is directly proportional to the genes that are responsible for the expression of the mRNA. Hence, the determination of the mRNA is important in the determination of the genes that are related to a particular organism. The process for the technique involves the RNA or the mRNA that has been collected to be separated by the process of gel electrophoresis.
The separation method usually involves running the RNA or the mRNA in an agarose gel (Cota GF et al. , 2012). The agarose gel is most preferred for this method because the particles are relatively large to be separated using the polyacrylamide gel. Otherwise of the particles would be small then the polyacrylamide gel would be preferred. After the separation then the RNA is then transferred to a sheet specially meant for blotting known as nitrocellulose membrane.
However, there are various types of membranes that can be used. The RNA transferred on the membrane is then incubated with the probe that has been designed with a complementary single strand of the desired gene. The mixture will then hybridize to form double-stranded fragments. The hybridization can be detected by the use of the probes that had been attached to the probes. The probes would either be radioactive or hence be determined using an x-ray film. The procedures for the hybridizations are the same for all the hybridization techniques except that the southern blotting is specific for DNA probes while in situ hybridization identification of the species takes place inside the cell. Restriction Polymorphism ribotyping This method involves the use of various techniques that have been combined for enhanced efficiency.
Some of the combined techniques are like PRC and electrophoresis. The method involves the use of the restriction enzymes that are obtained after the complete digestion of the DNA. The DNA fragments that have been obtained from the culture are then separated into different sizes with the technique of electrophoresis.
The separated DNA fragments are then hybridized with a single-stranded probe of a complementary sequence on nylon filament. The technique is relatively similar to the northern and southern blotting but the difference comes in that this technique is used in the identification of the very minute differentiation of the species genome. The differences might have been caused by mutations. These techniques can be used in the identification of mutations that even point to forms (Kudva et. Al. 2013). Another technique that can be used in the identifoac6tion of the micro-organisms in the molecular applications is the restriction genome pattern analysis by pulse-field gel electrophoresis.
The principle behind this technique is the digestion of the Genomic DNA. The digestion of the DNA is carried out using a restriction enzyme that has the characteristic of cutting the DNA at specified points. The whole length of the genomic DNA is cut into small fragments by the enzyme (Toyoizumi& Tsubouchi, 2013). This is done so that a clear, as well as comprehensive differentiation and identification of the various strains of the organisms, can be obtained.
The technique begins by collecting cultures of the suspected micro-organisms. This is done by centrifugation methods that separate the components of the culture on the basis of their weights. The cells of the micro-organisms are then lysed. This is then followed by DBA restriction for the whole genome. The method is carried out in blocks so as to facilitate the cutting of the DNA in specified regions and not in a random manner. The gel blocks are then transferred to an agarose gel.
The fragments are then separated using the PFGE electrophoresis. The fragments are separated into different sizes with the application of the pulsed electric (Rolph et al, 2011). There are various restriction endonucleases that can be used in the identification of specific strains of various organisms. Like the ApaI as well as the NotI have always been used in the identification of the various polymorphism traits in the O. oeni species. Additionally, the enzyme SfiI and the SmaI have been used in the identification of the various strains of Lactobacillus micro-organism. This technique has been very significant in the identification of micro-organisms at the strain level.
On the other hand, the technique provides assurance by the availability of two enzymes to be used for given specie. When there is doubt in the results that have been obtained then a second enzyme can be used. The technique has also been identified as the most appropriate in the analysis of starter survival as well as the establishment of species after the immunization of the malolactic fermentation (Rolph et al, 2011). However, this technique has one major downfall.
The technique is time-consuming. This is because of the initiation procedures that involve the cultures take a lot of time. This is because the cultures need to be carried out repeatedly over fresh cultures over and over. This is done in this manner so as to give the growing cells fresh mediums on which they grow on.
Cota GF, de Sousa MR, Demarqui FN, Rabello A (2012) The Diagnostic Accuracy of Serologic and Molecular Methods for Detecting Visceral Leishmaniasis in HIV Infected Patients: Meta-Analysis. PLoS Negl Trop Dis 6(5), 198-321.
Kudva, I. T., Smole, S., Griffin, R. W., Garren, J., Kalia, N., Murray, M., ...& Calderwood, S. B. (2013). Polymorphic Amplified Typing Sequences (PATS) Strain Typing System Accurately Discriminates a Set of Temporally and Spatially Disparate Escherichia coli O157 Isolates Associated with Human Infection. The open microbiology journal, 7, 123.
Rolph, H.J., Lennon, A., Riggio, M.P., Saunders, W.P., Mackenzie, D., Coldero, L. & Bagg, J. (2011). Molecular Identification of Microorganisms from Endodontic Infections.
Journal of Clinical Microbiology, 52(4), 126-348.
Sivaganensan, M., Siefring, S., Varma, M., &Haugland, R. A. (2014). Comparison of< i> Enterococcus quantitative polymerase chain reaction analysis results from Midwest US river samples using EPA Method 1611 and Method 1609 PCR reagents. Journal of Microbiological Methods.
Toyoizumi, H., &Tsubouchi, H. (2013, March).Analyzing Meiotic DSB Interference by Combining Southern Blotting and Microarray Analysis. In BIOTECHNO 2013, The Fifth International Conference on Bioinformatics, Bio computational Systems and Biotechnologies (pp. 44-47).