Free Essay About Molecular Identification Of Unknown Organism

Medicine

Introduction
Identifying the causative agent of infection is essential for successful diagnosis, targeted antibiotic treatment and epidemiological investigations (Mullis and Faloona, 1987). Most of the microorganisms are slow-growing or nonculturable microorganisms and thus make their identification difficult (Mullis and Faloona, 1987; Li et al, 2008). The process of genotyping using molecular based techniques provide a suitable alternate and allow the identification of the entire microorganism based on their genetic constituents (Tenover et al, 1997). Nonspecific molecular analysis using broad range PCR is based on the conserved sequence thus detect any kind of bacterial DNA present in a sample through targeting conserved bacterial sequences followed by the modern sequencing techniques efficiently and cost effectively (Zucol et al., 2006). The process of bacterial strain identification is sequential and focused to some important molecular techniques. The process begins with the PCR with degenerate primers to amplify the unknown sample sequence followed by the DNA sequencing with Sangar method8 (Sanger et al., 1977), pyrosequencing (Ahmedian, 2006) or next generation sequencing (Metzker 2010). Pyosequencing and next generation sequencing are much faster and less expensive than traditional Sanger’s dideoxy sequencing (Metzker 2010). Once the sequence is achieved, BLAST analysis is necessary to find out the identity of the unknown gene as well as the unknown organism (Prasad and Turner, 2011).
In Polymerase Chain Reaction (PCR), we need to use a primer to bind to the DNA to start the DNA replication which will amplify a specific piece of DNA, so we need to design your primers to only bind around that piece of DNA. This is accomplished by degenerate base pairing with deoxyinosine (can bind to any other bases) and using a low annealing temperature (stabilizes the binding of the primer to the DNA) in the first two or three cycles (Jensen et al, 1993).
The conserved regions are the target for designing PCR primer pairs for amplifying many bacterial species. The primers are often referred to as universal primers. The choice of primer pair have an impact on the bacterial species that are being detected from microbial community (Fredriksson et al., 2013). The 16S rRNA gene possesses several conserved regions common to diverse of bacterial species along with variable regions shared by fewer species. Targeting the 23S rRNA gene conserved sequences can be used in minority of cases compared to 16S rRNA gene (Zucol et al., 2006). Conserved regions in genes other than 16rRNA are used includes RNA polymerase beta subunit (rpoB), gyrase beta subunit (gyrB), recombinase A (recA), and heat shock protein (hsp60) for microbial investigations and differentiation of bacterial species (Ghebremedhin et al. 2008). Various web based sources and software are available for selection of appropriate primer such as CODEHOP, Gene Fisher and Primo Degenerate 3.4 (Abd-Elsalam, 2003). Using the degenerate primer suggests the use of a population of specific primers covering all the possible combinations of nucleotide sequences coding for assumed protein sequence (Iserte et al., 2013).

Universal-PCR protocol

The concentration of MgCl2 and the annealing temperature may be varied to optimize the reaction. The extension time may sometimes need to be increased for larger fragments. A hot-start reaction is required to reduce non-specific annealing of primers For example the Taq polymerase is not added until the PCR tubes have been heated to 94ºC (Ha˚kan Telenius et al., 1992).

Material and Method

DNA sequencing is essential, which will produce the DNA map of the unknown organism which can then be used in BLAST analysis to identify the unknown organism (Prasad and Turner, 2011). Pyrosequencing method requires primers and four enzymes for the pyrosequencing system. Those are klenow fragment of DNA polymerase I, ATP sulfurylase, Luciferase, and Apyrase. ATP sulfurylase converts the pyrophosphate into ATP and Luciferase produce light by utilizing the generated ATP.
DNA pyrosequencing technology is found to be much more effective than conventional biochemical processes to identify bacterial pathogens. The assay is based on the highly conserved nature of 16S rRNA genes by placing amplification and sequencing the primers in conserved target sequences flanking the variable V1 and V3 domains (Luna RA, 2007).
The last stage of strain typing is the DNA analysis using bioinformatics like running the BLAST algorithm to search for the DNA sequence match in databases to find DNA similarities with other bacteria (Lobo, 2008).

Application of non-specific DNA based identification

The PCR highly valuable technique, pathogen identification and determining their relationship based on genetic sequence. The method directly to positive blood culture broths and can identify mixtures of organisms. Enterobacteriaceae and Proteus, Klebsiella, Citrobacter, Enterobacter, Serratia, and Salmonella were identified as group (Anthony et al., 2000).
Using 16S rDNA almost 72 bacterial species both gram negative and positive bacteria including streptococcus, staphylococcus, listeria monocytogenes, Haemophilus influenza, Mycobacterium gordonae etc. are identified by comparison with known sequences deposited at Genbank or other databases (Negoro et al., 2013). Novel pathogens can be discovered due to universal nature of probes/primers. Thus, the high sensitivity for identifying the presence of short copy number objectives (Procop, 2007).
RNA-dependent RNA polymerase (RdRp) and Nsp14 are used for generating broad spectrum primers for coronavirus. This technique thus presents the way to identify rapidly emerging pathogens (Sampath, 2005).
Universal primers are on the conserved sequences, which evolve gradually overtime (Jackson, 2006). Nonspecific molecular technique may be able to classify antibiotic resistance because of the transmutations in preserved regions like gyrA/B (Fluit, 2001). Thus, it identifies β-lactam and aminoglycoside resistance genetic factor in gut microbiota of the healthy adults that serve as basin for these genes (Fouhy et al., 2014). It can be used so as to strengthen DNA orders of unknown origin since DOP-PCR is naturally species-independent (Ha˚kan Telenius et al., 1992; Panneerchelvam, 2003).
Eleven bacteria that often cause prosthetic infections may be identified quickly through universal PCR (Sauer, J. et al, 2005). A DOP-PCR feature has its capacity to increase viral sequences minus the previous sequences knowledge, which will increase the probability of novel viruses (Uhlenhaut, McClenahan, and Krause, 2011).
A general primer with NL-4 and ITS-1 combination that rims the D1/D2 and ITS regions may determine the unknown fungus identity with a total of 519, which is 390 mould and 129 yeast. Additionally, this primer may enable the prompt appropriate treatment and quick diagnosis for complete mycoses that late diagnosis may lead to life fatal consequences particularly in patients that are immunocompromised (Romanelli et al, 2014).
Furthermore, DOP-PCR enables complete genome exposure within a one reaction that generates microgram measures of genome-representative DNA from nanogram or picogram quantities (Nona Arneson et al, 2008). Thus, these approaches to amplify the entire genome were proposed for forensic, taxonomic, and cancer research (Lao k., et al, 2008).

CONCLUSION-

This process’ most beneficial feature is this system degeneracy. Along with low primary strengthening temperature within the PCR protocol, it ensures the priming starting from the multiple (e.g., approximately 10(5) in human) equally dispersed sites in a specified genome. Also, this process seems species-independent. Consequently, for the overall amplification of specified DNA, the degenerate PCR holds the advantages as to compare with normal PCR or multiplex PCR through specific primers that cannot detect the non-target pathogens.
When it comes to using the detailed primer based technology, it is essential to have knowledge of gene order of such organism. Therefore, we can use general DNA technologies in order to classify unknown bacteria species, fungi, or even virus (Sibley, et al., 2012; Pujol et al. 2005).

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