Identifying the causative agent of infection is essential for successful diagnosis, targeted antibiotic treatment and epidemiological investigations (Mullis and Faloona 1987).

Universal PCR -Biomedical Sciences

Identifying the causative agent of infection is essential for successful diagnosis, targeted antibiotic treatment and epidemiological investigations (Mullis and Faloona 1987). This is known as microbial strain typing. Most of the microorganisms are slow-growing or non-cultivable microorganisms and thus make their identification difficult. The process of genotyping using molecular based techniques provides an alternative that allows the identification of the entire microorganism based on their genetic constituents (Tenover et al 1997). In this review, I attempt to sum up the basic principle of non-specific molecular analyses such as Degenerate PCR and its use in diagnostic and research laboratories such as geno¬typing of bacterial species.
The process of bacterial strain identification is sequential and begins with the PCR where degenerate primers amplify the unknown sample sequence followed by the DNA sequencing using the Sangar methods. Pyrosequencing and next generation sequencing are faster and cheaper than traditional Sanger’s dideoxy sequencing. Once the sequence is achieved, BLAST analysis is necessary to find out the identity of the unknown gene as well as the unknown organism.
In Polymerase Chain Reaction, a primer is used to bind the DNA to start replication which will amplify a specific piece of DNA. The primer have to be designed to bind specifically to that piece of the DNA. In contrast, in degenerate PCR, one uses primers that bind to many different sequences. This is accomplished by degenerate base pairing with deoxyinosine and using a low annealing temperature in the first two or three cycles.
The primers are often referred to as universal primers. It is also reported that single universal primer pair are unable to target all bacteria as different conserve sequences are distributed in different fractions of a community. The choice of primer pair has an impact on the bacterial species that are being detected from microbial community (Fredriksson et al 2013).
Universal-PCR protocol
The degenerate PCR is pretty standard, with the main difference from standard PCR being that a higher concentration of primer is used. 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.

Table 1: Protocol for DOP PCR set up stage on ice (Ha˚kanTelenius et al., 1992).
1. Set up PCR, on ice:
Materials Concentration in µl
genomic DNA 50-100

10 x PCR buffer 2.5
MgCl2 (25 mM) 1-2.5
dNTPs (10 mM) 0.5
each primer (100 mM) 0.5
dH2O 24.5
Taq 0.25

Table 2: Protocol for DOP-PCR (Running phase) (Ha˚kanTelenius et al., 1992).

2. Run PCR:
Temperature in Celsius Duration in seconds
94 120 (add Taqpol for the hot-start)
then 35 rounds of:
94 30
45-55 30
72 60
followed by:
72 600

Material and Method
DNA sequencing is a must after the amplification is done to produce the DNA map of the unknown organism that can be used in BLAST analysis to identify the organism(Prasad and Turner, 2011 Pyrosequencing method requires primers and four enzymes. Pyrophosphate is released when dNTPs are added into the DNA strand by klenow Polymerase. ATP sulfurylase converts pyro¬phosphate into ATP while Luciferase produces light by utilizing the generated ATP. The nucleotides are added one at a time and the fluorescence detector detects the produced light as evidence of the incorporated nucleotide while generating a map based on nucleotide type (Ahmedian 2006).
DNA pyrosequencing technology is more effective than conventional biochemical processes in identifying 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.
Application of non-specific DNA based identification
Using DNA based methods, organisms responsible for bacteria can now directly be identiﬁed from the blood by ampliﬁcation of bacterial 23S rDNA. Extensive range of clinically signiﬁcant bacterial species can be identified. The method can identify mixtures of organisms as a group when applied directly to positive blood culture. (Anthony et al., 2000).Using 16S rDNA almost 72 bacterial species both gram negative and positive bacteria are identified by comparison with known sequences deposited at Genbank or other databases. The universal nature of the primers and the sensitivity of detecting the presence of low copy number targets allows for identification of novel pathogens.
RNA-dependent RNA polymerase and Nsp14 are used for generating broad spectrum primers allowing for diverse identification of coronavirus species hence a technique to identify rapidly growing viruses. Universal method using primers is preferred over the specific primer for routine practices. The single target PCR and the phylogenetic relatedness of a new emerging organism cannot be evaluated currently as it depends on the region. However, the use of universal primers based upon the conserved sequences that slowly evolve can help in identification of the relatedness and evolution of the emerging pathogens.
Degenerate primer amplifies more sequences in the genome compared to the regular PCR hence DOP-PCR is used in amplification of multiple loci. It can also be used in chromosome painting and amplification of large sections of the genome. Moreover, its species-independent nature is also used to amplify DNA sequences of uncharacterized origin. DOP-PCR assay is also used to identify DNA or RNA and viruses, either directly from biological specimens or from cell culture due to its ability to amplify viral sequences without prior knowledge of those sequences. This makes it capable of determining the novel or mutated viruses. Faster diagnosis that could prompt for appropriate treatment of the systemic mycoses where delays can lead to life threatening fatalities is therefore possible.
Furthermore, DOP-PCR enables complete genome coverage in a one reaction generating microgram quantities of genome-representative DNA from the starting material in amounts of nonograms or pictograms. This methods have been proposed for forensic, taxonomic and cancer research.

CONCLUSION
Degeneracy is the most vital component of microbe typing methods. When combined with the low initial annealing temperature in PCR protocol, it ensures that there is priming from evenly dispersed multiple sites within a given genome. The process is also independent of the species which gives it an advantage over other PCR methods which cannot detect non-target pathogens. For using specific primer based DNA technology we need to have an understanding of the gene sequence of the organism which is not required in case of non-specific techniques like DOP PCR. That is why we can use these non-specific DNA technologies to identify unknown species of bacteria, virus or fungus.

References
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