PCR-denaturing gradient gel electrophoresis

PCR-denaturing gradient gel electrophoresis (PCR-DGGE) involves amplification of DNA using PCR with genus-specific primers that target 16S rDNA sequences from bacteria. Following amplification, the DNA products are separated using electrophoresis on a DGGE gel. Once separated, the segments are removed from the gel, purified, and sequenced. Then the resultant sequence is compared to a very comprehensive database and the identity given. This technique is based on the premise that the sequence of the 16s rDNA fragment is unique for each species of bacteria.

Several researchers have used molecular techniques similar to PCR- DGGE to examine the microbial ecology of chronic wounds and have identified more than ten different species of bacteria in most chronic wound samples, including strict anaerobic bacteria not isolated on culture. Light and scanning electron microscopy techniques also used to analyse chronic and acute wound specimens have showed that 60% of chronic wounds contained a biofilm, whereas only 6% of acute wounds contained a biofilm (James et al. 2008).

There are numerous other molecular methods used in the research arena but these are not used in routine microbiology. This is because the techniques are either more expensive or more time-consuming compared to the traditional culture methods. Until these methods become more automated or less complex, they are unlikely to be introduced into routine diagnostic laboratories and will remain research tools.

Further reading

Ford M. Medical Microbiology (2nd ed). Oxford: Oxford University Press; 2014. Standard bacteriological procedures and methods are issued by Public Health England, and give details about specimen collection and transport, laboratory methods, culture media, and so on. They are available at the following Internet site: https://www.gov.uk/government/collections/ standards-for-microbiology-investigations-smi

References

Anonymous. Wound watch: three techniques for collecting wound specimens.

LPN2009. 2008. 4(1):24-25. Available from: http://www.nursingcenter.com/lnc/ static?pageid=811925

James GA, Swogger E, Wolcott R, et al. Biofilms in chronic wounds. Wound Repair Regen. 2008;16(1):37-44.

Levine NS, Lindberg RB, Mason AD, et al. The quantitative swab culture and smear: a quick simple method for determining the number of viable aerobic bacteria on open wounds. J Trauma. 1976;16(2):89-94.

 
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