In addition to the applications in human genetic studies, DHPLC has been used in the investigation of microbes because the method is cost-effective and time-saving. One application is the characterization of drug resistance in different bacterial pathogens. DHPLC was first applied to mutation screening in Staphylococcus aureus.
The study showed that DHPLC provided a rapid detection platform of identifying the quinolone resistance alleles of gyrA, gyrB, grlA, and grlB genes. Similarly, mutations in the quinolone resistancedetermining regions of Salmonella enterica (gyrA, gyrB, parC, and parE) and Yersinia pestis (gyrA) could also be detected by DHPLC. Multiplex PCR together with DHPLC analysis has also been developed for the detection of plasmid-mediated ampC β-lactamase gene mutations in Gramnegative bacteria.
For the investigation of the drug-resistance genes of Mycobacterium tuberculosis, mutations were detected by DHPLC in six genes including katG, rpoB, emB, gyrA, pncA, and rpsL, which are responsible for isoniazid, rifampicin, ethambutol, fluoroquinolone, pyrazinamide, and streptomycin resistance respectively. Besides, the DHPLC analysis system was also introduced to high-throughput bacterial identification.
Through analysis of the prokaryotic 16S rRNA gene, different species could be differentiated by the corresponding distinctive DHPLC peak profiles. This application showed an overall specificity of 100% and a sensitivity of 91.7%. An enhanced version of the WAVE HPLC system known as the WAVE Microbial Analysis System (Transgenomic) has been developed specifically for the purpose of microbial analysis. One recent application is the highthroughput typing of M. tuberculosis strains based on twelve loci of variable number of tandem repeat present in mycobacteria.
Typing results based on nondenaturing HPLC showed 100% concordance with those generated by agarose gel electrophoresis. It should be noted that such applications do not fully utilize the benefits of nondenaturing HPLC because the amounts of PCR products are not measured. DHPLC can also be used for the detection and identification of fungal species in blood culture and fecal samples. This approach provides a simpler and quicker method than the culture-based approach.