Introduction

Molecular diagnostic methods are gaining acceptance in clinical microbiological laboratories. Development of the PCR technique initiated a large number of reports on the use of DNA-based molecular methodology. These methods have proven to be useful for microbiological detection, strain typing, drug resistance, and epidemiology.

Currently, diagnosis of parasitic infections rely on clinical symptoms, travel history, geographical location of the patient, and several detection methods depending on the parasite or the specimen being tested. If culture or animal inoculation is required for identifying the parasite and introducing therapy, PCR may offer an advantage. When direct microscopy is sufficiently good to detect and species differentiate the parasites, PCR only offers an advantage by being able to process a large number of samples with an automated assay. However, when the parasite load is low, PCR may increase the sensitivity above that of microscopy.

For diagnostic purposes, a number of commercial kits for viral and bacterial detection by molecular methods are available, however, no kit for detection of parasites has been developed. This is, in part, due to the expense of new technology as well as a scarcity of these parasites in countries where this research is ongoing.

The PCR method requires the presence of specific key (primer) DNA sequences, complementary to DNA sequences flanking a DNA fragment of interest (target DNA) and a thermostable DNA polymerase. Following sample preparation, PCR amplification itself is a fast and automated method, simply requiring a programmable heating-block and addition of a standard reaction mixture. In general, detection of microbial DNA requires three procedure steps: (i) target DNA preparation; (ii) PCR

amplification (subsequently re-amplification, if nested or semi-nested PCR is used); and (iii) identification of the amplified DNA fragments (amplimers or amplicons), usually by gel-electrophoresis. Two main approaches for parasite diagnostic PCR assays are conducted: either a one-step species-specific (single) PCR, or a two-step procedure by a generic PCR, followed by DNA hybridization with a species-specific probe, a species-specific nested PCR or a species-specific detection by endonuclease restriction digest of the amplified PCR product.

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