Multiple Sequencing Primers Method for Detection and Identification of Microbial and Viral Pathogens

DNA sequencing is the gold standard method for accurate genotyping of microbial and viral pathogens, and is more reliable than the hybridization-based techniques, which might be subjected to false hybridization during discrimination of closely related types. A type-specific multiple sequencing primers DNA sequencing strategy, suitable for typing of microbial and viral pathogens has been developed. In the new method type-specific sequencing primers combined in a pool, are added to the DNA sample.

The new method is especially suited for typing of samples harboring different species or genotypes (multiple infections) and non-specific amplification products, eliminating the need for nested PCR, stringent PCR conditions and PCR cloning. Furthermore, the method has proved to be useful for samples containing subdominant types/species, and samples with low PCR yield, which avoids re-performing unsuccessful PCRs. We also introduce the sequence pattern recognition when there is a plurality of species in the sample, which facilitates typing of more than one target DNA in the sample. The multiple sequencing primers could be easily modeled and tailored according to the distribution and prevalence of varying bacteria, fungi and viruses by geographic regions or other demographic factors. This new approach strengthens the DNA sequencing technologies in diagnostics and research.