In a paper published online in the Journal of Molecular Diagnostics, a group led by Richard Willson from the University of Houston describe using dengue virus as a model to test a novel method that combines highly sensitive RT-PCR amplification of virus strains with serotype and finer-level identification by mass spectrometry. The researchers analyzed RNA fragments produced by guanosine-specific RNase T1 digestion using matrix-assisted laser desorption/ionization-time of flight mass spec. After comparing the pattern of fragment masses to a mass database, a database was created by computationally fragmenting 2,517 dengue strains. "Computationally, all 2,517 strains in the mass database were correctly identified at the serotype level from the predicted PCR product," they write. "The methodology was successfully demonstrated experimentally by identifying the serotypes of eight test strains using mosquito cell cultures infected with strains of all four serotypes and with full-length cDNA clones."
Also in the Journal of Molecular Diagnostics, a South Korean research team presents an innovative multiplex genotyping method called GTPlex-PyroSeq. Their new platform is a two-step process comprised of multiplex PCR which is then followed by a single reaction of pyrosequencing. In the paper, the authors show how GTPlex-PyroSeq can be effectively used for determining multiple human papillomavirus genotypes by comparing their assay with direct sequencing and HPV DNA chip analysis of 80 samples from high-risk, HPV-positive patients. "We found in single-type infections, 100 percent concordance with direct sequencing (70 of 80 perfect matches) and 97.5 percent concordance with HPV DNA chip data (50 of 80 perfect matches)," they write. "Additionally, our system was superior to direct sequencing in detection of multiple infections (12 of 80), with a limit of detection of 100 copies. The scalability of this multiplex system, with its open-platform design and ability to use various sample types, makes the GTPlex applicable for use in multiple settings."