Astrovirus RNA Detection Using Real-Time Reverse Transcription-Polymerase Chain Reaction

Abstract

The TaqMan-based real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay we developed is sensitive and detects seven (1-7) human astrovirus (HAstV) serotypes. We chose conserved regions at the 5' end of the open reading frame2 (ORF2) was chosen at the assay targets for designing new primers and the TaqMan MGB probe that detects all HAstV serotypes. Real-time RT-PCR reactivity was confirmed with HAstV serotype 1 through 7 control plasmids and efficiency ranged from 30 to 3.0 x 10(7)copies/tube. The assay developed detected HAstV sequences from clinical HAstV-positive samples. The assay was 10 to 10(3) times more sensitive than Conventional RT-PCR assay and free of crossreactivity against other enteric viruses, including norovirus, sapovirus, rotavirus, and adenovirus. These results indicate that our real-time RT-PCR assay rapidly detects and quantitatively analyzes HAstV serotype 1 through 7.