Development of an efficient entire capsid-coding region amplification method for direct detection of poliovirus from stool extracts.

Arita M1, Kilpatrick DR2, Nakamura T3, Burns CC2, Bukbuk D4, Oderinde SB4, Oberste MS2, Kew OM2, Pallansch MA2, Shimizu H3.

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Abstract

Laboratory diagnosis has played a critical role in the Global Polio Eradication Initiative (GPEI) since 1988 by isolating and identifying poliovirus (PV) from stool specimens by using cell culture, as a highly sensitive system to detect PV. In the present study, we aimed to develop a molecular method to detect PV directly from stool extracts with a high efficiency comparable to that of cell culture. We developed a method to efficiently amplify the entire capsid-coding region of human enteroviruses (EV) including PV. cDNAs of the entire capsid-coding region (3.9 kb) were obtained from as few as 50 copies of PV genomes. PV was detected from the cDNAs by an improved PV-specific real-time RT-PCR system and nucleotide sequence analysis of the VP1-coding region. For assay validation, we analyzed 84 stool extracts that were positive for PV in cell culture and detected PV genome from 100% of the extracts (84/84 samples) by this method in combination with a PV-specific extraction method. PV could be detected from 2/4 samples of stool extracts that were negative for PV in cell culture. In PV-positive samples, EV species C viruses were also detected with a high frequency (27%, 23/86 samples). This method would be useful for direct detection of PV from the stool extracts without using cell culture.