S George1, V K Menon, S Ramani, G Kang. 1. Department of Gastrointestinal Sciences, Christian Medical College, Vellore - 632 004, India.
Abstract
PURPOSE: Noroviruses (NoV) are increasingly recognized as an important cause for acute gastroenteritis, worldwide. Reverse transcription polymerase chain reaction (RT-PCR) and sequencing are the methods of choice for the detection of NoVs, but there is currently no consensus about the primers to be used in these assays. MATERIALS AND METHODS: In this study, five published primer sets were evaluated for the detection of genogroup II (GII) NoVs in India. The primers target different regions of the NoV genome. Three primer sets detect an NoV in a single round RT-PCR platform, while the remaining two primer sets are based on a nested RT-PCR platform. RESULT: A panel of 100 samples from previous studies on norovirus diarrhoea in children were tested by all five primer sets. Of them, 74 samples were identified as positive for NoV, by at least one primer set. Subsets of positive amplicons were sequenced to check for specificity. CONCLUSION: The most sensitive primer set was Girish 2002, which detected GII NoV by nested RT-PCR, and was modified from the previously published primers. This study demonstrates that higher detection can be obtained by either using multiple primer sets or using a sensitive nested RT-PCR assay. It also demonstrates the differences in primer sensitivity for detection of Genogroup II (GII) NoVs in India.
PURPOSE: Noroviruses (NoV) are increasingly recognized as an important cause for acute gastroenteritis, worldwide. Reverse transcription polymerase chain reaction (RT-PCR) and sequencing are the methods of choice for the detection of NoVs, but there is currently no consensus about the primers to be used in these assays. MATERIALS AND METHODS: In this study, five published primer sets were evaluated for the detection of genogroup II (GII) NoVs in India. The primers target different regions of the NoV genome. Three primer sets detect an NoV in a single round RT-PCR platform, while the remaining two primer sets are based on a nested RT-PCR platform. RESULT: A panel of 100 samples from previous studies on norovirus diarrhoea in children were tested by all five primer sets. Of them, 74 samples were identified as positive for NoV, by at least one primer set. Subsets of positive amplicons were sequenced to check for specificity. CONCLUSION: The most sensitive primer set was Girish 2002, which detected GII NoV by nested RT-PCR, and was modified from the previously published primers. This study demonstrates that higher detection can be obtained by either using multiple primer sets or using a sensitive nested RT-PCR assay. It also demonstrates the differences in primer sensitivity for detection of Genogroup II (GII) NoVs in India.
Authors: Mayuko Saito; Sonia Goel-Apaza; Susan Espetia; Daniel Velasquez; Lilia Cabrera; Sebastian Loli; Jean E Crabtree; Robert E Black; Margaret Kosek; William Checkley; Mirko Zimic; Caryn Bern; Vitaliano Cama; Robert H Gilman Journal: Clin Infect Dis Date: 2013-12-02 Impact factor: 9.079