Short communicationIncreased sensitivity for various rotavirus genotypes in stool specimens by amending three mismatched nucleotides in the forward primer of a real-time RT-PCR assay
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Acknowledgement
We thank Dr. Jutta Preiksaitis for editing our manuscript.
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2020, Infection, Genetics and EvolutionCitation Excerpt :The samples were incubated at 25 °C for 10 min, at 37 °C for 60 min and 70 °C for 15 min. After cDNA synthesis, screening of species A rotaviruses in the clinical samples and quantification of the genome in the river water samples were performed by real-time PCR using NSP3 gene-specific primers (Pang et al., 2011) and TaqMan Fast Advanced master mix in an ABI 7500 Fast Real-Time PCR system (Life Technologies, Carlsbad, CA). The cut-off threshold cycle (Ct) value for clinical samples was set at 37.
Evaluation of different genomic regions of Rotavirus A for development of real time PCR
2019, Journal of Virological MethodsCitation Excerpt :Being speedy, sensitive and specific, molecular assays extend several advantages over other conventional methods. Among these, several qRT-PCR assays have been developed focusing VP2, VP4, VP6, VP7, NSP3, and NSP4 genes for detection of RVA specific RNA (Kang et al., 2004; Pang et al., 2004; Logan et al., 2006; Min et al., 2006; Freeman et al., 2008; Gutiérrez-Aguirre et al., 2008; Jothikumar et al., 2009; Adlhoch et al., 2011; Pang et al., 2011; Kottaridi et al., 2012). However, due to the continuous accumulation of point mutations in rotavirus genome, reassortment of related genomic segments in rotavirus strains and zoonotic transmission of animal rotavirus strains that may introduce novel genotypes and/or lineages in humans (Matthijnssens et al., 2009; Martella et al., 2010), it is important to explore different genes of RVAs for their comparative utility in qRT-PCR assays from time to time.
Real-time RT-PCR, a necessary tool to support the diagnosis and surveillance of rotavirus in Mexico
2018, Diagnostic Microbiology and Infectious DiseaseCitation Excerpt :But, there is variability on these gene segments. Therefore, the studies suggest using primers from highly conserved region of NSP3 gene (Freeman et al., 2008; Jothikumar et al., 2009; Mijatovic-Rustempasic et al., 2013; Pang et al., 2004, 2011; Zeng et al., 2008). To detect group A rotavirus NSP3 gene, the first real-time RT-PCR protocol was developed by Pang et al. (2004), which showed a higher sensitivity than the conventional PCR (Pang et al., 2004).
Quantitative reverse transcription PCR to determine the inactivation of Human Rotavirus by chlorine
2017, International Journal of Hygiene and Environmental HealthCitation Excerpt :HRVs are non-enveloped viruses that contain genomes composed of 11 segments of double-stranded RNA comprising approximately 18,000 base pairs (bp) in total. To date, several RT-qPCR assays targeting a small portion of the viral genome have been developed to detect HRVs in environmental water samples (Adlhoch et al., 2011; Kottaridi et al., 2012; Pang et al., 2011; Plante et al., 2011); however, few studies have used RT-qPCR to determine HRV infectivity after disinfection. To overcome these limitations, the entire HRV genome was explored through conventional RT-PCR amplification of small fragments using 16 primer sets, and damage to the 1227–2354 bp region of the VP4 gene was associated with the disappearance of HRV infectivity by chlorine treatment (Xue et al., 2013).