Real-Time Reverse Transcription-PCR Assay for Comprehensive Detection of Human Rhinoviruses

Abstract

Human rhinoviruses (HRVs) are important contributors to respiratory disease, but their healthcare burden remains unclear, primarily because of the lack of sensitive, accurate, and convenient means of determining their causal role. To address this, we developed and clinically validated the sensitivity and specificity of a real-time reverse transcription-PCR (RT-PCR) assay targeting the viral 5 noncoding region defined by sequences obtained from all 100 currently recognized HRV prototype strains and 85 recently circulating field isolates. The assay successfully amplified all HRVs tested and could reproducibly detect 50 HRV RNA transcript copies, with a dynamic range of over 7 logs. In contrast, a quantified RNA transcript of human enterovirus 68 (HEV68) that showed the greatest sequence homology to the HRV primers and probe set was not detected below a concentration of 5 ؋ 10 5 copies per reaction. Nucleic acid extracts of 111 coded respiratory specimens that were culture positive for HRV or HEV were tested with the HRV real-time RT-PCR assay and by two independent laboratories that used different in-house HRV/HEV RT-PCR assays. Eighty-seven HRVculture-positive specimens were correctly identified by the real-time RT-PCR assay, and 4 of the 24 HEVpositive samples were positive for HRV. HRV-specific sequences subsequently were identified in these four specimens, suggesting HRV/HEV coinfection in these patients. The assay was successfully applied in an investigation of a coincidental outbreak of HRV respiratory illness among laboratory staff.

Molecular characterization of human rhinovirus field strains isolated during surveillance of enteroviruses

“…On: Thu, 10 May 2018 10:00:05 sequencing primers (Lu et al, 2008), those for the VP4/VP2 capsid protein region were 9895 and 9565 (Savolainen et al, 2002b) and those for the VP1 coding region were as described previously (Vlasak et al, 2003). The primers for an approximately 600 nt region spanning the 39 part of the 3D polymerase gene were as described previously (Savolainen et al, 2004).…”

A One-Step, Real-Time PCR Assay for Rapid Detection of Rhinovirus

“…In contrast, the two previously described one-step, realtime PCR assays have been designed for the iCycler real-time detection system (Bio-Rad; Hercules, CA). 8,9 The authors of one of these studies, 8 observed a reduction in amplification efficiency when the Applied Biosystems TaqMan One Step PCR Master Mix reagents kit was used with the iCycler real-time detection system underscoring the importance of establishing compatibility of all components of the PCR assay platform. Furthermore, most real-time assays for rhinovirus detection are twostep assays requiring a manual transfer of cDNA into PCR reactions and are consequently more cumbersome and more susceptible to contamination than a one-step PCR assay.…”

“…One-step assays have been developed for a limited number of PCR amplification platforms and chemistries and may not necessarily perform optimally with other platforms. 8 In addition, cross-reactivity with genetically similar enteroviruses has been reported with some assays. 8,9 A one-step, real-time PCR assay has not been described for the ABI Prism Sequence Detection System (Applied Biosystems; Foster City, CA) though this platform is widely used in clinical and research laboratories.…”

“…8 In addition, cross-reactivity with genetically similar enteroviruses has been reported with some assays. 8,9 A one-step, real-time PCR assay has not been described for the ABI Prism Sequence Detection System (Applied Biosystems; Foster City, CA) though this platform is widely used in clinical and research laboratories. High PCR efficiency with this platform generally requires the use of primers and a TaqMan probe (Applied Biosystems) with melting temperatures of 58°C to 60°C and 68°C to 70°C, respectively, and an amplicon size of 50 to 150 bp.…”

“…4 -7 However, only a few published PCR assays have combined reverse transcription and PCR in the same real-time reaction (ie, one-step assay). 8,9 The advantages of the one-step assay over the two-step assay include improved workflow, reduction in assay preparation time, and elimination of cross contamination from the transfer of cDNA from the reverse transcription reaction into the PCR reaction. One-step assays have been developed for a limited number of PCR amplification platforms and chemistries and may not necessarily perform optimally with other platforms.…”