Development of real-time reverse transcription-polymerase chain reaction assay for detection of noroviruses using induced flourescence resonance energy transfer technology

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Development of real-time reverse transcription-polymerase chain reaction assay for detection of noroviruses using induced flourescence resonance energy transfer technology

 

Author: Tang, Pui-san
Title: Development of real-time reverse transcription-polymerase chain reaction assay for detection of noroviruses using induced flourescence resonance energy transfer technology
Degree: M.Sc.
Year: 2010
Subject: Hong Kong Polytechnic University -- Dissertations
RNA viruses
Polymerase chain reaction
Fluorescence spectroscopy
Department: Dept. of Health Technology and Informatics
Pages: ix, 70 leaves : ill. (some col.) ; 31 cm.
InnoPac Record: http://library.polyu.edu.hk/record=b2356835
URI: http://theses.lib.polyu.edu.hk/handle/200/5845
Abstract: Noroviruses (NoVs) are RNA viruses belonging to the Caliviridae family. The viruses can be classified into Genogroups (G) I to V, only GI and GII are human pathogens. NoVs are responsible for the majority of acute gastroenteritis outbreaks and have a significant impact on public health worldwide. A rapid definitive diagnosis is the crucial factor to aid patient management and treatment guidance. Real-time reverse transcription polymerase chain reaction (RT-PCR) assay has many advantages over conventional PCR assay such as rapidity, lower risk of cross-contamination, higher sensitivity, being quantitative, does not require post-amplification processing. It is regarded as the reference method for detection of infectious agents. An induced fluorescence resonance energy transfer (iFRET) system-based real-time RT-PCR using degenerate primers for rapid detection and typing of NoV GI and GII was developed. iFRET is a variation of FRET between a donor intercalating dye and an acceptor fluorophore covalently linked to an oligonucleotide primer. In this study, DNA hybridization between NoV GII reverse primer and NoV template induces energy transfer from the SYBR Green I dye to the acceptor fluorophore (Cy5.5). Presumptive amplicon identification was established by measuring the change in acceptor fluorescence intensity during DNA denaturation in channel F1 (530 nm) and F3 (610 nm). The performance of the real-time RT-PCR using iFRET technology for NoV detection was assessed. Using serial dilution of a NoV RNA standard, the detection limits for GII.4 and GI.3 were found to be 2.35 x 10³ RNA copies/μl and 2.08 x 10⁵ RNA copies/μl, respectively. No cross-reaction was observed in samples positive for other gastroenteritis viruses such as Rotavirus and Adenovirus. The clinical sensitivity and specificity of the assay were evaluated against a commercial TaqMan RT-PCR assay. A total of 131 stool specimens collected from patients presenting with sporadic gastroenteritis were tested. The sensitivity and specificity of the iFRET-based assay were 94 % and 100 %, respectively. The findings in this study demonstrated that the real time RT-PCR assay would be feasible for laboratory diagnosis of NoV infection.

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