High resolution melting assay of dengue virus detection and identification

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High resolution melting assay of dengue virus detection and identification

 

Author: Wong, Wan-chi
Title: High resolution melting assay of dengue virus detection and identification
Degree: M.Sc.
Year: 2010
Subject: Hong Kong Polytechnic University -- Dissertations
Dengue viruses -- Identification
Bacterial genetics
Microorganisms -- Effect of temperature on.
Department: Dept. of Health Technology and Informatics
Pages: xii, 74 leaves : ill. ; 31 cm.
InnoPac Record: http://library.polyu.edu.hk/record=b2391079
URI: http://theses.lib.polyu.edu.hk/handle/200/5836
Abstract: Quick and accurate laboratory diagnosis is significant for the early launch of specific preventive health measures to restrict epidemic spread of dengue fever. High resolution melting (HRM) assay has been investigated for the rapid identification of bacteria and for influenza A serotyping. For that reason, applying HRM assay on serotyping of dengue viruses is also possible to be investigated. Saturating fluorescent dye, Syto 9, was utilized in this study. This dye binds to any dsDNA and is without ‘jumping effect’ during melting, therefore a high resolution signal can be achieved and detected by using a high resolution analyser such as LightCycler 480 (LC480). Since all serotypes are from the same genus, Flavivirus, their genome RNA may have some highly conserved region. HRM assay can distinguish the little variation in the selected conserved region by small differences in melting temperature (Tm), allow the technique to be applied for rapid serotyping and identification. In this study, use of HRM after PCR targeting of two different regions was investigated. 1) PCR of selected 3’ untranslated region (3’UTR): 3’UTR was first selected as the target region to be investigated. Recent serotyping of dengue virus using real-time RT-PCR requires one universal primer, 4 serotype-specific primers and serotype-specific probes to differentiate the unknown serotypes. However, only one pair of primers is required if using high resolution melting assay, the forward and reverse primers are named as DVF and DVR here, to perform serotyping for this approach. For applying in the clinical laboratory, dengue RNAs in clinical samples would only be required to undergo reverse transcription, and the cDNA directly undergoes PCR and HRM to be identified the serotype. Unfortunately, this approach failed, so the following approach was tried. 2) PCR of selected C-prM region: The applied theory is same as the previous approach targeting 3’UTR, but instead of newly designed primers, the pair of consensus primers developed by Lanciotti and indicated as D1 and D2 in the original article, were used to amplify the sequence within C-prM region. They can amplify the corresponding sequences of other flaviviruses. These primers are re-named as DF and DR for this study respectively. Low sensitivity and appearance of non-specific amplification products lead the approach of 3’ UTR as target fails to do the serotyping. The approach that PCR targeting C-prM had better performance between 2 approaches, but the performance is not stable. In conclusion, the investigated approaches by using high resolution melting assay for selected C-prM can be further improved to become suitable for serotyping.

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