Evaluation and adaptation of molecular approaches for detection and characterization of viruses of the respiratory tract

This study was designed for the development of novel methods for isothermal amplification of nucleic acids using phi29 and Bst DNA polymerases in the detection and characterization of pathogenic viruses of the respiratory tract. High Resolution Melting (HRM) analysis was used in this study for screening similar sequences to avoid unnecessary sequencing from genomic libraries and detection of common pathogenic viruses of the respiratory tract. Four different structures of DNA linear double stranded DNA (dsDNA, 48 kb), linear single stranded DNA (ssDNA, 3.6 kb), small circular dsDNA (2.6 kb) and a large circular dsDNA (8.6 kb) were used as representatives of different types of viral nucleic acids for the evaluation of phi29-amplification at limiting concentrations. Phi29-amplification of linear ds, large and small circular dsDNA could be amplified 10-100-fold but ssDNA could not be amplified at all. Alternative protocols were adapted for ligating ssDNA and dsDNA to construct large concatemers for efficient phi29-amplification. Phi29-amplification of ssDNA was found to increase >10-fold when it was ligated using T4 RNA ligase in the presence of a large excess of background ssDNA compared to that in the absence of background ssDNA. T4 DNA ligase-mediated phi29-amplification of double stranded cDNA (dscDNA) was found to increase >105-fold compared to non-ligated dscDNA. CircLigaseä II ssDNA ligase was used for circularization of ssDNA and aid phi29-amplification. CircLigaseä II ssDNA ligase-mediated phi29-amplification of ssDNA was found to increase >107-fold compared to non-ligated sscDNA. A method named RT-Bst was developed for simultaneous reverse transcription and Bst DNA polymerase amplification of cDNA in the same reaction. In a qualitative PCR assay, RT-Bst was found to be more efficient than the QuantiTectä reverse transcription kit (QIAGEN, Crawley, UK). The RT-Bst method was used for amplification of cDNA in 70 nasopharyngeal samples for detection of a panel of 12 pathogenic viruses of the respiratory tract. The performance of multiplex RT-Bst PCR detection of pathogenic viruses was comparable to that of multiplex one-step RT-PCR (performed in this study) and more efficient than immunofluorescence (performed by a hospital diagnostic laboratory). The RT-Bst protocol was further modified using tailed random primers to develop another protocol for whole genome amplification designated RT-Bst Single Primer Amplification (RT-Bst SPA) as a cheaper alternative to sequence independent single primer amplification. HRM analysis was used in this study to demonstrate its application for low cost screening of similar sequences to avoid unnecessary sequencing task from whole genome libraries prepared from MS2 RNA and tissue culture media positive for influenza B and hRSV. HRM analysis was also used for rapid and low cost detection of common pathogenic viruses of the respiratory tract. It is proposed that RT-Bst and RT-Bst SPA have the potential for sequence independent amplification of RNA sequences for subsequent multiplex PCR detection, and other downstream applications. HRM analysis can be used for rapid and cost effective detection of pathogenic viruses of the respiratory tract. However, a more detailed study will be required for further optimization and validation of the developed protocols for appropriate commercialization as kits.