Optimization of Pcr and Automated Sequencing of Clinical Isolates of Respiratory Syncytial Virus

A streamlined protocol was developed to carry out nucleotide sequence analysis on PCR products obtained from regions of the RNA genome of respiratory syncytial virus (RSV). Whole cell RNA extracted from tissue culture cells inoculated with RSV-infected nasopharyngeal aspirates was used as a template for single-tube cDNA synthesis and PCR amplification of specific regions of the RSV fusion (F) protein gene. The products were then purified using Sephacryl spin columns and digested using lambda-exonuclease to obtain single-stranded templates for analysis using dye-primer chemistry in an automated sequencer. Optimization of a range of parameters and comparison of a number of alternate procedures resulted in a final protocol that allowed the comparison of the sequences of a panel of local RSV subgroup A and B clinical isolates