Journal Article

A Sensitive Assay for Virus Discovery in Respiratory Clinical Samples

Deijs, Martin, Jebbink, Maarten F., van de Garde, Martijn D. B., Jakobs, Marja, Ieven, Margareta, de Vries, Michel, Koekkoek, Sylvie M., Molenkamp, Richard, Leus, Frank, Faria, Nuno R., Claas, Eric C. J., Baas, Frank, van der Hoek, Lia, Coenjaerts, Frank E. J., Goossens, Herman, van Schaik, Barbera D. C., Lammens, Christine, Luyf, Angela C. M., Canuti, Marta, Jachimowski, Loes C. M.

2011

Overview

In 5-40% of respiratory infections in children, the diagnostics remain negative, suggesting that the patients might be infected with a yet unknown pathogen. Virus discovery cDNA-AFLP (VIDISCA) is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors and subsequent amplification by PCR. However, direct discovery of unknown pathogens in nasopharyngeal swabs is difficult due to the high concentration of ribosomal RNA (rRNA) that acts as competitor. In the current study we optimized VIDISCA by adjusting the reverse transcription enzymes and decreasing rRNA amplification in the reverse transcription, using hexamer oligonucleotides that do not anneal to rRNA. Residual cDNA synthesis on rRNA templates was further reduced with oligonucleotides that anneal to rRNA but can not be extended due to 3'-dideoxy-C6-modification. With these modifications >90% reduction of rRNA amplification was established. Further improvement of the VIDISCA sensitivity was obtained by high throughput sequencing (VIDISCA-454). Eighteen nasopharyngeal swabs were analysed, all containing known respiratory viruses. We could identify the proper virus in the majority of samples tested (11/18). The median load in the VIDISCA-454 positive samples was 7.2 E5 viral genome copies/ml (ranging from 1.4 E3-7.7 E6). Our results show that optimization of VIDISCA and subsequent high-throughput-sequencing enhances sensitivity drastically and provides the opportunity to perform virus discovery directly in patient material.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Adapters / Amplification / Amplified fragment length polymorphism / Annealing / Bioinformatics / Biology / Children / Complementary DNA / Deoxyribonucleic acid / Diagnostic Techniques, Respiratory System - standards / DNA / DNA, Complementary - genetics / DNA, Viral - analysis / Enzymes / Epidemiology / Genomes / Genomics / High-Throughput Nucleotide Sequencing / Infections / Infectious diseases / Influenza / Kawasaki disease / Laboratories / Medicine / Next-generation sequencing / Oligonucleotides / Optimization / Pandemics / Pathogens / Polymerase Chain Reaction - methods / Respiratory syncytial virus / Respiratory Tract Infections - diagnosis / Respiratory Tract Infections - virology / Reverse transcription / Ribonucleic acid / Ribosomal RNA / RNA / RNA, Ribosomal - genetics / rRNA / Sensitivity / Sensitivity and Specificity / Sensitivity enhancement / Teaching hospitals / Transcription (Genetics) / Viral infections / Viral Load - methods / Virology / Viruses / Viruses - genetics / Viruses - isolation & purification