A Sensitive Assay for Virus Discovery in Respiratory Clinical Samples
| Autor: | Marja E. Jakobs, Martijn D. B. van de Garde, Herman Goossens, Michel de Vries, Frank E. J. Coenjaerts, Christine Lammens, Martin Deijs, Richard Molenkamp, Barbera D. C. van Schaik, Loes C. M. Jachimowski, Maarten F. Jebbink, Margareta Ieven, Marta Canuti, Lia van der Hoek, F.R. Leus, Frank Baas, Nuno R. Faria, Sylvie M. Koekkoek, Angela C. M. Luyf, Eric C. J. Claas |
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| Přispěvatelé: | Faculteit der Geneeskunde, Medical Microbiology and Infection Prevention, AII - Amsterdam institute for Infection and Immunity, ACS - Amsterdam Cardiovascular Sciences, AGEM - Amsterdam Gastroenterology Endocrinology Metabolism, ANS - Amsterdam Neuroscience, CCA -Cancer Center Amsterdam, Epidemiology and Data Science, Other departments, Human Genetics, Neurology, Genome Analysis |
| Jazyk: | angličtina |
| Rok vydání: | 2011 |
| Předmět: |
Viral Diseases
Pulmonology Gene Identification and Analysis Common Cold lcsh:Medicine Polymerase Chain Reaction Biochemistry law.invention DNA amplification law lcsh:Science Respiratory Tract Infections Polymerase chain reaction 0303 health sciences Multidisciplinary High-Throughput Nucleotide Sequencing Viral Load Clinical Laboratory Sciences 3. Good health Nucleic acids Lower Respiratory Tract Infections Infectious Diseases Viruses Medicine Research Article Test Evaluation DNA Complementary Diagnostic Techniques Respiratory System Biology Sensitivity and Specificity Microbiology DNA sequencing Virus Molecular Genetics 03 medical and health sciences Diagnostic Medicine Virology Complementary DNA Genetics 030304 developmental biology SARS 030306 microbiology infections community children disease identification coronavirus pneumonia diagnosis etiology pcr lcsh:R RNA DNA Ribosomal RNA Molecular biology Reverse transcriptase Viral Disease Diagnosis Restriction enzyme Viral Pneumonia RNA Ribosomal DNA Viral Respiratory Infections lcsh:Q Human medicine |
| Zdroj: | PLoS ONE, 6(1):e16118. Public Library of Science PLoS ONE PLoS ONE, 6(1) PLoS ONE; Vol 6 PLoS ONE, Vol 6, Iss 1, p e16118 (2011) PLoS ONE, 6(1). Public Library of Science |
| ISSN: | 1932-6203 |
| Popis: | In 540% 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 E37.7 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. |
| Databáze: | OpenAIRE |
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