Detection of Hepatitis A Virus by the Nucleic Acid Sequence-Based Amplification Technique and Comparison with Reverse Transcription-PCR
| Autor: | Burton W. Blais, Julie Jean, Ismail Fliss, André Darveau |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
viruses
Blueberry Plants Dot blot Biology Sensitivity and Specificity Waste Disposal Fluid Applied Microbiology and Biotechnology Methods Self-Sustained Sequence Replication DNA Primers Ecology Reverse Transcriptase Polymerase Chain Reaction virus diseases RNA Hepatitis A Lettuce biochemical phenomena metabolism and nutrition Amplicon Virology Molecular biology NASBA Reverse transcription polymerase chain reaction Hepatitis A virus Water Microbiology Oligomer restriction Food Science Biotechnology Waste disposal |
| Zdroj: | Applied and Environmental Microbiology. 67:5593-5600 |
| ISSN: | 1098-5336 0099-2240 |
| DOI: | 10.1128/aem.67.12.5593-5600.2001 |
| Popis: | A nucleic acid sequence-based amplification (NASBA) technique for the detection of hepatitis A virus (HAV) in foods was developed and compared to the traditional reverse transcription (RT)-PCR technique. Oligonucleotide primers targeting the VP1 and VP2 genes encoding the major HAV capsid proteins were used for the amplification of viral RNA in an isothermal process resulting in the accumulation of RNA amplicons. Amplicons were detected by hybridization with a digoxigenin-labeled oligonucleotide probe in a dot blot assay format. Using the NASBA, as little as 0.4 ng of target RNA/ml was detected per comparison to 4 ng/ml for RT-PCR. When crude HAV viral lysate was used, a detection limit of 2 PFU (4 × 10 2 PFU/ml) was obtained with NASBA, compared to 50 PFU (1 × 10 4 PFU/ml) obtained with RT-PCR. No interference was encountered in the amplification of HAV RNA in the presence of excess nontarget RNA or DNA. The NASBA system successfully detected HAV recovered from experimentally inoculated samples of waste water, lettuce, and blueberries. Compared to RT-PCR and other amplification techniques, the NASBA system offers several advantages in terms of sensitivity, rapidity, and simplicity. This technique should be readily adaptable for detection of other RNA viruses in both foods and clinical samples. |
| Databáze: | OpenAIRE |
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