Typing of Dengue Viruses in Clinical Specimens and Mosquitoes by Single-Tube Multiplex Reverse Transcriptase PCR
AUTOR(ES)
Harris, Eva
FONTE
American Society for Microbiology
RESUMO
In recent years, dengue viruses (serotypes 1 to 4) have spread throughout tropical regions worldwide. In many places, multiple dengue virus serotypes are circulating concurrently, which may increase the risk for the more severe form of the disease, dengue hemorrhagic fever. For the control and prevention of dengue fever, it is important to rapidly detect and type the virus in clinical samples and mosquitoes. Assays based on reverse transcriptase (RT) PCR (RT-PCR) amplification of dengue viral RNA can offer a rapid, sensitive, and specific approach to the typing of dengue viruses. We have reduced a two-step nested RT-PCR protocol to a single-tube reaction with sensitivity equivalent to that of the two-step protocol (1 to 50 PFU) in order to maximize simplicity and minimize the risk of sample cross-contamination. This assay was also optimized for use with a thermostable RT-polymerase. We designed a plasmid-based internal control that produces a uniquely sized product and can be used to control for both reverse transcription or amplification steps without the risk of generating false-positive results. This single-tube RT-PCR procedure was used to type dengue viruses during the 1995 and 1997-1998 outbreaks in Nicaragua. In addition, an extraction procedure that permits the sensitive detection of viral RNA in pools of up to 50 mosquitoes without PCR inhibition or RNA degradation was developed. This assay should serve as a practical tool for use in countries where dengue fever is endemic, in conjunction with classical methods for surveillance and epidemiology of dengue viruses.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=105176Documentos Relacionados
- Direct genotypic detection of Mycobacterium tuberculosis rifampin resistance in clinical specimens by using single-tube heminested PCR.
- Single-Tube Single-Enzyme Reverse Transcriptase PCR Assay for Detection of Bovine Viral Diarrhea Virus in Pooled Bovine Serum
- Rapid Identification of Nine Microorganisms Causing Acute Respiratory Tract Infections by Single-Tube Multiplex Reverse Transcription-PCR: Feasibility Study
- Sensitive Detection of RNA Viruses Associated with Gastroenteritis by a Hanging-Drop Single-Tube Nested Reverse Transcription-PCR Method
- Single-tube nested PCR in the diagnosis of tuberculosis.