Development of real time PCR for detection and quantitation of Dengue Viruses
Autor(es): Gurukumar K R, Priyadarshini D, Patil J A, Bhagat A, Singh A, Shah P S, Cecilia D
Resumo: Dengue virus (DENV), a mosquito borne flavivirus is an important pathogen causing more than 50 million infections every year around the world. Dengue diagnosis depends on serology, which is not useful in the early phase of the disease - virus isolation, which is laborious - time consuming. There is need for a rapid, sensitive - high throughput method for detection of DENV in the early stages of the disease. Several real-time PCR assays have been described for dengue viruses, but there is scope for improvement. The new generation TaqMan Minor Groove Binding (MGB) probe approach was used to develop an improved real time RT-PCR (qRT-PCR) for DENV in this study. The 3'UTR of thirteen Indian strains of DENV was sequenced - aligned with 41 representative sequences from GenBank. A region conserved in all four serotypes was used to target primers - probes for the qRT-PCR. A single MGB probe - a single primer pair for all the four serotypes of DENV were designed. The sensitivity of the two step qRT-PCR assay was10 copies of RNA molecules per reaction. The specificity - sensitivity of the assay was 100% when tested with a panel of 39 known positive - negative samples. Viral RNA could be detected - quantitated in infected mouse brain, cell cultures, mosquitoes - clinical samples. Viral RNA could be detected in patients even after seroconversion till 10 days post onset of infection. There was no signal with Japanese Encephalitis (JE), West Nile (WN), Chikungunya (CHK) viruses or with Leptospira, Plasmodium vivax, Plasmodium falciparum - Rickettsia positive clinical samples. We have developed a highly sensitive - specific qRT-PCR for detection - quantitation of dengue viruses. The assay will be a useful tool for differential diagnosis of dengue fever in a situation where a number of other clinically indistinguishable infectious diseases like malaria, Chikungunya, rickettsia - leptospira occur. The ability of the assay to detect DENV-2 in inoculated mosquitoes makes it a potential tool for detecting DENV in field-caught mosquitoes.
Imprenta: Virology Journal, v. 6, p. 10, 2009
Identificador do objeto digital: 10.1186/1743-422X-6-10
Descritores: Chikungunya virus - Cell ; Chikungunya virus - DNA ; Chikungunya virus - Flaviviridae ; Chikungunya virus - Genome ; Chikungunya virus - Pathogenesis ; Chikungunya virus - RNA ; Chikungunya virus - Infectious diseases ; Chikungunya virus - Serology ; Chikungunya virus - Viral infections ; Chikungunya virus - PCR detection ; Chikungunya virus - Real Time PCR ; Chikungunya virus - RT-PCR ; Chikungunya virus - Serology ; Chikungunya Virus - Virus ; Chikungunya virus - Chikungunya fever ; Chikungunya virus - Dengue ; Chikungunya virus - Public health
Data de publicação: 2009
