Rapid Detection and Quantification of RNA of Ebola and Marburg Viruses, Lassa Virus, Crimean-Congo Hemorrhagic Fever Virus, Rift Valley Fever Virus, Dengue Virus, and Yellow Fever Virus by Real-Time Reverse Transcription-PCR

Abstract

Viral hemorrhagic fevers (VHFs) are acute infections with high case fatality rates. Important VHF agents are Ebola and Marburg viruses (MBGV/EBOV), Lassa virus (LASV), Crimean-Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), dengue virus (DENV), and yellow fever virus (YFV). VHFs are clinically difficult to diagnose and to distinguish; a rapid and reliable laboratory diagnosis is required in suspected cases. We have established six one-step, real-time reverse transcription-PCR assays for these pathogens based on the Superscript reverse transcriptase-Platinum Taq polymerase enzyme mixture. Novel primers and/or 5-nuclease detection probes were designed for RVFV, DENV, YFV, and CCHFV by using the latest DNA database entries. PCR products were detected in real time on a LightCycler instrument by using 5-nuclease technology (RVFV, DENV, and YFV) or SybrGreen dye intercalation (MBGV/EBOV, LASV, and CCHFV). The inhibitory effect of SybrGreen on reverse transcription was overcome by initial immobilization of the dye in the reaction capillaries. Universal cycling conditions for SybrGreen and 5-nuclease probe detection were established. Thus, up to three assays could be performed in parallel, facilitating rapid testing for several pathogens. All assays were thoroughly optimized and validated in terms of analytical sensitivity by using in vitro-transcribed RNA. The >95% detection limits as determined by probit regression analysis ranged from 1,545 to 2,835 viral genome equivalents/ml of serum (8.6 to 16 RNA copies per assay). The suitability of the assays was exemplified by detection and quantification of viral RNA in serum samples of VHF patients.

Curcumin Inhibits Rift Valley Fever Virus Replication in Human Cells

“…Experiments carried out using the virulent ZH501 strain demonstrate that curcumin can inhibit replication of the fully virulent virus as well. Finally, our experiments using the INFAR Ϫ/Ϫ murine model (28,29) provide preliminary proofof-concept validation that curcumin can down-regulate virus in the livers of infected animals as well, thus paving the way for further development of novel curcumin-based therapeutic options.…”

“…Viral RNA from cell culture supernatants was extracted using Ambion's MagMAX TM -96 viral RNA isolation kit, and RNA was analyzed by qRT-PCR. The primers and probe used for amplification of viral RNA were originally described by Drosten et al (29). qRT-PCR assays were performed using the ABI Prism 7000 and Invitrogen's RNA UltraSense TM one-step quantitative RT-PCR system.…”

Essentials of filoviral load quantification

“…50 The EZ1 Real-time RT-PCR Assay (US Department of Defense, USA) for Ebola virus, which uses a synthetic RNA standard, was validated by good laboratory practices for quantitative measurement of viral load in non-human primate plasma. However, this feature was omitted in the emergency use authorisation version because of increased regulatory hurdles.…”

Dengue virus type 4 in Niterói, Rio de Janeiro: the role of molecular techniques in laboratory diagnosis and entomological surveillance

“…Real-time RT-PCR methods have been established as a more rapid and sensitive technique for the detection and quantification of DENV in clinical samples (Drosten et al 2002, Lai et al 2007. In this study, we used quantitative real-time RT-PCR to quantify the DENV-4 viral titre (1.08 x 10 3 copies/mL) from a single Ae.…”

“…aegypti mosquitoes was subjected to a quantitative realtime RT-PCR according to the protocol described by Drosten et al (2002).…”