Single-tube and nested reverse transcriptase–polymerase chain reaction for detection of Rift Valley fever virus in human and animal sera

Sall, Amadou Alpha; Thonnon, J.; Sène, O. K.; Ameth, Fall; Ndiaye, Mignane; Baudez, Bertrand; Mathiot, C; Bouloy, Michèle

doi:10.1016/s0166-0934(00)00252-4

Cited by 51 publications

(38 citation statements)

References 23 publications

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“…The high mean viral load of 1.31 ϫ 10 7 molecules/ml of serum is typical for infections with Bunyaviridae. In sheep, Rift Valley fever virus, for example, produces a high viremia of up to 10 5 viruses/ml of serum in the early days of an infection (31). An early diagnostic window for this virus is therefore amenable to RT-PCR and has also been documented for Crimean-Congo hemorrhagic fever virus (5).…”

Section: Discussionmentioning

confidence: 99%

Rapid Detection of Human Pathogenic Orthobunyaviruses

et al. 2003

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Modern detection and identification tools can help to provide answers to urgent questions about the incidence, prevalence, and epidemiology of currently emerging diseases. We developed highly sensitive one-step TaqMan reverse transcription-PCR assays with sensitivities ranging from 10 4 to 10 1 molecules for 11 human pathogens of the orthobunyaviruses. We compared the performances of these assays on three currently available cyclers (ABI-PRISM 7700, LightCycler, and SmartCycler). The assay for Oropouche virus (OROV) was tested using sera collected from days 1 to 5 after onset of OROV disease and was found to be greatly superior to an established nested PCR system. A mean copy number of 1.31 ؋ 10 7 OROV RNA/ml of serum was detected. Diagnostic RNA detection can be used as early as day 1 after onset of OROV disease. The use of a mobile SmartCycler and a hands-on time of less than 3 h could help to intensify outbreak surveillance and control, especially in field studies.

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Section: Discussionmentioning

confidence: 99%

Rapid Detection of Human Pathogenic Orthobunyaviruses

et al. 2003

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“…A traditional RT-PCR was already developed for the detection of RVFV in different specimens and proved to be efficient for the diagnosis of human cases during the 1998 Mauritanian outbreak (35). A high level of sensitivity was obtained in a nested RT-PCR assay, which detected 0.5 PFU of MP12 virus and was approximately 100-fold more sensitive than the simple one.…”

Section: Real-time Rtd-pcr Of Rvfvmentioning

confidence: 99%

“…Currently, diagnosis is based on detection of specific antibodies or virus isolation in animal and mosquito cells (4). Reverse transcription (RT)-PCR techniques have been described and used to detect the RVFV genome in mosquitoes (11) and, recently, in clinical samples (35). In this work we developed a real-time RT-PCR method in order to detect and specifically quantify the virus either from cells or from sera and evaluated the potential of this assay for the diagnosis and screening of antiviral compounds.…”

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confidence: 99%

Quantitative Real-Time PCR Detection of Rift Valley Fever Virus and Its Application to Evaluation of Antiviral Compounds

et al. 2001

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The Rift Valley fever virus (RVFV), a member of the genus Phlebovirus (family Bunyaviridae) is an enveloped negative-strand RNA virus with a tripartite genome. Until 2000, RVFV circulation was limited to the African continent, but the recent deadly outbreak in the Arabian Peninsula dramatically illustrated the need for rapid diagnostic methods, effective treatments, and prophylaxis. A method for quantifying the small RNA segment by a real-time detection reverse transcription (RT)-PCR using TaqMan technology and targeting the nonstructural protein-coding region was developed, and primers and a probe were designed. After optimization of the amplification reaction and establishment of a calibration curve with synthetic RNA transcribed in vitro from a plasmid containing the gene of interest, real-time RT-PCR was assessed with samples consisting of RVFV from infected Vero cells. The method was found to be specific for RVFV, and it was successfully applied to the detection of the RVFV genome in animal sera infected with RVFV as well as to the assessment of the efficiency of various drugs (ribavirin, alpha interferon, 6-azauridine, and glycyrrhizin) for antiviral activity. Altogether, the results indicated a strong correlation between the infectious virus titer and the amount of viral genome assayed by real time RT-PCR. This novel method could be of great interest for the rapid diagnosis and screening of new antiviral compounds, as it is sensitive and time saving and does not require manipulation of infectious material.The Rift Valley fever (RVF) virus (RVFV), a member of the genus Phlebovirus, belongs to the Bunyaviridae family and possesses a negative-stranded, tripartite RNA genome composed of a large, a medium, and a small (S) segment (for reviews, see references 9 and 37). Like other phleboviruses, the S segment utilizes an ambisense strategy to code for two proteins, the nucleocapsid protein and the nonstructural protein (NS s ), which are synthesized from subgenomic viral complementary and viral sense mRNA, respectively.RVF is a mosquito-borne zoonosis predominantly provoking the death of young animals and abortion (e.g., sheep and goats) (for reviews, see references 24, 39 and 41). The disease was first identified in sheep by Daubney et al. in Kenya in 1931, and it is endemic almost everywhere in subtropical Africa (6). Transmission to humans occurs primarily by contact with infected animal body fluids and by mosquito bites. Infection is usually asymptomatic or associated with a brief self-limited febrile illness. However, complications such as retinitis, encephalitis, or hemorrhagic fever occur in some patients with mortality rates of up to 10 to 12% (21, 28).The potential of RVF as a disease emerging in new areas was first documented in Egypt in 1977 (16), and since then, epidemics have occurred in Mauritania (1987Mauritania ( to 1988Mauritania ( and 1998, Madagascar (1990to 1991), Egypt (1993, and eastern Africa (in Kenya, Somalia, and Tanzania) (references 33 and 34 and references therein). Recently, the...

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“…The reverse applied to sera where cell culture isolations of RVFV were successful in 13 (5%) cases and RT-PCR identified RVFV in just 4 (1.5%) of the cases. The nested RT-PCR procedure we employed targeting the S segment was reportedly more sensitive than virus isolation by cell culture for detection of RVFV in the sera of infected mice and lambs (Sall et al, 2001) but was certainly less sensitive than cell culture for detecting a single RVFV-infected mosquito in a pool of 50 (Ibrahim et al, 1997). Sang et al (2010) reported 18 isolations of RVFV from mosquito pools of 25 that had tested negative by a nested RT-PCR screening.…”

Section: Discussionmentioning

confidence: 99%

“…Extracted RNA was tested for RVFV using a nested reverse transcriptase-polymerase chain reaction (RT-PCR) that targets the NSs coding region of the small (S) segment (∼750 bp) according to the procedure and primer sequences published by Sall et al (2001). Amplified products were loaded onto 2% agarose gel, electrophoresed, and visualized by ethidium bromide staining against a molecular weight reference ladder.…”

Section: Analyses Of Human Clinical Samplesmentioning

confidence: 99%