Real-Time Reverse-Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of Rift Valley Fever Virus

Abstract

The development and validation of a one-step, single-tube, real-time accelerated reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of the L RNA segment of Rift Valley fever virus (RVFV) are described. The assay was performed at a constant temperature (63°C), with a real-time follow-up using a LightCycler and a double-stranded-DNA-intercalating fluorochrome. The assay is highly sensitive and comparable to real-time RT-PCR, with a detection limit of ϳ10 RNA copies per assay. However, the RT-LAMP assay is much faster than traditional RT-PCR and generates results in <30 min for most diluted samples. The specificity of the primers was established using other, related arboviruses as well as viruscontaining and virus-free sera. The RT-LAMP assay reported here is thus a valuable tool for the rapid detection of RVFV in field diagnostic laboratories.

Recombinase polymerase amplification assay for rapid detection of Rift Valley fever virus

“…10 Two LAMP protocols targeting the RVFV L-gene have been published. 24,25 In general LAMP assays need 6 primers leading to longer amplicons and possibly more difficult design in the case of highly variable RNA viruses, whereas the RPA design with three oligonucleotides offers almost the same flexibility as real time PCR. However the longer TwistAmp TM Exo probes may be a design obstacle and here the use of locked nucleotides may help to reduce probe length.…”

Molecular diagnosis of infections and resistance in veterinary and human parasites

“…A change from polymerase chain reaction to loop-mediated isothermal amplication (LAMP) has certain advantages because temperature cycling is not necessary. A number of other amplification systems which do not require temperature cycling have also been developed (Compton, 1991;Guatelli et al, 1990;Walker et al, 1992a,b;Abramowitz, 1996;Vrana, 1996) and applied to parasite detection (Matovu et al, 2010a), but LAMP is currently the most widely used alternative to PCR (Kuboki et al, 2003;Thekisoe et al, 2005;Alhassan et al, 2007;Han et al, 2007;Karanis et al, 2007;Guan et al, 2008;Njiru et al, 2008a,b;Peyrefitte et al, 2008;Aonuma et al, 2009;Liang et al, 2009;Nkouawa et al, 2009;Takagi et al, 2009;Abbasi et al, 2010;Adams et al, 2010;Iseki et al, 2010;Kumagai et al, 2010;Lau et al, 2010;Matovu et al, 2010b;Ndung'u et al, 2010;Polley et al, 2010;Poschl et al, 2010;Thekisoe et al, 2010;Wang et al, 2010;Njiru, 2011). LAMP and these other alternatives to PCR are still based on enzymatic reactions however, so the transport and storage of reagents at below room temperature is a requirement and a challenge for field application.…”

Rapid Genome Detection of Schmallenberg Virus and Bovine Viral Diarrhea Virus by Use of Isothermal Amplification Methods and High-Speed Real-Time Reverse Transcriptase PCR

“…Real-time monitoring using an intercalating dye was chosen as the detection strategy for LAMP, in order to prevent contamination and to enable a direct comparison to RT-qPCR and RPA. This approach has previously been described and successfully applied (18,37,38). However, the LAMP reaction can also be performed using a simple heat block or a water bath.…”