Schematic diagram of primers used in the RT-LAMP assay and the nucleotide sequence of the Nucleoprotein gene used to generate the inner, outer and loop primers (GenBank accession No. M13215). The arrows indicate the location of primers and the direction of primer extension. Sph1 restriction enzyme site was highlighted 

Monitoring of LAMP Amplification: A) Real-time turbidity of RT-LAMP reaction detected by real-time turbidimeter, RV1 and 2: Rabies clinical samples; B) Agarose gel analysis of RT-LAMP amplified products: Lane 1 and 2: Rabies clinical samples; Lane P: Positive; Lane N: Negative control without template RNA; Lane M: 1 Kb DNA ladder; C: Visualization of RT-LAMP products in the presence of HNB dye 

Sensitivity of the RT-LAMP assay: A) Agarose gel electrophoresis analysis with extracted RNA from known FFU of the rabies virus and visual inspection with HNB dye: Lane M: 1 Kb DNA ladder; Lane N: Negative control without template RNA; Lane 1: 104 FFU; Lane 2: 103 FFU; Lane 3: 102 FFU; Lane 4: 101 FFU; Lane 5: 10-1 FFU; B) Agarose gel electrophoresis analysis for copy number determination using in vitro transcribed RNA and visual inspection with HNB dye: Lane M: 100 bp DNA ladder; Lane N: Negative control without template RNA; Lane 1 to 5: tenfold dilutions of RNA from 107 to 103 copies/reaction; C) One Step RT-PCR with in vitro transcribed RNA shows a 605 bp amplification product: Lane M: 100 bp DNA ladder: Lane N: Negative control without template RNA: Lane 1 to 5: tenfold dilutions of RNA from 107 to 103 copies/reaction 

Rabies RT-LAMP evaluation was performed using extracted RNA from infected brain samples. RV1 to RV5 (Lane 1 to 5) are the rabies clinical samples. Negative: Normal-mouse brain tissue; Positive: rabies positive sample; (A) Real-time monitoring of RT-LAMP amplification by turbidimeter; B) Agarose gel analysis revealing the typical electrophoresis pattern of LAMP amplified product; C) Tube containing the amplified RT-LAMP products in the presence of HNB dye