Evaluation of a real-time mobile PCR device (PCR 1100) for the detection of the rabies gene in field samples

Abstract

Background: The Philippines is ranked among the top countries with 200–300 annual deaths due to rabies. Most human rabies cases have been reported in remote areas, where dog surveillance is inadequate. Therefore, a strategy to effectively improve surveillance in remote areas will increase the number of detections. Detecting pathogens using portable real-time reverse transcription-polymerase chain reaction (RT-PCR) has the potential to be accepted in these areas. Thus, we aimed to develop an assay to detect the rabies virus (RABV) genome by combining the robust primer system LN34 with the PicoGene PCR1100 portable rapid instrument targeting RABV RNA (PCR1100 assay). Methods: Procedures were optimised using an LN34 primer/probe set, KAPA3G Plant PCR Kit (KAPA Biosystems), FastGene Scriptase II (NIPPON Genetics), and an artificial positive control RNA. Results: Positive control RNA showed an analytical limit of detection of 10 copies/µL without false positivity, generating results in approximately 32 min. Compared to dFAT or RT-qPCR using field samples, the sensitivity and specificity of the PCR1100 assay were 100%, and even lower copy numbers (approximately 10 copies/µL) were detected. Conclusions: This study demonstrated that the developed assay can detect rabies RNA in field samples. Because dog-mediated rabies is endemic in remote areas, the rapidity, mobility, and practicality of the PCR1100 assay as well as the high sensitivity of the LN34 system make it an ideal tool for the confirmation of rabies in these areas.