Development of a Recombinase Polymerase Amplification Assay for the Rapid Isothermal Detection of Human Norovirus

Introduction: Reverse transcriptase qPCR (RT-qPCR) is commonly used for human norovirus (HuNoV) detection, however it is sensitive to matrix-associated inhibitors; not readily field deployable; and produces results in 1-2 hours. Recombinase polymerase amplification (RPA) uses a recombinase protein to anneal DNA for extension and amplification under isothermal conditions rapidly with potentially less sensitivity to inhibitors. Reverse transcriptase RPA (RT-RPA) has been developed for detection of other viruses but not HuNoV.

Purpose: To develop an RT-RPA assay specific to HuNoV. 

Methods: Multiple primer sets corresponding to the HuNoV GII.4 New Orleans genome were identified. Using different primer combinations, the capability of amplification of purified HuNoV RNA was screened by RT-RPA (TwistDx, Cambridgeshire, UK) and a probe designed to accommodate candidate primer sets. The RT-RPA assay was used to identify the optimal primer sets that were then tested for efficiency (time to result) and limit of detection using serially diluted GII.4 New Orleans.

Results: Forty-eight primer combinations (8 candidate forward primers and 12 reverse primers) were subjected to initial screening. Eight primer pairs selectively amplified the desired product and one corresponding probe was designed. Two primer pairs (G2F5-G2R11 and G2F5-G2R12) produced significantly faster results (P < 0.05).  At 40°C, time to results ranged from <1 minute to 7.0 minutes when amplifying purified RNA in the range of 1.5 x 10¹⁰ to 1.5 x 10⁶ genomic copies. When applied to dilutions of briefly heated 20% outbreak stool suspensions, signals in the range of 1.5 x 10¹⁰ to 1.5 x 10⁷genome equivalent copies in 6.6 to 14.7 minutes were produced. 

Significance: This is the first report of a RT-RPA assay for detection of HuNoV in relatively unpurified samples under isothermal conditions in minutes. The RT-RPA assay has potential for near “real-time” detection of HuNoV contamination in complex samples like foods and feces.