Purpose: To evaluate the performance of a reverse transcription (RT)-qRPA assay for rapid detection of HuNoV in multiple samples.
Methods: A previously developed primer-probe set (G2F5/G2R11/G2P1) used in RT-qRPA (TwistDx, Cambridge, UK) developed for detection of HuNoV GII.4 was applied to multiple serially diluted, heated stool samples – or purified RNA thereof – derived from foodborne outbreaks.
Results: The G2F5/G2R11/G2P1 primer-probe set was successfully used to detect HuNoV GII.4 New Orleans by RT-qRPA in all six patient stool samples (2 - 20% suspended in PBS) and RNA extracted from these samples. The average rates of detection [time in min of signal development per log genomic copy (LGC) of template in the reaction] for RNA and stool samples were 1.44 ± 0.29 min/LGC and 1.45 ± 0.38 min/LGC, respectively. There was no significant difference in rate of signal development when comparing RT-qRPA done using heated stool suspension dilutions with purified RNA (P > 0.05). Direct detection of HuNoV in dilutions of all heated HuNoV stools was achieved between 7.2 and 16.7 min. The limit of detection (LOD) of the assay was 5.5 ± 1.6 LGC for stool and 4.7 ± 0.5 LGC for purified RNA.
Significance: HuNoV could be detected using the RT-qRPA method in 20% fecal suspensions of the majority of patients and 2% suspensions of all of the patients screened – both being relatively dirty matrices – in less than 20 min with reasonable detection limits. This rapid, portable, isothermal method has potential for use in direct detection of HuNoV in food, food handler and environmental samples.