Use of TaqMan real-time reverse transcription-PCR for rapid detection, quantification, and typing of norovirus

Abstract

Noroviruses (NoVs) are the most commonly identified cause of outbreaks and sporadic cases of acute gastroenteritis. We evaluated and optimized NoV-specific TaqMan real-time reverse transcription (RT)-PCR assays for the rapid detection and typing of NoV strains belonging to genogroups GI and GII and adapted them to the LightCycler platform. We expanded the detection ability of the assays by developing an assay that detects the GIV NoV strain. The assays were validated with 92 clinical samples and 33 water samples from confirmed NoV outbreaks and suspected NoV contamination cases. The assays detected NoV RNA in all of the clinical specimens previously confirmed positive by conventional RT-PCR and sequencing. Additionally, the TaqMan assays successfully detected NoV RNA in water samples containing low viral concentrations and inhibitors of RT and/or PCR, whereas the conventional method with region B primers required dilution of the inhibitors. By means of serially diluted NoV T7 RNA transcripts, a potential detection limit of <10 transcript copies per reaction mixture was observed with the GII assay and a potential detection limit of <100 transcript copies per reaction mixture was observed with the GI assay. These results and the ability to detect virus in water that was negative by RT-PCR demonstrate the higher sensitivity of the TaqMan assay compared with that of a conventional RT-PCR assay. The TaqMan methods dramatically decrease the turnaround time by eliminating post-PCR processing. These assays have proven useful in assisting scientists in public health and diagnostic laboratories report findings quickly to outbreak management teams.

FIG. 1.

Phylogenetic dendrogram of strains belonging to clusters of human NoVs. Underlined clusters denote GI, GII, and GIV strains tested by real-time RT-PCR (43).

FIG. 2.

CNs detected from archived specimens of water and stool. A comparison of water (A) (n = 33) and stool (B) (n = 72) samples is shown. Specimens with CNs of >40 or not detected were considered negative (Neg).

FIG. 3.

Relationship between known copy numbers of T7 RNA (GI and GII) or viral RNA from stool extract (GIV) and the detection threshold. T7 RNA transcripts were made for representative GI and GII strains, and a fecal specimen was used for GIV. (A) Serial dilution of a GI/3 NoV RNA transcript demonstrating a wide dynamic range. The detection limit was 9.836 × 10¹ copies/μl, the slope was −3.370, the y intercept was 43.97, and the R² value was −1.00. (B) GII/4 serial dilution of a NoV RNA transcript showing a wide dynamic range. The detection limit was 9.05 copies/μl, the slope was −3.324, the y intercept was 39.20, and the R² value was −1.00. The mean squared error was 0.195. (C) (GIV) NoV RNA stool standard serial dilutions. The slope was −3.310, the y intercept was 58.96, and the R² value was −1.00. The mean squared error was 0.0763. The detection limit of the GIV stool standard curve was 8.644 × 10⁵ copies/μl. The slope, y intercept, and mean squared error were 3.392, 59.27, and 0.0764, respectively, and the R² value was −1.00.

FIG. 4.

Detection of NoV in water samples from cruise ship A by conventional (A) versus real-time (B) RT-PCR assay. (A) A conventional RT-PCR assay failed to detect NoV RNA from potable water (lanes 2 and 3) compared with a positive control (lane 4, GII strain) and a negative control (lane 5, water). Lanes 1 and 6 were 123-bp ladders in a 3% agarose gel with 0.5 μg of ethidium bromide run at 120 V for 60 min. (B) A real-time RT-PCR assay identified 2 positive water samples (▴, ⋄) among 12 samples tested. Controls included a GII/4 strain T7 RNA transcript (▪), a GII/4 fecal specimen (○), and a no-template negative control water sample (−). (C) Conventional RT-PCR detected NoV RNA in potable water after samples were diluted 1:100. Lane 1 was a 123-bp ladder in a 3% agarose gel with 0.5 μg of ethidium bromide run at 120 V for 60 min. Lanes 2 and 5 were positive controls (GII strain); lanes 3 and 7 were water samples 1 and 2, respectively; and lanes 4 and 6 were negative controls.