Rapid detection of norovirus from fecal specimens by real-time reverse transcription-loop-mediated isothermal amplification assay

Abstract

In this study, we developed a one-step, single-tube genogroup-specific reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of norovirus (NoV) genomes targeting from the C terminus of the RNA-dependent RNA polymerase gene to the capsid N-terminal/shell domain region. This is the first report on the development of an RT-LAMP assay for the detection of NoV genomes. Because of the diversity of NoV genotypes, we used 9 and 13 specially designed primers containing mixed bases for genogroup I (GI) and II (GII), respectively. The RT-LAMP assay had the advantages of rapidity, simplicity, specificity, and selectively and could obtain results within 90 min, generally even within 60 min, under isothermal conditions at 62 degrees C. The detection limits for NoV genomes were between 10(2) and 10(3) copies/tube for GI and GII with differentiation by genotype, and no cross-reactions among NoV GI and GII and other gastroenteritis viruses, such as sapovirus, human astrovirus, adenovirus type 40 and 41, and group A and C rotavirus, were found. In the evaluation tests with fecal specimens obtained from gastroenteritis outbreaks, the sensitivity and specificity of the RT-LAMP assay with regard to RT-PCR were 100 and 94% for GI and 100 and 100% for GII, respectively. These findings establish that the RT-LAMP assay is potentially useful for the rapid detection of NoV genomes from fecal specimens in outbreaks of food-borne and person-to-person-transmitted gastroenteritis.

FIG. 1.

Location of primers used for genogroup I (A) and II (B) for genogroup-specific RT-LAMP assay. (A) Sequence of Norwalk/68/US (GenBank accession number M87661); (B) sequence of Lordsdale/93/UK (X86557). Numbers at the beginning and end of the sequences denote nucleotide positions. Primers FIP and BIP in Table 1 consist of F1C plus TTTT plus F2 and B1C plus TTTT plus B2, respectively, where TTTT is a spacer. The B3 primers for genogroups I and II were primers G1SKR and G2SKR, respectively, as described by Kojima et al. (13).

FIG. 2.

Sensitivity of genogroup-specific RT-LAMP assay for standard RNAs of genotype GI/2 for genogroup I (A) and GII/12 for genogroup II (B). Lanes: M, 100-bp DNA ladder; 1, 10⁴ copies/tube; 2, 10³ copies/tube; 3, 10² copies/tube; 4, 10¹ copies/tube. Positive reaction shows a ladder-like pattern in 3% agarose gel electrophoresis. The turbidity was measured using an LA-320C (Teramecs), and RT-PCR was carried out with primers G1SKF and G2SKR for genogroup I and G2SKF and G2SKR for genogroup II (13). +, positive; −, negative.

FIG. 3.

Evaluation of cross-reactivity of genogroup-specific RT-LAMP assay between genogroups I and II. Standard RNAs of genotypes GI/2 and GII/12 at 10⁸ copies/tube were used in genogroups I and II, respectively. The real-time turbidity was determined using an LA-320C (Teramecs).