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°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.

Efficient Identification of Clinically Relevant Candida Yeast Species by Use of an Assay Combining Panfungal Loop-Mediated Isothermal DNA Amplification with Hybridization to Species-Specific Oligonucleotide Probes

“…3A), which may be ascribed to the five mismatches between the FIP primer and the respective C. krusei target site. An alternative to circumvent this lower sensitivity could be to utilize degenerated primers or a mixture of primers, which has proved successful in other LAMP assays (10,38). We confirmed that the LAMP reaction proceeds without a previous thermal denaturation of the template DNA (30), making this technique really isothermal (Fig.…”

Simultaneous Detection and Genogroup‐Screening Test for Norovirus Genogroups I and II from Fecal Specimens in Single Tube by Reverse Transcription‐Loop‐Mediated Isothermal Amplification Assay

“…These specimens gave negative results in the sRT-LAMP assay within 90 min (data not shown). The specificity and selectivity of the RT-LAMP assay has also been demonstrated in previous studies (3,10,11).…”

“…The sRT-LAMP assay was performed in 25 µl of reaction mixture with an RNA amplification kit (Eiken Chemical, Tochigi, Japan). The conditions for sRT-LAMP assay were the same as in the previous study (3), but the format of the reaction mixture was slightly changed. Briefly, 20 pmol of each of the three newlydesigned primers were added into the reaction mixture and seven forward loop primers were used as fluorescence-labeled primers.…”

Noroviruses Surrogate Detection Using Loop-Mediated Isothermal Amplification (LAMP), Conventional RT-PCR and Quantitative RT-PCR in Sg. Tekala and Sg. Gabai Stream Water

“…A study based on human volunteer showed the NoV infectious dose may be as low as 10to 100 viruses particles [6]emphasizes the need on highly sensitive method to detect their prevalence in water. NoV is usually detected in 3to 4h by RT-PCR method [22]. In latest innovation in DNA detection by amplification techniques, loop-mediated isothermal amplification (LAMP) has gain attention for it versatility in shorten the DNA amplification time to around one hour with results visible by naked eye using fluorescent dye.…”