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.2004 Oct;42(10):4679-85.
doi: 10.1128/JCM.42.10.4679-4685.2004.

Evaluation and validation of real-time reverse transcription-pcr assay using the LightCycler system for detection and quantitation of norovirus

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Evaluation and validation of real-time reverse transcription-pcr assay using the LightCycler system for detection and quantitation of norovirus

Xiaoli Pang et al. J Clin Microbiol.2004 Oct.

Abstract

We developed an assay for the detection and quantitation of norovirus with the LightCycler SYBR Green-based real-time reverse transcription-PCR (real-time LC RT-PCR) and previously published primers in the capsid and the polymerase gene. One hundred thirty-two stool specimens from the Provincial Laboratory for Public Health (Microbiology), Alberta, Canada, and the Centers for Disease Control and Prevention, Atlanta, Ga., were used to validate the new assay. The samples were collected from patients involved in outbreaks of acute gastroenteritis or children who presented with sporadic gastroenteritis. The real-time LC RT-PCR assay detected norovirus strains from three genogroup I (G-I) clusters (G-I/1, G-I/2, and G-I/3) and 10 genogroup II (G-II) clusters (G-II/1, G-II/2, G-II/3, G-II/4, G-II/6, G-II/7, G-II/10, G-II/12, G-II/15, and G-II/16). There was 100% concordance with the results from 58 stool specimens which tested positive by conventional RT-PCR assays. By dilution analysis, the real-time LC RT-PCR was 10,000 times more sensitive than the conventional RT-PCR. The new assay increased the number of samples in which noroviruses were detected by 19%. The real-time LC RT-PCR had a wide dynamic range, detecting from 5 to 5 x 10(6) copies of RNA per reaction, resulting in a theoretical lower limit of detection of 25,000 copies of RNA per g of stool. No cross-reactions were found with specimens containing sapovirus, rotavirus, astrovirus, and adenovirus. Because of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, the real-time LC RT-PCR will be useful as a routine assay for the clinical diagnosis of norovirus infection.

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Figures

FIG. 1.
FIG. 1.
Two curves fitted by plotting the percentage of specimens that tested positive for norovirus by both the real-time LC RT-PCR and conventional RT-PCR assays against theCt (threshold cycle) of the samples and by plotting the percentage of specimens that tested positive only by the real-time RT-PCR against theCt values (the sigmoid curves). TheCt values were also plotted against the RNA copies per gram of stool (dashed line). Specimens detectable by both RT-PCR assays contained high concentration of norovirus RNA (median, 106.5 at the cross point), and the specimens that tested positive only by the real-time LC RT-PCR contained low copy number of the norovirus RNA (median, 105 at the cross point). When the norovirus RNA copy number per gram of stool is ≤105 and theCt value is >31, the specimen will only test positive by the real-time LC RT-PCR assay.
FIG.2.
FIG.2.
Comparison of sensitivity of norovirus detection by real-time LC RT-PCR and conventional RT-PCR in a series of 10-fold dilutions of norovirus RNA (neat to 10−9). A. Melting curve analysis of norovirus PCR fragments and primer-dimers. The melting temperature (Tm) for the norovirus PCR fragment is 82.50°C, and theTm for the primer-dimmer is less than 78°C. B. Amplification profile of real-time LC RT-PCR. SYBR Green dye intensity was plotted against the cycle number (top), and the linear regression is shown (bottom). C. The RT-PCR products by real-time (top) and conventional (bottom) RT-PCR were separated on a 3% agarose gel stained with ethidium bromide. Lanes 2 to 11 correspond to dilutions from neat to 10−9, respectively.
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