Evaluation of a broadly reactive nucleic acid sequence based amplification assay for the detection of noroviruses in faecal material

Moore, Catherine; Clark, Eina M.; Gallimore, Christopher; Corden, Sally; Gray, Jim; Westmoreland, D.

doi:10.1016/s1386-6532(03)00170-7

Cited by 40 publications

(30 citation statements)

References 22 publications

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“…Norovirus NASBA. Noroviruses were detected using primers Ni, JV12Y, and JV13i, the last of which was labeled with a T7 RNA polymerase promoter to increase specificity (18,36). Different combinations of molecular beacons were used for detection: mix A, containing MBNVG2.1, MBNVG2.2, and MBNVG2.4; mix B, containing MBNVG1.1, MBNVG1.2, and MBNVG1.3; and mix C, containing MBGGIc, MBGGIIi, MBUK3, and MBJV5 (Table 1).…”

Section: Methodsmentioning

confidence: 99%

“…Moreover, NASBA does not use Taq polymerase enzymes that are sensitive to inhibitory factors present in the environment, such as humic acids and heavy metals (43), which might make NASBA less prone to environmental inhibitors. Norovirus NASBA assays by end-point detection have been described previously for detection of noroviruses in clinical samples (17,36) and in food (23). Recently, we described the successful real-time detection of enteroviruses in large-volume water samples by NASBA (40), indicating that a real-time NASBA assay for the detection of norovirus RNA might be applicable to water samples as well.…”

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confidence: 96%

“…With the increase in knowledge of norovirus sequences, many molecular detection assays that detect a wide variety of norovirus strains have been developed, e.g., reverse line blot hybridization (47), reverse transcriptase PCR (RT-PCR) (1,13,46), and nucleic acid sequence-based amplification (NASBA) (17,36). In the last decade, several different regions of the norovirus genome have been analyzed for their potential to detect the large variety of norovirus strains as well as newly occurring norovirus variants.…”

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confidence: 99%

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Real-Time Detection of Noroviruses in Surface Water by Use of a Broadly Reactive Nucleic Acid Sequence-Based Amplification Assay

Rutjes

Berg

Lodder

et al. 2006

Appl Environ Microbiol

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Noroviruses are the most common agents causing outbreaks of viral gastroenteritis. Outbreaks originating from contaminated drinking water and from recreational waters have been described. Due to a lack of cell culture systems, noroviruses are detected mostly by molecular methods. Molecular detection assays for viruses in water are often repressed by inhibitory factors present in the environment, like humic acids and heavy metals. To study the effect of environmental inhibitors on the performance of nucleic acid sequence-based amplification (NASBA), we developed a real-time norovirus NASBA targeting part of the RNA-dependent RNA polymerase (RdRp) gene. Specificity of the assay was studied with 33 divergent clones that contained part of the targeted RdRp gene of noroviruses from 15 different genogroups. Viral RNA originated from commercial oysters, surface waters, and sewage treatment plants in The Netherlands. Ninety-seven percent of the clones derived from human noroviruses were detected by real-time NASBA. Two clones containing animal noroviruses were not detected by NASBA. We compared the norovirus detection by real-time NASBA with that by conventional reverse transcriptase PCR (RT-PCR) with large-volume river water samples and found that inhibitory factors of RT-PCR had little or no effect on the performance of the norovirus NASBA. This consequently resulted in a higher sensitivity of the NASBA assay than of the RT-PCR. We show that by combining an efficient RNA extraction method with real-time NASBA the sensitivity of norovirus detection in water samples increased at least 100 times, which consequently has implications for the outcome of the infectious risk assessment.Noroviruses belong to the family Caliciviridae and contain a positive-sense, single-stranded RNA genome of approximately 7.6 kb (14). Based on sequence information of the genes encoding the viral RNA-dependent RNA polymerase (RdRp) and the capsid protein, the norovirus genus is divided into five genogroups (genogroup I [GGI] to GGV) that can be divided further into genetic clusters, each represented by a prototype virus. The diversity of norovirus variants increases continually due to the generation of new variants, with two groups of strains predominating in the past 5 years (22,29,33,34,39). Noroviruses that cluster within GGI and GGII are mostly human pathogens. They are the most common agents involved in outbreaks of gastroenteritis. Outbreaks originating from contaminated drinking water as well as from recreational water have been described previously (4,11,18,21,37).Due to the lack of a cell culture system, noroviruses are currently detected mainly by molecular techniques. With the increase in knowledge of norovirus sequences, many molecular detection assays that detect a wide variety of norovirus strains have been developed, e.g., reverse line blot hybridization (47), reverse transcriptase PCR (RT-PCR) (1,13,46), and nucleic acid sequence-based amplification (NASBA) (17,36). In the last decade, several different regions of the norovirus ...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 96%

mentioning

confidence: 99%

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Real-Time Detection of Noroviruses in Surface Water by Use of a Broadly Reactive Nucleic Acid Sequence-Based Amplification Assay

Rutjes

Berg

Lodder

et al. 2006

Appl Environ Microbiol

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“…In addition to the wide variety of RT-PCR-based amplification assays, isothermal PCR alternatives have also been developed for norovirus detection. Several groups have designed nucleic acid sequence-based amplification (NASBA) strategies by using previously reported primer pairs modified for NASBA compatibility (225)(226)(227)(228). A small study by Houde et al (226) determined that NASBA and RT-PCR using the GII primer sets described by Kageyama et al (198) showed equivalent analytical sensitivities but that the NASBA assay provided less consistent signals.…”

Section: Molecular Diagnostic Testsmentioning

confidence: 99%

Norovirus

2015

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SUMMARY Norovirus, an RNA virus of the family Caliciviridae , is a human enteric pathogen that causes substantial morbidity across both health care and community settings. Several factors enhance the transmissibility of norovirus, including the small inoculum required to produce infection (<100 viral particles), prolonged viral shedding, and its ability to survive in the environment. In this review, we describe the basic virology and immunology of noroviruses, the clinical disease resulting from infection and its diagnosis and management, as well as host and pathogen factors that complicate vaccine development. Additionally, we discuss overall epidemiology, infection control strategies, and global reporting efforts aimed at controlling this worldwide cause of acute gastroenteritis. Prompt implementation of infection control measures remains the mainstay of norovirus outbreak management.

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“…NASBA with detection by ECL-labeled probes is a highly sensitive and specific amplification method for enteroviruses and other RNA targets (2,6,8,15). In this study, we compared NASBA-ECL with real-time NASBA using molecular beacons and found excellent agreement.…”

Section: Discussionmentioning

confidence: 74%

Real-Time Nucleic Acid Sequence-Based Amplification Using Molecular Beacons for Detection of Enterovirus RNA in Clinical Specimens

2005

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Real-time nucleic acid sequence-based amplification (NASBA) using molecular beacon technology (NASBAbeacon) was compared to standard NASBA with postamplification hybridization using electrochemiluminescently labeled probes (NASBA-ECL) for detection of enteroviruses (EV) in 133 cerebrospinal fluid and 27 stool samples. NASBA-ECL and NASBA-beacon were similar in sensitivity, detecting 55 (100%) and 52 (94.5%) EV-positive samples, respectively. There were no false positives. Both NASBA assays were significantly more sensitive than culture. Real-time NASBA-beacon reagents and equipment rental were more expensive than those for NASBA-ECL; however, time to result was shortened by 1.5 h, hands-on time was reduced by 25 min, and the assay was much simpler for technologists to learn and perform.The detection of enteroviruses (EV) in clinical specimens, particularly spinal fluid, by molecular amplification techniques has important advantages over cell culture, namely, greater sensitivity and faster results. The impact on clinical decisionmaking as well as overall health care costs can be substantial (11,14). Although reverse transcription-PCR (RT-PCR) is the most commonly used test, nucleic acid sequence-based amplification (NASBA) using the Nuclisens Basic Kit has proved of equal or greater sensitivity for detection of enteroviruses (2, 6, 7).In the Nuclisens Basic Kit, amplified RNA products are detected by hybridization using electrochemiluminscently (ECL) labeled probes, a highly sensitive methodology. Recently, real-time RT-PCR using TaqMan chemistries has been applied to further shorten both technical hands-on time and time to result (3, 10). In this report, we compare the Nuclisens EasyQ Enterovirus Test, which utilizes real-time molecular beacons as probes (NASBA-beacon), to our standard NASBA with ECL probes (NASBA-ECL) for detection of EV in clinical specimens. MATERIALS AND METHODSSamples and patients. Samples submitted to the Clinical Virology Laboratory at Yale New Haven Hospital (YNHH) for EV diagnosis were used. For the retrospective study, RNA extracts from 53 samples stored at Ϫ70°C for 1 to 8 months were used. For the prospective study, 107 samples submitted for EV diagnosis from mid-May through August 2004 were tested within 0 to 3 days by NASBA with ECL detection as the standard clinical test. The RNA extracts were then retested by real-time NASBA-beacon (Nuclisens EasyQ Enterovirus Test) within 0 to 10 days. Extracts not tested the same day were stored at Ϫ70°C.The retrospective study included 53 cerebrospinal fluid (CSF) samples from 53 patients. The prospective study included 107 samples from 107 patients: 80 CSF and 27 stool samples. The stool samples were submitted as part of an epidemiologic investigation of an outbreak of meningitis, nausea, and vomiting in a church youth group returning from Mexico. Spinal fluid and stool samples were submitted in sterile containers. Patients' ages ranged from 5 days to 93 years, and 34 percent were over 18 years of age.Virus isolation. Viral cultures on CSF we...

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Evaluation of a broadly reactive nucleic acid sequence based amplification assay for the detection of noroviruses in faecal material

Cited by 40 publications

References 22 publications

Real-Time Detection of Noroviruses in Surface Water by Use of a Broadly Reactive Nucleic Acid Sequence-Based Amplification Assay

Real-Time Detection of Noroviruses in Surface Water by Use of a Broadly Reactive Nucleic Acid Sequence-Based Amplification Assay

Norovirus

Real-Time Nucleic Acid Sequence-Based Amplification Using Molecular Beacons for Detection of Enterovirus RNA in Clinical Specimens

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