Human noroviruses are widespread and contagious viruses causing nonbacterial gastroenteritis. Real-time reverse transcription quantitative PCR (real-time RT-qPCR) is currently the gold standard for the sensitive and accurate detection of these pathogens and serves as a critical tool in outbreak prev...
Human noroviruses are widespread and contagious viruses causing nonbacterial gastroenteritis. Real-time reverse transcription quantitative PCR (real-time RT-qPCR) is currently the gold standard for the sensitive and accurate detection of these pathogens and serves as a critical tool in outbreak prevention and control. Different surveillance teams, however, may use different assays, and variability in specimen conditions may lead to disagreement in results. Furthermore, the norovirus genome is highly variable and continuously evolving. These issues necessitate the re-examination of the real-time RT-qPCR's robustness in the context of accurate detection as well as the investigation of practical strategies to enhance assay performance. Four widely referenced real-time RT-qPCR assays (Assays A-D) were simultaneously performed to evaluate characteristics such as PCR efficiency, detection limit, and sensitivity and specificity with RT-PCR, and to assess the most accurate method for detecting norovirus genogroups I and II. Overall, Assay D was evaluated to be the most precise and accurate assay in this study. A ZEN internal quencher, which decreases nonspecific fluorescence during the PCR, was added to Assay D's probe, which further improved the assay performance. This study compared several detection assays for noroviruses, and an improvement strategy based on such comparisons provided useful characterizations of a highly optimized real-time RT-qPCR assay for norovirus detection.
Human noroviruses are widespread and contagious viruses causing nonbacterial gastroenteritis. Real-time reverse transcription quantitative PCR (real-time RT-qPCR) is currently the gold standard for the sensitive and accurate detection of these pathogens and serves as a critical tool in outbreak prevention and control. Different surveillance teams, however, may use different assays, and variability in specimen conditions may lead to disagreement in results. Furthermore, the norovirus genome is highly variable and continuously evolving. These issues necessitate the re-examination of the real-time RT-qPCR's robustness in the context of accurate detection as well as the investigation of practical strategies to enhance assay performance. Four widely referenced real-time RT-qPCR assays (Assays A-D) were simultaneously performed to evaluate characteristics such as PCR efficiency, detection limit, and sensitivity and specificity with RT-PCR, and to assess the most accurate method for detecting norovirus genogroups I and II. Overall, Assay D was evaluated to be the most precise and accurate assay in this study. A ZEN internal quencher, which decreases nonspecific fluorescence during the PCR, was added to Assay D's probe, which further improved the assay performance. This study compared several detection assays for noroviruses, and an improvement strategy based on such comparisons provided useful characterizations of a highly optimized real-time RT-qPCR assay for norovirus detection.
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문제 정의
This study provides further evidence for the need of routine evaluation of real-time RT-qPCR and RT-PCR in consideration of their risk for false predictions.
제안 방법
However, the accuracy of absolute quantification is determined by the LOQ’s accuracy, which is defined by the PCR efficiency and R2 value. Assays A and D were characterized with sensitive LOQ, allowing quantification of virus copies to at least 1 log genomic copies per reaction (Table 3), and so were evaluated as the more accurate assays for detecting low concentrations of norovirus.
base from the 5’ end, as an insertion in the phosphate-pentose backbone. Fresh RNA extractions were used to simultaneously run Assay D and the modified Assay D (Assay D-zen) for GII, each twice consecutively using the Norovirus RNA Positive Control (AccuPower Norovirus Real-Time RT-PCR Kit; Bioneer) as the standard control. Assay D-zen followed the same primer concentrations and cycling conditions as Assay D (Table 2).
Positive results of both real-time RT-qPCR and RT-PCR were further compared by Venn diagram analysis to determine the most efficient assay with the greatest detection rate.
RNA samples were subjected to conventional RT-PCR using a semi-nested procedure, using COG1F, G1SKF, G1SKR [19] and GI-F1M, GI-F2, GI-R1M primer sets [20] for GI, and COG2F, G2SKF, G2SKR [19] and GII-F1M, GII-F3M, GII-R1M primer sets [20] for GII detection (Table 1). One-step RT was carried out using the OneStep RT-PCR Kit (Qiagen).
The accuracy of each real-time RT-qPCR assay according to RT-PCR results was determined by the following equations for sensitivity, specificity, positive predictive value, and negative predictive value.
The evaluation and comparison of different norovirus detection assays will provide useful insight to examine the consistency between assays. The aim of this study was to investigate characteristics, such as PCR efficiency and detection limit, as well as sensitivity and specificity with RT-PCR. Based on such assessment, the effect of an internal quencher on assay sensitivity was investigated.
대상 데이터
9, which were detected in this study. Eleven GII sequences were used for alignment with GII primers and probes and included the Lordsdale GII.4 reference strain, GII.2, GII.6, GII.13, and GII.17 detected in this study (Fig. 3). Sequence alignment showed that the primers and probes of the four assays had essentially the same target region.
Seven GI sequences were used for alignment with GI primer and probe sets and included the Norwalk GI.1 reference strain, GI.4, GI.6, GI.8, and GI.9, which were detected in this study.
이론/모형
Positive results of real-time RT-qPCR assays according to LOD, and positive results of RT-PCR assays were overlaid on Venn diagrams to observe agreement between the two methods (Fig. 1).
성능/효과
Results of the real-time RT-qPCR assays were compared with the results of semi-nested RT-PCR assays [19, 20]. Analysis with RT-PCR confirmed that, out of a total of 61 samples tested, 14 were positive for GI (22.9%) and 25 were positive for GII (40.9%), of which 10 (16.4%) were samples with mixed genogroups. Multiple sequence alignment of these RT-PCR amplicon sequences using the BLASTN program confirmed 7 GI genotypes (GI.
1). By combining positive results of both real-time RT-qPCR and RT-PCR, there were a total of 24 positives for GI and 34 positives for GII. Comparison of real-time RT-qPCR assays suggested that Assays A and D were more in agreement with RT-PCR than Assays B and C.
4%) were samples with mixed genogroups. Multiple sequence alignment of these RT-PCR amplicon sequences using the BLASTN program confirmed 7 GI genotypes (GI.1, GI.3, GI.4, GI.5, GI.6, GI.8, GI.9) and 5 GII genotypes (GII.2, GII.4, GII.6, GII.13, GII.17).
The total number of positive results by RT-PCR assays [19, 20] are indicated in yellow. Positive results of the four real-time RT-qPCR assays are colored respectively in orange, green, blue, and purple.
The performance of real-time RT-qPCR assays was evaluated by their PCR efficiency, limit of quantification (LOQ), and limit of detection (LOD). PCR efficiency was determined by the following equation:
참고문헌 (31)
Ahmed SM, Hall AJ, Robinson AE, Verhoef L, Premkumar P, Parashar UD, et al. 2014. Global prevalence of norovirus in cases of gastroenteritis: a systematic review and meta-analysis. Lancet Infect. Dis. 14: 725-730.
Patel MM, Widdowson MA, Glass RI, Akazawa K, Vinje J, Parashar UD. 2008. Systematic literature review of role of noroviruses in sporadic gastroenteritis. Emerg. Infect. Dis. 14: 1224-1231.
Cheesbrough JS, Green J, Gallimore CI, Wright PA, Brown DWG. 2000. Widespread environmental contamination with Norwalk-like viruses (NLV) detected in a prolonged hotel outbreak of gastroenteritis. Epidemiol. Infect. 125: 93-98.
Stals A, Mathijs E, Baert L, Bottledoorn N, Denayer S, Mauroy A, et al. 2012. Molecular detection and genotyping of noroviruses. Food Environ. Virol. 4: 153-167.
Green K. 2013. Caliciviridae: The Noroviruses, pp. 583-609. In Knipe DM, Howley PM, Cohen JI, Griffin DE, Lamb RA, Martin MA, Racaniello VR, Roizman B (eds). Fields Virology, 6th Ed. Vol 1. Lippincott Williams & Wilkins, Philadelphia, PA, USA.
Kageyama T, Kojima S, Shinohara M, Uchida K, Fukushi S, Hoshino FB, et al. 2003. Broadly reactive and highly sensitive assay for Norwalk-like viruses based on real-time quantitative reverse transcription-PCR. J. Clin. Microbiol. 41: 1548-1557.
Jothikumar N, Lowther JA, Henshilwood K, Lees DN, Hill VR, Vinje, J. 2005. Rapid and sensitive detection of noroviruses by using TaqMan-based one-step reverse transcription-PCR assays and application to naturally contaminated shellfish samples. Appl. Environ. Microbiol. 71: 1870-1875.
Park YB, Cho YH, Ko GP. 2011. A duplex real-time RT-PCR assay for the simultaneous genogroup-specific detection of noroviruses in both clinical and environmental specimens. Virus Genes 43: 192-200.
Ministry of Food and Drug Safety. 2013. Guideline for Investigation on the Cause of Food Poisoning, Ch. 5. Ministry of Food and Drug Safety, Chungcheonbuk-do, Republic of Korea. [In Korean].
Mattison K, Grudeski E, Auk B, Brassard J, Charest H, Dust K, et al. 2011. Analytical performance of norovirus real-time RT-PCR detection protocols in Canadian laboratories. J. Clin. Virol. 50: 109-113
Lee SG, Lee SH, Park SW, Suh CI, Jheong WH, Oh S, Paik SY. 2011. Standardized positive controls for detection of norovirus by reverse transcription PCR. Virol. J. 8: 260-267.
Martinez-Martinez M, Diez-Valcarce M, Hernandez M, Rodriguez-Lazaro D. 2011. Design and application of nucleic acid standards for quantitative detection of enteric viruses by real-time PCR. Food Environ. Virol. 3: 92-98.
Xia H, Gravelsina S, Ohrmalm C, Ottoson J, Blomberg J. 2016. Development of single-tube nested real-time PCR assays with long internally quenched probes for detection of norovirus genogroup II. Biotechniques 60: 28-34.
Kojima S, Kageyama T, Fukushi S, Hoshino FB, Shinohara M, Uchida K, et al. 2002. Genogroup-specific PCR primers for detection of Norwalk-like viruses. J. Virol. Methods 100: 107-114.
Kim SH, Cheon DS, Kim JH, Lee DH, Jheong WH, Heo YJ, et al. 2005. Outbreaks of gastroenteritis that occurred during school excursions in Korea were associated with several waterborne strains of norovirus. J. Clin. Microbiol. 43: 4836-4839.
Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. 2009. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55: 611-622.
Lopman B, Simmons K, Gambhir M, Vinje J, Parashar U. 2014. Epidemiological implications of asymptomatic reinfection: a mathematical modeling study of norovirus. Am. J. Epidemiol. 179: 507-512.
Kabue JP, Meader E, Hunter PR, Potgieter N. 2016. Norovirus prevalence and estimated viral load in symptomatic and asymptomatic children from rural communities of Vhembe district, South Africa. J. Clin. Virol. 84: 12-18.
Stals A, Van Collie E, Uyttendaele M. 2013. Virus genes everywhere: public health implications of PCR-based testing of foods. Curr. Opin. Virol. 3: 69-73.
Park GW, Collins N, Barclay L, Hu L, Prasad BVV, Lopman BA, Vinje J. 2016. Strain-specific virolysis patterns of human noroviruses in response to alcohols. PLoS One 11: e0157787
Vega E, Barclay L, Gregoricus N, Williams K, Lee D, Vinje J. 2011. Novel surveillance network for norovirus gastroenteritis outbreaks, United States. Emerg. Infect. Dis. 17: 1389-1395.
Gonzalez MD, Langley LC, Buchan BW, Faron ML, Maier M, Templeton K, et al. 2016. Multicenter evaluation of the Xpert norovirus assay for detection of norovirus genogroups I and II in fecal specimens. J. Clin. Microbiol. 54: 142-147.
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