Rapid and sensitive lateral flow biosensor for the detection of GII human norovirus based on immunofluorescent nanomagnetic microspheres.

Human norovirus (HuNoV) is the most predominant viral agents of acute gastroenteritis. Point-of-care testing (POCT) based on lateral flow immunochromatography (LIFC) has become an important tool for rapid diagnosis of HuNoVs. However, low sensitivity and lack of quantitation are the bottlenecks of traditional LIFC. Thus, we established a rapid and accurate technique that combined immunomagnetic enrichment (IM) with LFIC to identify GII HuNoVs in fecal specimens. Before preparing immunofluorescent nanomagnetic microspheres and achieving the effect of HuNoV enrichment in IM and fluorescent signal in LFIC, amino-functionalized magnetic beads (MBs) and carboxylated quantum dots (QDs) were coupled at a mass ratio of 4:10. Anti-HuNoV monoclonal antibody was then conjugated with QDs-MB. The limit of detection was 1.56 × 104 copies/mL, and the quantitative detection range was 1.56 × 104 copies/mL-1 × 106 copies/mL under optimal circumstances. The common HuNoV genotypes GII.2, GII.3, GII.4, and GII.17 can be detected, there was no cross-reaction with various enteric viruses, including rotavirus, astrovirus, enterovirus, and sapovirus. A comparison between IM-LFIC and RT-qPCR for the detection of 87 fecal specimens showed a high level of agreement (kappa = 0.799). This suggested that the method is rapid and sensitive, making it a promising option for point-of-care testing in the future.

Bayesian evaluation of temporal changes in sensitivity and specificity of three serological tests for multiple circulating strains of rabbit haemorrhagic disease virus.

Competition and indirect ELISAs are currently being used to monitor rabbit haemorrhagic disease viruses (RHDV1 and RHDV2) in rabbits worldwide. Temporal changes in the sensitivity (Se) and specificity (Sp) of RHDV1 competition-ELISA (cELISA1), RHDV2 competition-ELISA (cELISA2), and RHDV1 Immunoglobulin G (IgG1) ELISA, were investigated using Bayesian Latent Class models (BCLM) in the Australian wild rabbit population where both viruses circulate simultaneously and a long-term serological dataset exists. When cELISA1 was compared to IgG1 ELISA, the Se of cELISA1 improved while the Sp of IgG1 ELISA declined over the 2011-21. This corresponded with a decline in the true RHDV1 prevalence in 2018-21, suggesting that a large proportion of RHDV1 exposed rabbits survived the introduction and dominance of RHDV2 up to approximately 2017/2018, after which they died and were not replaced. The Se and Sp estimates for 2014-15 for both cELISA1 and IgG1 ELISA, and the true prevalence when analysing all three tests together were similar to those obtained from the analysis of cELISA1/IgG1 ELISA. The same was also true for the Se and Sp of cELISA2 and IgG1 ELISA estimates from 2018 onwards. This suggests that RHDV1 was the dominant infection type in 2014-15, but RHDV2 was the dominant infection type in 2018-21. Further, the increase in Se of cELISA2 and the low Sp of IgG1 ELISA in the cELISA2/IgG1 ELISA analysis, compared to the Se of cELISA2 and Sp of IgG1 ELISA when analysing all three tests together suggests that the underlying infection status was more influenced by RHDV2 and that the higher Se of IgG1 ELISA is due to cross-reaction of RHDV2 antibodies on IgG1 ELISA. The true prevalence data suggest that RHDV2 exposure peaked in 2017. Our findings show that test characteristics changed in response to the changing virus prevalences over time. IgG1 ELISA, currently having a high Se, should be used to monitor both viruses and will perform better than both cELISAs.

Evaluation of a novel triplex immunochromatographic test for rapid simultaneous detection of norovirus, rotavirus, and adenovirus on a single strip test.

BACKGROUND: Acute gastroenteritis is one of the major causes of morbidity and mortality in young children worldwide. Among these, rotavirus, norovirus, and adenovirus have been reported as the primary viral pathogens associated with the disease. Rapid diagnosis of viral pathogens is crucial when diarrhea outbreaks occur to ensure the timely administration of appropriate treatment and control measures. METHODS: We evaluated three immunochromatographic test kits designed for the detection of norovirus, rotavirus, and adenovirus in 71 stool specimens collected from children with diarrhea who visited clinics in Japan. The first kit is a triplex immunochromatographic test kit designed for simultaneous detections of norovirus, rotavirus, and adenovirus on a single strip (this kit was referred to as IC-A). The other two immunochromatographic test kits are a dual detection kit for rotavirus and adenovirus, and a single detection kit for norovirus (IC-B). The RT-PCR/PCR was used as the gold standard method. RESULTS: The results revealed that both IC-A and IC-B kits exhibited the same level of sensitivity of detection for rotavirus (72.7%) and adenovirus (22.7%), although the detection rate was lower than that of the RT-PCR/PCR method. However, there was a slight difference in the sensitivity of detection for norovirus between IC-A and IC-B, at 86.7% and 93.3%, respectively. The sensitivity of detection for adenovirus of both kits was relatively lower than those of RT-PCR method. This could be due to low viral load of adenovirus in clinical specimens below the detection limit of IC-A and IC-B kits. However, both immunochromatographic test kits (IC-A and IC-B) exhibited 100% specificity for norovirus, rotavirus, and adenovirus. CONCLUSIONS: The triplex immunochromatographic test kit (IC-A) designed for simultaneous detection of norovirus, rotavirus, and adenovirus has been proved to be more practical and convenient than the use of single or dual detection kits with more or less the same sensitivity and specificity of detections.

Sapovirus infection as another cause of persistent viral diarrhea: case series and review of the literature.

Human sapovirus (HuSaV) is a common cause of gastroenteritis worldwide and is responsible for approximately 4% of acute gastroenteritis episodes in Europe. As reported with norovirus, patients with immunocompromised states are at increased risk of developing HuSaV infection, which can lead to persistent diarrhea and chronic viral shedding in some individuals. Chronic infections are incompletely investigated in these patients, and, due to the lack of specific treatment for HuSaV infection, different clinical approaches were carried out in order to provide further evidence on clinical evolution of these patients with different treatments. In this retrospective study, we report five immunocompromised pediatric patients with recurrent diarrhea caused by HuSaV and long-term viral shedding. Stool samples were analyzed by real-time PCR and tested for enteropathogenic viruses and bacteria and protozoa. Among transplant recipients, reduction of immunosuppressant therapy led to clinical improvement and relief of symptoms, maintaining a balance between managing the infection and preventing graft rejection. Nitazoxanide for 14 days was only used in one of these patients, showing to be an effective therapy to achieve reduction in time to resolution of symptoms. Neither nitazoxanide nor modification of immunosuppressant therapy could avoid recurrences. Further investigations are needed to develop new approaches that can both clear the infection and avoid persistent diarrhea in these patients.

Norovirus-Associated Gastroenteritis Vesikari Score and Pre-Existing Salivary IgA in Young Children from Rural South Africa.

Norovirus (NoV) is the leading cause of viral gastroenteritis, mostly affecting young children worldwide. However, limited data are available to determine the severity of norovirus-associated AGE (acute gastroenteritis) and to correlate it with the NoV-specific IgA antibodies' level. Between October 2019 and September 2021, two hundred stool samples were randomly collected from symptomatic cases for the vesikari score and NoV-specific IgA assessment in young children from rural South Africa. Additionally, one hundred saliva specimens were concomitantly sampled within the same cohort to evaluate the NoV-specific salivary IgA levels. In addition, 50 paired saliva and stool samples were simultaneously collected from asymptomatic children to serve as controls. NoV strains in stool samples were detected using real-time RT-PCR, amplified, and genotyped with RT-PCR and Sanger sequencing. ELISA using NoV VLP (virus-like particles) GII.4 as antigens was performed on the saliva specimens. Dehydrated children were predominantly those with NoV infections (65/74, 88%; p < 0.0001). NoV-positive infections were significantly associated with the severe diarrhea cases having a high vesikari score (55%, 33/60) when compared to the non-severe diarrheal score (29.3%, 41/140; p < 0.0308). NoV of the GII genogroup was mainly detected in severe diarrhea cases (50.9%, 30/59; p = 0.0036). The geometric means of the NoV-specific IgA level were higher in the asymptomatic NoV-infected group (0.286) as compared to the symptomatic group (0.174). This finding suggests that mucosal immunity may not protect the children from the NoV infection. However, the findings indicated the contribution of the pre-existing NoV-specific IgA immune response in reducing the severity of diarrheal disease. A high vesikari score of AGE associated with the NoV GII genogroup circulating in the study area underscores the need for an appropriate treatment of AGE based on the severity level of NoV-associated clinical symptoms in young children.

High-resolution melting analysis for simultaneous detection and discrimination between wild-type and vaccine strains of feline calicivirus.

High-resolution melting (HRM) analysis, a post-polymerase chain reaction (PCR) application in a single closed tube, is the straightforward method for simultaneous detection, genotyping, and mutation scanning, enabling more significant dynamic detection and sequencing-free turnaround time. This study aimed to establish a combined reverse-transcription quantitative PCR and HRM (RT-qPCR-HRM) assay for diagnosing and genotyping feline calicivirus (FCV). This developed method was validated with constructed FCV plasmids, clinical swab samples from living cats, fresh-frozen lung tissues from necropsied cats, and four available FCV vaccines. We performed RT-qPCR to amplify a 99-base pair sequence, targeting a segment between open reading frame (ORF) 1 and ORF2. Subsequently, the HRM assay was promptly applied using Rotor-Gene Q® Software. The results significantly revealed simultaneous detection and genetic discrimination between commercially available FCV vaccine strains, wild-type Thai FCV strains, and VS-FCV strains within a single PCR reaction. There was no cross-reactivity with other feline common viruses, including feline herpesvirus-1, feline coronavirus, feline leukemia virus, feline immunodeficiency virus, and feline morbillivirus. The detection limit of the assay was 6.18 × 101 copies/µl. This study, therefore, is the first demonstration of the uses and benefits of the RT-qPCR-HRM assay for FCV detection and strain differentiation in naturally infected cats.

An approach to zwitterionic peptide design for colorimetric detection of the Southampton norovirus SV3CP protease.

Noroviruses are highly contagious and are one of the leading causes of acute gastroenteritis worldwide. Due to a lack of effective antiviral therapies, there is a need to diagnose and surveil norovirus infections to implement quarantine protocols and prevent large outbreaks. Currently, the gold standard of diagnosis uses reverse transcription polymerase chain reaction (RT-PCR), but PCR can have limited availability. Here, we propose a combination of a tunable peptide substrate and gold nanoparticles (AuNPs) to colorimetrically detect the Southampton norovirus 3C-like protease (SV3CP), a key protease in viral replication. Careful design of the substrate employs a zwitterionic peptide with opposite charged moieties on the C- and N- termini to induce a rapid color change visible to the naked eye; thus, this color change is indicative of SV3CP activity. This work expands on existing zwitterionic peptide strategies for protease detection by systematically evaluating the effects of lysine and arginine on nanoparticle charge screening. We also determine a limit of detection for SV3CP of 28.0 nM with comparable results in external breath condensate, urine, and fecal matter for 100 nM of SV3CP. The key advantage of this system is its simplicity and accessibility, thus making it an attractive tool for qualitative point-of-care diagnostics.

Point-of-Care Testing for Norovirus Typing Using CRISPR/Cas12a Combined with Reverse Transcription Recombinase Polymerase Amplification.

Noroviruses (NoVs) are one of the leading causes of acute gastroenteritis in humans. This study combined reverse transcription recombinase polymerase amplification (RT-RPA) with a clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) nucleic acid detection system to develop a point-of-care testing (POCT) technology for typing NoVs. The detection can be completed within 35 min at 37 °C, covering each genotype of genogroup I (GI) and II (GII) NoVs. The sensitivity of this method is 10 copies/µL for GI and 1 copy/µL for GII NoV plasmids. For the detection of clinical samples, the detection results of this method for NoV infected samples are consistent with the RT-qPCR detection method in the laboratory, and this detection method has no cross-reactivity with rotavirus and adenovirus. Therefore, the detection method established in this study enables the diagnosis and screening of suspected patients and close contacts by POCT, which is important for the timely identification and control of NoV outbreaks. In addition, the typing detection of GI and GII NoVs can achieve a precise diagnosis and treatment of patients infected with NoVs.

Comparison of magnetic bead and rapid swab RNA extraction methods for detecting rabbit hemorrhagic disease virus 2 in rabbit liver samples.

We compared a bead RNA extraction method with a one-tube method that required only a heat block and ice. RNA was first extracted from liver samples from nine rabbits dying from rabbit hemorrhagic disease virus 2 (RHDV2) using magnetic beads, and RT-PCR was used to detect RHDV2 sequence. Following freezing, RNA was extracted a second time using the SwiftX™ Swabs Viral RNA Extraction Reagent. RHDV2 was detected in all nine samples. Cycle threshold values were higher in the RT-PCR following SwiftX extraction (mean: 3.79), indicating that the second extraction method resulted in approximately a 1 log10 reduction in sensitivity. A second freeze-thaw for the samples and less tissue extracted using SwiftX may have contributed additionally to the loss in sensitivity.

Acute rotavirus infection causes significant activation of the IL-33/IL-13 axis.

AIM: Overactivation of the IL-33/IL-13 axis is the main step in initializing allergic inflammation and promoting allergic diseases. Data on viral pathogens as risk factors for subsequent allergic disease are contradictory. The strongest associations have been made between upper respiratory tract virus infections and asthma. Intestinal viral infections also activate IL-33 and IL-13 as part of the innate antiviral response. The aim of this study was to test whether there are differences in IL-13 and IL-33 concentrations in pediatric patients with acute rotavirus- and norovirus infections and healthy controls. MATERIAL AND METHODS: Forty children with acute rotavirus, 27 with acute norovirus intestinal infections and 17 control children were enrolled in this study. Blood IL-33 and IL-13 detection was performed with enzyme-linked immunosorbent assays (ELISAs). RESULTS: Acute rotavirus infection caused a significant elevation in IL-33 and IL-13 compared to acute norovirus infection (63.85 pg/ml vs. 0, P = 0.0026, and 94.24 pg/ml vs. 0.88 pg/ml, P = 0.0003, respectively) and healthy controls (63.85 pg/ml vs. 9.89 pg/ ml, P = 0.0018, and 94.24 pg/ml vs. 0.14 pg/ml, P < 0.0001, respectively). There was no significant difference in IL-33 and IL-13 concentrations between the acute norovirus group and healthy controls (0 vs. 9.89 pg/ml, P = 0.8276 and 0.88 pg/ml vs. 0.14 pg/ml, P = 0.1652, respectively). CONCLUSION: Acute rotavirus infection causes a significant elevation in IL-33 and IL-13, compared to norovirus and healthy control children.

Rapid two-stage amplification in a single tube for simultaneous detection of norovirus GII and group a rotavirus.

The most prevalent viruses currently causing diarrhea are norovirus and rotavirus, and rapid and sensitive detection methods are essential for the early diagnosis of disease. The purpose of this study was to establish a sensitive single-tube two-stage nucleic acid amplification method-reverse transcription recombinase-assisted PCR (RT-RAP)-for simultaneous detection of norovirus GII and group A Rotavirus, with the first stage consisting of isothermal reverse transcription recombinase-aided amplification (RT-RAA) and the second stage consisting of qPCR (quantitative PCR). RT-RAP is more sensitive than either RT-RAA or qRT-PCR (quantitative RT-PCR) alone. And the addition of a barrier that can be disassembled after heating enabled the detection of samples within 1 h in a single closed tube. Sensitivity was 10 copies/reaction of norovirus (Novs) GII and group A rotavirus (RVA). In parallel, two hundred fecal specimens were used to evaluate the method and compare it with a commercial fluorescent quantitative RT-PCR. The data showed kappa values of 0.957 and 0.98 (p < 0.05) for detecting Novs GII and RVA by the two methods, indicating the potential of the newly established assay to be applied to clinical and laboratory testing.

Treatment options for chronic norovirus infection in immunocompromised patients.

Norovirus is generally an acute infection causing symptoms such as diarrhea, nausea, and vomiting lasting for 24-48 hours. However, for immunocompromised patients, norovirus gastroenteritis can last for several years and result in villous atrophy and lead to severe malnutrition, dehydration, electrolyte imbalance and continuous viral shedding. Several treatment strategies have been suggested in case reports: nitazoxanide, ribavirin and enterally administered immunoglobulin with varying results. Favipiravir is also suggested but not tested on humans, highlighting the need for further research.

Follow-up evaluation for norovirus asymptomatic infection among healthy adults: a prospective matched cohort study.

OBJECTIVES: No study has evaluated the follow-up of asymptomatic norovirus infection among healthy adults. This study compared norovirus detection rates between previously known norovirus-positive and norovirus-negative adults without diarrheal symptoms, and evaluated reinfection risk among the previously-norovirus-positive group, and new asymptomatic norovirus infection risk among the previously-norovirus-negative group after 600 days. METHODS: This prospective age- and sex-matched cohort study, conducted in 2019 at a Japan teaching hospital, included apparently healthy asymptomatic adults with a positive norovirus result and those with a negative result (ratio 1:3) during the 2016-2017 screening. The primary outcome was real-time reverse-transcription polymerase chain reaction-confirmed norovirus in stool specimens. We evaluated descriptive statistics and associated factors, including demographics, social habits, and clinical parameters. RESULTS: Of 288 participants [mean age, 59.9 (standard deviation: 12.6) years; male, 143 (49.7%)], 73 [genogroup (G) I 35; GII 37; both included 1 each] were positive for norovirus previously, while 215 were negative. After a median of 599 (interquartile range 515-799) days between baseline screening and follow-up, 14 (4.9%; GI 0; GII 14) tested positive for norovirus (2.7% and 5.6% among positive- and negative-norovirus groups at baseline, respectively). Among previously norovirus-negative participants, being older, having elevated blood pressure and haemoglobin A1c level, and drinking Japanese sake at baseline were associated with positive results at follow-up. CONCLUSIONS: Genogroup homotypic protective effect may exist for subsequent asymptomatic infection. There may be higher risks of future asymptomatic norovirus infection in previously no-norovirus asymptomatic infection people with specific lifestyles or medical histories. SUMMARYThe detection follow-up rates of norovirus were 2.7% and 5.6% among asymptomatic adults with positive- and negative-norovirus status at baseline, respectively. Specific lifestyles or medical histories may confer higher risk of norovirus detection.

Development and Characterization of Synthetic Norovirus RNA for Use in Molecular Detection Methods.

Background: Reference materials are essential for the quality assurance of molecular detection methods. We developed and characterized synthetic norovirus GI and GII RNA reference materials. Methods: Norovirus GI and GII RNA sequences including the ORF1-ORF2 junction region were designed based on 1,495 reported norovirus sequences and synthesized via plasmid preparation and in vitro transcription. The synthetic norovirus GI and GII RNAs were evaluated using six commercial norovirus detection kits used in Korea and subjected to homogeneity and stability analyses. A multicenter study involving five laboratories and using four commercial real-time PCR norovirus detection assays was conducted for synthetic norovirus RNA characterization and uncertainty measurements. Results: The synthetic norovirus GI and GII RNAs were positively detected using the six commercial norovirus detection kits and were homogeneous and stable for one year when stored at -20°C or -70°C. All data from the five laboratories were within a range of 1.0 log copies/µL difference for each RNA, and the overall mean concentrations for norovirus GI and GII RNAs were 7.90 log copies/µL and 6.96 log copies/µL, respectively. Conclusions: The synthetic norovirus GI and GII RNAs are adequate for quality control based on commercial molecular detection reagents for noroviruses with high sequence variability. The synthetic RNAs can be used as reference materials in norovirus molecular detection methods.

Timing and genotype distribution of symptomatic and asymptomatic sapovirus infections and re-infections in a Nicaraguan birth cohort.

OBJECTIVES: To characterize the timing and genotype distribution of symptomatic and asymptomatic sapovirus infections and re-infections in a Nicaraguan birth cohort. METHODS: Infants (N = 444) were enrolled at 10-14 days of life and observed weekly until 2 years of age. Stool samples were collected for each acute gastroenteritis (AGE) episode, and routine stool samples were collected monthly. Stool samples were tested for sapovirus using RT-qPCR, and positive samples were genotyped. RESULTS: A total of 348 children completed 2 years of AGE weekly surveillance; 93 (26.7%) of them experienced sapovirus AGE. Most infections occurred after 5 months of age and mainly during the second year of life (62.4%, 58/93) and early in the rainy season. Sapovirus screening in all stools from a subset of 67 children who consistently provided samples showed sapovirus infections in 91 of 330 (27.6%) AGE episodes and in 39 of 1350 (2.9%) routine stools. In this subset, the median age at the first sapovirus AGE was 11.2 months (95% CI, 9.3-15.9 months); 38 of 67 (57%) children experienced re-infections, 19 symptomatic and 19 asymptomatic. On average, sapovirus re-infections were reported 7.2 months after symptomatic and 5.3 months after asymptomatic infections. Genogroup GI (64%, 69/108) was the most common detected. Sapovirus GI.1 was more frequently detected in AGE stool samples than in routine stool samples (47.2%, 43/91 vs. 25.6%, 10/39; p 0.005), and re-infection with the same genotype was uncommon. DISCUSSION: The first sapovirus infections occurred at approximately 11 months of age, whereas the median time to symptomatic re-infection was 7.2 months. Re-infections with the same sapovirus genotype were rare during 2 years of life suggesting genotype-specific protection after natural infection.

Early detection of norovirus outbreak using machine learning methods in South Korea.

BACKGROUND: The norovirus is a major cause of acute gastroenteritis at all ages but particularly has a high chance of affecting children under the age of five. Given that the outbreak of norovirus in Korea is seasonal, it is important to try and predict the start and end of norovirus outbreaks. METHODS: We predicted weekly norovirus warnings using six machine learning algorithms using test data from 2017 to 2018 and training data from 2009 to 2016. In addition, we proposed a novel method for the early detection of norovirus using a calculated norovirus risk index. Further, feature importance was calculated to evaluate the contribution of the estimated weekly norovirus warnings. RESULTS: The long short-term memory machine learning (LSTM) algorithm proved to be the best algorithm for predicting weekly norovirus warnings, with 97.2% and 92.5% accuracy in the training and test data, respectively. The LSTM algorithm predicted the observed start and end weeks of the early detection of norovirus within a 3-week range. CONCLUSIONS: The results of this study show that early detection can provide important insights for the preparation and control of norovirus outbreaks by the government. Our method provides indicators of high-risk weeks. In particular, last norovirus detection rate, minimum temperature, and day length, play critical roles in estimating weekly norovirus warnings.

Development and validation of an efficient nomogram for risk assessment of norovirus infection in pediatric patients.

This study aimed to establish a predictive model and nomogram based on routine laboratory blood indicators and clinical symptoms, subsequently providing a rapid risk assessment of norovirus (NoV) infection in children. This retrospective study enrolled 307 pediatric patients with symptoms of acute gastroenteritis and detected NoV using real-time quantitative polymerase chain reaction. Significant indicators selected by multivariate logistic regression, including routine blood tests and consultation symptoms, were used to develop the nomogram. We divided the sample into training and internal validation sets and performed external validation of the final model. Furthermore, we evaluated the clinical performance using the Akaike information criterion (AIC), area under the curve (AUC), calibration curve, decision curve analysis (DCA), sensitivity, specificity, concordance rate, positive predictive value, and negative predictive value. Overall, 153 cases were NoV-PCR-positive, and 154 were negative. The multivariate logistic regression included five predictors of NoV infection, including symptoms of vomiting, upper respiratory tract infection, and indicators of white blood cells, lymphocyte absolute counts, and platelet counts. The nomogram showed a significant predictive value with overall internal set diagnosis, with an AUC of 0.827 (95% confidence interval (CI): 0.785-0.868), and 0.812 (95% CI: 0.755-0.869) with 0.799 (95% CI: 0.705-0.894) in the training and internal validation sets, respectively. Nevertheless, the AUC in the external validation set was higher (0.915; 95% CI: 0.862-0.968). This nomogram is a useful tool for risk assessment for NoV infection. Moreover, the evaluated indicators are accessible, substantially reducing the time for laboratory testing.

Ultrasensitive and visual detection of human norovirus genotype GII.4 or GII.17 using CRISPR-Cas12a assay.

BACKGROUND: Integrating CRISPR-Cas12a sensors with isothermal signal amplification can be exploited to develop low-cost, disposable, and ultrasensitive assays for the diagnostics of human pathogens. METHODS: RT-RAA-Cas12a-mediated real-time or end-point fluorescent and lateral flow strip (LFS) assays for direct detection of norovirus (NOV) genotype GII.4 or GII.17 were explored. RESULTS: The results showed that our RT-RAA-Cas12a-mediated fluorescent and LFS assay could detect NOV GII.4 or GII.17 by targeting the viral protein 1 gene. Our RT-RAA-Cas12a-mediated fluorescent and LFS assay can specifically detect NOV GII.4 or GII.17 with no cross-reactivity for other related viruses. The low limit of detection could reach 0.1 copies/µL within approximately 30-40 min, and the results were visualized using an ultraviolet light illuminator or on a LFS without complex equipment. In addition, our RT-RAA-Cas12a-mediated fluorescent and LFS assay provided a visual and faster alternative to real-time RT-PCR assay, with 95.7% and 94.3% positive predictive agreement and 100% negative predictive agreement. CONCLUSIONS: Together, our RT-RAA-Cas12a-mediated approach would have a great potential for point-of-care diagnostics of NOV GII.4 and/or GII.17 in resource-limited settings.

Colorimetric detection of norovirus by helicase-dependent amplification method based on specific primers integrated with HRPzyme.

Noroviruses (NoVs) are the most common causes of epidemic gastroenteritis, responsible for at least 50% of all gastroenteritis outbreaks worldwide and significant causes of foodborne illness. In the USA, approximately 21 million illnesses attributable to NoVs have annually occurred. Therefore, there is a great demand to develop a rapid, low-cost, and accurate detection method for NoVs. This study first reported colorimetric helicase-dependent amplification (HDA) methods based on specific primers integrated with HRPzyme for the rapid and sensitive detection of NoV GI and GII. The colorimetric HDA methods exhibited a detection limit of 10 copies mL-1 of each NoV GI and GII and were confirmed to be specific to each NoV GI and GII. The period required to complete the HDA method was 2 h, including a step of RNA extraction and cDNA synthesis without expensive instruments such as a thermal cycler and detector. The cutoff value of the method for the oyster artificially inoculated with a known amount of NoV was all 102 copies g-1 for NoV GI and GII. Therefore, the HDA method developed in this study can be useful tool for the on-site detection of NoVs in food samples.