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Adaptation of NoV LAMP Primers by PCR for Highly Sensitive Detection of Noroviruses in Water
Abstract
A fast and highly sensitive detection of noroviruses was established by Reverse Transcriptase PCR amplification using primers adapted from loop-mediated isothermal amplification (LAMP) of noroviruses (RT-L-NoV PCR amplification). The amplification was carried out in 30-min early incubation in 50 °C for reverse transcription activity and further 35 PCR short cycles for total finishing time within 60 min with no cross-reactivity with other common environmental species and strains. The LOD of this method was 10−15 × 100 ng/ulor 1zg/ul of pIDTSMART-NoV gene per reaction which is reported to be the highest sensitivity conventional PCR-based detection method for noroviruses. These early findings give hope for a potentially useful RT-L-NoV PCR amplification assay for a highly sensitive detection of NoV genomes especially in diluted concentration sample like water.
... As NASBA only requires a forward and reverse primer, the work may easily be translated to PCR or other isothermal techniques, like RPA. This translation may not work so easily for other isothermal techniques that require numerous primers like LAMP; however, one group does report successful adaptation of LAMP primers for a PCR norovirus assay (Khairuddin et al., 2016). ...
Foodborne and enteric viruses continue to impose a significant public health and economic burden globally. As many of these viruses are highly transmissible, the ability to detect them portably, sensitively, and rapidly is critical to reduce their spread. Although still considered a gold standard for detection of these viruses, real time polymerase chain reaction (PCR)-based technologies have limitations such as limited portability, need for extensive sample processing/extraction, and long time to result. In particular, the limitations related to the susceptibility of real time PCR methods to potential inhibitory substances present in food and environmental samples is a continuing challenge, as the need for extensive nucleic acid purification prior to their use compromises the portability and rapidity of such methods. Isothermal amplification methods have been the subject of much investigation for these viruses, as these techniques have been found to be comparable to or better than established PCR-based methods in portability, sensitivity, specificity, rapidity, and simplicity of sample processing. The purpose of this review is to survey and compare reports of these isothermal amplification methods developed for foodborne and enteric viruses, with a special focus on the performance of these methods in the presence of complex matrices.
... Specificity test for the primers used in this study were validated using 15environmental DNA strains as described in our previous study [17]: Burkholderia cepacia, B. thailandensis, B. pseudomallei, Bacillus subtilis, B. macerans, B. circulans, B. megaterium, Staphylococcus sp., S. epiderminis, S. heamolyticus, E. coli BL21, E. coli Nova Blue, Shigella sp., Salmonella sp. and Shinella granuli. All strains, positive control (DNA plasmid of NoV) and negative control (steril dH 2 O) were assessed simultaneously in optimized LAMP condition. ...
A preliminary study was carried out in order to evaluate a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for detecting Noroviruses (NoV) RNA surrogates as external standard for NoV.The detection limit of the NoV RT-LAMP assay was observed to be 22 copies/μL. This RT-LAMP assay sensitivity was comparablewith the quantitative reverse transcriptase-Polymerase Chain Reaction (PCR) and shown to be 10-fold more sensitive than end-point conventional reverse transcriptase-PCR (RT-PCR). The NoV RT-LAMP assay showed high specificity to NoV targeted gene when specificity test was completed with no cross-reactivity with other 17 environmental strains. The assay also was performed with 11 spiked recreational stream water randomly picked from two recreational areas in Hulu Langat Malaysia, Sg. Tekala and Sg. Gabai. The RT-LAMP assay is simpler compared to the conventional PCR and real-time PCR, which in optimum isothermal temperature of 63°C, the amplification can be completed in 40 minutes. Results of spiked recreational stream water samples suggested that the NoV RT-LAMP assay can be used as monitoring tool for NoV surveillance in recreational stream water.
Rotavirus and recently norovirus have been described as important and most common cause of acute gastroenteritis in children. The mode of transmission is fecal-oral. Diagnosis of both of these viruses can be made by rapid antigen detection of the viruses in stool specimens and strains can be further characterized by enzyme immunoassay or reverse transcriptase polymerase chain reaction. Stool specimens collected from various hospitals in Malaysia were examined for norovirus by both immunochromatography and reverse-transcription polymerase chain reaction techniques. Rotavirus antigen was screened by a commercially available latex agglutination test kit. Altogether, 168 stool samples were collected for both norovirus and rotavirus screening. Out of these, 77/168 (45.8%) were examined for rotavirus with 17/77 (22%) rotavirus antigen positive. Due to very small amount of fecal materials obtained in some cases, only 151/168 (89.8%) were sufficient for norovirus and 14/151 (9.3%) were positive for norovirus genogroup II (GII). This study highlights that rotavirus remains the main agent for acute gastroenteritis and identification for emerging norovirus among the children is becoming important for proper patient management.
Background
Despite significant reduction of rotavirus (RV) infections following implementation of RotaTeq vaccination in Nicaragua, a large burden of patients with diarrhea persists.
Methods
We conducted a community- and hospital-based study of the burden of RV, norovirus (NV) and sapovirus (SV) infections as cause of sporadic acute gastroenteritis (GE) among 330 children ≤ 5 years of age between September 2009 and October 2010 in two major cities of Nicaragua with a RotaTeq coverage rate of 95%.
Results
We found that NV, SV and RV infections altogether accounted for 45% of cases of GE. Notably, NV was found in 24% (79/330) of the children, followed by SV (17%, 57/330) and RV (8%, 25/330). The detection rate in the hospital setting was 27%, 15% and 14% for NV, SV and RV respectively, whereas in the community setting the detection rate of RV was < 1%. Among each of the investigated viruses one particular genogroup or genotype was dominant; GII.4 (82%) for NV, GI (46%) for SV and G1P[8] (64%) in RV. These variants were also found in higher proportions in the hospital setting compared to the community setting. The GII.4.2006 Minerva strain circulating globally since 2006 was the most common among genotyped NV in this study, with the GII.4-2010 New Orleans emerging in 2010.
Conclusions
This study shows that NV has become the leading viral cause of gastroenteritis at hospital and community settings in Nicaragua after implementation of RV vaccination.
A simple, rapid, specific, and sensitive colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay with hydroxynaphthol blue dye (HNB) was established, targeting RNA-dependent RNA polymerase and capsid protein gene for the detection of the dominant norovirus genogroup in China-NoV GII. The assay was carried out at 65 °C for 60 min with no cross-reactivity with other common gastroenteritis viruses. The sensitivity of this assay was 10(3) copies per reaction which is equivalent to the conventional RT-PCR test. The clinical test showed 94.83 % coincidence rate for NoV genogroup II detection compared with the results, confirmed by the Department of Viral Diarrhea of Chinese Center for Disease Control and Prevention via conventional RT-PCR. The HNB dye-based RT-LAMP could be a novel rapid screening method for prevalent norovirus genogroup II in China, especially in those resource-limited hospitals and rural local clinics.
Molecular techniques and virus concentration methods have shown that previously unknown viruses are shed by humans and animals, and may be transmitted by sewage-contaminated water. In the present study, 10-L river-water samples from urban areas in Barcelona, Spain and Rio Janeiro, Brazil, have been analyzed to evaluate the viral dissemination of human viruses, validating also a low-cost concentration method for virus quantification in fresh water. Three viral groups were analyzed: (i) recently reported viruses, klassevirus (KV), asfarvirus-like virus (ASFLV), and the polyomaviruses Merkel cell (MCPyV), KI (KIPyV) and WU (WUPyV); (ii) the gastroenteritis agents noroviruses (NoV) and rotaviruses (RV); and (iii) the human fecal viral indicators in water, human adenoviruses (HAdV) and JC polyomaviruses (JCPyV). Virus detection was based on nested and quantitative PCR assays. For KV and ASFLV, nested PCR assays were developed for the present study. The method applied for virus concentration in fresh water samples is a one-step procedure based on a skimmed-milk flocculation procedure described previously for seawater. Using spiked river water samples, inter- and intra-laboratory assays showed a viral recovery rate of about 50% (20-95%) for HAdV, JCPyV, NoV and RV with a coefficient of variation ≤50%. HAdV and JCPyV were detected in 100% (12/12) of the river samples from Barcelona and Rio de Janeiro. Moreover, NoV GGII was detected in 83% (5/6) and MCPyV in 50% (3/6) of the samples from Barcelona, whereas none of the other viruses tested were detected. NoV GGII was detected in 33% (2/6), KV in 33% (2/6), ASFLV in 17% (1/6) and MCPyV in 50% (3/6) of the samples from Rio de Janeiro, whereas KIPyV and WUPyV were not detected. RV were only analyzed in Rio de Janeiro and resulted positive in 67% (4/6) of the samples. The procedure applied here to river water represents a useful, straightforward and cost-effective method that could be applied in routine water quality testing. The results of the assays expand our understanding of the global distribution of the viral pathogens studied here and their persistence in the environment.
Norovirus is a common cause of gastroenteritis, and is thought to be the causative agent in 68-90% of all gastroenteritis outbreaks. The seasonality of disease occurrence is sufficiently stereotyped to result in this disease being dubbed "winter vomiting disease." The genesis of this seasonality has been obscure. We sought to identify environmental factors associated with Norovirus outbreaks in Toronto, Canada. We evaluated 253 outbreaks of gastroenteritis linked to Norovirus between November 2005 and March 2008. Poisson regression models were constructed to evaluate associations between average environmental exposures and case counts. A case-crossover approach was used to evaluate associations between acute changes in environment and outbreak risk. Case-crossover analysis indicated an association between low Lake Ontario temperature (<or=4 degrees C) (hazard ratio [HR], 5.61 [95% CI, 2.81-11.12]) and high flow (>2.5 m(3)/s) in the Don River (HR, 3.17 [95% CI, 2.30-4.36]), 1-7 days prior to case occurrence. For both exposure variables, the highest hazard ratios were found 24-48 h prior to case onset. Regression models provided further support for these patterns. The association between local watershed conditions and Norovirus outbreak risk suggest a source-water reservoir for this pathogen. We hypothesize that the reservoir may be maintained through the discharge of wastewater containing virus particles; wintertime seasonality may be explained by enhanced viral persistence at low temperatures.
The prevalence and genotypes of norovirus genogroup I (GI) and GII in tropical urban catchment waters and an estuarine bay were studied. A comparative analysis was performed with environmental isolates of noroviruses and concurrently identified clinical isolates in Singapore during gastroenteritis outbreaks between August 2006 to January 2007. Noroviruses in environmental water samples were concentrated by using ultrafiltration techniques and then analyzed by reverse transcription-seminested PCR assay targeting the partial capsid region of noroviruses and DNA sequencing. Among the 60 water samples collected, noroviruses were detected in 43 (71.7%) of these samples. Of these 43 norovirus-positive samples, the coexistence of both GI and GII strains was identified in 23 (53.5%) water samples. The phylogenetic analysis revealed multiple genotypes of noroviruses GI and GII in environmental water samples. GI and GII strains were clustered into seven and nine (including two unclassified) genotypes, respectively. The major norovirus genotypes in environmental water samples were GI/2 and GI/4 and GII/4. Genotyping of the 21 norovirus-positive clinical samples showed that all of the strains belonged to the GII/4 cluster. The environmental and clinical norovirus GII/4 isolates showed high levels of nucleotide sequence identity to each other and to the novel GII/4 variant associated with global epidemics of gastroenteritis during 2006. This study suggests the emergence and circulation of multiple novel norovirus GI and GII genotypes in water environments. Further comprehensive surveillance of water environments for noroviruses and routine clinical reporting is warranted.
We conducted a systematic review of studies that used reverse transcription-PCR to diagnose norovirus (NoV) infections in patients with mild or moderate (outpatient) and severe (hospitalized) diarrhea. NoVs accounted for 12%(95% confidence interval [CI] 10%-15%) of severe gastroenteritis cases among children <5 years of age and 12% (95% CI 9%-15%) of mild and moderate diarrhea cases among persons of all ages. Of 19 studies among children <5 years of age, 7 were in developing countries where pooled prevalence of severe NoV disease (12%) was comparable to that for industrialized countries (12%). We estimate that each year NoVs cause 64,000 episodes of diarrhea requiring hospitalization and 900,000 clinic visits among children in industrialized countries, and up to 200,000 deaths of children <5 years of age in developing countries. Future efforts should focus on developing targeted strategies, possibly even vaccines, for preventing NoV disease and better documenting their impact among children living in developing countries, where >95% of the deaths from diarrhea occur.
Information about norovirus (NoV) infections in Central America is limited. Through a passive community and hospital pediatric diarrhea surveillance program, a total of 542 stool samples were collected between March 2005 and February 2006 in León, Nicaragua. NoV was detected in 12% (65/542) of the children; of these, 11% (45/409) were in the community and 15% (20/133) were in the hospital, with most strains (88%) belonging to genogroup II. NoV infections were age and gender associated, with children of <2 years of age (P < 0.05) and girls (P < 0.05) being most affected. Breast-feeding did not reduce the number of NoV infections. An important proportion (57%) of NoV-infected children were coinfected with diarrheagenic Escherichia coli. A significant proportion (18/31) of NoV-positive children with dehydration required intravenous rehydration. Nucleotide sequence analysis (38/65) of the N-terminal and shell region in the capsid gene revealed that at least six genotypes (GI.4, GII.2, GII.4, GII.7, GII.17, and a potentially novel cluster termed "GII.18-Nica") circulated during the study period, with GII.4 virus being predominant (26/38). The majority (20/26) of those GII.4 strains shared high nucleotide homology (99%) with the globally emerging Hunter strain. The mean viral load was approximately 15-fold higher in children infected with GII.4 virus than in those infected with other G.II viruses, with the highest viral load observed for the group of children infected with GII.4 and requiring intravenous rehydration. This study, the first of its type from a Central American country, suggests that NoV is an important etiological agent of acute diarrhea among children of <2 years of age in Nicaragua.
To investigate the actual fluctuations in the concentrations of noroviruses (NoVs) GI and GII, and enteric adenoviruses (EAdVs) in river water and its relationship with the number of acute infectious gastroenteritis patients, one-year weekly quantitative monitoring of NoVs GI and GII and EAdVs was performed in the Tone River in Japan where the surface water is utilized for the main production of drinking water for the Tokyo Metropolitan Area from October 2009 to September 2010. Noroviruses GI and GII and EAdVs were detected in 28 (54%), 33 (63%), and 23 (44%) of the 52 samples (1 L each), respectively. The concentrations of NoVs GI and GII and EAdVs fluctuated strongly and were more abundant in winter and early spring. The concentration of NoVs GI was transiently greater than 10,000 copies/L. The number of acute infectious gastroenteritis patients in the upper river basin was highly correlated with all the viral concentrations, while general microbial indicator data such as turbidity and heterotrophic plate count were independent of viral concentration as suggested in previous studies. To the best of our knowledge, this is the first study that clearly shows the strong correlation of the number of gastroenteritis with virus contamination in lower river basin.
Noroviruses (NoVs) are recognized as a significant cause of acute gastroenteritis in children and adults. A 14-month study, from January 2006 to February 2007, was undertaken in a hospital in Thailand to determine the prevalence and genetic characterization of NoVs in patients of all ages with acute gastroenteritis. Based on reverse transcription-nested polymerase chain reaction (RT-nested PCR), NoVs were detected in 122 of 273 (44.7%) collected stool samples. Of the 122 NoV-positive samples, 28 (23%) belonged to GI, 79 (64.8%) belonged to GII, and 15 (12.2%) were mixed infections of GI and GII strains. Three NoV GI-positive and 42 NoV GII-positive samples were characterized successfully by DNA sequencing of the RT-nested PCR products and phylogenetic analysis. For NoV GI, two genotypes were identified: GI-2 (one sample) and GI-6 (two samples). NoV GII could be classified further into five distinct genotypes: GII-2 (1 sample), GII-3 (3 samples), GII-4 (14 samples), GII-6 (3 samples), and GII-17 (2 samples), and one unclassified genotype (19 samples). All NoV GII-4 strains showed 88-98% nucleotide identity with NoV GII-4 2006b variants reported worldwide. Among genotypes of NoV characterized, one co-infected stool sample exhibited NoVs GI-6 and GII-4 2006b. This study suggests that there is an important role of NoVs as etiologic agents in patients with acute gastroenteritis. The predominant circulating genotype of NoV infections is GII-4 2006b variant.
We studied the genetic diversity of human noroviruses in river waters by RT-nested PCR and phylogenetic analysis. During 2002-2003, water samples were collected from four rivers in Gyeonggi Province, South Korea. Among the 58 samples, 32 (55.2%) and 26 (44.8%) showed positive results with noroviruses belonging to genogroups I (GI) and II (GII), respectively. The phylogenetic analysis grouped 8 and 7 genotypes in GI and GII, respectively. The major types were GI/1, GI/13, and GII/15, and GI/1 and GI/3 were temporarily distributed. Most GI- and GII-grouped strains were closely related to the reference strains from neighboring countries, China and Japan, and GII/4-related strains had similar sequences to strains recognized as worldwide epidemic outbreaks. The strains circulating between countries are of particular concern to the outbreaks of noroviral diseases in Korea and must be periodically monitored in the natural environments.
We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity,
efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed
primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense
and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer
primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers
that hybridize to the other end of the target, which produces a stem–loop DNA structure. In subsequent LAMP cycling one inner
primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem–loop DNA
and a new stem–loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 109 copies of target in less than an hour. The final products are stem–loop DNAs with several inverted repeats of the target
and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target
in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences
afterwards, it is expected to amplify the target sequence with high selectivity.
Noroviruses (NV) are transmitted by fecally contaminated food, vomit, and person-to-person contact. They are one of the main causes of non-bacterial acute gastroenteritis in nursing, old people and children's homes. NV outbreaks are characterized by a short incubation period (12-48 h), nausea, vomiting and diarrhea, and high secondary attack rates. The illness is generally mild and self-limiting. The aim of diagnostic procedures in viral gastroenteritis is to avoid nosocomial infections on the one hand and unnecessary antibiotic treatment on the other. Diagnostic procedures for NV are based on the detection of virus in stool samples by (immune) transmission electron microscopy (TEM), antigen ELISA, or polymerase chain reaction (PCR). In our study, a total of 244 stool samples obtained from 227 patients between March and May 2002 were tested by TEM, antigen ELISA and in-house PCR. Our data showed that PCR has the highest sensitivity (94.1%), followed by TEM (58.3%), and ELISA (31.3%), while specificity was highest for TEM (98.0%), followed by ELISA (94.9%), and PCR (92.4%). All three methods tested (TEM, ELISA and PCR) are useful for epidemiological investigations in gastroenteritis outbreaks; however, to maximize diagnostic validity for individual cases, at least two of the methods should be combined.
This report describes norovirus (NoV) and sapovirus (SaV) infections in hospitalized children with acute sporadic gastroenteritis in Ho Chi Minh City, Vietnam. Stool specimens collected between December 1999 and November 2000 were examined for NoV and SaV using reverse transcription-PCR and phylogenetic analysis. NoVs were detected in 72 of 448 rotavirus-negative specimens, counted as part of an overall annual detection rate of 5.4% (72 of 1,339 children). This included four NoV genogroup I (GI) strains and 68 NoV GII strains. Only one SaV GI strain was detected in the rotavirus-negative specimens. Over 73% of the NoV sequences belonged to GII/4 (Lordsdale cluster) and were detected in all months except March. We also detected GII/3 strains (Saitama U201 cluster), a naturally occurring recombinant NoV, between January 2000 and March 2000 but not after this period. Other NoV strains belonging to GI/4, GI/8, GII/1, and GII/7 were also detected but were infrequent. In addition, two almost identical NoV GII strains (strains 026 and 0703) collected six months apart were classified into a new genotype that includes the Mc37 strain, which was previously shown to be a recombinant NoV. During this one-year study, the NoV prevailed at the end of the rainy season and the beginning of the dry season. Further epidemiological studies may be necessary to determine whether the GII/4 strains continue to dominant in this region.
Diarrhea, over the years, has killed millions of people and continues to be a major threat in Bangladesh.
To determine the incidence of norovirus infection in infants and young children with acute gastroenteritis in Dhaka City, Bangladesh and to determine the genogroup and genotype in norovirus-positive stool specimens.
Fecal specimens were collected from infants and children with acute gastroenteritis in Dhaka City, Bangladesh from October 2004 to September 2005, and examined for norovirus by reverse transcription-polymerase chain reaction.
Noroviruses were detected in 41 of 917 fecal specimens. Molecular analysis of norovirus was carried out by sequencing methods. Only norovirus GII/4 strains were detected during this study. The dominant genotype throughout the study period was GII/4. Norovirus infections were most commonly observed in winter and rainy seasons in Dhaka City. The common clinical symptoms in norovirus-infected patients were diarrhea (90%), vomiting (75%) and abdominal pain (46%).
This is the first epidemiological research of norovirus in Bangladesh. Norovirus is an important enteropathogen responsible for viral gastroenteritis among infants and children in Bangladesh.
Diarrhoea: why children are still dying and what can be done?
- World Health Organisation
- W Tessa
- S Peter
- Clarissa B Mickey