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Sequence-independent amplification coupled with DNA microarray analysis for detection and genotyping of noroviruses.

Hu Y, Yan H, Mammel M, Chen H - AMB Express (2015)

Bottom Line: By testing 22 clinical fecal specimens obtained from acute gastroenteritis cases as blinded samples, 2 were GI positive and 18 were GII positive as well as 2 negative for NoVs.The study showed that there was 100 % concordance for positive NoV detection at genogroup level between the results of Ribo-SPIA/microarray and the phylogenetic analysis of viral sequences of the capsid gene.In addition, 85 % genotype agreement was observed for the new assay compared to the results of phylogenetic analysis.

View Article: PubMed Central - PubMed

Affiliation: Northeast Region Laboratory, Office of Regulatory Affairs, Food and Drug Administration, Jamaica, NY, USA. yuan.hu@fda.hhs.gov.

ABSTRACT
Noroviruses (NoVs) have high levels of genetic sequence diversities, which lead to difficulties in designing robust universal primers to efficiently amplify specific viral genomes for molecular analysis. We here described the practicality of sequence-independent amplification combined with DNA microarray analysis for simultaneous detection and genotyping of human NoVs in fecal specimens. We showed that single primer isothermal linear amplification (Ribo-SPIA) of genogroup I (GI) and genogroup II (GII) NoVs could be run through the same amplification protocol without the need to design and use any virus-specific primers. Related virus could be subtyped by the unique pattern of hybridization with the amplified product to the microarray. By testing 22 clinical fecal specimens obtained from acute gastroenteritis cases as blinded samples, 2 were GI positive and 18 were GII positive as well as 2 negative for NoVs. A NoV GII positive specimen was also identified as having co-occurrence of hepatitis A virus. The study showed that there was 100 % concordance for positive NoV detection at genogroup level between the results of Ribo-SPIA/microarray and the phylogenetic analysis of viral sequences of the capsid gene. In addition, 85 % genotype agreement was observed for the new assay compared to the results of phylogenetic analysis.

No MeSH data available.


Related in: MedlinePlus

Phylogenetic dendrogram was constructed based on nucleic acid sequences of HAV VP1/P2A region of 15 strains using neighbour-joining method with ClustalX algorithm and MEGA5 program. Numbers on each branch indicate supporting bootstrap value of 1000 resampled data sets. HAV strain (106) obtained in present study was indicated with black square
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Fig4: Phylogenetic dendrogram was constructed based on nucleic acid sequences of HAV VP1/P2A region of 15 strains using neighbour-joining method with ClustalX algorithm and MEGA5 program. Numbers on each branch indicate supporting bootstrap value of 1000 resampled data sets. HAV strain (106) obtained in present study was indicated with black square

Mentions: The RT-PCR detected GI or GII NoV in 20 specimens except in 10,016 and 184. This was in line with the positive microarray results. With 20 NoV-positive samples, partial viral capsid genes were amplified by RT-PCR and sequenced. Based on the phylogenetic result, samples 120 and 101 were clustered to genotype GI.8 and GI.1, respectively. The remaining 18 samples were categorized into 5 GII genotypes including GII.4 (11), GII.8 (2), GII.1 (2), GII.5 (2) and GII.2 (1) as shown in Fig. 3. As a result, there was 100 % concordance for positive NoV detection at genogroup level between the results of microarray and phylogenetic analysis (Table 1). Three samples 120,123 and 102 which were genotyped as GI.1, GII.12 and GII.3, respectively, in microarray analysis were identified as GI.8, GII.1 and GII.1 in the phylogenetic result. Thus, 85 % (17/20) genotype agreement was observed between the results of Ribo-SPIA/microarray and phylogenetic analysis (Table 1). No statistically significant difference was detected between the two results (McNemar’s test; P = 0.2482). In addition, HAV RNA was also detected by nested RT-PCR in 106 which was NoV GII positive. Sequence comparison between the nested RT-PCR product with other HAV strains revealed that the virus displayed the highest sequence similarity to a subgenotype IB strain HM175/18f (Fig. 4).Fig. 3


Sequence-independent amplification coupled with DNA microarray analysis for detection and genotyping of noroviruses.

Hu Y, Yan H, Mammel M, Chen H - AMB Express (2015)

Phylogenetic dendrogram was constructed based on nucleic acid sequences of HAV VP1/P2A region of 15 strains using neighbour-joining method with ClustalX algorithm and MEGA5 program. Numbers on each branch indicate supporting bootstrap value of 1000 resampled data sets. HAV strain (106) obtained in present study was indicated with black square
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4641146&req=5

Fig4: Phylogenetic dendrogram was constructed based on nucleic acid sequences of HAV VP1/P2A region of 15 strains using neighbour-joining method with ClustalX algorithm and MEGA5 program. Numbers on each branch indicate supporting bootstrap value of 1000 resampled data sets. HAV strain (106) obtained in present study was indicated with black square
Mentions: The RT-PCR detected GI or GII NoV in 20 specimens except in 10,016 and 184. This was in line with the positive microarray results. With 20 NoV-positive samples, partial viral capsid genes were amplified by RT-PCR and sequenced. Based on the phylogenetic result, samples 120 and 101 were clustered to genotype GI.8 and GI.1, respectively. The remaining 18 samples were categorized into 5 GII genotypes including GII.4 (11), GII.8 (2), GII.1 (2), GII.5 (2) and GII.2 (1) as shown in Fig. 3. As a result, there was 100 % concordance for positive NoV detection at genogroup level between the results of microarray and phylogenetic analysis (Table 1). Three samples 120,123 and 102 which were genotyped as GI.1, GII.12 and GII.3, respectively, in microarray analysis were identified as GI.8, GII.1 and GII.1 in the phylogenetic result. Thus, 85 % (17/20) genotype agreement was observed between the results of Ribo-SPIA/microarray and phylogenetic analysis (Table 1). No statistically significant difference was detected between the two results (McNemar’s test; P = 0.2482). In addition, HAV RNA was also detected by nested RT-PCR in 106 which was NoV GII positive. Sequence comparison between the nested RT-PCR product with other HAV strains revealed that the virus displayed the highest sequence similarity to a subgenotype IB strain HM175/18f (Fig. 4).Fig. 3

Bottom Line: By testing 22 clinical fecal specimens obtained from acute gastroenteritis cases as blinded samples, 2 were GI positive and 18 were GII positive as well as 2 negative for NoVs.The study showed that there was 100 % concordance for positive NoV detection at genogroup level between the results of Ribo-SPIA/microarray and the phylogenetic analysis of viral sequences of the capsid gene.In addition, 85 % genotype agreement was observed for the new assay compared to the results of phylogenetic analysis.

View Article: PubMed Central - PubMed

Affiliation: Northeast Region Laboratory, Office of Regulatory Affairs, Food and Drug Administration, Jamaica, NY, USA. yuan.hu@fda.hhs.gov.

ABSTRACT
Noroviruses (NoVs) have high levels of genetic sequence diversities, which lead to difficulties in designing robust universal primers to efficiently amplify specific viral genomes for molecular analysis. We here described the practicality of sequence-independent amplification combined with DNA microarray analysis for simultaneous detection and genotyping of human NoVs in fecal specimens. We showed that single primer isothermal linear amplification (Ribo-SPIA) of genogroup I (GI) and genogroup II (GII) NoVs could be run through the same amplification protocol without the need to design and use any virus-specific primers. Related virus could be subtyped by the unique pattern of hybridization with the amplified product to the microarray. By testing 22 clinical fecal specimens obtained from acute gastroenteritis cases as blinded samples, 2 were GI positive and 18 were GII positive as well as 2 negative for NoVs. A NoV GII positive specimen was also identified as having co-occurrence of hepatitis A virus. The study showed that there was 100 % concordance for positive NoV detection at genogroup level between the results of Ribo-SPIA/microarray and the phylogenetic analysis of viral sequences of the capsid gene. In addition, 85 % genotype agreement was observed for the new assay compared to the results of phylogenetic analysis.

No MeSH data available.


Related in: MedlinePlus