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Detection of rotavirus using padlock probes and rolling circle amplification.

Mezger A, Öhrmalm C, Herthnek D, Blomberg J, Nilsson M - PLoS ONE (2014)

Bottom Line: Rotavirus is often diagnosed by latex agglutination assay, chromatography immunoassay, or by electron microscopy, which are all quite insensitive.Reverse transcription polymerase chain reaction, on the other hand, is very sensitive to variations at the genomic level.We developed a novel assay based on a set of 58 different padlock probes with a detection limit of 1,000 copies.

View Article: PubMed Central - PubMed

Affiliation: Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Solna, Sweden.

ABSTRACT
Rotavirus infections are one of the most common reasons for hospitalizations due to gastrointestinal diseases. Rotavirus is often diagnosed by latex agglutination assay, chromatography immunoassay, or by electron microscopy, which are all quite insensitive. Reverse transcription polymerase chain reaction, on the other hand, is very sensitive to variations at the genomic level. We developed a novel assay based on a set of 58 different padlock probes with a detection limit of 1,000 copies. Twenty-two patient samples were analyzed and the assay showed high concordance with a PCR-based assay. In summary, we present a new assay for sensitive and variation tolerant detection of rotavirus.

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Related in: MedlinePlus

Padlock probe design.(A) The sequence of the six padlock probes with the haplotyped degenerations and ligation site. (B) Alignment of 214 Human Rotaviruses. The variations in Rotavirus were mapped using BLASTn and ConSort. The length of the black bars represents the frequency of variation as an average percentage conservation at each nt position (y-axis).
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pone-0111874-g001: Padlock probe design.(A) The sequence of the six padlock probes with the haplotyped degenerations and ligation site. (B) Alignment of 214 Human Rotaviruses. The variations in Rotavirus were mapped using BLASTn and ConSort. The length of the black bars represents the frequency of variation as an average percentage conservation at each nt position (y-axis).

Mentions: All Human Rotavirus A types that were available on NCBI blastn (National Library of Medicine) were used to design the padlock probes. Using the Consort program [12], sequence conservation and frequencies of nucleotide variation were compared for all eleven human Rotavirus A segments for all available sequences in NCBI blastn (National Library of Medicine). This resulted in that the VP6 gene, coding for the major capsid protein, was chosen as a target region for the padlock probes based on its relative conserved regions. The chosen region, nt 96–125 of the 1356 nt long VP6 gene (Genebank nr AB022768), in which the padlock probes are designed against is based on the alignment of 214 human rotavirus A sequences (Figure 1B). A query of VP6 gene rotavirus in BLASTn was analyzed in the ConSort program [12] to construct a primer and the 5′ and 3′ arms of padlock probes targeting rotavirus. The design was made according to the principle of NucZip [13]. The consensus sequence for the target region in the NIH database is CARTTYAAYCWRATGATARTWACHATGAA, causing a degeneration of 384. Due to that specific nucleotide positions vary together, Consort settings of 1–10 degeneration were used to haplotype the padlock target region into a minimum of sequences. The final design consisted of six padlock probe designs (degeneration 32, 8, 8, 8, 1, and 1) that together create a mix of 58 unique padlock probes (Figure 1A) needed to cover 95% of the published sequences in the NIH database targeting both genogroups, I and II, of VP6. Further, the padlock probes (69 nt) sequence consist of a 5′ arm of 15 nt Rotavirus targeting probe, followed by the AluI restriction site, the target region for the detection probe, and 3′ arm of 14 nt Rotavirus targeting probe, and were ordered from IDT. All oligonucleotide sequences used in this assay are listed in Table 1.


Detection of rotavirus using padlock probes and rolling circle amplification.

Mezger A, Öhrmalm C, Herthnek D, Blomberg J, Nilsson M - PLoS ONE (2014)

Padlock probe design.(A) The sequence of the six padlock probes with the haplotyped degenerations and ligation site. (B) Alignment of 214 Human Rotaviruses. The variations in Rotavirus were mapped using BLASTn and ConSort. The length of the black bars represents the frequency of variation as an average percentage conservation at each nt position (y-axis).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219791&req=5

pone-0111874-g001: Padlock probe design.(A) The sequence of the six padlock probes with the haplotyped degenerations and ligation site. (B) Alignment of 214 Human Rotaviruses. The variations in Rotavirus were mapped using BLASTn and ConSort. The length of the black bars represents the frequency of variation as an average percentage conservation at each nt position (y-axis).
Mentions: All Human Rotavirus A types that were available on NCBI blastn (National Library of Medicine) were used to design the padlock probes. Using the Consort program [12], sequence conservation and frequencies of nucleotide variation were compared for all eleven human Rotavirus A segments for all available sequences in NCBI blastn (National Library of Medicine). This resulted in that the VP6 gene, coding for the major capsid protein, was chosen as a target region for the padlock probes based on its relative conserved regions. The chosen region, nt 96–125 of the 1356 nt long VP6 gene (Genebank nr AB022768), in which the padlock probes are designed against is based on the alignment of 214 human rotavirus A sequences (Figure 1B). A query of VP6 gene rotavirus in BLASTn was analyzed in the ConSort program [12] to construct a primer and the 5′ and 3′ arms of padlock probes targeting rotavirus. The design was made according to the principle of NucZip [13]. The consensus sequence for the target region in the NIH database is CARTTYAAYCWRATGATARTWACHATGAA, causing a degeneration of 384. Due to that specific nucleotide positions vary together, Consort settings of 1–10 degeneration were used to haplotype the padlock target region into a minimum of sequences. The final design consisted of six padlock probe designs (degeneration 32, 8, 8, 8, 1, and 1) that together create a mix of 58 unique padlock probes (Figure 1A) needed to cover 95% of the published sequences in the NIH database targeting both genogroups, I and II, of VP6. Further, the padlock probes (69 nt) sequence consist of a 5′ arm of 15 nt Rotavirus targeting probe, followed by the AluI restriction site, the target region for the detection probe, and 3′ arm of 14 nt Rotavirus targeting probe, and were ordered from IDT. All oligonucleotide sequences used in this assay are listed in Table 1.

Bottom Line: Rotavirus is often diagnosed by latex agglutination assay, chromatography immunoassay, or by electron microscopy, which are all quite insensitive.Reverse transcription polymerase chain reaction, on the other hand, is very sensitive to variations at the genomic level.We developed a novel assay based on a set of 58 different padlock probes with a detection limit of 1,000 copies.

View Article: PubMed Central - PubMed

Affiliation: Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Solna, Sweden.

ABSTRACT
Rotavirus infections are one of the most common reasons for hospitalizations due to gastrointestinal diseases. Rotavirus is often diagnosed by latex agglutination assay, chromatography immunoassay, or by electron microscopy, which are all quite insensitive. Reverse transcription polymerase chain reaction, on the other hand, is very sensitive to variations at the genomic level. We developed a novel assay based on a set of 58 different padlock probes with a detection limit of 1,000 copies. Twenty-two patient samples were analyzed and the assay showed high concordance with a PCR-based assay. In summary, we present a new assay for sensitive and variation tolerant detection of rotavirus.

Show MeSH
Related in: MedlinePlus