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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

Analytical sensitivity of the method.Short synthetic 5′ biotinylated DNA templates are serially diluted to assess the analytical sensitivity of our assay. The y-axis shows the number of rolling circle products (RCPs) and the x-axis the copy number of a synthetic biotinylated DNA target. The negative sample is a no template control. Error bars ±1 s.d.; n = 3.
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pone-0111874-g003: Analytical sensitivity of the method.Short synthetic 5′ biotinylated DNA templates are serially diluted to assess the analytical sensitivity of our assay. The y-axis shows the number of rolling circle products (RCPs) and the x-axis the copy number of a synthetic biotinylated DNA target. The negative sample is a no template control. Error bars ±1 s.d.; n = 3.

Mentions: Before testing the assay on patient samples the analytical sensitivity was estimated by preparing a 1∶10 dilution series from 106 to 103 copies of a short synthetic biotinylated target (5′ Biotin -TTCATAGTAATTATCATTTGGTTAAATTG-3′). This target is a not an ideal substitute for the real viral genome, but should be a relatively good substitute for the cDNA copy of the genome. Thus, any loss in cDNA synthesis efficiency is not taken into account with this target. C2CA was performed as described in materials and methods and 2.5 µl were analyzed in the microfluidic detection system. With the current setup the assay has a limit of detection of 1,000 molecules and a dynamic range of at least four magnitudes (Figure 3). The amount of target was shown to be linearly correlated to the number of RCPs. These properties allow for a quantitative assay with sufficient sensitivity for analysis of clinical samples, since clinical samples from rotavirus patients usually contain viral loads exceeding this limit of detection.


Detection of rotavirus using padlock probes and rolling circle amplification.

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

Analytical sensitivity of the method.Short synthetic 5′ biotinylated DNA templates are serially diluted to assess the analytical sensitivity of our assay. The y-axis shows the number of rolling circle products (RCPs) and the x-axis the copy number of a synthetic biotinylated DNA target. The negative sample is a no template control. Error bars ±1 s.d.; n = 3.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111874-g003: Analytical sensitivity of the method.Short synthetic 5′ biotinylated DNA templates are serially diluted to assess the analytical sensitivity of our assay. The y-axis shows the number of rolling circle products (RCPs) and the x-axis the copy number of a synthetic biotinylated DNA target. The negative sample is a no template control. Error bars ±1 s.d.; n = 3.
Mentions: Before testing the assay on patient samples the analytical sensitivity was estimated by preparing a 1∶10 dilution series from 106 to 103 copies of a short synthetic biotinylated target (5′ Biotin -TTCATAGTAATTATCATTTGGTTAAATTG-3′). This target is a not an ideal substitute for the real viral genome, but should be a relatively good substitute for the cDNA copy of the genome. Thus, any loss in cDNA synthesis efficiency is not taken into account with this target. C2CA was performed as described in materials and methods and 2.5 µl were analyzed in the microfluidic detection system. With the current setup the assay has a limit of detection of 1,000 molecules and a dynamic range of at least four magnitudes (Figure 3). The amount of target was shown to be linearly correlated to the number of RCPs. These properties allow for a quantitative assay with sufficient sensitivity for analysis of clinical samples, since clinical samples from rotavirus patients usually contain viral loads exceeding this limit of detection.

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