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Comparison of Three Adenovirus Quantitative PCR Assays with ATCC Reference Strains and Clinical Samples.

ABSTRACT: Adenoviruses (AdV) have been associated with a variety of human diseases and are recognized as causing significant morbidity and mortality in immunocompromised or transplant patients. Quantification of AdV DNA in plasma is notoriously difficult due to the genetic diversity of the 71 different serotypes identified to date. There is no World Health Organization standard available to harmonize quantitative data, so results between labs vary widely. In this study, we compared a laboratory-developed multiplex PCR assay with primers and probes specific for each group (A to G) and subgroup E4 (Octaplex) to one with a single primer and probe set (modified from N. Jothikumar et al., Appl Environ Microbiol 71:3131-3136, 2005) and one utilizing bisulfite pretreatment of DNA to reduce variation prior to amplification (Genetic Signatures). Our Octaplex assay detected all low-copy-number clinical samples, while the other two assays had subsets of samples that did not amplify. The modified Jothikumar assay failed to efficiently amplify three of the high-copy-number cultured strains, while the Genetic Signatures 3base assay had a positive bias, resulting in higher copies/ml (>0.5 log10) for all culture fluids tested. All three assays resulted in endpoint detection of the available 51 AdV types. Using two different materials to generate a standard curve revealed that the Octaplex TaqMan assay and the modified Jothikumar assay both consistently gave adenovirus levels lower than the commercial platform for AdV culture fluids but not patient samples. This study highlights the differences in detection of AdV between laboratories that can be attributed to both the PCR method, as well as the reference material used for quantitation.

SUBMITTER: Starr K

PROVIDER: S-EPMC6813019 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Comparison of Three Adenovirus Quantitative PCR Assays with ATCC Reference Strains and Clinical Samples.

Starr Kimberly K Greninger Alexander L AL Makhsous Negar N Jerome Keith R KR Cook Linda L

Journal of clinical microbiology 20191023 11

Adenoviruses (AdV) have been associated with a variety of human diseases and are recognized as causing significant morbidity and mortality in immunocompromised or transplant patients. Quantification of AdV DNA in plasma is notoriously difficult due to the genetic diversity of the 71 different serotypes identified to date. There is no World Health Organization standard available to harmonize quantitative data, so results between labs vary widely. In this study, we compared a laboratory-developed ...[more]

PMID: 31434723

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Project description:BackgroundPCR allelic discrimination technologies have broad applications in the detection of single nucleotide polymorphisms (SNPs) in genetics and genomics. The use of fluorescence-tagged probes is the leading method for targeted SNP detection, but assay costs and error rates could be improved to increase genotyping efficiency. A new assay, rhAmp, based on RNase H2-dependent PCR (rhPCR) combined with a universal reporter system attempts to reduce error rates from primer/primer and primer/probe dimers while lowering costs compared to existing technologies. Before rhAmp can be widely adopted, more experimentation is required to validate its effectiveness versus established methods.ResultsThe aim of this study was to compare the accuracy, sensitivity and costs of TaqMan, KASP, and rhAmp SNP genotyping methods in sugar beet (Beta vulgaris L.). For each approach, assays were designed to genotype 33 SNPs in a set of 96 sugar beet individuals obtained from 12 parental lines. The assay sensitivity was tested using a series of dilutions from 100 to 0.1 ng per PCR reaction. PCR was carried out on the QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific, USA). The call-rate, defined as the percentage of genotype calls relative to the possible number of calls, was 97.0, 97.6, and 98.1% for TaqMan, KASP, and rhAmp, respectively. For rhAmp SNP, 24 of the 33 SNPs demonstrated 100% concordance with other two technologies. The genotype concordance with either technologies for the other 9 targets was above 99% (99.34-99.89%).ConclusionThe sensitivity test demonstrated that TaqMan and rhAmp were able to successfully determine SNP genotypes using as little as 0.2 ng DNA per reaction, while the KASP was unable to ascertain SNP states below 0.9 ng of DNA per reaction. Comparative cost per reaction was also analyzed with rhAmp SNP offering the lowest cost per reaction. In conclusion, rhAmp produced more calls than either TaqMan or KASP, higher signal to NTC data while offering the lowest cost per reaction.

Dataset Information

Comparison of Three Adenovirus Quantitative PCR Assays with ATCC Reference Strains and Clinical Samples.

Publications

Comparison of Three Adenovirus Quantitative PCR Assays with ATCC Reference Strains and Clinical Samples.

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