Comparison of droplet digital PCR and quantitative real-time PCR in mcrA-based methanogen community analysis

Abstract BACKGROUND The WHO recently released a BK virus (BKV) international standard. This study evaluated the WHO international standard and commercially available BKV standards by quantitative real-time PCR (qPCR) and droplet digital PCR (ddPCR). METHODS WHO, Exact Diagnostics, Acrometrix, and Zeptometrix BKV standards were tested by qPCR and ddPCR. Two preparations of NIST BKV clones were also tested. Nucleic acid was extracted with the Roche MP96 and MPLC, followed by quantification in duplicate. To resolve discrepancies, we sequenced the WHO and NIST materials. RESULTS Manufacturers' expected copies/mL were close to WHO IU/mL: linear regression of qPCR data revealed 1.12 Exact copies/IU, 0.76 Acrometrix copies/IU, and 0.70 Zeptometrix copies/IU. For ddPCR, similar concentrations were measured when either the VP1 region or the T region was targeted, and concentrations were almost 2-fold higher when both regions were targeted simultaneously. ddPCR results for the VP1 and T regions were similar for all commercial standards, but targeting the T region of the WHO standard led to a 4-fold lower result than the VP1 region. Next-generation sequencing revealed no primer or probe mismatches. However, large differences in coverage across the WHO standard and junctional reads were observed, indicating subpopulations of the WHO standard with deletions in the T region. CONCLUSIONS BKV standards showed concordance among providers, but the WHO standard contains subpopulations of viruses with various deletions in the T region. PCR results will vary depending on which region of the WHO standard is targeted.

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Development of droplet digital PCR assays to quantify genes involved in nitrification and denitrification, comparison with quantitative real-time PCR and validation of assays in vineyard soil

Canadian Journal of Microbiology ◽

10.1139/cjm-2020-0033 ◽

2020 ◽

pp. 1-14

Author(s):

Tanja M. Voegel ◽

Melissa M. Larrabee ◽

Louise M. Nelson

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Environmental Samples ◽

Soil Bacteria ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Quantitative Real Time Pcr ◽

Soil Dna ◽

Soil C ◽

Total Bacteria

Quantifying genes in soil is important to relate the abundance of soil bacteria to biogeochemical cycles. Quantitative real-time PCR is widely used for quantification, but its use with environmental samples is limited by poor reaction efficiencies or by PCR inhibition through co-purified soil substances. Droplet digital PCR (ddPCR) is a technology for absolute, sensitive quantification of genes. This study optimized eight ddPCR assays to quantify total bacteria and archaea as well as the nitrification (bacterial and archaeal amoA) and denitrification (nirS, nirK, nosZI, nosZII) genes involved in the generation or reduction of the greenhouse gas nitrous oxide. Detection and quantification thresholds were compared with those of quantitative real-time PCR and were equal to, or improved, in ddPCR. To validate the assays using environmental samples, soil DNA was isolated from two vineyards in the Okanagan valley in British Columbia, Canada, over the 2017 growing season. Soil properties related to the observed gene abundances were determined. Total bacteria, nirK, and nosZII increased with time and the soil C/N ratio and NH4+-N concentration affected total archaea and archaeal amoA negatively. The results, compared with those of other studies, showed that ddPCR is a valid alternative to qPCR to quantify genes involved in nitrification or denitrification.

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