Development of Human Rhinovirus RNA Reference Material Using Digital PCR

Figures

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  Figure 1. A schematic overview of the procedures RM production.

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  Figure 2. Human RV RM serial dilution. Detection of human RV based on assays by qPCR and ddPCR. (A) Ct value (qPCR) and (B) copy number (ddPCR) of rhinovirus using the assay. All experiments were conducted three times and the data presented represent the average values obtained.

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  Figure 3. RMs homogeneity test. A homogeneity experiment was performed by randomly selecting 10 of the produced RMs. Error bars represent the standard deviation at each data point, calculated based on the mean of the replicated measurements (n = 3). Homogeneity values for the gene among bottles are presented as percentages. As a result, the average value is 1.6 × 105 copies/mL, and the calculation results of the relative standard deviation (RSD) of about 11% and the standard uncertainty of 3.2% are shown. Therefore, it was confirmed that the produced RM was made homogeneously.

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  Figure 4. Short-term and long-term stability of RV RMs. (A) The short-term stability of the RM was confirmed after 4 days and 7 days at 4 °C and −20 °C. As a result, the results showed an almost negligible difference at both 4 °C and −20 °C. (B) Long-term stability after 1, 3, 6, and 12 months was confirmed. RM was stored at −80 °C.

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  Figure 5. RV RM freeze–thawing repeated test. An experiment was conducted to confirm the change in the number of copies when the freezing and thawing of the RM were repeated. The experiment was repeated in the order of thawing at 4 °C and freezing at −80 °C. As a result, it shows the result that the change in the number of copies appears insignificant until the third repetition. Therefore, it shows results that can be used stably even in repeated freeze and thawing.