Water contamination by viruses has an increasing worldwide impact on human

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Water contamination by viruses has an increasing worldwide impact on human health and has led to requirements for accurate and quantitative molecular tools. particularly low concentrations usually found in water samples. Electronic supplementary material The online version of this article (doi:10.1007/s00216-013-7476-y) contains supplementary material which is available to authorized users. target numbers recognized per sample quantified by electron microscopy (RoV particles/ml). RT-qPCR assay; RT-ddPCR assay. The marks the threshold … Table 1 Sensitivity of the RT-ddPCR and RT-qPCR assays With the ddPCR instrument used in this study the PCR reaction mixture is separated into 20 0 droplets enabling a theoretical dynamic range of approximately five orders of magnitude [11]. To determine the dynamic range of the Rivaroxaban RT-ddPCR assay exactly a decimal dilution series of viral RNA (Fig.?1) was analyzed. The correlation coefficient (R2) acquired by linear regression analysis showed a good linearity of amplification for both RT-qPCR (R2?=?0.9961) and RT-ddPCR (R2?=?0.9981) assays demonstrating a satisfactory dynamic range of at least four orders of magnitude. Interestingly in the lower part of this dynamic range RT-ddPCR showed excellent measurement repeatability that was unequaled by RT-qPCR as indicated from the coefficient of variance of the measured rotavirus RNA copy figures between replicates (below 15?% for RT-ddPCR between 107 and 103 RoV particles/ml related to concentrations of Rivaroxaban 54 400 632 and 6?±?1 rotavirus copies/10?μL reaction) (Fig.?1). This result demonstrates the higher precision and repeatability of RT-ddPCR for quantifying waterborne viruses at the low concentrations present in most samples analyzed routinely for water quality [23]. Such higher precision of digital PCR approach vs. qPCR technology for low target concentration herein proved for RNA focuses on was previously only shown with DNA focuses on [16 17 24 Another important advantage observed with ddPCR is definitely that it provides an absolute quantity of viral RNA copies present in the sample. The concentration of RoV particles in the initial suspension utilized for the experiments was estimated to be 1.1?×?1011 particles/ml based on TEM counting with latex beads. After applying a correction factor taking into account the dilutions used in RNA extraction and amplification and presuming no deficits in the extraction procedure the amount of RoV focuses on per 10?μl reaction volume is definitely estimated to be approximately 6.2?×?107 in the 1010 RoV particles/ml sample and 6.2 in the 103 RoV particles/ml sample. Considering that electron microscope (EM) estimation for rotavirus concentration was carried out for the viral particles and that RT-ddPCR and RT-qPCR detect RNA the EM estimation is in good correlation with the quantification carried out by both methods (Table?1). Theoretically the dynamic range Mouse monoclonal antibody to CDK4. The protein encoded by this gene is a member of the Ser/Thr protein kinase family. This proteinis highly similar to the gene products of S. cerevisiae cdc28 and S. pombe cdc2. It is a catalyticsubunit of the protein kinase complex that is important for cell cycle G1 phase progression. Theactivity of this kinase is restricted to the G1-S phase, which is controlled by the regulatorysubunits D-type cyclins and CDK inhibitor p16(INK4a). This kinase was shown to be responsiblefor the phosphorylation of retinoblastoma gene product (Rb). Mutations in this gene as well as inits related proteins including D-type cyclins, p16(INK4a) and Rb were all found to be associatedwith tumorigenesis of a variety of cancers. Multiple polyadenylation sites of this gene have beenreported. of a ddPCR reaction is limited by the number of droplets that are analyzed. With the instrument used for this study a value of 5.9 average target copies per droplet (118 0 targets per 20?μl of reaction) equivalent to 99.5?% of the droplets hosting target RNA was defined as the theoretical upper limit that can guarantee quantitative data acquisition [17]. Consequently for the highest concentrations tested (samples comprising 1010 to 108 RoV particles/ml) the number of rotavirus copies was above the instrument upper range of quantification near saturation with almost 100?% of the analyzed droplets comprising rotavirus cDNA copies (Table?1). At these levels of concentration despite the presence of rotavirus becoming recognized the Poisson regulation can no longer be applied and the concentration of focuses on cannot be identified. In water samples viruses are usually less abundant than these limits. In Rivaroxaban most cases the Rivaroxaban concentration of viruses in a sample can be quantified by RT-ddPCR. However for higher disease loads samples have to be diluted before measurement with RT-ddPCR. On the other hand a ddPCR instrument enabling creation of a larger quantity of droplets would increase the available dynamic range and allow direct quantification of samples with higher levels of disease [10]. RT-ddPCR and RT-qPCR assays were also carried out on spiked environmental samples. The rotavirus copy numbers measured with the two assays were similar but lower variability (as determined by the coefficient of variance of the disease copy numbers measured between replicates) was.