Identification and optimization of parameters for accurate quantification of RNA by RT-dPCR.

Reverse transcription-digital PCR (RT-dPCR) is attracting attention as a method that enables SI-traceable RNA quantification without calibration, but its accuracy and bias have not been thoroughly studied. In this study, the accurate quantification of RNA by the RT-dPCR method was investigated using NMIJ CRM 6204-b, an RNA certified reference material whose certified value was assigned by orthogonal chemical measurement methods. Moreover, a two-step RT-dPCR method was adopted to examine in detail the conditions for the RT reaction process, which was expected to be the major uncertainty component in the RT-dPCR measurement. Optimization experiments revealed that the type of reverse transcriptase, the concentration of template RNA, and the type and concentration of primers in the RT reaction affected the value quantified by RT-dPCR. Under the optimal conditions, the value quantified by RT-dPCR, 76.4 ng/µL ± 6.7 ng/µï»¿L (the quantified value ± expanded uncertainty (k = 2)), was consistent with the certified value, 68.2 ng/µï»¿L ± 5.8 ng/µï»¿L, of NMIJ CRM 6204-b RNA 1000-A within the expanded uncertainty. From the results of the uncertainty evaluation, the relative combined uncertainty of the RT-dPCR method was 4.42%, and the major uncertainty components in the RT-dPCR method were the preparation of RT solution (3.68%), the inter-day difference (1.80%), and the RT reaction (1.30%). Together, the results suggested that the contribution of the RT reaction process to the total uncertainty was greater than that of the dPCR process.

Optimizing virus inactivation methods for molecular detection techniques: Implications for viral protein and RNA measurements.

To facilitate the development of effective viral detection techniques, a positive control material is required for validating their quantitative performance. Inactivated viruses serve as viable control materials, as they can be handled without the constraints of biohazard safety facilities. However, inactivation alters the structure of viral component molecules, necessitating the selection of inactivation methods that have minimal effects on the target molecules relevant to molecular detection techniques. Only a limited number of studies have investigated inactivation methods to produce viral control materials. Therefore, the aim of this study was to investigate various virus inactivation methods and evaluate their impact on molecular detection techniques, with a specific focus on viral proteins and RNA. We evaluated the effects of ultraviolet (UV) irradiation, heat, beta-propiolactone (BPL), hydrogen peroxide (H2O2), and perchloric acid (HClO4) inactivation methods to identify the most effective technique and its optimal conditions. Enzyme-linked immunosorbent assay (ELISA) and reverse transcription-digital polymerase chain reaction (RT-dPCR) were employed as model assays to assess the effects of these treatments on protein and RNA measurements. Among the evaluated methods, UV and heat treatments demonstrated minimal interference with ELISA, while heat treatment had the least impact on RT-dPCR measurements. Consequently, our findings revealed that heat inactivation holds the potential for producing inactivated viruses that can be effectively used in molecular detection techniques targeting both viral protein and RNA.

Quantitative performance of digital ELISA for the highly sensitive quantification of viral proteins and influenza virus.

A single-molecule assay (SiMoA) using a digital enzyme-linked immunosorbent assay (ELISA) has been attracting attention as a promising method that can detect viruses with ultra-high sensitivity. However, the quantitative application of digital ELISA has not been adequately reported. Therefore, in this study, we first evaluated the linearity and sensitivity of digital ELISA using a Certified Reference Material of C-reactive protein (NMIJ CRM 6201-c) as a quality control material. Next, we originally screened those antibody pair that are suitable for detecting recombinant viral proteins of influenza A virus, nucleoprotein (NP), and hemagglutinin (HA), and established the measurement system. Under optimized conditions, the limit of detection (LOD) of NP and HA was 0.59 fM and 0.99 fM, and the coefficient of determination, R2, was 0.9998 and 0.9979, respectively. Two subtypes of influenza virus, A/Puerto Rico/8/1934 (H1N1) [PR8] and A/Panama/2007/99 (H3N2) [Pan99], were also quantified under established conditions, and the LOD of PR8 was 3.1 × 102 PFU/mL on targeting NP and 7.4 × 102 PFU/mL on targeting HA. The LOD of Pan99 was 5.3 × 102 PFU/mL on targeting NP. The specificity and robustness of the recombinant viral protein and influenza virus measurements using digital ELISA were also evaluated. Our measurement system showed enough specificity to discriminate the viral subtypes properly and showed sufficient inter- and intra-assay variations for both measurements of recombinant viral proteins and viruses, except for NP-targeting virus measurement.

International co-validation on absolute quantification of single nucleotide variants of KRAS by digital PCR.

The precise quantification of KRAS single nucleotide variant (SNV) is critical for the treatment and prognosis of lung and colorectal cancer. Validation of digital PCR (dPCR) as a method for accurate quantification of KRAS SNV has great clinical importance. An international co-validation on absolute quantification of KRAS SNV by dPCR was conducted among three national measurement institutes (NMIs) from China (NIM), South Korea (KRISS), and Japan (NMIJ). A candidate reference material (RM) was provided by NIM and three measurands were reported: copy number concentration (Tc) of KRAS G12A mutation and wild type and KRAS G12A fractional abundance (FA). Homogeneity and stability assessment showed that the study materials provided by NIM were sufficiently homogeneous and stable during the study period. En number performance statistics was used to evaluate equivalence of the study among the three NMIs. All En values for both Tc and KRAS G12A FA≤1 showed good agreement and consistency with the reference value within the expanded uncertainty. This indicates that dPCR with full uncertainty evaluation can serve as a candidate primary reference measurement procedure (PRMP) for the KRAS SNV measurement and value assignment of reference materials.

Development of certified reference material NMIJ CRM 6205-a for the validation of DNA quantification methods: accurate mass concentrations of 600-bp DNA solutions having artificial sequences.

Two 600-bp DNA solutions (DNA600-G and DNA600-T) were developed as certified reference material, NMIJ CRM 6205-a, for the validation of DNA quantification methods. Both DNA600-G and DNA600-T are ideal as "spike-in control" because these materials have artificial nucleic acid sequences. The certified values were determined as the mass concentration of total DNA (whole DNA materials in sample solution regardless of sequence) at 25 °C by formic acid hydrolysis/liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) and inductively coupled plasma-mass spectrometry (ICP-MS) based on the amount of phosphorus. DNAs were synthesized, and plasmids including the synthesized DNAs were cloned into Escherichia coli DH5α. The amplified plasmids were digested with a restriction enzyme and highly purified. Then, the purified DNAs were diluted with water to approximately 1 ng/µL. By using the CRM-validated methods in fields where DNA quantification is required, the reliability of DNA quantification could be improved. Graphical abstract.

Practical determination of LODP (limit of detection for microarray platform) for the evaluation of microarray platforms.

The performance indicator called limit of detection for microarray platform (LODP) was defined in ISO 16578:2013. The methods to determine practical LODP were explored. In general, + 3 SD of the background is used as the signal strength of limit of detection and criteria for dividing positive and negative results. Since the negative signal had been defined differently for each microarray platform, signals obtained from Non-Probe Spots (NPS) installed on the microarrays were defined as the "background" of microarrays. LODP was determined as the lowest concentration of which the average signal exceeded Avg. + 3 SD of the background (NPS) and the signal was significantly different from those of the lower and higher adjacent concentration points measured with a diluted series of reference materials. For reliable qualitative analysis, the positive results can be defined as signals higher than those corresponding to LODP and negative results as lower signals, without determining limit of detection for all target probes. The use of LODP also enables comparisons of platform performances without checking sequence dependencies, and assists to select reliable and fitting platforms for experimental purposes.

Formic acid hydrolysis/liquid chromatography isotope dilution mass spectrometry: An accurate method for large DNA quantification.

Liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) with formic acid hydrolysis was established for the accurate quantification of λDNA. The over-decomposition of nucleobases in formic acid hydrolysis was restricted by optimizing the reaction temperature and the reaction time, and accurately corrected by using deoxynucleotides (dNMPs) and isotope-labeled dNMPs as the calibrator and the internal standard, respectively. The present method could quantify λDNA with an expanded uncertainty of 4.6% using 10fmol of λDNA. The analytical results obtained with the present method were validated by comparing with the results of phosphate-base quantification by inductively coupled plasma-mass spectrometry (ICP-MS). The results showed good agreement with each other. We conclude that the formic acid hydrolysis/LC-IDMS method can quantify λDNA accurately and is promising as the primary method for the certification of DNA as reference material.

Metal free columns for determination of deoxynucleotide monophosphate by liquid chromatography/mass spectrometry and application to oligonucleotide.

We have developed a highly sensitive method for the analysis of deoxynucleotide monophosphates (dNMPs), which involves the use of liquid chromatography/mass spectrometry (LC/MS) and a new metal-free column. The new column solves the problem that the phosphate group in dNMPs interacts with the metal portion of the device or column. After optimization of the analytical conditions, the limits of detection (LODs) of dNMPs were from 5.4ng/g to 6.3ng/g. Those values were 10 times lower than the LODs of previous methods. We applied the method to the determination of the base composition and the quantification of 20-mer oligonucleotide. Despite use of a very small sample amount of 14.5ng, we were able to determine the base composition, and the result was consistent with theoretical values. We were also able to quantify the mass fraction of oligonucleotide with 8.2% expanded uncertainty (k=2). By means of the developed method, we were able to analyze dNMPs with high sensitivity as well as determine the base composition and quantify the mass fraction of oligonucleotide despite use of a small amount of sample.