Clinical chemistry and laboratory medicine · 2026
Pseudovirus reference material and RT-digital PCR reference method for the quantification of human rhinovirus RNA with metrological traceability.
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Li Huijie, Liu Yang, Cai Wenyi, Jin Xiaohua, Huang Wenfeng, Wang Shangjuan, Zhu Lingxiang, Yang Yi, Zhang Yu, Zhou Shujun, Hong Tian, Zhao Lulu, Chen Xun, Chen Haofeng, Guo Yong, Gao Huafang, Du Meihong, Dong Lianhua
Аннотация
OBJECTIVES: This study developed a pseudoviral human rhinovirus (HRV) reference material using pseudovirus technology and integrating RT-dPCR with HPLC-IDMS to achieve accurate HRV RNA quantification, aiming to standardize HRV detection across labs and platforms.
METHODS: We developed an RT-digital PCR (RT-dPCR) reference method for HRV, incorporating reverse transcription efficiency (RTE) correction using HPLC-isotope dilution mass spectrometry (HPLC-IDMS), which enables value assignment of the well-characterized pseudoviral reference material (RM) containing the conserved 5'UTR gene.
RESULTS: The developed RT-dPCR method demonstrated high analytical sensitivity with a limit of detection (LoD) of eight copies/reaction and a limit of quantification (LoQ) of 11 copies/reaction. RTE was accurately determined as 101.25 % using HPLC-IDMS, enabling metrologically traceable RNA quantification. The pseudoviral RM was characterized using the traceable RT-dPCR method; its reference value and expanded uncertainty were determined to be (3.12 ± 0.69) × 103 copies/μL (k=2). The RM exhibited sufficient homogeneity and stability for 12 months at -80 °C, and its non-infectious nature coupled with its ability to simulate the entire workflow including nucleic acid extraction offers significant advantages.
CONCLUSIONS: This study provided essential tools for standardizing HRV detection across different laboratories and platforms. This work enables traceable RNA quantification in SI units and simulates the full analytical process, thereby enhancing the accuracy and comparability of results. Furthermore, this approach serves as a transferable model for developing reference materials and traceable quantification methods for other respiratory pathogens, contributing to advancements in molecular diagnostics and public health.
METHODS: We developed an RT-digital PCR (RT-dPCR) reference method for HRV, incorporating reverse transcription efficiency (RTE) correction using HPLC-isotope dilution mass spectrometry (HPLC-IDMS), which enables value assignment of the well-characterized pseudoviral reference material (RM) containing the conserved 5'UTR gene.
RESULTS: The developed RT-dPCR method demonstrated high analytical sensitivity with a limit of detection (LoD) of eight copies/reaction and a limit of quantification (LoQ) of 11 copies/reaction. RTE was accurately determined as 101.25 % using HPLC-IDMS, enabling metrologically traceable RNA quantification. The pseudoviral RM was characterized using the traceable RT-dPCR method; its reference value and expanded uncertainty were determined to be (3.12 ± 0.69) × 103 copies/μL (k=2). The RM exhibited sufficient homogeneity and stability for 12 months at -80 °C, and its non-infectious nature coupled with its ability to simulate the entire workflow including nucleic acid extraction offers significant advantages.
CONCLUSIONS: This study provided essential tools for standardizing HRV detection across different laboratories and platforms. This work enables traceable RNA quantification in SI units and simulates the full analytical process, thereby enhancing the accuracy and comparability of results. Furthermore, this approach serves as a transferable model for developing reference materials and traceable quantification methods for other respiratory pathogens, contributing to advancements in molecular diagnostics and public health.