Pseudovirus reference material and RT-digital PCR reference method for the quantification of human rhinovirus RNA...

Area of Science:

• Molecular Diagnostics
• Metrology in Diagnostics
• Respiratory Pathogen Detection

Background:

• Accurate quantification of human rhinovirus (HRV) RNA is crucial for diagnostics and public health.
• Current HRV detection methods lack standardization across laboratories and platforms.
• Development of reliable reference materials is needed to improve HRV detection accuracy.

Purpose of the Study:

• To develop a pseudoviral HRV reference material (RM) for accurate RNA quantification.
• To establish a standardized method for HRV detection using RT-digital PCR (RT-dPCR) integrated with HPLC-isotope dilution mass spectrometry (HPLC-IDMS).
• To enable metrologically traceable quantification of HRV RNA.

Main Methods:

• Development of a pseudoviral HRV reference material using pseudovirus technology.
• Implementation of a reverse transcription digital PCR (RT-dPCR) reference method for HRV.
• Correction for reverse transcription efficiency (RTE) using HPLC-isotope dilution mass spectrometry (HPLC-IDMS).

Main Results:

• The RT-dPCR method achieved high analytical sensitivity (LoD: 8 copies/reaction, LoQ: 11 copies/reaction).
• HPLC-IDMS accurately determined RTE as 101.25%, enabling traceable RNA quantification.
• The pseudoviral RM was characterized with a reference value of (3.12 ± 0.69) × 10³ copies/μL, showing stability for 12 months.

Conclusions:

• A standardized pseudoviral HRV reference material and traceable quantification method were successfully developed.
• This approach enhances the accuracy and comparability of HRV detection results across different platforms.
• The methodology serves as a transferable model for developing reference materials for other respiratory pathogens.