Abstract:

Objective: To develop a reference material for astrovirus used in nucleic acid amplification testing. Methods:
Purified astrovirus cRNAs were prepared by applying gene cloning and in vitro transcription methods. On the base of
preliminary quantitation, the purified cRNAs were diluted to the appropriate concentration and sub-packaged with equal
volume. Two types of astrovirus reference material sample were obtained after some of the sub-packages were treated with
RNAsafe, while the rest were not treated. The homogeneity and stability were tested in both types of reference material
sample by applying real-time quantitative (RT-qPCR). The concentrations of astrovirus reference material were determined
by combined use of RT-qPCR and digital PC, and the uncertainty was analyzed. Results: There were no significant
differences between intra-class variance and inter-class variance (P > 0.05). Two types of astrovirus reference material were
stable at 20–25 ℃ for 10 days, at 4 ℃ for 2 weeks, at -20 ℃ for 3 months, and at -80 ℃ or in liquid nitrogen for half a year.
The reference material samples without RNAsafe were valued as (1.652 ± 0.143) ×108 copies/μL (preserved at -80 ℃) or
(1.652 ± 0.135) ×108 copies/μL (preserved in liquid nitrogen). Conclusion: The samples without RNAsafe could be used as a
reference material for astrovirus used in nucleic acid amplification testing.

Key words: astrovirus, nucleic acid, real-time quantitative polymerase chain reaction (RT-qPCR), digital polymerase chain reaction, reference material