Citation: LIANG Long-Hui, CHENG Xi, YANG Yang, YAN Long, YU Hui-Lan, DU-Bin, LIU Chang-Cai, LIU Shi-Lei. An in Vitro Detection Method for Depurination Activity of Ricin Based on A Novel RNA Substrate and Its Application[J]. Chinese Journal of Analytical Chemistry, ;2021, 49(10): 1694-1703. doi: 10.19756/j.issn.0253-3820.211117 shu

An in Vitro Detection Method for Depurination Activity of Ricin Based on A Novel RNA Substrate and Its Application

LIANG Long-Hui ¹ ,
CHENG Xi ² ,
YANG Yang ¹ ,
YAN Long ¹ ,
YU Hui-Lan ¹ ,
DU-Bin ¹ ,
LIU Chang-Cai ^1,, ,
LIU Shi-Lei ^1,,

1.
State Key Laboratory of NBC Protection for Civilian, Beijing 102205, China;
2.
College of Liberal Arts and Sciences, National University of Defense Technology, Changsha 410073, China

Corresponding author: LIU Chang-Cai, LIU Shi-Lei,
Received Date: 11 February 2021
Revised Date: 9 July 2021

Fund Project: Supported by the State Key Laboratory of NBC Protection for Civilian (No.SKLNBC2020-08).

An in vitro detection method for active ricin based on depurination of a novel RNA substrate was developed with no adenine interference in the negative control sample. The problem that the substrate was prone to self-hydrolysis and caused false positive results in the traditional depurination activity detection method was solved. The depurination activities of single-stranded DNA and RNA substrates with the same sequence were systematically compared. It was found that RNA was more suitable as the substrate for detection of ricin depurination activity. The optimization of reaction conditions showed that the pH values, temperature, and concentration of RNA substrate were the key factors for detection of ricin depurination activity. Under the optimal conditions, there was no adenine interference in the negative control sample. The influence of RNA stem-loop composition on substrate activity was validated by screening a series of RNA substrates. A novel RNA substrate was obtained with the optimal depurination activity, and its depurination activity was twice that of the reported substrate. By combining with the immunocapture purification, a quantitative detection method for the active ricin by isotope-labeled internal liquid chromatography-triple quadrupole mass spectrometry with multiple reaction monitoring using the novel RNA as the depurination substrate was established. This method had good specificity, the linear range of active ricin ranged from 5.0 to 300 ng/mL, and the limit of detection was 1.0 ng/mL (S/N=3). This method was applied to the quantitative detection of active ricin in tap water, milk, and plasma samples. The recoveries of spiked samples were between 91.0% and 116.5%, and the relative standard deviations (RSDs) were between 3.2% and 9.3%. This method was helpful for the screening and identification of active ricin in the fields of food safety and chemical weapons inspection.
- Ricin,
- RNA substrates,
- Depurination activity,
- Type Ⅱ ribosome inactivating protein,
- Multiple reaction monitoring
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