Development of national reference of Yersinia pestis nucleic acid detection reagents
10.13200/j.cnki.cjb.004320
- VernacularTitle:鼠疫耶尔森菌核酸检测试剂国家参考品的研制
- Author:
ZHANG Yuanyuan
- Publication Type:Journal Article
- Keywords:
Yersinia pestis;
Nucleic acid detection reagents;
National reference
- From:
Chinese Journal of Biologicals
2024;37(10):1185-1189+1199
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop a national reference for nucleic acid detection reagents of Yersinia pestis, so as to use it for the evaluation and quality control of Yersinia pestis nucleic acid detection reagents. Methods Five strains of Yersinia pestis[(EV strain, 0614F strain, otten strain, Tjiusidej(R) strain and Microtus vole 201 strain)] and 10 strains of negative bacteria(3 Yersinia enterocolitica strains, Brucella bovis 104M strain, Bacillus anthracis, Salmonella typhi, Francis tulage LVS strain,Yersinia pseudotuberculosis, Vibrio cholerae and Shigella) were cultured, harvested and inactivated. The stock solution of the reference was obtained and verified by ordinary PCR amplification, and a set of national references composed of 5 positive references, 10 negative references, 1 reference of minimum detection limit(EV strain positive reference was prepared by freeze-drying) and 1 repetitive reference(the same preparation method as the minimum detection limit reference) was prepared. Fluorescence quantitative PCR was used to detect the coincidence rate, uniformity and stability of the national reference products, and four laboratories were organized for collaborative calibration. Results After ordinary PCR amplification, the corresponding target gene bands were found in the stock solution of all the positive references, and no corresponding gene bands were amplified in the stock solution of negative references. The coincidence rates of the national references detected by fluorescence quantitative PCR were 100%. There was no significant difference in Ct values of 3a gene in 10 minimum detection limit references in three repeated detections(F = 1. 567, P = 0. 193). Compared with the references without freezing and thawing, there was no significant difference in Ct values of 3a gene of the minimum detection limit references and positive references after freezing and thawing for three times(t = 0. 416 and 0. 079, respectively, each P > 0. 05). Compared with the references without high temperature preservation, the Ct value of the minimum detection limit reference stored at37 ℃ for 5 d was significantly different(t = 7. 109, P = 0. 002), while there was no significant difference at 4 and 25 ℃(t = 0. 341and 0. 751, respectively, each P > 0. 05). The Ct values of positive references had no significant difference after preservation at 4, 25 and 37 ℃ for 5 d(t = 2. 442, 0. 373 and-0. 043, respectively, each P > 0. 05). The coincidence rates of positive and negative references in four laboratories were all 100%. The minimum detection limit of the minimum detection limit reference detected by two laboratories was 1 × 10~2/mL, and that by the other two laboratories was 1 × 10~3/mL. The CVs of the repetitive reference detection results in four laboratories were all less than 10. 0%. Conclusion This set of national reference for Yersinia pestis nucleic acid detection reagents developed in this study has good uniformity and freeze-thaw stability, and has good accelerated stability at 4 and 25 ℃, which can be used for the evaluation and quality control of Yersinia pestis nucleic acid detection reagents.
