Establishment of a digital PCR detection method for Staphylococcus aureus in laboratory animals using microdroplet technique
10.3969/j.issn.1005-4847.2025.03.011
- VernacularTitle:实验动物金黄色葡萄球菌微滴式数字PCR检测方法建立和优化
- Author:
Yuyu LI
1
;
Jiying YAO
;
Yonglu TIAN
;
Tiantian SUN
;
Yusheng WEI
;
Xiaying LI
Author Information
1. 北京大学生命科学学院实验动物中心,北京 100871
- Publication Type:Journal Article
- Keywords:
Staphylococcus aureus;
droplet digital PCR;
laboratory animals
- From:
Acta Laboratorium Animalis Scientia Sinica
2025;33(3):430-439
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a rapid and accurate droplet digital PCR(ddPCR)detection method for detecting Staphylococcus aureus(SA)in laboratory animals and the environment.Methods Using the heat-stable nuclease gene(nuc)of SA as the target gene,a pair of specific primers and probes are designed within its conserved region.Optimize the reaction conditions,test the dynamic range,and evaluate the specificity and stability of the method.Using the same template,test reactions were performed with both ddPCR and real-time quantitative PCR(qPCR)method to assess the interchangeability between the two approaches.Finally,the method is applied to the detection of various clinical samples.Results The kinetic range of the established SA ddPCR method is 100～15 000 copies/μL,with a detection limit of 2.5 copies and a quantification limit of 10 copies;The specificity of this method was tested,and only SA showed positive droplets,while no positive droplets were found for other pathogens;After measuring three parallel samples,the standard deviation and relative standard deviation were calculated.It was found that within the dynamic detection interval of ddPCR,as the target copy number gradually decreased,the relative standard deviation showed an upward trend,but remained below 25％.This result indicates that the detection method has good stability.Conclusions The established ddPCR method for detecting SA has the advantages of high sensitivity,strong specificity,good stability,and good reproducibility.This method can be applied for the detection of SA in laboratory animals.
