Establishment of visualized rapid detection method for Klebsiella pneumoniae based on RPA-LFD and its effect
10.11816/cn.ni.2025-250362
- VernacularTitle:基于RPA-LFD可视化快速检测肺炎克雷伯菌方法的建立及评价
- Author:
Yuxuan ZANG
1
;
Yanxia XING
;
Yanhua LI
;
Guobin SONG
;
Guoping XI
;
Yunfeng BAI
Author Information
1. 山西大同大学医学院,山西大同 037009;山西大同大学化学生物传感山西省重点实验室,山西大同 037009
- Publication Type:Journal Article
- Keywords:
Klebsiella pneumoniae;
Recombinase polymerase amplification;
Lateral flow dipstick;
Rapid detection
- From:
Chinese Journal of Nosocomiology
2025;35(17):2603-2607
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE T o establish a simple,rapid,highly sensitive and highly specific visualized detection meth-od for Klebsiella pneumoniae based on recombinase polymerase amplification(RPA)technique combined with the lateral flow dipstick(LFD)technique.METHODS The specific primers targeting the khe gene of K.pneumoniae were designed for RPA,the reaction temperature and reaction time for RPA were optimized,the optimal experi-mental conditions for the LFD were determined based on the colorimetric results.The sensitivity,stability,speci-ficity and clinical practicability of RPA-LFD in detection of the K.pneumoniae were evaluated.RESULTS RPA could achieved the optimal detection result for simplification of K.pneumoniae at 34 ℃ for 10 min.The LFD showed the best color development when Milipore HF 135s was used as the nitrocellulose membrane,phosphate buffered saline containing Tween-20(PBST)was used as the sample developing solution,and the concentrations of streptavidin and secondary antibody were 0.8 mg/ml and 0.1 mg/ml,respectively.The detection limit of the RPA-LFD was 5 CFU/ml for detection of K.pneumoniae,the relative standard deviation of the band color inten-sity was less than 5％in six parallel experiments,and there was no cross reactions with other bacterial strains.In addition,the sensitivity of the RPA-LFD in detection of K.pneumoniae was not affected by the serum compo-nents such as proteins and lipids.CONCLUSION The rapid detection method for K.pneumoniae that is established based RPA-LFD is characterized by the simple operation,high sensitivity and high specificity,and it does not need complicated equipment or strict technical requirement for operators and provides a new technique for early diagno-sis and epidemiological survey of the K.pneumoniae.
