Establishment of visualized LAMP assay targeting rcsA gene for rapid detection of Klebsiella pneumoniae
10.11816/cn.ni.2025-241819
- VernacularTitle:针对rcsA基因建立可视化LAMP方法快速检测肺炎克雷伯菌
- Author:
Yushan CHEN
1
;
Qian HU
;
Lin XIANG
;
Jia YAN
;
Jiawei LIU
;
Qiguang LI
;
Linhai LI
;
Lingqing XU
Author Information
1. 广州中医药大学研究生院,广东 广州 510006;广州医科大学附属清远医院(清远市人民医院)检验科,广东清远 511500
- Publication Type:Journal Article
- Keywords:
Klebsiella pneumoniae;
Loop-mediated isothermal amplification;
rcsA gene;
Visualized result;
Rapid detection
- From:
Chinese Journal of Nosocomiology
2025;35(10):1450-1454
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE To establish a visualized loop-mediated isothermal amplification(LAMP)for rapid detec-tion of Klebsiella pneumoniae and evaluate the sensitivity and specificity of the detection method.METHODS To-tally4 specific LAMP primers were designed by targeting to rcsA gene of the K.pneumoniae.The reaction condi-tions(temperature,time)and the parameters of the reaction system were optimized,the reaction was made visu-alized by using chimeric fluorescent dye SYBR Green Ⅰ.The optimal reactions conditions and reaction systems were determined based on the results of visualization and agarose gel electrophoresis.The optimized conditions and systems were used to test the sensitivity by diluting DNA template on a 10-fold gradient,meanwhile,the specifici-ty of the method was evaluated by detecting K.pneumoniae and other ten species of common bacteria and fungi.RESULTS The optimal reaction temperature was 63 ℃ after the optimization,with the reaction time 35 min;the concentration of buffer solution in the reaction system was determined as 0.8×,with the concentration of magne-sium ion 8 mmol/L,the concentration of dNTPs 1.4 mmol/L,the ratio of internal to external primers 6∶1,the concentration of Bst DNA polymerase 0.32 U/μl,the concentration of betaine 0.75 mmol/L.For the test of sensi-tivity,the method could detect the template with the concentration of 1.5 ng/μl.For the test of specificity,only the detection of K.pneumoniae could display positive visualized result and positive electrophoretic band.CONCLUSIONS The visualized LAMP with high specificity and sensitivity for rapid detection of K.pneumoniae is successfully established,facilitating the observation of the detection result without the use of precise instruments.It is suitable for the detection in grass-root laboratories after successful preliminary application in clinical detection.
