Evaluation of LAMP assay using phenotypic tests and PCR for detection of blaKPC gene among clinical samples. Issue 4 (26th February 2022)

• Record Type: Journal Article
• Authors: Chen, Nianzhen; Li, Gen; Si, Yuying; Zhang, Wenyan; Ye, Yangqin; Wang, Yuchao; Wang, Keli; Zong, Ming; Fan, Lieying
• Is Part Of: Journal of clinical laboratory analysis. Volume 36:Issue 4(2022)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2022-02-26
• Subjects: CRE -- KPC -- LAMP -- PCR -- rapid detection; Diagnosis, Laboratory -- Periodicals; Medical laboratory technology -- Periodicals; 616
• DOI: 10.1002/jcla.24310
• Languages: English
• ISSNs: 0887-8013

Abstract

Abstract: Background: Carbapenem‐resistant Enterobacteriaceae (CRE) infection constitutes a public health threat, which bla KPC was the major carbapenemases concerned in China. Timely and efficient diagnosis is of paramount importance for controlling the spread of drug‐resistant bacteria. Here, we develop an approach based on loop‐mediated isothermal amplification (LAMP) for rapid confirmation of bla KPC within 60 min from samples collected. Methods: We designed primers specific to detect bla KPC and evaluated it for its sensitivity and specificity of detection using real‐time monitoring. Five hundred forty‐six clinical specimens were analyzed by the LAMP assay and compared with the phenotypic tests and PCR. The samples with inconsistent results were further verified by Sanger sequencing. Results: The LAMP assay displayed a detection limit of 1 × 10 2 CFU/ml, which was 10‐fold more sensitive than the PCR. No cross‐reactivity was observed for strains that produced other types of β‐lactamase. Furthermore, we demonstrated concordant results (Kappa > 0.75) between the genotypic method and phenotypic tests for the 546 clinical samples. The data presented in this study suggested that the genotypic method is a reliable assay for identifying bla KPC‐induced CRE in China. The results of the Sanger sequencing indicate that the developed method not only has high accuracy but also meets the need for rapid diagnosis, while the PCR method is prone to false negatives. Conclusions: We successfully constructed a LAMP technique that can be used for auxiliary diagnosis of CRE, which is faster, cheaper, and more accurate than the PCR. It may therefore be routinely applied for detection of bla KPC producers in routine clinical laboratories. Abstract : Carbapenem‐resistant Enterobacteriaceae (CRE) infection constitutes a public health threat, which bla KPC was the major carbapenemases concerned in China. However, conventional identification and susceptibility testing methods of microorganisms usually require at least 2 days from specimen collection. Timely and efficient diagnosis is of paramount importance for controlling the spread of drug‐resistant bacteria. We develop an approach based on loop‐mediated isothermal amplification (LAMP) for rapid confirmation of bla KPC within 60 min from samples collected. Five hundred forty‐six clinical specimens were analyzed by the LAMP assay and compared with the phenotypic tests and PCR. The LAMP assay displayed a detection limit of 1 × 10 2 CFU/ml, which was 10‐fold more sensitive than the PCR. The results obtained with 546 clinical samples indicate that the genotypic method and phenotypic tests have a good agreement (Kappa > 0.75). It is a reliable assay for identifying blaKPC induced CRE in China. The inconsistent results were verified by Sanger sequencing furtherly. The results of the Sanger sequencing indicate that the developed method not only has high accuracy but also meets the need for rapid diagnosis, while the PCR method is prone to false negatives. It may therefore be routinely applied for detection of bla KPC producers in routine clinical laboratories.