Open AccessEvaluation of LAMP assay using phenotypic tests and PCR for detection of bla KPC gene among clinical samples
Author(s) -
Chen Nianzhen,
Li Gen,
Si Yuying,
Zhang Wenyan,
Ye Yangqin,
Wang Yuchao,
Wang Keli,
Zong Ming,
Fan Lieying
Publication year - 2022
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.24310
Subject(s) - sanger sequencing , loop mediated isothermal amplification , biology , detection limit , polymerase chain reaction , genotype , real time polymerase chain reaction , microbiology and biotechnology , computational biology , gene , genetics , dna sequencing , chromatography , dna , chemistry
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.