Rapid in vitro detection of CTX-M groups 1, 2, 8, 9 resistance genes by LAMP assays

Abstract

BackgroundThe prevalence of bacteria producing CTX-M Extended-Spectrum β-lactamases (ESBLs) has increased around the world and some of them became a major cause of infections such as bloodstream or urinary tract infections (UTI). We developed a loop-mediated isothermal amplification (LAMP) assay for a simple, rapid and sensitive detection of the four most common CTX-M groups, namely CTX-M groups 1, 2, 8 and 9.MethodsLAMP primers targeting the four ESBLs CTX-M groups were designed using the Primer Explorer V4 software. The detection limit of the method was tested by serial dilution of reference DNAs. The primer specificity of the LAMP reaction was tested on DNA extracted from six strains producing various group of CTX-M and validated using DNA extracted from CTX-M-resistant clinical isolates (isolated from pus, urine, or blood). Results were compared with those of conventional PCR.ResultsWe were able to detect down to 0.1 pg/ul of DNA using the newly developed LAMP assays whereas the minimal amount detectable for conventional PCR was 50 to 100pg/ul, indicating that the LAMP assay was found to have a detection limit at least 500 to 1000 times lower than the PCR. Additionally, representative genes from the CTX-M groups 1, 2, 8 and 9 were amplified using the designed assay and no cross amplification was observed between the four CTX-M groups, demonstrating the specificity of the LAMP assay. Of the 37 clinical strains tested, the four LAMP assays showed 100% sensitivity and 87%, 97%, 100%, 100% specificity for the CTX-M groups 1, 2, 8 and 9 respectively.ConclusionBeing sensitive, specific, rapid and standard methods, the LAMP assays developed in this study have a potential to be beneficial tools in molecular epidemiology and surveillance studies of the four prevalent EBSLs CTX-M groups even in low cost laboratory.

Multiple-Antibiotic Resistance and Presence of CTX-M Genes among Enterobacteriaceae Isolates from Different Sources in Iwo, Osun State, Nigeria

“…The distribution of the isolated enterobacteria genera at the different sampling site is represented in Table 2. Serratia sp had the highest occurrence (13) followed by Enterobacter sp (11), Klebsiella sp (6). E. coli (5) and Proteus sp (5) had the same number of isolates followed by Salmonella sp 3 1 shows the presence of CTX-M genes at 415bp on four isolates (isolates 3, 6, 14 and 16).…”

“…PCR assays were performed to determine the presence of β-lactamase resistance genes in the MDR Enterobacteriaceae isolates according to [11]. Amplification of the DNA was performed in a PCR apparatus with Taq kit.…”

Development and evaluation of loop-mediated isothermal amplification for detection of Yersinia pestis in plague biological samples

“…DNA extracts from biological samples were obtained using a method previously described by Rivoarilala et al [ 23 ] with slight modification. Samples were placed into a boiling water bath for 10 min and centrifuged at 12,000 rpm for 5 min.…”

Behavior of Antibiotic-Resistant Fecal Coliforms in the Stream of a Sewage Treatment Plant in Tokyo

“…First, the ESBL production test was performed for the multi-ARB strains by conducting double disc synergy test (DDST), using a Sensi Disc of AMPC/CVA along with CTX, CAZ, and ATM (Japan Becton-Dickinson). Second, in the ESBL-producing bacteria-like strains, ESBL-encoding genes were amplified by PCR using specific primers for the CTX-M-1 group, CTX-M-2 group, CTX-M-9 group, TEM group, SHV group, and ampC [16] [17] [18] under the conditions shown in Table 1. The sequencing was carried out by Eurofins Genomics.…”