Multiplex PCR amplification assay for the detection of ^blaSHV, ^blaTEM and ^blaCTX‐M genes in Enterobacteriaceae

Abstract

Extended-spectrum beta-lactamases (ESBLs) are often mediated by (bla-)SHV, (bla)TEM and (bla)CTX-M genes in Enterobacteriaceae and other Gram-negative bacteria. Numerous molecular typing methods, including PCR-based assays, have been developed for their identification. To reduce the number of PCR amplifications needed we have developed a multiplex PCR assay which detects and discriminates between (bla-)SHV, (bla)TEM and (bla)CTX-M PCR amplicons of 747, 445 and 593 bp, respectively. This multiplex PCR assay allowed the identification of (bla-)SHV, (bla)TEM and (bla)CTX-M genes in a series of clinical isolates of Enterobacteriaceae with previously characterised ESBL phenotype. The presence of (bla)SHV, (bla)TEM and (bla)CTX-M genes was confirmed by partial DNA sequence analysis. Apparently, the universal well-established CTX-M primer pair used here to reveal plasmid-encoded (bla)CTX-M genes would also amplify the chromosomally located K-1 enzyme gene in all Klebsiella oxytoca strains included in the study.

Nosocomial infection and characterization of extended-spectrum β-lactamases-producing Enterobacteriaceae in Northeast Brazil

“…The frequency of the bla CTX-M gene was 90.4% (104/115). Other researchers also reported high prevalence rates of 92% 33 , 82% 14 , 72% 34 and 70% 10 . The frequency of the bla TEM was 75% (86/115), which resembles the results of the studies conducted in Sweden and Brazil 10,33 .…”

“…Detection of the β-lactamase genes was carried out with the following components in a 25µl reaction volume: 1µl of DNA, 10pmol of the specific primers, 0.3µl of Taq DNA polymerase (0.5U/µl) (Invitrogen, Brazil), 1µl of MgCl 2 (50mM) (Invitrogen, Brazil), 2.5µl of buffer (10x) (Invitrogen, Brazil), 2.5µl of dNTPs (2mM) (Invitrogen, Brazil) and Milli-Q water. PCR amplification conditions were as follows: initial denaturation step at 95°C for 15min, 30 cycles of denaturation at 94°C for 30s, annealing at 60°C for 30s, extension at 72°C for 2min, followed by a final extension step at 72°C for 10min 10 .…”

Development of a multiplex PCR system and its application in detection of blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaOXA-1 group genes in clinical Klebsiella pneumoniae and Escherichia coli strains

“…In 2007, another M-PCR system was described that could detect the bla TEM , bla SHV and bla CTX-M genotypes using universal PCR primers for the bla CTX-M group. 19 That system requires extra singleplex assays to identify the five groups of the bla CTX-M genotype. Three multiplex assays were later described by Woodford in 2010 30 for the detection of the five CTX-M groups, metallo-β-lactamases and OXA-type carbapenemases.…”

“…For instance, two assays were previously constructed including one that detects the five phylogenetic groups of the bla CTX-M genotype, 18 and another that targets the bla TEM , bla SHV and bla CTX-M genotypes. 19 Recently, a group of workers developed six M-PCR assays that can detect most of the bla genes in different combinations. 17 Others have constructed M-PCR assays for detecting carbapenemase 20 and AmpC 21 genes.…”

Molecular identification of blaSHV, blaLEN and blaOKP β-lactamase genes in Klebsiella pneumoniae by bi-directional sequencing of universal SP6- and T7-sequence-tagged blaSHV-PCR amplicons

“…Numerous molecular typing methods have been developed for identification of the growing number of bla SHV-genes [4,[14][15][16][17][18], including specific PCR assays of SHV, LEN, and OKP genes [9,19]. However, identification of a SHV genotype at the nucleotide level often requires cloning of the amplicons, followed by sequencing.…”