A High Resolution DNA Melting Curve Analysis for the Rapid and Efficient Molecular Diagnostics of Extended Spectrum β-Lactamase Determinants from Foodborne Escherichia coli

Abstract

The accurate identification of Extended-Spectrum β-Lactamase (ESBL) genes in Gram-negative bacteria is necessary for surveillance and epidemiological studies of transmission through foods. We report a novel rapid, cheap, and accurate closed tube molecular diagnostic tool based on two multiplex HRM protocols for analysis of the predominant ESBL families encountered in foods. The first multiplex PCR assay targeted blaCTX-M including phylogenetic groups 1 (CTX-M-1-15, including CTX-M-1, CTX-M-3 and CTX-M-15), 2 (CTX-M-2), and 9 (CTX-M-9-14, including CTX-M-9 and CTX-M-14). The second assay involved blaTEM /bla CTX-M /blaSHV, including TEM variants (TEM-1 and TEM-2), SHV-1-56 (SHV-1, SHV-2 and SHV-56), and CTX-M-8-41 (CTX-M-8, CTX-M-25, CTX-M-26 and CTX-M-39 to CTX-M-41). The individual melting curves were differentiated by a temperature shift according to the type of ESBL gene. The specificity and sensitivity of the first assay were 100% and 98%, respectively. For the second assay, the specificity and sensitivity were 87% and 89%, respectively. The detection of ESBL variants or mutations in existing genes was also demonstrated by the subtyping of a variant of the CTXM-1-15. The HRM is a potential tool for the rapid detection of present β-lactamase genes and their characterization in a highly sensitive, closed-tube, inexpensive method that is applicable in high throughput studies.

High-resolution melting analysis identifies reservoir hosts of zoonotic Leishmania parasites in Tunisia

“…In the last few years, HRM PCR has become more widely applied for epidemiological purposes related to infectious diseases in addition to genotyping yeasts, viruses, or bacteria [23][24][25][26]. Indeed, this technique allows distinction of the different viral or bacterial strains [23][24][25], detection of mutated and wild-type strains [13], mutations located on resistance genes, such as resistance to rifampicin in Mycobacterium tuberculosis [27], or differentiation between Brucella species [28].…”

<p>Distribution of Class B and Class A β-Lactamases in Clinical Strains of <em>Pseudomonas aeruginosa</em>: Comparison of Phenotypic Methods and High-Resolution Melting Analysis (HRMA) Assay</p>

“…For the identification of MBL-producing strains, the susceptibility and specificity of the HRMA method were 99.2% and 99.7%, respectively, and were 100% reported for the detection of carbapenemase-producing. Studies in UK, 19 Denmark, 34 and USA 35 have shown that the sensitivity and specificity of HRMA to detect class A and class B β-lactamase are more than all phenotypic methods. However, in these studies, some phenotypic methods were used to detect strains producing ESBL and bla KPC with false positives and false negatives.…”

Portable Differential Detection of CTX-M ESBL Gene Variants, bla CTX-M-1 and bla CTX-M-15 , from Escherichia coli Isolates and Animal Fecal Samples Using Loop-Primer Endonuclease Cleavage Loop-Mediated Isothermal Amplification

“…Typically, CTX-M-1 and CTX-M-15 phenotyping and genotyping is performed by antibiotic susceptibility testing followed by CTX-M group specific PCR and DNA sequencing ( 29 – 32 ), a considerably lengthier and more expensive process than the LEC-LAMP assay approach. Various studies have reported the use of PCR followed by melt analysis for CTX-M-1 and CTX-M-15 differentiation ( 33 – 35 ), however, these methods require postamplification analysis producing ambiguous results that require discrimination of near identical melt curves often only differing by less than 0.5°C. Further highlighting the utility of this LEC-LAMP assay, Leflon-Guibout and colleagues previously reported a CTX-M-15 specific PCR assay that cannot effectively differentiate CTX-M-15 from CTX-M-1 based on available related nucleotide sequence information ( 36 ).…”