Optimization and development of high-resolution melting curve analysis (HRMA) assay for detection of New Delhi metallo-β-lactamase (NDM) producing <i>Pseudomonas aeruginosa</i>

Abstract

&lt;abstract&gt; &lt;p&gt;New Delhi metallo-β-lactamase-1 (NDM-1) producing &lt;italic&gt;Pseudomonas aeruginosa&lt;/italic&gt; strain detection plays a vital role in confirming bacterial disease diagnosis and following the source of an outbreak for public health. However, the standard method for NDM-1 determination, which relies on the features of the colony of the bacteria cultured from the patient's specimen, is time-consuming and lacks accuracy and sensitivity. This study aimed to standardize a high-resolution melting curve analysis (HRMA) assay to detect NDM producing &lt;italic&gt;P. aeruginosa&lt;/italic&gt;. For optimization and development of the HRMA method, a reference strain of &lt;italic&gt;P. aeruginosa&lt;/italic&gt; was used. For evaluating the broad range PCR data, ABI Step One-Plus Manager Software version 3.2 and Precision Melt Analysis Software 3.02 (Applied Biosystems) were used.&lt;/p&gt; &lt;p&gt;Based on the results, expected results were obtained for all tested strains, with high analytical sensitivity and specificity. Temperature melting analyses of the HRMA time PCR assays showed the Tm at 89.57 °C, 76.92 °C and 82.97 °C for N-1, N-2 and N-3 genes, respectively. Also, melting point temperatures of the &lt;italic&gt;bla&lt;/italic&gt;&lt;sub&gt;VIM&lt;/sub&gt;, &lt;italic&gt;bla&lt;/italic&gt;&lt;sub&gt;SPM&lt;/sub&gt; and &lt;italic&gt;bla&lt;/italic&gt;&lt;sub&gt;SIM&lt;/sub&gt; amplicons for isolates identified as MBL strains were 84.56 °C, 85.35 °C and 86.62 °C, respectively. The amplification results using negative control genomes as templates were negative, showing the specificity of the designed assays. Our study's data indicated that the sensitivity and specificity of the HRMA method are linked to the primer length and the fluorescent dye. We can further identify antibiotic resistance in NDMproducing &lt;italic&gt;P. aeruginosa&lt;/italic&gt; by software analysis and melting curve analysis.&lt;/p&gt; &lt;/abstract&gt;

Simple Sequence Repeat Fingerprint Identification of Essential-Oil-Bearing Rosa rugosa via High-Resolution Melting (HRM) Analysis

“…DNA template, primers and annealing temperature were important for accurate HRM curve profiling [26,27]. Most studies recommended higher annealing temperature for melting curve profiling [26][27][28]. Our optimization of SSR-HRM indicated that a low annealing temperature (56 • C, Groups 1, 4, 7) resulted in nonspecific amplification which generated incorrect SSR genotyping.…”