Development of high-resolution melting curve analysis in rapid detection of vanA gene, Enterococcus faecalis, and Enterococcus faecium from clinical isolates

Dehbashi, Sanaz; Tahmasebi, Hamed; Sedighi, Parinaz; Davarian, Faeze; Arabestani, Mohammad Reza

doi:10.1186/s41182-020-00197-9

Cited by 6 publications

(5 citation statements)

References 24 publications

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“…When comparing the results obtained on the MR platform with the conventional culture-based methodologies used for the detection of bacterial pathogens, our platform offers rapidity and sensitivity, not requiring lengthy bacterial culturing enrichment steps (Rajapaksha et al, 2018). Also, contrarily to molecular techniques that need laboratory equipment and specialized personal (Dehbashi et al, 2020;Galia et al, 2019;Parcell & Orange, 2013;Waar et al, 2005), the MR platform can be used at CUNHA ET AL.…”

Section: Discussionmentioning

confidence: 99%

“…When comparing the results obtained on the MR platform with the conventional culture‐based methodologies used for the detection of bacterial pathogens, our platform offers rapidity and sensitivity, not requiring lengthy bacterial culturing enrichment steps (Rajapaksha et al, 2018). Also, contrarily to molecular techniques that need laboratory equipment and specialized personal (Dehbashi et al, 2020; Galia et al, 2019; Parcell & Orange, 2013; Waar et al, 2005), the MR platform can be used at PoC due to its portability, automation, and ability to provide real‐time measurements. In relation to other biosensing platforms (Hu & Bohn, 2017; Menon et al, 2020), the MR sensors do not present signal interference and background noise for biological samples (Freitas et al, 2012; Gaster et al, 2009).…”

Section: Discussionmentioning

confidence: 99%

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Rapid and multiplex detection of nosocomial pathogens on a phage‐based magnetoresistive lab‐on‐chip platform

Cunha

Henriques

Cardoso

et al. 2021

Biotech & Bioengineering

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Nosocomial or hospital-acquired infections (HAIs) have a major impact on mortality worldwide. Enterococcus and Staphylococcus are among the leading causes of HAIs and thus are important pathogens to control mainly due to their increased antibiotic resistance. The gold-standard diagnostic methods for HAIs are time-consuming, which hinders timely and adequate treatment. Therefore, the development of fast and accurate diagnostic tools is an urgent demand. In this study, we combined the sensitivity of magnetoresistive (MR) sensors, the portability of a lab-on-chip platform, and the specificity of phage receptor binding proteins (RBPs) as probes for the rapid and multiplex detection of Enterococcus and Staphylococcus. For this, bacterial cells were firstly labelled with magnetic nanoparticles (MNPs) functionalized with RBPs and then measured on the MR sensors. The results indicate that the RBP-MNPS provided a specific individual and simultaneous capture of more than 70% of Enterococcus and Staphylococcus cells. Moreover, high signals from the MR sensors were obtained for these samples, providing the detection of both pathogens at low concentrations (10 CFU/ml) in less than 2 h. Overall, the lab-on-chip MR platform herein presented holds great potential to be used as a point-of-care for the rapid, sensitive and specific multiplex diagnosis of bacterial infections.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Rapid and multiplex detection of nosocomial pathogens on a phage‐based magnetoresistive lab‐on‐chip platform

Cunha

Henriques

Cardoso

et al. 2021

Biotech & Bioengineering

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“…The mecA and mecC were both included in UTI-HMGS for the surveillance of MRSA, which is a leading cause of deadly hospital-acquired infections (Lamontagne Boulet et al, 2018). For the resistance genes related to VRE, the most widely distributed in clinical strains is vanA in a lot of studies, especially in China (Dehbashi et al, 2020). Apart from the most common and important pathogens or resistance genes above, the number of remaining uncommon species and resistance genes were too large that it is not feasible to cover all of them in UTI-HMGS.…”

Section: Discussionmentioning

confidence: 99%

The Direct Semi-Quantitative Detection of 18 Pathogens and Simultaneous Screening for Nine Resistance Genes in Clinical Urine Samples by a High-Throughput Multiplex Genetic Detection System

Sun

Liu

Zhang

et al. 2021

Front. Cell. Infect. Microbiol.

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BackgroundUrinary tract infections (UTIs) are one the most common infections. The rapid and accurate identification of uropathogens, and the determination of antimicrobial susceptibility, are essential aspects of the management of UTIs. However, existing detection methods are associated with certain limitations. In this study, a new urinary tract infection high-throughput multiplex genetic detection system (UTI-HMGS) was developed for the semi-quantitative detection of 18 pathogens and the simultaneously screening of nine resistance genes directly from the clinical urine sample within 4 hours.MethodsWe designed and optimized a multiplex polymerase chain reaction (PCR) involving fluorescent dye-labeled specific primers to detect 18 pathogens and nine resistance genes. The specificity of the UTI-HMGS was tested using standard strains or plasmids for each gene target. The sensitivity of the UTI-HMGS assay was tested by the detection of serial tenfold dilutions of plasmids or simulated positive urine samples. We also collected clinical urine samples and used these to perform urine culture and antimicrobial susceptibility testing (AST). Finally, all urine samples were detected by UTI-HMGS and the results were compared with both urine culture and Sanger sequencing.ResultsUTI-HMGS showed high levels of sensitivity and specificity for the detection of uropathogens when compared with culture and sequencing. In addition, ten species of bacteria and three species of fungi were detected semi-quantitatively to allow accurate discrimination of significant bacteriuria and candiduria. The sensitivity of the UTI-HMGS for the all the target genes could reach 50 copies per reaction. In total, 531 urine samples were collected and analyzed by UTI-HMGS, which exhibited high levels of sensitivity and specificity for the detection of uropathogens and resistance genes when compared with Sanger sequencing. The results from UTI-HMGS showed that the detection rates of 15 pathogens were significantly higher (P<0.05) than that of the culture method. In addition, there were 41(7.72%, 41/531) urine samples were positive for difficult-to-culture pathogens, which were missed detected by routine culture method.ConclusionsUTI-HMGS proved to be an efficient method for the direct semi-quantitative detection of 18 uropathogens and the simultaneously screening of nine antibiotic resistance genes in urine samples. The UTI-HMGS could represent an alternative method for the clinical detection and monitoring of antibiotic resistance.

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“…However, HRMA takes it one step further in its ability to capture much more detail. It has an increased resolving power, as melting curves from different amplicons may be differentiated based on the melt curve's shape even when the melt temperature values are the same [14] , [15] .…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2022

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<abstract> New Delhi metallo-β-lactamase-1 (NDM-1) producing <italic>Pseudomonas aeruginosa</italic> 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 <italic>P. aeruginosa</italic>. For optimization and development of the HRMA method, a reference strain of <italic>P. aeruginosa</italic> 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. 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 <italic>bla</italic>VIM, <italic>bla</italic>SPM and <italic>bla</italic>SIM 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 <italic>P. aeruginosa</italic> by software analysis and melting curve analysis. </abstract>

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Development of high-resolution melting curve analysis in rapid detection of vanA gene, Enterococcus faecalis, and Enterococcus faecium from clinical isolates

Cited by 6 publications

References 24 publications

Rapid and multiplex detection of nosocomial pathogens on a phage‐based magnetoresistive lab‐on‐chip platform

Rapid and multiplex detection of nosocomial pathogens on a phage‐based magnetoresistive lab‐on‐chip platform

The Direct Semi-Quantitative Detection of 18 Pathogens and Simultaneous Screening for Nine Resistance Genes in Clinical Urine Samples by a High-Throughput Multiplex Genetic Detection System

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>

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