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>

Sanaz Dehbashi; Hamed Tahmasebi; Mohammad Yousef Alikhani; Fariba Keramat; Mohammad Reza Arabestani; Sanaz Dehbashi; Hamed Tahmasebi; Mohammad Yousef Alikhani; Fariba Keramat; Mohammad Reza Arabestani

doi:10.3934/microbiol.2022015

AIMS Microbiology

2022, Volume 8, Issue 2: 178-192. doi: 10.3934/microbiol.2022015

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Research article

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

1.
Department of Laboratory Sciences, Varastegan Institute of Medical Sciences, Mashhad, Iran
2.
School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran
3.
Microbiology Department, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran
4.
Brucellosis Research Center, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran

Received: 02 March 2022 Revised: 22 April 2022 Accepted: 04 May 2022 Published: 09 May 2022

New Delhi metallo-β-lactamase-1 (NDM-1) producing Pseudomonas aeruginosa 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 P. aeruginosa. For optimization and development of the HRMA method, a reference strain of P. aeruginosa 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 bla_VIM, bla_SPM and bla_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 P. aeruginosa by software analysis and melting curve analysis.
- Pseudomonas aeruginosa,
- high-resolution melting curve analysis (HRMA),
- New Delhi metallo-β-lactamase (NDM)
Citation: Sanaz Dehbashi, Hamed Tahmasebi, Mohammad Yousef Alikhani, Fariba Keramat, Mohammad Reza Arabestani. Optimization and development of high-resolution melting curve analysis (HRMA) assay for detection of New Delhi metallo-β-lactamase (NDM) producing Pseudomonas aeruginosa[J]. AIMS Microbiology, 2022, 8(2): 178-192. doi: 10.3934/microbiol.2022015

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Abstract

New Delhi metallo-β-lactamase-1 (NDM-1) producing Pseudomonas aeruginosa 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 P. aeruginosa. For optimization and development of the HRMA method, a reference strain of P. aeruginosa 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 bla_VIM, bla_SPM and bla_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 P. aeruginosa by software analysis and melting curve analysis.

Acknowledgments

The authors would like to acknowledge the Vice Chancellor of Hamadan University of Medical Sciences for the funding and support of the study. The manuscript has been presented as a preprint at the following link: https://www.researchsquare.com/article/rs-7632/v1.

Funding information

This study has been adapted from a research fund at Hamadan University of Medical Sciences (Project No: 9808145924).

Conflict of interest

The authors declare that they have no conflict of interest.

Author contributions

MRA and HT proposed, designed and carried out the study. HT and SD analyzed the generated data, drafted the manuscript and performed the data analysis. MRA provided some of the strains, and HT participated in proofreading of the manuscript and critical revision. MYA and FK on editing the article. All authors read and approved the final manuscript.

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