High resolution melting analysis for the differentiation of Mycobacterium species

Issa, Rahizan; Abdul, Hatijah; Hashim, Siti Hasmah; Seradja, Valentinus H.; Shaili, Nurul ‘Aishah; Hassan, Nurul Akma Mohd

doi:10.1099/jmm.0.072611-0

Cited by 15 publications

(13 citation statements)

References 10 publications

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“…Another limitation is that the test may detect silent or other mutations not actually associated with drug resistance. An advantage of this technique is that it can be implemented to detect fluoroquinolone, pyrazinamide and streptomycin resistance [ 24 – 26 ].…”

Section: Discussionmentioning

confidence: 99%

High-resolution melting analysis for molecular detection of multidrug resistance tuberculosis in Peruvian isolates

et al. 2016

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BackgroundThe emergence of multidrug-resistant strains is a major health problem especially for countries with high TB incidence such as Peru. In this study, we evaluated High Resolution Melting (HRM) assay in Peruvian isolates for the detection of mutations within rpoB, katG genes and promoter region inhA to determine isoniazid and rifampicin resistance in Mycobacterium tuberculosis (Mtb).MethodsDNA samples extracted from a total of 167 clinical isolates of Mtb, 89 drug-sensitive and 78 multidrug-resistant, were blindly analyzed by HRM analysis and verified by DNA sequencing.ResultsThe HRM analysis generated patterns that were specific to distinguish between sensitive and resistance isolates. The sensitivity and specificity of the HRM assays in comparison with drug susceptibility testing (DST) for detection of rifampicin resistance were 98.7 % and 97.5 %, and for isoniazid resistance were 98.7 % and 100 %.ConclusionThis study suggests that HRM Analysis could help with rapid diagnosis of MDR-TB cases in Peru.

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Section: Discussionmentioning

confidence: 99%

High-resolution melting analysis for molecular detection of multidrug resistance tuberculosis in Peruvian isolates

et al. 2016

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“…On the other hand, the weakness of the technique lies in the fact that monitoring the melting temperature of PCR products requires special equipment and software. However, it appears that equipment for performing qPCR with the possibility of HRM analysis has now become basic equipment for many labs, so the use of this method for standard bacterial genotyping is becoming increasingly available [37,38].…”

Section: High-resolution Melting Analysismentioning

confidence: 99%

Molecular Routes to Specific Identification of the Lactobacillus Casei Group at the Species, Subspecies and Strain Level

Jarocki

Komoń-Janczara

Glibowska

et al. 2020

IJMS

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The genus Lactobacillus includes, among others, Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus, species that are collectively referred to as the Lactobacillus casei group. Many studies have shown that strains belonging to this group may decrease lactose intolerance, the effects of inflammatory bowel disease, diarrhea, constipation, food allergies and even colon cancer. Moreover, evidences exists of positive effects of these bacteria on mucosal immunity and blood cholesterol level. Because of their beneficial influence on human health, many of them are used as food additives and probiotic pharmaceuticals. It should be stressed that health-promoting properties are not attributed at the species level, but to specific strains. Therefore, procedures are necessary to allow specific identification at each phylogenetic level—genus, species and strain. In this paper we present a practical overview of molecular methods for the identification and differentiation of L. casei bacteria. The research included 30 bacterial strains belonging to three species: L.casei, L. paracasei and L. rhamnosus. Among the tested procedures were genus- and species-specific PCR, multiplex-PCR, Real-Time HRM analysis, RFLP-PCR, rep-PCR, RAPD-PCR, AFLP-PCR, and proteomic methods such as MALDI-TOF MS typing and SDS-PAGE fingerprinting. The obtained results showed that multiplex-PCR and MALDI-TOF MS turned out to be the most useful methods to identify the tested bacteria at the species level. At the strain level, the AFLP-PCR method showed the highest discriminatory power. We hope that the presented results will allow for the easy selection of an appropriate procedure, depending on the experiment conducted and the equipment capabilities of any given laboratory.

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“…In compare to our results, they could differentiate fewer distinct groups, occasionally with lower sensitivity and specificity. In Malaysia, Issa et al (2014) , developed a qPCR-HRM analysis using 16S rRNA as target gene for the differentiation of the Mycobacterium species. However, they could not identify some of the species that are frequently present in clinical specimens such as M. simiae, M. fortuitum, M. abscessus , and M. kansasii by their applied method.…”

Section: Discussionmentioning

confidence: 99%

“…This assay is reported previously as a robust technique with high discriminatory power ( Dai et al, 2011 ). In contrast, all other similar studies employed 16S rRNA , ITS (Internal Transcribed spacer) or hsp65 genes as a target gene for PCR-HRM analysis ( Douarre et al, 2012 ; Perng et al, 2012 ; Phung et al, 2013 ; Thomson et al, 2013 ; Issa et al, 2014 ; Chen et al, 2017 ).…”

Section: Discussionmentioning

confidence: 99%

“…High-resolution melting curve (HRM) analysis is a homogeneous, closed-tube post-PCR method for identifying single nucleotide polymorphisms (SNPs), novel mutations, methylation patterns, and species identification. Recently, HRM assay has been used as a simple, low cost and rapid method in mycobacterial research works such as investigation of drug resistance among M. tuberculosis ( Pietzka et al, 2009 ; Ramirez et al, 2010 ), or mycobacterial species identification ( Perng et al, 2012 ; Issa et al, 2014 ; Chen et al, 2017 ). However, most of the latter analyses could only discriminate NTM into group or complex level.…”

Section: Introductionmentioning

confidence: 99%

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Differential Identification of Mycobacterial Species Using High-Resolution Melting Analysis

et al. 2017

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Infections caused by non-tuberculous mycobacteria (NTM) is increasing wordwide. Due to the difference in treatment of NTM infections and tuberculosis, rapid species identification of mycobacterial clinical isolates is necessary for the effective management of mycobacterial diseases treatment and their control strategy. In this study, a cost-effective technique, real-time PCR coupled with high-resolution melting (HRM) analysis, was developed for the differentiation of Mycobacterial species using a novel rpoBC sequence. A total of 107 mycobacterial isolates (nine references and 98 clinical isolates) were subjected to differentiation using rpoBC locus sequence in a real-time PCR-HRM assay scheme. From 98 Mycobacterium clinical isolates, 88 species (89.7%), were identified at the species level by rpoBC locus sequence analysis as a gold standard method. M. simiae was the most frequently encountered species (41 isolates), followed by M. fortuitum (20 isolates), M. tuberculosis (15 isolates), M. kansassi (10 isolates), M. abscessus group (5 isolates), M. avium (5 isolates), and M. chelonae and M. intracellulare one isolate each. The HRM analysis generated six unique specific groups representing M. tuberculosis complex, M. kansasii, M. simiae, M. fortuitum, M. abscessus–M. chelonae group, and M. avium complex. In conclusion, this study showed that the rpoBC-based real-time PCR followed by HRM analysis could differentiate the majority of mycobacterial species that are commonly encountered in clinical specimens.

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High resolution melting analysis for the differentiation of Mycobacterium species

Cited by 15 publications

References 10 publications

High-resolution melting analysis for molecular detection of multidrug resistance tuberculosis in Peruvian isolates

High-resolution melting analysis for molecular detection of multidrug resistance tuberculosis in Peruvian isolates

Molecular Routes to Specific Identification of the Lactobacillus Casei Group at the Species, Subspecies and Strain Level

Differential Identification of Mycobacterial Species Using High-Resolution Melting Analysis

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