High-resolution melt curve analysis: A real-time based multipurpose approach for diagnosis and epidemiological investigations of parasitic infections

ABSTRACT Drug-resistant tuberculosis (TB) is a major threat to TB control worldwide. Globally, only 40% of the 340,000 notified TB patients estimated to have multidrug-resistant-TB (MDR-TB) were detected in 2015. This study was carried out to evaluate the utility of high-resolution melt curve analysis (HRM) for the rapid and direct detection of MDR-TB in Mycobacterium tuberculosis in sputum samples. A reference plasmid library was first generated of the most frequently observed mutations in the resistance-determining regions of rpoB , katG , and an inhA promoter and used as positive controls in HRM. The assay was first validated in 25 MDR M. tuberculosis clinical isolates. The assay was evaluated on DNA isolated from 99 M. tuberculosis culture-positive sputum samples that included 84 smear-negative sputum samples, using DNA sequencing as gold standard. Mutants were discriminated from the wild type by comparing melting-curve patterns with those of control plasmids using HRM software. Rifampin (RIF) and isoniazid (INH) monoresistance were detected in 11 and 21 specimens, respectively, by HRM. Six samples were classified as MDR-TB by sequencing, one of which was missed by HRM. The HRM-RIF, INH- katG , and INH- inhA assays had 89% (95% confidence interval [CI], 52, 100%), 85% (95% CI, 62, 97%), and 100% (95% CI, 74, 100%) sensitivity, respectively, in smear-negative samples, while all assays had 100% sensitivity in smear-positive samples. All assays had 100% specificity. Concordance of 97% to 100% (κ value, 0.9 to 1) was noted between sequencing and HRM. Heteroresistance was observed in 5 of 99 samples by sequencing. In conclusion, the HRM assay was a cost-effective (Indian rupee [INR]400/US$6), rapid, and closed-tube method for the direct detection of MDR-TB in sputum, especially for direct smear-negative cases.

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Rapid and specific detection of Salmonella in water samples using real-time PCR and High Resolution Melt (HRM) curve analysis

Water Science & Technology ◽

10.2166/wst.2011.838 ◽

2011 ◽

Vol 64 (12) ◽

pp. 2453-2459 ◽

Cited By ~ 2

Author(s):

G. N. van Blerk ◽

L. Leibach ◽

A. Mabunda ◽

A. Chapman ◽

D. Louw

Keyword(s):

Surface Water ◽

High Resolution ◽

Real Time ◽

Water Samples ◽

Real Time Pcr ◽

Curve Analysis ◽

Pcr Assay ◽

High Resolution Melt ◽

Gene Target ◽

Enrichment Step

A real-time PCR assay combined with a pre-enrichment step for the specific and rapid detection of Salmonella in water samples is described. Following amplification of the invA gene target, High Resolution Melt (HRM) curve analysis was used to discriminate between products formed and to positively identify invA amplification. The real-time PCR assay was evaluated for specificity and sensitivity. The assay displayed 100% specificity for Salmonella and combined with a 16–18 h non-selective pre-enrichment step, the assay proved to be highly sensitive with a detection limit of 1.0 CFU/ml for surface water samples. The detection assay also demonstrated a high intra-run and inter-run repeatability with very little variation in invA amplicon melting temperature. When applied to water samples received routinely by the laboratory, the assay showed the presence of Salmonella in particularly surface water and treated effluent samples. Using the HRM based assay, the time required for Salmonella detection was drastically shortened to less than 24 h compared to several days when using standard culturing methods. This assay provides a useful tool for routine water quality monitoring as well as for quick screening during disease outbreaks.

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Evaluation of PCR and high-resolution melt curve analysis for differentiation of Salmonella isolates

Avian Pathology ◽

10.1080/03079457.2016.1268676 ◽

2017 ◽

Vol 46 (3) ◽

pp. 319-331 ◽

Cited By ~ 3

Author(s):

Mohammad Sadegh Saeidabadi ◽

Hassan Nili ◽

Habibollah Dadras ◽

Hassan Sharifiyazdi ◽

Joanne Connolly ◽

...

Keyword(s):

High Resolution ◽

Curve Analysis ◽

High Resolution Melt ◽

Melt Curve Analysis

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Evaluation of high-resolution melt curve analysis for rapid differentiation of Campylobacter hepaticus from other species in birds

PLoS ONE ◽

10.1371/journal.pone.0251328 ◽

2021 ◽

Vol 16 (5) ◽

pp. e0251328

Author(s):

Petrina Young ◽

Pol Tarce ◽

Sadhana Adhikary ◽

Joanne Connolly ◽

Tim Crawshaw ◽

...

Keyword(s):

High Resolution ◽

Dna Sequence ◽

Egg Production ◽

Curve Analysis ◽

Bacterial Disease ◽

Campylobacter Coli ◽

High Resolution Melt ◽

Melt Curve Analysis ◽

Clinical Specimens ◽

Bile Samples

Spotty liver disease (SLD) is a bacterial disease of chicken, causing mortalities and reduction in egg production, hence, contributing to economic loss in the poultry industry. The causative agent of SLD has only recently been identified as a novel Campylobacter species, Campylobacter hepaticus. Specific primers were designed from the hsp60 gene of Campylobacter hepaticus and PCR followed by high-resolution melt curve analysis was optimised to detect and differentiate three species of Campylobacter (Campylobacter coli, Campylobacter jejuni and Campylobacter hepaticus). The three Campylobacter species produced a distinct curve profile and was differentiated using HRM curve analysis. The potential of the PCR-HRM curve analysis was shown in the genotyping of 37 Campylobacter isolates from clinical specimens from poultry farms. PCR-HRM curve analysis of DNA extracts from bile samples or cultures from bile samples, were identified as Campylobacter hepaticus and confirmed by DNA sequencing. The DNA sequence analysis of selected samples from each of the three HRM distinctive curves patterns showed that each DNA sequence was associated with a unique melt profile. The potential of the PCR-HRM curve analysis in genotyping of Campylobacter species was also evaluated using faecal specimens from 100 wild birds. The results presented in this study indicate that PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping of Campylobacter species using either bacterial cultures or clinical specimens.

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