High-resolution Melting Curve Profiles Research Articles

Direct detection of Corynebacterium striatum, Corynebacterium propinquum, and Corynebacterium simulans in sputum samples by high-resolution melt curve analysis

BackgroundPulmonary infections caused by non-diphtheriae corynebacteria are increasing. However, rapid identification of Corynebacterium species poses a challenge due to the low genetic variation within the genus.MethodsThree reference strains and 99 clinical isolates were used in this study. A qPCR followed by high-resolution melting (HRM) targeting ssrA was performed to simultaneously identify C. striatum, C. propinquum and C. simulans. To further evaluate this assay’s performance, 88 clinical sputum samples were tested by HRM and the detection results were compared with those of the traditional culture method and multiple cross-displacement amplification (MCDA) assay.ResultsThe melting curve produced by a pair of universal primers generated species-specific HRM curve profiles and could distinguish the three target species from other related bacteria. The limit of detection of HRM assay for DNA from the three purified Corynebacterium species was 100 fg. Compared with the culture method, HRM detected 22 additional positive specimens, representing a 23.9% relative increase in detection rate. The HRM assay had 98.4% (95% confidence interval [CI], 90.5–99.9%) sensitivity and 100% (95% CI, 82.8–100%) specificity. Additionally, 95.5% concordance between HRM and MCDA (κ = 0.89 [95% CI, 0.79–0.99]) was noted.ConclusionsThe HRM assay was a simple, rapid, sensitive, and specific diagnostic tool for detecting C. striatum, C. propinquum, and C. simulans, with the potential to contribute to early diagnosis, epidemiological surveillance, and rapid response to outbreak.

HRM and 16S rRNA gene sequencing reveal the cultivable microbiota of the European sea bass during ice storage

The total cultivable microbiota of the ice-stored European sea bass (Dicentrarchus labrax), the most important commercial fish species of the Mediterranean aquaculture, was determined using 16S rRNA gene sequence analysis. High Resolution Melting (HRM) curve profiles and sequencing analysis (V3–V4 region of the 16S rRNA gene) were used respectively for the differentiation and identification of the collected isolates from six time intervals (day 0, 4, 8, 12, 14 and 16) while fish were stored in ice. HRM analysis differentiated the unknown microbiota in ten groups (208 isolates) and in two single isolates based on their HRM curve profiles. The isolates with HRM profiles which were >91% similar within each group were found to belong to the same species using sequencing analysis. Thus, the ten groups consist of representatives of Psychrobacter glacincola, Ps. alimentarius, Ps. cryohalolentis, Ps. maritimus, Ps. fozii, Pseudomonas sp., Paeniglutamicibacter sp., Carnobacterium sp., Leucobacter aridicolis and Bacillus thuringiensis. Based on this approach, Ps. cryohalolentis was found to be the most dominant phylotype at the beginning of fish shelf-life compared to other species. The abundance of this bacterium decreased throughout storage, while Ps. glacincola increased and dominated at the time of the sensory minimum acceptability (day 14) and rejection (day 16). To conclude, HRM could be used for the rapid determination of sea bass microbiota, using the representatives of each group as reference bacterial strains, in order for scientists to solve rapidly stakeholders problems related with microbial quality or safety of fish.

HRM analysis as a tool to facilitate identification of bacteria from mussels during storage at 4 °C

High-resolution melting (HRM) analysis followed by sequencing was applied for determination of bacteria grown on plates isolated from farmed mussels (Mytilus galloprovincialis) during their storage at 4 °C. The V3–V4 region of the 16S rRNA gene from the isolates was amplified using 16S universal primers. Melting curves (peaks) and high resolution melting curves (shape) of the amplicons and sequencing analysis were used for differentiation and identification of the isolated bacteria, respectively. The majority of the isolates (a sum of 101 colonies, from five time intervals: day 0, 2, 4, 6 and 8) from non-selective solid medium plates were classified in four bacterial groups based on the melting curves (peaks) and HRM curves (shape) of the amplicons, while three isolates presented distinct HRM curve profiles (single). Afterwards, sequencing analysis showed that the isolates with a) the same melting peak temperature and b) HRM curves that were >95% similar grouped into the same bacterial species. Therefore, based on this methodology, the cultivable microbial population of chill-stored mussels was initially dominated by Psychrobacter alimentarius against others, such as Psychrobacter pulmonis, Psychrobacter celer and Klebsiella pneumoniae. P. alimentarius was also the dominant microorganism at the time of the sensory rejection (day 8). Concluding, HRM analysis could be used as a useful tool for the rapid differentiation of the bacteria isolated from mussels during storage, at species level, and then identification is feasible by the sequencing of one only representative of each bacterial species, thus reducing the cost of required sequencing.

Identification of Phytophthora species by a high resolution melting analysis: an innovative tool for rapid differentiation

A new molecular method via the high resolution melting (HRM) analysis of the Ypt1 gene non-coding regions was validated for ten Phytophthora species with a broad host range from forest trees to crop species. The melting curve analysis of the amplicons specifically grouped all species into 10 respective unique and distinct HRM curve profiles. The analysis of the normalised HRM melting curves, assigning P. nicotianae as a normalised reference genotype, revealed that the genotype similarities among all the species were adequately low, indicating that Ypt1 marker was sufficient to identify and differentiate the tested species. This HRM method is rapid and reproducible allowing the identification of Phytophthora species and the screening of eventual variants eliminating the separate steps and reducing the risk of contamination.

Chemical and Molecular Characterization of Crude Oil Obtained by Olive-Pomace Recentrifugation

In oil-mills, olive-pomace recentrifugation is a common way to reduce pomace moisture and, at the same time, to recover the oil therein. According to current rules, the obtained oil is defined as “crude olive-pomace oil.” The aim of this work is to verify the effect of recentrifugation on specific chemical and molecular parameters of the crude olive-pomace oil, by comparing it with the corresponding virgin olive oil obtained from the same olive lots. In particular, the following were considered: (i) the polar compounds of the oils that include compounds originated from oxidative and hydrolytic degradation, analyzed by high-performance size exclusion chromatography (HPSEC), and (ii) the profile of DNA microsatellite molecular markers that was analyzed by using the High Resolution Melting (HRM) technique. The obtained results evidenced the significantly higher hydrolytic degradation of crude olive-pomace oil, compared with the corresponding virgin olive oil, but at an extent unlikely able to allow the detection of fraudulent admixtures with virgin olive oils. In addition, the findings demonstrated the feasibility of the application of the HRM analysis of DNA microsatellites to crude olive-pomace oil, able to reveal the alteration of the declared varietal profile of a virgin olive oil sample by simply checking the HRM curve profiles.

High-throughput genome editing and phenotyping facilitated by high resolution melting curve analysis.

With the goal to generate and characterize the phenotypes of null alleles in all genes within an organism and the recent advances in custom nucleases, genome editing limitations have moved from mutation generation to mutation detection. We previously demonstrated that High Resolution Melting (HRM) analysis is a rapid and efficient means of genotyping known zebrafish mutants. Here we establish optimized conditions for HRM based detection of novel mutant alleles. Using these conditions, we demonstrate that HRM is highly efficient at mutation detection across multiple genome editing platforms (ZFNs, TALENs, and CRISPRs); we observed nuclease generated HRM positive targeting in 1 of 6 (16%) open pool derived ZFNs, 14 of 23 (60%) TALENs, and 58 of 77 (75%) CRISPR nucleases. Successful targeting, based on HRM of G0 embryos correlates well with successful germline transmission (46 of 47 nucleases); yet, surprisingly mutations in the somatic tail DNA weakly correlate with mutations in the germline F1 progeny DNA. This suggests that analysis of G0 tail DNA is a good indicator of the efficiency of the nuclease, but not necessarily a good indicator of germline alleles that will be present in the F1s. However, we demonstrate that small amplicon HRM curve profiles of F1 progeny DNA can be used to differentiate between specific mutant alleles, facilitating rare allele identification and isolation; and that HRM is a powerful technique for screening possible off-target mutations that may be generated by the nucleases. Our data suggest that micro-homology based alternative NHEJ repair is primarily utilized in the generation of CRISPR mutant alleles and allows us to predict likelihood of generating a null allele. Lastly, we demonstrate that HRM can be used to quickly distinguish genotype-phenotype correlations within F1 embryos derived from G0 intercrosses. Together these data indicate that custom nucleases, in conjunction with the ease and speed of HRM, will facilitate future high-throughput mutation generation and analysis needed to establish mutants in all genes of an organism.

Identification of lactic acid bacteria isolated from poultry carcasses by high-resolution melting (HRM) analysis

Lactic acid bacteria (LAB) are a complex group of Gram-positive bacteria belonging to different genera with common morphological, metabolic and physiological characteristics. Their classification was initially based on biochemical methods, but nowadays molecular methods are usually applied for the identification of LAB. Herein, real-time PCR assay coupled with high-resolution melting (HRM) analysis was developed for identifying and distinguishing LAB isolates coming from poultry carcasses. The 16S rRNA gene from these isolates was amplified using primers that annealed to conserved regions. The melting curve analysis of the amplicons classified all isolates into ten LAB species and generated ten distinct HRM curve profiles. The results from HRM analysis were compared to those produced by API 50 CH biochemical microkits and ribosomal DNA sequencing, suggesting the superiority of HRM against API. In conclusion, HRM was proven to be a fast, reliable and cost-effective method for identification of LAB isolates. HRM analysis could be used in order to reduce the time needed for the identification assay and the cost of sequencing the entire group of LAB isolates. The melting profile of known LAB species could be used as a reference for the rapid identification of unknown LAB isolates without the need of sequencing.

High-resolution melting analysis allowed fast and accurate closed-tube genotyping of Fusarium oxysporum formae speciales complex

The fungus Fusarium oxysporum is a highly complex species composed by many strains put together into groups called formae speciales. As it is difficult and laborious to discriminate Fusarium formae specials via biochemical or phenotypic methods, it is very important to develop novel, rapid, and simple to perform identification methods. Herein, real-time PCR assay [using universal internal transcribed spacer (ITS) primers] coupled with high-resolution melting (HRM) analysis was developed for identifying and distinguishing F. oxysporum formae speciales complex. The melting curve analysis of these amplicons specifically classified all isolates into seven F. oxysporum formae speciales and generated seven HRM curve profiles. The smallest DNA sequence difference recognized in this study was one nucleotide. The results presented show that HRM curve analysis of Fusarium ITS sequences is a simple, quick, and reproducible method that allows both the identification of seven F. oxysporum formae speciales and at the same time their screening for variants. Our genotyping assay uses the combined information of simultaneously acquired HRM data from an unlabeled probe and the full-length amplicon. Finally, the completion of both reaction and analysis in a closed tube saves time by eliminating the separate steps and reduces the risk of contamination.

High-resolution melting analysis of the single nucleotide polymorphism hot-spot region in the rpoB gene as an indicator of reduced susceptibility to rifaximin in Clostridium difficile

Clostridium difficile, a Gram-positive, spore-forming, anaerobic bacterium, is the main causative agent of hospital-acquired diarrhoea worldwide. In addition to metronidazole and vancomycin, rifaximin, a rifamycin derivative, is a promising antibiotic for the treatment of recurring C. difficile infections (CDI). However, exposure of C. difficile to this antibiotic has led to the development of rifaximin-resistance due to point mutations in the β-subunit of the RNA polymerase (rpoB) gene. In the present study, 348 C. difficile strains with known PCR-ribotypes were investigated for respective single nucleotide polymorphisms (SNPs) within the proposed rpoB hot-spot region by using high-resolution melting (HRM) analysis. This method allows the detection of SNPs by comparing the altered melting behaviour of dsDNA with that of wild-type DNA. Discrimination between wild-type and mutant strains was enhanced by creating heteroduplexes by mixing sample DNA with wild-type DNA, leading to characteristic melting curve shapes from samples containing SNPs in the respective rpoB section. In the present study, we were able to identify 16 different rpoB sequence-types (ST) by sequencing analysis of a 325 bp fragment. The 16 PCR STs displayed a total of 24 different SNPs. Fifteen of these 24 SNPs were located within the proposed 151 bp SNP hot-spot region, resulting in 11 different HRM curve profiles (CP). Eleven SNPs (seven of which were within the proposed hot-spot region) led to amino acid substitutions associated with reduced susceptibility to rifaximin and 13 SNPs (eight of which were within the hot-spot region) were synonymous. This investigation clearly demonstrates that HRM analysis of the proposed SNP hot-spot region in the rpoB gene of C. difficile is a fast and cost-effective method for the identification of C. difficile samples with reduced susceptibility to rifaximin and even allows simultaneous SNP subtyping of the respective C. difficile isolates.

Differentiation of Mycoplasma gallisepticum strains using PCR and high-resolution melting curve analysis

Mycoplasma gallisepticum (MG) is an economically important pathogen of poultry worldwide, causing chronic respiratory disease in chickens and turkeys. Differentiation of MG strains is critical, especially in countries where poultry flocks are vaccinated with live vaccines. In this study, oligonucleotide primers were designed based on a region preceding the trinucleotide repeat of a member of the vlhA gene family, and amplicons of 145-352 bp were generated from cultures of 10 different MG strains, including the ts-11, F and 6/85 vaccine strains. High-resolution melting (HRM) curve analysis of the resultant amplicons could differentiate all MG strains. Analysis of the nucleotide sequences of the amplicons from each strain revealed that each melting curve profile related to a unique DNA sequence. The HRM curve profiles (for ts-11) remained consistent after at least five passages under laboratory conditions. PCR-HRM curve analysis of 33 DNA extracts derived from respiratory swabs, or mycoplasma cultures grown from respiratory swabs, of ts-11-vaccinated commercial or specific pathogen-free chickens identified all these specimens, according to their sequences, as ts-11. The potential of the PCR-HRM curve analysis was also shown in the genotyping of 30 additional MG isolates from Europe, the USA and Israel. The results presented in this study indicate that PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping of MG isolates/strains using both MG cultures and clinical swabs.