High Resolution Melting Curve Assay Research Articles

High-resolution melting (HRM) curve analysis as a potential tool for the identification of earthworm species and haplotypes.

BackgroundEarthworm communities are an important component of soil biodiversity and contribute to a number of ecosystem functions such as soil-nutrient cycling. Taxonomic identification is an essential requirement to assess earthworm biodiversity and functionality. Although morphological identification of species is labour-intensive, it is the most commonly used method due to a lack of cost-efficient alternatives. Molecular approaches to identify earthworms at species and haplotype level such as DNA barcoding are gaining popularity in science but are rarely applied in practice. In contrast to barcoding, the differentiation of PCR products based on their thermal denaturation properties using high-resolution melting (HRM) curve analysis is a fast and cost-efficient molecular closed-tube, post-PCR tool that allows identification of taxa.MethodsWe developed a HRM curve assay to identify eight earthworm species common to agricultural soils in Central Europe (Allolobophora chlorotica, Aporrectodea caliginosa, Apo. limicola, Apo. longa, Apo. rosea, Lumbricus castaneus, L. rubellus, and L. terrestris). For this, a new primer pair targeting a 158-bp long subregion of the cytochrome c oxidase I (COI) gene was designed. Our HRM assay was further tested for the differentiation of COI haplotypes using 28 individuals of the earthworm species Allo. chlorotica. Furthermore, we developed a novel extraction method for DNA from earthworm tissue that is fast and requires minimal consumables and laboratory equipment.ResultsThe developed HRM curve assay allowed identifying all eight earthworm species. Performing the assay on 28 individuals of the earthworm species Allo. chlorotica enabled the distinction among different COI haplotypes. Furthermore, we successfully developed a rapid, robust, scalable, and inexpensive method for the extraction of earthworm DNA from fresh or frozen tissue.ConclusionsHRM curve analysis of COI genes has the potential to identify earthworm species and haplotypes and could complement morphological identification, especially for juvenile or damaged individuals. Our rapid and inexpensive DNA extraction method from earthworm tissue helps to reduce the costs of molecular analyses and thereby promote their application in practice.

Genotyping of the Galactosemia GALT 591 A>G Mutation by Pyrosequencing

Abstract Introduction/Objective This project aimed to design a pyrosequencing assay capable of genetically analyzing the presence of GALT 591 A>G mutation for the diagnosis of galactosemia. Galactosemia is an autosomal recessive disorder that affects enzyme activity of galactose-1-phosphate uridylytransferase (GALT). This enzyme is responsible for proper processing of galactose to glucose; if not broken down the accumulation of galactose in the body results in developmental delays, clouding of the eyes, speech difficulties, and intellectual disabilities. Pyrosequencing offers a valuable platform in assessing whether patients are wild type unaffected (A:A), heterozygous carrier (A:G), or homozygous affected (G:G) at the alleles within the GALT 591 locus by measuring percentage of the wild type A allele and the mutant G allele. Methods PCR primers were designed for the mutation locus, and PCR amplification was optimized for the target 77 bp product, with verification by gel electrophoresis. The pyrosequencing assay was designed and validated on the Qiagen PyroMark Q24 instrument. Control samples included commercially purchased DNA from the Coriell Cell Repository. Results One hundred and thirty-three reactions were utilized to establish the following assay validation parameters: accuracy (100% for G:G, 95% for A:G and A:A), precision (determined by mean and standard deviation with a standard deviation of 0.6 for G:G, 4.55 for A:G and 4.85 for A:A), and limit of detection (10 ng DNA pre-PCR and 2 µL of PCR product loaded). Twenty-six blinded samples were utilized to test assay clinical performance, as compared against a high resolution melt curve assay. Conclusion It was determined that detection of GALT 591 A>G via pyrosequencing is highly sensitive and specific with a clinical sensitivity of 100% and a clinical specificity of 95.83%. It was concluded that this assay could be applied in a clinical environment for GALT 591 A>G genotyping to aid in galactosemia diagnosis.

High-resolution melting curve analysis: a novel method for identification of Mycoplasma species isolated from clinical cases of bovine and porcine respiratory disease.

Mycoplasma species cause wide ranges of infectious diseases in human and animals. The aim of the present study was to evaluate a real-time polymerase chain reaction (RT-PCR) followed by a high-resolution melting curve assay (HRM) for rapid differentiation of Mycoplasma species isolated from clinical cases of bovine and porcine respiratory disease. Lung samples from suspected cases to respiratory infections from cows and pigs were cultured on specific media, and the extracted DNA were tested by conventional polymerase chain reaction (PCR) assays for Mycoplasma. A set of universal primers specific for the 16S ribosomal RNA gene was designed and used for RT-PCR and HRM. The HRM analysis was able to differentiate between five different species of Mycoplasmas, namely, M. hyopneumoniae, M. bovis, M. hyorhinis, M. hyosynoviae and other uncultured Mycoplasma. All results were confirmed based on 16S rRNA gene sequencing. This rapid and reliable assay was as a simple alternative to PCR and sequencing, differentiating bovine and porcine mycoplasmas in species level.

The Clinical Utility of Analysis High Resolution Melting Curve Assay for Simultaneous Identification of Methicillin and Mupirocin Resistant in Coagulase-Negative Staphylococci.

Resistance to methicillin and mupirocin in coagulase-negative staphylococci (CoNS) is considered as one of the most important issues. Meanwhile, use of phenotypic methods in detecting the resistance to methicillin and mupirocin has a lower accuracy and speed compared to other molecular methods, such as High-Resolution Melting Analysis (HRM). In this study, HRM technique was used to identify CoNS resistance stains. This experimental study was done on 86 isolates of CoNS strains isolated at Hamadan Hospital. Pheno-typic tests were used to identify Staphylococcus saprophyticus and S. epidermidis. Methicillin and Mupirocin resistant strains were tested using the MIC and HRM methods, and sensitivity and specificity of the primers were determined based on melting curve temperature range. In addition, data was analyzed by Applied Biosystems StepOne v 2.3 and Applied Biosystems HRM v 3.0.1 software. Eighty-six (86) coagulase-negative isolates were isolated from different clinical specimens. Among these, 69 isolates of S. epidermidis and 17 isolates of S. saprophyticus were identified. Of the 69 S. epidermidis isolates, 19 isolates with oxacillin MIC ≥ 0.5 µg/mL and methicillin-resistant, and 11 isolates with mupirocin MIC ≥ 32 µg/mL and resistant to mupirocin. Of the 17 S. saprophyticus isolates, three isolates with oxacillin MIC ≥ 0.5 µg/mL were also methicillin-resistant, and one isolate with mupirocin MIC ≥ 32 µg/mL was resistant to mupirocin. Melting curve analysis for mecA and mupA primers was determined 81.7 ± 0.5°C and 74 ± 0.5°C, respectively. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of HRM assay for detection of methicillin and mupirocin strains testing were 100%. Identification of methicillin and mupirocin resistance in CoNSs using the HRM method has high sensitivity and specificity. Molecular methods are more accurate and faster than phenotypic methods and can identify a large number of resistant isolates in a short time.

Using a High Resolution Melt Curve Assay to Examine the Single Nucleotide Polymorphisms of the HFE Gene

Pharmacogenetics is the study of how inherited genetic variations correspond to an individual's specific reaction to medication. Pharmacogenetic testing determines an individual's genotype. This is beneficial in the medical field because it informs doctors and patients about drug efficacy, drug dosage recommendations, and the risk of adverse side effects for their genotype. The purpose of this project is to determine how to use high resolution melting analysis to characterize the different alleles caused by single nucleotide polymorphisms (SNPs). The SNP being tested is in the HFE gene which codes for an iron‐sensing protein which also regulates iron uptake. This SNP has been shown to predict the effectiveness of treatment with Humira® (adalimumab) in patients with Crohn's disease. We are presently developing a protocol for high resolution melting analysis of this SNP using the Roche LightCycler 480. The high resolution melting analysis should be specific enough to differentiate between the different SNPs. We will verify our SNPs results with DNA sequencing. The results of this study will be presented.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A novel HRM assay for differentiating classical strains and highly pathogenic strains of type 2 porcine reproductive and respiratory syndrome virus

Differentiation of classical strains and highly pathogenic strains of porcine reproductive and respiratory syndrome virus is crucial for effective vaccination programs and epidemiological studies. We used nested PCR and high resolution melting curve analysis with unlabeled probe to distinguish between the classical and the highly pathogenic strains of this virus. Two sets of primers and a 20 bp unlabeled probe were designed from the NSP3 gene. The unlabeled probe included two mutations specific for the classical and highly pathogenic strains of the virus. An additional primer set from the NSP2 gene of the highly pathogenic vaccine strain JXA1-R was used to detect its exclusive single nucleotide polymorphism. We tested 107 clinical samples, 21 clinical samples were positive for PRRSV (consistent with conventional PCR assay), among them four were positive for the classical strain with the remainder 17 for the highly pathogenic strain. Around 10 °C difference between probe melting temperatures showed the high discriminatory power of this method. Among highly pathogenic positive samples, three samples were determined as positive for JXA1-R vaccine-related strain with a 95% genotype confidence percentage. All these genotyping results using the high resolution melting curve assay were confirmed with DNA sequencing. This unlabeled probe method provides an alternative means to differentiate the classical strains from the highly pathogenic porcine reproductive and respiratory syndrome virus strains rapidly and accurately.

Use of high-resolution melting curve analysis to differentiate vaccine and wild type strains of grass carp reovirus genotype II

Grass carp reovirus (GCRV) is the causative agent of a hemorrhagic disease that causes severe economic losses in the grass carp (Ctenopharyngodon idella) farming industry in China. Discrimination between wild-type field and vaccine strains of GCRV is crucial for meaningful disease diagnosis and epidemiological investigation, yet current detection methods do not discriminate between these different viruses. This study exploited sequence differences between vaccine viruses and the virulent and field strains in the S6 gene that is present in all GCRV strains to develop a high resolution melting curve assay to differentiate between virus strains. The high resolution melting curve analysis was as analytically sensitive as real-time quantitative-Polymerase Chain Reaction (qPCR) detection and at least 10 times sensitive than the conventional PCR. This one-step assay will facilitate grass carp hemorrhagic disease outbreak responses and control.

Speciation of common Gram-negative pathogens using a highly multiplexed high resolution melt curve assay

The identification of the bacterial species responsible for an infection remains an important step for the selection of antimicrobial therapy. Gram-negative bacteria are an important source of hospital and community acquired infections and frequently antimicrobial resistant. Speciation of bacteria is typically carried out by biochemical profiling of organisms isolated from clinical specimens, which is time consuming and delays the initiation of tailored treatment. Whilst molecular methods such as PCR have been used, they often struggle with the challenge of detecting and discriminating a wide range of targets. High resolution melt analysis is an end-point qPCR detection method that provides greater multiplexing capability than probe based methods. Here we report the design of a high resolution melt analysis assay for the identification of six common Gram-negative pathogens; Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas aeruginosa, Salmonella Sp, and Acinetobacter baumannii, and a generic Gram-negative specific 16S rRNA control. The assay was evaluated using a well characterised collection of 113 clinically isolated Gram-negative bacteria. The agreement between the HRM assay and the reference test of PCR and sequencing was 98.2% (Kappa 0.96); the overall sensitivity and specificity of the assay was 97.1% (95% CI: 90.1–99.7%) and 100% (95% CI: 91.78–100%) respectively.

High-resolution melting-curve (HRM) analysis for C. meleagridis identification in stool samples

BackgroundCryptosporidiosis represents a major public health problem. This infection, caused by a protozoan parasite of the genus Cryptosporidium, has been reported worldwide as a frequent cause of diarrhoea. In the immunocompetent host, the typical watery diarrhea can be self-limiting. However, it is severe and chronic, in the immunocompromised host and may cause death. Cryptosporidium spp. are coccidians, which complete their life cycle in both humans and animals. The two species C. hominis and C. parvum are the major cause of human infection. Compared to studies on C. hominis and C. parvum, only a few studies have developed methods to identify C. meleagridis. AimTo develop a new real time PCR-coupled High resolution melting assay allowing the detection for C. meleagridis, in addition of the other dominant species (C. hominis and C. parvum). MethodsThe polymorphic sequence on the dihydrofolate reductase gene (DHFR) of three species was sequenced to design primers pair and establish a sensitive real-time PCR coupled to a high-resolution melting-curve (HRM) analysis method, allowing the detection of Cryptosporidium sp. and discrimination between three prevalent species in Tunisia. We analyzed a collection of 42 archived human isolates of the three studied species. ResultsReal-time PCR coupled to HRM assay allowed detection of Cryptosporidium, using the new designed primers, and basing on melting profile, we can distinguish C. meleagridis species in addition to C. parvum and C. hominis. ConclusionWe developed a qPCR-HRM assay that allows Cryptosporidium genotyping. This method is sensitive and able to distinguish three Cryptosporidium species.

Effective pragmatic approach of diagnosis of multidrug-resistant tuberculosis by high-resolution melt curve assay.

Effective management of multidrug-resistant tuberculosis (MDR-TB) requires cost-effective and rapid screening of rifampicin (RIF) and isoniazid (INH) resistance. Accordingly, a highly promising high-resolution melting (HRM) analysis was evaluated in the detection of mutation in rpoB, katG gene and inhA promoter region in Mycobacterium tuberculosis isolates. A total of 143 M. tuberculosis isolates comprising phenotypically confirmed 94 MDR and 49 sensitive isolates were analyzed by HRM following real-time-polymerase chain reaction in comparison to gold standard of targeted DNA sequencing of rpoB, katG gene and inhA promoter region. HRM correctly identified MDR-TB by rapid and accurate detection of predominantly and infrequently occurring specific single nucleotide polymorphism in rpoB, katG gene and inhA promoter region. rpoB HRM showed sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 98% each respectively. Predominantly, S531 L/W (TCG → TTG/TGG) mutation accounted for 68.47% of RIF resistance followed by H526Y/R (13.04%, CAC → TAC/CGC), D516Y/V/G (10.86%, GAC → TAC/GTC/GGC), Q513P (4.34%, CAA → CCA), and one rare mutation at codon position L533A (CTG → CGG). Combined KatG and inhA HRM sensitivity, specificity, PPV, and NPV were 90%, 100%, 100%, and 84.48% respectively and detected frequent mutation at codon position S315T/I/N (70%, AGC → ACC, AGC → ACT, AGC → AAC) and rare mutation at codon position T314P (3.3%, ACC → CCC) and 329 (2.2%, GAC → GCC) of katG gene. In inhA, mutations were recorded at mostly promoter position - 15 (10%, C → T) and infrequently at - 8 (3.3%, T → G, T → C). HRM assay limitation noticed in recognizing silent mutation in rpoB as a mutant, nondetection of infrequent mutation S310A in katG, and the inability of detecting mutation outside the targeted region of investigated genes. HRM may prove to be a vital molecular assay in rapid screening of TB cases for early detection of MDR TB, leading to early evidenced-based initiation of antitubercular treatment that will significantly reduce MDR transmission.

Clinical application of high resolution melting curve assay in bacteria identification and antimicrobial susceptibility testing

High resolution melting (HRM) analysis is a real-time PCR based technique which can detect DNA sequence polymorphism. Immediately after routine PCR with saturated fluorescent dye, melting transition of the amplicon was monitored and fluorescence was collected. According to the different melting curve, single base differences can be distinguished. HRM is a low-cost, single-step, closed-tube, accurate and rapid method, which has aroused general concern. Rapid and sensitive detection of the clinical pathogens is critical to diagnosis of infectious diseases. However, clinical research on application of HRM in infectious disease pathogen identification and drug resistance, especially melting curve analysis based bedside technology integration will provide strong support for the diagnosis and treatment of infectious diseases. (Chin J Lab Med, 2017, 40: 84-87) Key words: Bacterial infections; Drug resistance, bacterial; Microbial sensitivity tests; High resolution melting

High resolution melt curve analysis based on methylation status for human semen identification.

A high resolution melt curve assay to differentiate semen from blood, saliva, urine, and vaginal fluid based on methylation status at the Dapper Isoform 1 (DACT1) gene was developed. Stains made from blood, saliva, urine, semen, and vaginal fluid were obtained from volunteers and DNA was isolated using either organic extraction (saliva, urine, and vaginal fluid) or Chelex® 100 extraction (blood and semen). Extracts were then subjected to bisulfite modification in order to convert unmethylated cytosines to uracil, consequently creating sequences whose amplicons have melt curves that vary depending on their initial methylation status. When primers designed to amplify the promoter region of the DACT1 gene were used, DNA from semen samples was distinguishable from other fluids by a having a statistically significant lower melting temperature. The assay was found to be sperm-significant since semen from a vasectomized man produced a melting temperature similar to the non-semen body fluids. Blood and semen stains stored up to 5months and tested at various intervals showed little variation in melt temperature indicating the methylation status was stable during the course of the study. The assay is a more viable method for forensic science practice than most molecular-based methods for body fluid stain identification since it is time efficient and utilizes instrumentation common to forensic biology laboratories. In addition, the assay is advantageous over traditional presumptive chemical methods for body fluid identification since results are confirmatory and the assay offers the possibility of multiplexing which may test for multiple body fluids simultaneously.

A Rapid Multiplex Real-Time PCR High-Resolution Melt Curve Assay for the Simultaneous Detection of Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus in Food

Three important foodborne pathogens, Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus, are of great concern for food safety. They may also coexist in food matrices and, in the case of B. cereus and S. aureus, the resulting illnesses can resemble each other owing to similar symptoms. Therefore, their simultaneous detection may have advantages in terms of cost savings and rapidity. Given this context, a rapid multiplex real-time PCR high-resolution melt curve assay for the simultaneous detection of these three pathogens in food was developed. The assay successfully detected B. cereus (gyrB), L. monocytogenes (hly), and S. aureus (nuc) in a single reaction, and the average melting temperatures were 76.23, 80.19, and 74.01°C, respectively. The application of SYTO9 dye and a slow melt curve analysis ramp rate (0.1°C/s) enabled the production of sharp, high-resolution melt curve peaks that were easily distinguishable from each other. The detection limit in food (milk, rice, and lettuce) was 3.7 × 10(3) CFU/g without an enrichment step and 3.7 × 10(1) CFU/g following the 10-h enrichment. Hence, the assay developed here is specific and sensitive, providing an efficient tool for implementation in food for the simultaneous detection of B. cereus, L. monocytogenes, and S. aureus .

A novel pentaplex real time (RT)- PCR high resolution melt curve assay for simultaneous detection of emetic and enterotoxin producing Bacillus cereus in food

Bacillus cereus causing emetic and diarrheal type of food poisoning is widely distributed in nature and is therefore considered a major foodborne pathogen. There is a growing demand for fast, accurate, reliable and economic detection of potentially toxigenic B. cereus. To improve differential diagnosis of toxigenic B. cereus, a highly sensitive pentaplex RT- PCR high resolution melt curve assay was developed for simultaneous detection of 4 major enterotoxim genes (cytK, entFM, hblD, nheA) and emetic toxin gene (ces). The average melting temperatures (Tm) of PCR products were 72.2 °C (ces), 74.23 °C (cytK), 76.55 °C (nheA), 78.42 °C (entFM) and 81.90 (hblD). The multiplex assay was evaluated using 71 bacterial strains including 17 emetic B. cereus reference strains, 9 enterotoxic B. cereus reference strains, 4 B. cereus group members, 23 wild B. cereus strains, 18 non-target strains, and was further tested on artificially inoculated foods. The detection limit in food samples was approximately 103 CFU/g without enrichment and 101 CFU/g was observed following 7 h enrichment. The DNA intercalating dye SYTO9 used in this study generated high resolution melt curve peaks for the target strains and genes in which the peaks were sharp and easily distinguishable from each other. Thus, the developed multiplex real-time (RT) PCR approach can be a reliable tool for the identification of emetic and enterotoxic strains of B. cereus present in food and food-related samples.

High resolution melting curve assay for detecting rs12979860 IL28B polymorphisms involved in response of Iranian patients to chronic hepatitis C treatment.

A recent genome-wide association study (GWAS) on patients with chronic hepatitis C (CHC) treated with peginterferon and ribavirin (pegIFN-α/RBV) identified a single nucleotide polymorphism (SNP) on chromosome 19 (rs12979860) which was strongly associated with a sustained virological response (SVR). The aim of this study was twofold: to study the relationship between IL28B rs12979860 and sustained virological response (SVR) to pegIFN-α/RVB therapy among CHC patients and to detect the rs12979860 polymorphism by high resolution melting curve (HRM) assay as a simple, fast, sensitive, and inexpensive method. The study examined outcomes in 100 patients with chronic hepatitis C in 2 provinces of Iran from December 2011 to June 2013. Two methods were applied to detect IL28B polymorphisms: PCR-sequencing as a gold standard method and HRM as a simple, fast, sensitive, and inexpensive method. The frequencies of IL28B rs12979860 CC, CT, and TT alleles in chronic hepatitis C genotype 1a patients were 10% (10/100), 35% (35/100), and 6% (6/100) and in genotype 3a were 13% (13/100), 31% (31/100), and 5% (5/100), respectively. In genotype 3a infected patients, rs12979860 (CC and CT alleles) and in genotype 1a infected patients (CC allele) were significantly associated with a sustained virological response (SVR). The SVR rates for CC, CT and TT (IL28B rs12979860) were 18%, 34% and 4%, respectively. Multiple logistic regression analysis identified two independent factors that were significantly associated with SVR: IL-28B genotype (rs 12979860 CC vs TT and CT; odds ratio [ORs], 7.86 and 4.084, respectively), and HCV subtype 1a (OR, 7.46). In the present study, an association between SVR rates and IL28B polymorphisms was observed. The HRM assay described herein is rapid, inexpensive, sensitive and accurate for detecting rs12979860 alleles in CHC patients. This method can be readily adopted by any molecular diagnostic laboratory with HRM capability and will be clinically beneficial in predicting treatment response in HCV genotype 1 and 3 infected patients. In addition, it was demonstrated that CC and CT alleles in HCV-3a and the CC allele in HCV-1a were significantly associated with response to pegIFN-α/RBV treatment. The present results may help identify subjects for whom the therapy might be successful.

Analysis of agouti signaling protein (ASIP) gene polymorphisms and association with coat color in Tibetan sheep (Ovis aries).

Tibetan sheep, an indigenous breed, have a wide variety of phenotypes and a colorful coat, which make this breed an interesting model for evaluating the effects of coat-color gene mutations on this phenotypic trait. The agouti signaling protein (ASIP) gene is a positional candidate gene, as was inferred based on previous study. In our research, ASIP gene copy numbers in genomic DNA were detected using a novel approach, and the exon 2 g.100-104 mutation and copy number variation (CNV) of ASIP were associated with coat color in 256 sheep collected from eight populations with different coat colors by high-resolution melting curve assay. We found that the relative copy numbers of ASIP ranged from one to eight in Tibetan sheep. All of the g.100-104 genotypes in the populations were in Hardy-Weinberg equilibrium, and there was no relationship between the g.100-104 genotype and coat color (P > 0.05). The single ASIP CNV allele was found to be almost entirely associated with solid-black coat color; however, not all solid-black sheep displayed the putative single ASIP CNV genotype. From our study, we speculate that the ASIP CNV is under great selective pressure and the single ASIP CNV allows selection for black coat color in Tibetan sheep, but this does not explain all black phenotypes in Tibetan sheep.

A PCR based protocol for detecting indel mutations induced by TALENs and CRISPR/Cas9 in zebrafish.

Genome editing techniques such as the zinc-finger nucleases (ZFNs), transcription activator-like effecter nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system Cas9 can induce efficient DNA double strand breaks (DSBs) at the target genomic sequence and result in indel mutations by the error-prone non-homologous end joining (NHEJ) DNA repair system. Several methods including sequence specific endonuclease assay, T7E1 assay and high resolution melting curve assay (HRM) etc have been developed to detect the efficiency of the induced mutations. However, these assays have some limitations in that they either require specific sequences in the target sites or are unable to generate sequencing-ready mutant DNA fragments or unable to distinguish induced mutations from natural nucleotide polymorphism. Here, we developed a simple PCR-based protocol for detecting indel mutations induced by TALEN and Cas9 in zebrafish. We designed 2 pairs of primers for each target locus, with one putative amplicon extending beyond the putative indel site and the other overlapping it. With these primers, we performed a qPCR assay to efficiently detect the frequencies of newly induced mutations, which was accompanied with a T-vector-based colony analysis to generate single-copy mutant fragment clones for subsequent DNA sequencing. Thus, our work has provided a very simple, efficient and fast assay for detecting induced mutations, which we anticipate will be widely used in the area of genome editing.

Su1197 High Resolution Melt Curve Assay for the Detection of Point Mutations Associated With Helicobacter pylori Resistance to Clarithromycin

Su1197 High Resolution Melt Curve Assay for the Detection of Point Mutations Associated With Helicobacter pylori Resistance to Clarithromycin

High resolution melting curve assay for rapid detection of drug-resistant Mycobacterium tuberculosis

We developed and evaluated a high resolution melting (HRM) curve assay by using real-time PCR for the detection of the most frequent mutations of Mycobacterium tuberculosis, which are responsible for the resistance of four anti-TB drugs: rifampicin, isoniazid, ethambutol, and streptomycin. The HRM assay was successfully used for the detection of dominant mutations: A516V, H526A, H526T, S531L, L533P, and A516G/S531L in rpoB; S315T, and S315A in katG; -15C/T, and -8T/C in mab-inhA; M306I in embB; K88Q and K43R in rpsL; and 513A/C in rrs. We were able to discriminate the mutant from the wild type by analyzing the melting-curve shape in 40 clinical M. tuberculosis isolates, and the results of the HRM assay were completely consistent with those of DNA sequencing. This HRM assay is a simple, rapid, and cost-effective method that can be performed in a closed tube. Therefore, our assay is a potentially useful tool for the rapid detection of drug-resistant M. tuberculosis.

Enhanced annealing of mismatched oligonucleotides using a novel melting curve assay allows efficient in vitro discrimination and restriction of a single nucleotide polymorphism

BackgroundMany SNP discrimination strategies employ natural restriction endonucleases to discriminate between allelic states. However, SNPs are often not associated with a restriction site and therefore, a number of attempts have been made to generate sequence-adaptable restriction endonucleases. In this study, a simple, sequence-adaptable SNP discrimination mechanism between a 'wild-type' and 'mutant' template is demonstrated. This model differs from other artificial restriction endonuclease models as cis- rather than trans-orientated regions of single stranded DNA were generated and cleaved, and therefore, overcomes potential issues of either inefficient or non-specific binding when only a single variant is targeted.ResultsA series of mismatch 'bubbles' that spanned 0-5-bp surrounding a point mutation was generated and analysed for sensitivity to S1 nuclease. In this model, generation of oligonucleotide-mediated ssDNA mismatch 'bubbles' in the presence of S1 nuclease resulted in the selective degradation of the mutant template while maintaining wild-type template integrity. Increasing the size of the mismatch increased the rate of mutant sequence degradation, until a threshold above which discrimination was lost and the wild-type sequence was degraded. This level of fine discrimination was possible due to the development of a novel high-resolution melting curve assay to empirically determine changes in Tm (~5.0°C per base-pair mismatch) and to optimise annealing conditions (~18.38°C below Tm) of the mismatched oligonucleotide sets.ConclusionsThe in vitro 'cleavage bubble' model presented is sequence-adaptable as determined by the binding oligonucleotide, and hence, has the potential to be tailored to discriminate between any two or more SNPs. Furthermore, the demonstrated fluorometric assay has broad application potential, offering a rapid, sensitive and high-throughput means to determine Tm and annealing rates as an alternative to conventional hybridisation detection strategies.