Amplification of rs9934438 and rs1057910 variants was carried out in a 96-well plate in the LightCycler® 480 Real-Time PCR System (Roche Diagnostics) under the same reaction system and conditions. The PCR reaction mix- ture was prepared in 20 μl total volume by add- ing 10 ng of genomic DNA, 10 μl Master Mix and 2.4 μl MgCl 2 (HighResolutionMelting Master, Roche), 5 pmol of forward and reverse primers and 4.8 μl dH 2 O. Thermocycling condi- tions for PCR included one cycle denaturation at 95°C for 10 min and 55 cycles consisted of denaturation at 95°C for 15 s, annealing at 63°C for 10 s and extension at 72°C for 10 s, and cooling at 40°C for 30 s. All primers were selected using the Primer5 software (CA, USA), synthesized by Introvogen (Shanghai, China). Primer sequences were as follows: VKORC-F, 5’-CCGAGAAAGGTGATTTCCAA-3’, VKORC-R, 5’- TGACATGGAATCCTGACGTG-3’, CYP2C9-F, 5’-CA- CGAGGTCCAGAGATAC-3’, CYP2C9-R, 5’-GGAAT- GAGATAGTTTCTGAATTTAAT-3’.
In this work, the functionality of starch branching enzyme IIa (SBEIIa) has been targeted in bread wheat by TILLING. An EMS-mutagenised wheat population has been screened using HighResolutionMelting of PCR products to identify functional SNPs in the three homoeologous genes encoding the target enzyme in the hexaploid genome. Results: This analysis resulted in the identification of 56, 14 and 53 new allelic variants respectively for SBEIIa-A, SBEIIa-B and SBEIIa-D. The effects of the mutations on protein structure and functionality were evaluated by a bioinformatic approach. Two putative null alleles containing non-sense or splice site mutations were identified for each of the three homoeologous SBEIIa genes; qRT-PCR analysis showed a significant decrease of their gene expression and resulted in increased amylose content. Pyramiding of different single null homoeologous allowed to isolate double null mutants showing an increase of amylose content up to 21% compared to the control. Conclusion: TILLING has successfully been used to generate novel alleles for SBEIIa genes known to control amylose content in wheat. Single and double null SBEIIa genotypes have been found to show a significant increase in amylose content.
Background: The emergence of drug resistance among Mycobacterium tuberculosis (MTB) strains is a serious health concern worldwide. The development of rapid molecular diagnostic methods in recent years has a significant impact on the early detection of resistance to major anti-TB drugs in MTB isolates, which helps in employing appropriate treatment regimen and prevents the spread of drug-resistant strains. This study was designed to evaluate the efficacy of real-time PCR and high-resolutionmelting (HRM) curve analysis for the determination of resistance to rifampin (RIF), isoniazid (INH), and ofloxacin (OFX) in MTB isolates and to investigate their resistance-related mutations.
Background: The 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 HighResolutionMelting (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 ).
Several molecular techniques have been described for the detection of anti-TB drug resistance-associated mutations, including the line probe assays GenoType MTBDR plus (Hain Lifescience GmbH, Nehren, Germany), GenoType MTBDRsl (Hain Lifescience GmbH, Nehren, Germany), INNO-LiPA Rif.TB (Innogenetics, Ghent, Belgium), and Xpert MTB/RIF (Cepheid, Sunnyvale, CA)  . However, these are probe based as- says and only suitable for mutations that are located in hotspot regions of specific genes. Mutations in the pnc A are dispersed throughout the gene and its upstream pro- moter; this makes the development of probe-based methods challenging . Direct se- quencing of pnc A amplicons remains the best genotypic strategy, however this is costly. High-resolutionmelting (HRM) curve analysis is a simple technique and has been widely used for many applications. PCR amplicons are heated and fluorescence loss is monitored in real-time. Variations in sequences are detected by difference in melting point (T m ) compared to reference DNA; it does not require the use specific probes  . In the TB field, HRM has been used for detecting rifampin (RIF), isoniazid (INH), streptomycin (STR), and fluoroquinolone resistant M. tuberculosis . In this study, we describe an HRM technique to detect pnc A mutations in M. tuberculosis clinical isolates and compare its results to the phenotypic PZA susceptibility testing (The Bac- tec MGIT 960). Isolates that had discordant results were sent for sequencing. This method uses the Roche LightCycler 480 ® and involves simultaneous amplification of
In this study, we used a high-resolutionmelting (HRM) analysis approach to perform allele-specific expression measurement in CHEK2 . As in currently used methods for investigating DAE, this approach is applied to individual subjects who are heterozygous for an exonic marker SNP, specifically targeted by a labelled probe, called SimpleProbe [18,19]. Data acquisition on HRM instruments consists of monitoring changes in the fluorescence intensity of the probe, as it dissociates from the two allelic templates, while the probe-target duplexes are continuously heated. We have already reported the use of this methodology to compare the relative abundance of allelic transcripts in a study inves- tigating mRNA degradation due to NMD in BRCA2 , and in a group of selected genes involved in the cellular response to the cytotoxic agent cytarabine . In these studies, DAE analysis was limited by the single- capillary throughput of the HRM device used, the HR- 1 ™ instrument, and allelic imbalance was quantified manually. Here, we report additional experiments and testing, as well as up-scaling possibilities with a high- throughput HRM device, the LightScanner ® instrument that uses a 384-well plate format. To improve the analy- sis of allelic expression, an analysis tool was developed using R in order to process data acquired with HRM commercialized software. Our script provides allelic imbalance estimates and subsequent statistical calcula- tions that are required to assess DAE.
The high-resolutionmelting (HRM) assay is a novel and powerful molecular method applied to mutation detection, genotype analysis and species identifica- tion [23–26]. During double strand DNA (dsDNA) dissociation to single stranded DNA with increasing temperature, melting curve and melting temperature (Tm) are generated by monitoring the fluorescence of the binding dye present in dsDNA . The changes in melting curve shape and Tm peaks are generated due to differences in amplicon composition. Currently, there are no reports on HRM assays for discrimina- tion of Babesia species infective to bovines. However, assays have been performed to identify Babesia caballi and Theileria equi, discriminate four Babeisa sp. infec- tive to humans and diagnose five Plasmodium spp. [27–29]. In the present study, we developed a reliable, rapid and powerful RT-PCR-HRM assay targeting the 18S rRNA gene to discriminate between four Babesia spp. in cattle.
In recent years, real-time PCR coupled with melting curve analysis, especially with high-resolutionmelting curve analysis (HRMA), has shown promise as a tool for the detection of DNA sequence variation because this novel method detects more mu- tations with few probes or without probes. HRMA is a rapid, sim- ple, cost-effective, and closed-tube method, and it has been ap- plied to a variety of diseases, such as inherited, infectious, and oncological diseases (14). This PCR-based method was success- fully used for rapid identification and susceptibility testing of My- cobacterium tuberculosis in previous studies (2, 4, 15, 16). On the other hand, previous studies have demonstrated that RIF resis- tance is a good surrogate marker for MDR-TB, especially when molecular methods are used for rapid detection of RIF-resistant M. tuberculosis isolates, as 90% of RIF-resistant M. tuberculosis
Real-time PCR, followed by high-resolutionmelting analysis (HRM), generates thermodynamic differences in the dissociation profile of amplicons resulting in specific signatures of polymorphisms due to small differences in nucleotide composition . HRM is rapid, comparatively little laborious and a relatively cheap method where the post-PCR treatment is contained in the tubes with small risk for lab-contamination. HRM has been used for iden- tification of other infectious agents [10 – 13]. In earlier work, we have already shown that HRM can be a valu- able tool for Leishmania genotyping, using hsp70 as a target . AAP3 is an amino acid transporter, which mediates uptake of lysine, histidine, phenylalanine, cit- rulline and arginine, with the highest affinity for the last one [15 – 20]. AAP3 is involved in the polyamine pathway, essential for parasite replication [17, 21 – 23]. The coding se- quence for AAP3 is conserved among Leishmania spp., in- dicating its value as the chosen target . We already demonstrated aap3 as an attractive target for detecting Leishmania, by a real-time PCR method, but this approach did not discriminate the species . In this paper, we de- scribe the use of the aap3 coding sequence as target for dif- ferentiation of Leishmania spp., using HRM analysis. The aap3-HRM method showed to be a specific and sensitive tool to differentiate Leishmania spp., using reference strain cultures and validated using clinical samples, naturally in- fected sand flies and experimentally infected mice samples.
We have developed a high-resolutionmelting (HRM) assay to scan for mutations in the rpoB, inhA, ahpC, and katG genes and/or promoter regions for the detection of rifampin and isoniazid resistance in Mycobacte- rium tuberculosis. For assay development, 23 drug-resistant isolates of M. tuberculosis having 29 different mutations, together with 40 drug-susceptible isolates, were utilized. All 29 mutations were accurately detected by our assay. We further validated the assay with a series of 59 samples tested in a blind manner. All sequence alterations that were within the regions targeted by the HRM assay were correctly identified. Compared against results of DNA sequencing, the sensitivity and specificity of our HRM assay were 100%. For the blinded samples, the specificities and sensitivities were 89.3% and 100%, respectively, for detecting rifampin resistance and 98.1% and 83.3%, respectively, for detecting isoniazid resistance, as isolates with mutations in regions not encompassed by our assay were not detected. A C-to-T sequence alteration at position ⴚ 15 of the ahpC regulatory region, which was previously reported to be associated with isoniazid resistance, may possibly be a polymorphism, as it was detected in an isoniazid-susceptible M. tuberculosis isolate. HRM is a rapid, accurate, simple, closed-tube, and low-cost method. It is thus an ideal assay to be used in countries with a high prevalence of drug-resistant M. tuberculosis and where cost-effectiveness is essential. As a mutation-scanning assay for detecting drug-resistant M. tuberculosis, it can potentially lead to better treatment outcomes resulting from earlier treatment with the appropriate antibiotics.
Stool specimens and RNA extraction. Stool specimens were collected from children under the age of 5 years with sporadic cases of gastroenteritis in Japan between June 1998 and December 1999. The 14 fecal samples used were all SRSV (small round structured virus) positive by transmission electron micros- copy (TEM). For the reference strains of the high-resolutionmelting (HRM) assay, we selected stool samples that were determined by RT-PCR and sequenc- ing to be positive for six noroviruses belonging to the dominant subtypes that have been circulating in Japan: genotypes 4, 8, and 9 from GI and genotypes 3, 4, and 10 from GII. Stool specimens were stored at ⫺ 80°C until RNA extraction. A 20% stool suspension was prepared in phosphate-buffered saline and homog- enized. This suspension was treated with an equal volume of 1,1,2-trichloro-1,2,2- trifluoroethane (Wako Chemical Co., Tokyo, Japan) and then centrifuged at 2,000 ⫻ g for 30 min at 4°C, and the aqueous layer was collected. The QIAamp viral RNA minikit (Qiagen, Hilden, Germany) was used to extract RNA from 140 l of the aqueous layer according to the manufacturer’s instructions.
Background: Complex traits like cancer, diabetes, obesity or schizophrenia arise from an intricate interaction between genetic and environmental factors. Complex disorders often cluster in families without a clear-cut pattern of inheritance. Genomic wide association studies focus on the detection of tens or hundreds individual markers contributing to complex diseases. In order to test if a subset of single nucleotide polymorphisms (SNPs) from candidate genes are associated to a condition of interest in a particular individual or group of people, new techniques are needed. High-resolutionmelting (HRM) analysis is a new method in which polymerase chain reaction (PCR) and mutations scanning are carried out simultaneously in a closed tube, making the procedure fast, inexpensive and easy. Preterm birth (PTB) is considered a complex disease, where genetic and environmental factors interact to carry out the delivery of a newborn before 37 weeks of gestation. It is accepted that inflammation plays an important role in pregnancy and PTB.
Next-generation sequencing (NGS) has recently been used for analysis of HIV diversity, but this method is labor-intensive, costly, and requires complex protocols for data analysis. We compared diversity measures obtained using NGS data to those ob- tained using a diversity assay based on high-resolutionmelting (HRM) of DNA duplexes. The HRM diversity assay provides a single numeric score that reflects the level of diversity in the region analyzed. HIV gag and env from individuals in Rakai, Uganda, were analyzed in a previous study using NGS (n ⴝ 220 samples from 110 individuals). Three sequence-based diversity measures were calculated from the NGS sequence data (percent diversity, percent complexity, and Shannon entropy). The ampli- con pools used for NGS were analyzed with the HRM diversity assay. HRM scores were significantly associated with sequence- based measures of HIV diversity for both gag and env (P < 0.001 for all measures). The level of diversity measured by the HRM diversity assay and NGS increased over time in both regions analyzed (P < 0.001 for all measures except for percent complexity in gag), and similar amounts of diversification were observed with both methods (P < 0.001 for all measures except for percent complexity in gag). Diversity measures obtained using the HRM diversity assay were significantly associated with those from NGS, and similar increases in diversity over time were detected by both methods. The HRM diversity assay is faster and less ex- pensive than NGS, facilitating rapid analysis of large studies of HIV diversity and evolution.
and detection of various Phytophthoras in different plant species, tissues, and environmental samples (Schena et al. 2013). However, these methods are inappropriate for the identification of Phytophthoras at culture level due to difficulties in differentiating species variants, interspecific hybrids or even previ- ously uncharacterised species due to the increasing number of Phytophthora species that have still been found and described worldwide (Kroon et al. 2012). A robust and sensitive molecular analytical tech- nique named highresolutionmelting (HRM) analysis has been employed, apart from other applications, also for fungal genotyping and differentiation (for review see Zambounis et al. 2015). Briefly, this technique exploits the dissociation of double to single stranded DNA by elevating temperatures and the release of a homogeneously intercalated fluorescent dye (Reed & Wittwer 2004). The thermodynamic properties of the amplicons, such as sequence lengths and GC contents, motivate specific fluctuations in fluores- cence and the revealing melting curve profiles (Zam- bounis et al. 2015). The possibility of contamination is astonishingly low as the entire process is fulfilled in a single step (Madesis et al. 2014). Finally, the significant elimination of post-handling steps, plus the exclusion of coming in touch with hazardous chemicals promotes this method as an excellent alternative molecular fungal diagnostic approach.
We assessed the use of high-resolutionmelting (HRM) analysis for the rapid identification of influenza A virus subtypes and the detection of newly emerging virus variants. The viral matrix gene was amplified by LightCycler real-time reverse transcription-PCR (RT-PCR) in the presence of the LCGreen I fluorescent dye. Upon optimization of the assay conditions, all the major influenza A virus subtypes, including H1N1, H3N2, H5N1, H7N3, and H9N2, were amplifiable by this method and had a PCR product length of 179 bp. Real-time RT-PCR of in vitro-transcribed H3N2 RNA revealed a standard curve for quantification with a linear range (correlation coefficient ⴝ 0.9935) across at least 8 log units of RNA concentrations and a detection limit of 10 3 copies of viral RNA. We performed HRM analysis of the PCR products with the HR-1 instrument and used the melting profiles as molecular fingerprints for virus subtyping. The virus subtypes were identified from the high-resolution derivative plot obtained by heteroduplex formation between the PCR products of the viral isolates tested and those of the reference viral isolates. The melting profiles were consistent with minimal interassay variability. Hence, an HRM database and a working protocol were established for the identification of these five influenza A virus subtypes. When this protocol was used to test 21 clinical influenza A virus isolates, the results were comparable to those obtained by RT-PCR with hemagglutinin-specific primer sets. Sequence variants of the clinical isolates (n ⴝ 4) were also revealed by our HRM analytical scheme. This assay requires no multiplexing or hybridization probes and provides a new approach for influenza A virus subtyping and genetic screening of virus variants in a clinical virology laboratory.
One of the challenges in the detection of SNPs in clini- cal samples is to have methods to identify the SNPs rapidly, accurately, and cost effectively. Many assays have been developed to detect SNPs such as direct sequencing, single-stranded conformation polymorphism analysis, denaturing gradient gel electrophoresis, temperature gra- dient capillary electrophoresis, restriction enzyme diges- tion of polymerase chain reaction (PCR) products and sequencing [11-13]. Each of these methods has a different trade off between complexity, sensitivity, turn-around time and cost. HighResolutionMelting analysis is a well estab- lished, closed tube, rapid and high throughput technique. The analysis depends on DNA melting and annealing in the presence of saturating DNA binding dye. The presence of a single base change in the amplicon influences the thermodynamic stability of the duplex resulting in a slight change in the melting temperature (Tm) and the shape of the melt curve [14,15]. The aim of this study was to evalu- ate the use of HRM as a tool for detecting known SNPs in the three human genes, RET , PAX2 and ALDH1A2 which affect kidney size in children.
The high-resolutionmelting analysis is a cost-effective and simple post-PCR technique that can be used for SNP genotyping. The objective of this study was to apply the highresolutionmelting analysis for detection of SNP polymorphism CAPN530 of bovine calpain gene (CAPN1) in Simmental bulls. A total 42 Simmental bulls were included in this study. Genomic DNA was extracted from sperm by commercial Nucleospin Tissue kit. The amplification and highresolutionmelting analysis (HRMA) was done by commercial SensiMix TM HRM kit in the
The goal of the paper was to identify - lactoglobulin gene polymorphism in bulls. The - lactoglobulin (LGB) is expressed in milk and is important in the evaluation of milk production potential and butterfat and protein content. LGB is localized on bovine chromosome 11. The AA genotype of LGB is associated with higher milk yield, the BB genotype with higher fat and casein content and is more desirable for cheese making. The material involved 46 bulls (Slovak spotted breed – 41 bulls, Pinzgau breed – 3 bulls and Holstein breed – 2 bulls). Bovine genomic DNA was isolated from sperm using commercial kit NucleoSpin Tissue and used in order to estimate LGB genotypes by means of PCR RFLP method and highresolutionmelting analysis (HRMA). In the population of Slovak spotted breed we detected all genotypes AA, AB and BB with frequency 0.3415, 0.4390 and 0.2195, subsequently. In Pinzgau breed was detected homozygote genotypes AA and BB with frequency 0.3333 and 0.6667. In Holstein breed was observed only heterozygote genotype AB with frequency 1.
Background: Fasciolosis is a shared disease between humans and livestock caused by hepatic trematodes; Fasciola hepatica and F. gigantica. Differentiate between the two species of this genus is essential. High-ResolutionMelting (HRM) Analysis represents a new approach to this issue. This method can be performed right after termination of Real-Time PCR. This technique has not been used for identification of adult F. hepatica and F. gigantica genotypes. The aim of this study was to determine Fasciola species by using HRM in isolates taken from Iran, respectively.