Application of a new designed high resolution melting analysis for mycobacterial species identification.

The Non-tuberculous mycobacterial (NTM) isolates should be distinguished from tuberculosis and identified at the species level for choosing an appropriate treatment plan. In this study, two molecular methods were used to differentiate NTM species, including a new designed High Resolution Melting (HRM) and Multilocus Sequence Analysis (MLSA). Seventy-five mycobacterial isolates were evaluated by sequencing four genes ( MLSA) and a HRM assay specifically targeting atpE was designed to rapidly and accurately identify and differentiate mycobacterium species. Out of 70 NTM isolates, 66 (94.3%), 65 (92.9%), 65 (92.9%) and 64 (91.4%) isolates were identified to the species level by PCR of atpE, tuf, rpoB and dnaK genes. We could identify 100% of the isolates to the species level (14 different species) by MLSA. By using HRM assay, all NTM isolates were identified and classified into eight groups, in addition, Mycobacterium tuberculosis and Nocardia were also detected simultaneously. The MLSA technique was able to differentiate all 14 species of NTM isolates. According to the results, the HRM assay is a rapid and beneficial method for identifying NTM, M. tuberculosis (MTB), and Nocardia isolates without sequencing.

High-Resolution Melting (HRM) analysis of DNA methylation using semiconductor chip-based digital PCR.

BACKGROUND: Digital PCR (dPCR) technology allows absolute quantification and detection of disease-associated rare variants, and thus the use of dPCR technology has been increasing in clinical research and diagnostics. The high-resolution melting curve analysis (HRM) of qPCR is widely used to distinguish true positives from false positives and detect rare variants. In particular, qPCR-HRM is commonly used for methylation assessment in research and diagnostics due to its simplicity and high reproducibility. Most dPCR instruments have limited fluorescence channels available and separate heating and imaging systems. Therefore, it is difficult to perform HRM analysis using dPCR instruments. OBJECTIVE: A new digital real-time PCR instrument (LOAA) has been recently developed to integrate partitioning, thermocycling, and imaging in a single dPCR instrument. In addition, a new technique to perform HRM analysis is utilized in LOAA. The aim of the present study is to evaluate the efficiency and accuracy of LOAA dPCR on HRM analysis for the detection of methylation. METHODS: In this study, comprehensive comparison with Bio-Rad qRT-PCR and droplet-based dPCR equipment was performed to verify the HRM analysis-based methylation detection efficiency of the LOAA digital PCR equipment. Here, sodium bisulfite modification method was applied to detect methylated DNA sequences by each PCR method. RESULTS: Melting curve analysis detected four different Tm values using LOAA and qPCR, and found that LOAA, unlike qPCR, successfully distinguished between different Tm values when the Tm values were very similar. In addition, melting temperatures increased by each methylation were about 0.5℃ for qPCR and about 0.2 ~ 0.6℃ for LOAA. The melting temperature analyses of methylated and unmethylated DNA samples were conducted using LOAA dPCR with TaqMan probes and EvaGreen, and the result found that Tm values of methylated DNA samples are higher than those of unmethylated DNA samples. CONCLUSION: The present study shows that LOAA dPCR could detect different melting temperatures according to methylation status of target sequences, indicating that LOAA dPCR would be useful for diagnostic applications that require the accurate quantification and assessment of DNA methylation.

Development of self-compatible Chinese cabbage lines of Chiifu through marker-assisted selection.

The continuously refined genome assembly of the Chinese cabbage accession Chiifu is widely recognized as the reference for Brassica rapa. However, the high self-incompatibility of Chiifu limits its broader utilization. In this study, we report the development of self-compatible Chiifu lines through a meticulous marker-assisted selection (MAS) strategy, involving the substitution of the Chiifu allele of MLPK (M-locus protein kinase) with that from the self-compatible Yellow Sarson (YS). A YS-based marker (SC-MLPK) was employed to screen 841 B. rapa accessions, confirming that all eight accessions with the mlpk/mlpk (mm) genotype exhibited self-compatibility. Additionally, we designed 131 High-Resolution Melting (HRM) markers evenly distributed across the B. rapa genome as genomic background selection (GBS) markers to facilitate the introgression of self-compatibility from YS into Chiifu along with SC-MLPK. Genome background screening revealed that the BC3S1 population had a proportion of the recurrent parent genome (PR) ranging from 93.9% to 98.5%. From this population, we identified self-compatible individuals exhibiting a high number of pollen tubes penetrating stigmas (NPT) (>25) and a maximum compatibility index (CI) value of 7.5. Furthermore, we selected two individuals demonstrating significant similarity to Chiifu in both genetic background and morphological appearance, alongside self-compatibility. These selected individuals were self-pollinated to generate two novel lines designated as SC-Chiifu Lines. The development of these self-compatible Chiifu lines, together with the SC-MLPK marker and the set of HRM markers, represents valuable tools for B. rapa genetics and breeding.

Temperature-Wise Calibration Increases the Accuracy of DNA Methylation Levels Determined by High-Resolution Melting (HRM).

High-resolution melting (HRM) is a cost-efficient tool for targeted DNA methylation analysis. HRM yields the average methylation status across all CpGs in PCR products. Moreover, it provides information on the methylation pattern, e.g., the occurrence of monoallelic methylation. HRM assays have to be calibrated by analyzing DNA methylation standards of known methylation status and mixtures thereof. In general, DNA methylation levels determined by the classical calibration approach, including the whole temperature range in between normalization intervals, are in good agreement with the mean of the DNA methylation status of individual CpGs determined by pyrosequencing (PSQ), the gold standard of targeted DNA methylation analysis. However, the classical calibration approach leads to highly inaccurate results for samples with heterogeneous DNA methylation since they result in more complex melt curves, differing in their shape compared to those of DNA standards and mixtures thereof. Here, we present a novel calibration approach, i.e., temperature-wise calibration. By temperature-wise calibration, methylation profiles over temperature are obtained, which help in finding the optimal calibration range and thus increase the accuracy of HRM data, particularly for heterogeneous DNA methylation. For explaining the principle and demonstrating the potential of the novel calibration approach, we selected the promoter and two enhancers of MGMT, a gene encoding the repair protein MGMT.

Development of a multiplex polymerase chain reaction assay for detection of hepatitis C virus, hepatitis B virus, and human immunodeficiency virus 1.

BACKGROUND: Hepatitis C virus (HCV), hepatitis B virus (HBV), and human immunodeficiency virus 1 (HIV-1) are the most epidemic blood-borne viruses, posing threats to human health and causing economic losses to nations for combating the infection transmission. The diagnostic methodologies that depend on the detection of viral nucleic acids are much more expensive, but they are more accurate than serological testing. AIM: To develop a rapid, cost-effective, and accurate diagnostic multiplex polymerase chain reaction (PCR) assay for simultaneous detection of HCV, HBV, and HIV-1. METHODS: The design of the proposed PCR assay targets the amplification of a short conserved region featured with a distinguishable melting profile and electrophoretic molecular weight inside each viral genome. Therefore, this diagnostic method will be appropriate for application in both conventional (combined with electrophoresis) and real-time PCR facilities. Confirmatory in silico investigations were conducted to prove the capability of the approached PCR assay to detect variants of each virus. Then, Egyptian isolates of each virus were subjected to the wet lab examination using the given diagnostic assay. RESULTS: The in silico investigations confirmed that the PCR primers can match many viral variants in a multiplex PCR assay. The wet lab experiment proved the efficiency of the assay in distinguishing each viral type through high-resolution melting analysis. Compared to related published assays, the proposed assay in the current study is more sensitive and competitive with many expensive PCR assays. CONCLUSION: This study provides a simple, cost-effective, and sensitive diagnostic PCR assay facilitating the detection of the most epidemic blood-borne viruses; this makes the proposed assay promising to be substitutive for the mistakable and cheap serological-based assays.

Gene polymorphisms of TACR1 serve as the potential pharmacogenetic predictors of response to the neurokinin-1 receptor antagonist-based antiemetic regimens: a candidate-gene association study in breast cancer patients.

PURPOSE: The current candidate gene association study aims to investigate tag SNPs from the TACR1 gene as pharmacogenetic predictors of response to the antiemetic guidelines-recommended, NK-1 receptor antagonist-based, triple antiemetic regimens. METHODS: A set of eighteen tag SNPs of TACR1 were genotyped in breast cancer patients receiving anthracycline and cyclophosphamide (with/without docetaxel) applying real-time PCR-HRMA. Data analysis for 121 ultimately enrolled patients was initiated by defining haplotype blocks using PHASE v.2.1. The association of each tag SNP and haplotype alleles with failure to achieve the defined antiemetic regimen efficacy endpoints was tested using PLINK (v.1.9 and v.1.07, respectively) based on the logistic regression, adjusting for the previously known chemotherapy-induced nausea and vomiting (CINV) prognostic factors. All reported p-values were corrected using the permutation test (n = 100,000). RESULTS: Four variants of rs881, rs17010730, rs727156, and rs3755462, as well as haplotypes containing the mentioned variants, were significantly associated with failure to achieve at least one of the defined efficacy endpoints. Variant annotation via in-silico studies revealed that the non-seed sequence variant, rs881, is located in the miRNA (hsa-miR-613) binding site. The other three variants or a variant in complete linkage disequilibrium with them overlap a region of high H3K9ac-promoter-like signature or regions of high enhancer-like signature in the brain or gastrointestinal tissue. CONCLUSION: Playing an essential role in regulating TACR1 expression, gene polymorphisms of TACR1 serve as the potential pharmacogenetic predictors of response to the NK-1 receptor antagonist-based, triple antiemetic regimens. If clinically approved, modifying the NK-1 receptor antagonist dose leads to better management of CINV in risk-allele carriers.

DNA melting analysis.

Melting is a fundamental property of DNA that can be monitored by absorbance or fluorescence. PCR conveniently produces enough DNA to be directly monitored on real-time instruments with fluorescently labeled probes or dyes. Dyes monitor the entire PCR product, while probes focus on a specific locus within the amplicon. Advances in amplicon melting include high resolution instruments, saturating DNA dyes that better reveal multiple products, prediction programs for domain melting, barcode taxonomic identification, high speed microfluidic melting, and highly parallel digital melting. Most single base variants and small insertions or deletions can be genotyped by high resolution amplicon melting. High resolution melting also enables heterozygote scanning for any variant within a PCR product. A web application (uMelt, http://www.dna-utah.org) predicts amplicon melting curves with multiple domains, a useful tool for verifying intended products. Additional applications include methylation assessment, copy number determination and verification of sequence identity. When amplicon melting does not provide sufficient detail, unlabeled probes or snapback primers can be used instead of covalently labeled probes. DNA melting is a simple, inexpensive, and powerful tool with many research applications that is beginning to make its mark in clinical diagnostics.

High-resolution melting real-time PCR assays for subtyping of five diarrheagenic Escherichia coli by a single well in milk.

Diarrheagenic Escherichia coli (DEC) is a kind of foodborne pathogen that poses a significant threat to both food safety and human health. To address the current challenges of high prevalence and difficult subtyping of DEC, this study developed a method that combined multiplex polymerase chain reaction (PCR) with high resolution melting (HRM) analysis for subtyping 5 kinds of DEC. The target genes are amplified by multiplex PCR in a single well, and HRM curve analysis was applied for distinct amplicons based on different melting temperature (Tm) values. The method enables discrimination of different DEC types based on characteristic peaks and distinct Tm values in the thermal melting curve. The assay exhibited 100% sensitivity and 100% specificity with a detection limit of 0.5-1 ng/µL. The results showed that different DNA concentrations did not influence the subtyping results, demonstrating this method owed high reliability and stability. In addition, the method was also used for the detection and subtyping of DEC in milk. This method streamlines operational procedures, shorts the detection time, and offers a novel tool for subtyping DEC.

Simultaneous detection and differentiation of DuCV-1 and DuCV-2 by high-resolution melting analysis.

Birds infected with duck circovirus (DuCV) can potentially cause immunosuppression by damaging lymphoid tissues, causing great losses in the duck breeding industry. Duck circovirus can be divided into two genotypes (DuCV-1 and DuCV-2), but simultaneous detection and differentiation of DuCV-1 and DuCV-2 by high-resolution melting (HRM) analysis is still lacking. Here, we designed specific primers according to the sequence characteristics of the newly identified ORF3 gene and then established a PCR-HRM method for the simultaneous detection and differentiation of DuCV-1 and DuCV-2 via high-resolution melting analysis. Our data showed that the established PCR-HRM assay had the advantages of specificity, with the lowest detection limits of 61.9 copies/µL (for DuCV-1) and 60.6 copies/µL (for DuCV-2). The melting curve of the PCR-HRM results indicated that the amplification product was specific, with no cross-reaction with common waterfowl origin pathogens and a low coefficient of variation less than 1.50% in both intra-batch and inter-batch repetitions, indicating the advantages of repeatability. We found that the percentage of DuCV-2-positive ducks was higher than that of DuCV-1-positive ducks, with 8.62% rate of DuCV-1 and DuCV-2 coinfection. In addition, we found DuCV-2-positive in geese firstly. In conclusion, this study provides a candidate PCR-HRM assay for the detection and accurate differentiation of DuCV-1 and DuCV-2 infection, which will help us for further epidemiological surveillance of DuCVs.

Characterization of Eighty-Eight Single-Nucleotide Polymorphism Markers in the Manila Clam Ruditapes philippinarum Based on High-Resolution Melting (HRM) Analysis.

Single-nucleotide polymorphisms (SNPs) are the most commonly used DNA markers in population genetic studies. We used the Illumina HiSeq4000 platform to develop single-nucleotide polymorphism (SNP) markers for Manila clam Ruditapes philippinarum using restriction site-associated DNA sequencing (RAD-seq) genotyping. Eighty-eight SNP markers were successfully developed by using high-resolution melting (HRM) analysis, with a success rate of 44%. SNP markers were analyzed for genetic diversity in two clam populations. The observed heterozygosity per locus ranged from 0 to 0.9515, while the expected heterozygosity per locus ranged from 0.0629 to 0.4997. The value of FIS was estimated to be from -0.9643 to 1.0000. The global Fst value was 0.1248 (p < 0.001). After Bonferroni correction, 15 loci deviated significantly from the Hardy-Weinberg equilibrium (p < 0.0006). These SNP markers provide a valuable resource for population and conservation genetics studies in this commercially important species.

Existing status and future advancements of adulteration detection techniques in herbal products.

BACKGROUND: Herbal products have been commonly used all over the world for centuries. Its products have gained remarkable acceptance as therapeutic agents for a variety of disorders. However, following recent research disclosing discrepancies between labeling and actual components of herbal products, there is growing concern about the efficacy, quality and safety of the products. The admixture and adulteration of herbal medicinal products pose a risk of serious health compromise and the well-being of the consumers. To prevent adulteration in raw ingredients and final herbal products, it is necessary to use approaches to assess both genomes as well as metabolomics of the products; this offers quality assurance in terms of product identification and purity. The combinations of molecular and analytical methods are inevitable for thorough verification and quality control of herbal medicine. METHODS AND RESULTS: This review discusses the combination of DNA barcoding, DNA metabarcoding, mass spectroscopy as well as HPLC for the authentication of herbal medicine and determination of the level of adulteration. It also discusses the roles of PCR and real-time PCR techniques in validating and ensuring the quality, purity and identity of the herbal products. CONCLUSIONS: In conclusion, each technique has its own pros and cons, but the cumulative of both the chemical and molecular methods is proven to be the best strategy for adulteration detection. Moreover, CRISPR diagnosis tools equipped with multiplexing techniques may be implemented for screening adulteration from herbal drugs, this will play a crucial role in herbal product authentication in the future.

Congenital septal defects in Karachi, Pakistan: an update of mutational screening by high-resolution melting (HRM) analysis of MTHFR C677T.

BACKGROUND: Congenital heart defects (CHDs) are the heart structural malformations present at birth. Septal defects account for 40% of CHD, including atrial, ventricular and atrioventricular septal defects. In Pakistan, the prevalence of CHD is 3.4 in 1000, and a study estimated that 60,000 babies are born with CHD annually. Methylenetetrahydrofolate reductase (MTHFR), a chief enzyme, involved in the folate metabolism. The missense mutation, C677T (rs1801133), exists in MTHFR gene, results in a MTHFR thermolabile variant having low enzymatic activity. The study is aim to identify the MTHFR C677T variant association with septal defects. METHODS: Samples of 194 CHD patients (age [Formula: see text]= 5.8 ± 5.1) and 50 normal echo controls (age [Formula: see text]= 6.0 ± 4.9), confirmed by pediatric consultant, were collected. Extracted DNA, quantified by agarose gel electrophoresis and nanodrop, was screened for SNP by high-resolution melting (HRM). Further, HRM results were confirmed using restriction analysis and sequencing. HRM was simply and precisely genotyped the samples within 3 h at low cost. RESULTS: Genotypic data suggested that heterozygous mutant (CT) was frequent in congenital septal defect patients (0.26) which was higher than controls (0.143), p > 0.05. Mutant (TT) genotype was not found in this study. CONCLUSIONS: rs1801133 has lack of significant association with congenital septal defects. The absence of TT genotype in this study suggesting the role of natural selection in targeted population. HRM is an easy, fast and next generation of PCR, which may be used for applied genomics.

Improving the efficiency of Y-chromosome detection and the quality of STR typing in forensic casework with an in-house made qPCR and HRM system based on SYTO™ 9 chemistry.

DNA quantification prior to STR amplification is a crucial step in forensic casework. Obtaining good-quality genetic STR profiles depends mainly on the amount and integrity of the DNA input in the PCR. In addition, the detection of male trace DNA provides key information for forensic investigation. AIM: To evaluate the correlation between the quantification results obtained with the previously developed Amel-Y system, and its ability to detect Y-chromosome DNA by HRM, with the resulting STR profiles, and to ultimately show that Amel-Y can be routinely used in forensic casework to improve STR and Y-STR results. MATERIAL & METHODS: Biological samples derived from forensic casework (85 reference and 391 evidence samples) were quantified by the Amel-Y system (a duplex qPCR/HRM based on SYTO™ 9 chemistry) using Rotor-Gene 6000. STRs were amplified and analyzed with GeneAmp™ PCR System 9700 or Veriti™ Thermal Cyclers and ABI 3500 Genetic Analyzer, respectively. RESULTS: After DNA normalization, a total of 386 STR profiles were obtained (305 full and 81 partial). Sex typing by HRM was 100% successful in reference samples. Male DNA was detected by HRM in 210 evidence samples. 80/201 were mixed with an excess of female DNA. In addition, Amel-Y was able to detect Y-chromosome DNA in mixed samples that did not amplify the Y-variant of Amelogenin marker with commercial STR kits. The reproducibility and precision of the Amel-Y system were demonstrated (CVCt% ≤ 9.55) within the dynamic range analyzed (0.016-50 ng/µL; 41 independent runs). Amel-Y also proved to be compatible with other real-time PCR platforms. CONCLUSION: We demonstrated that Amel-Y is a robust quantification system that can be routinely used in forensic casework to obtain reliable autosomal STR profiles and can be suitable as a predictor for Y-STR typing success when male DNA is detected. HRM can be used as a rapid screening tool for male DNA detection in mixed samples. Alternative designs like Amel-Y offer independence from commercial quantification kits in forensic labs.

A rapid molecular diagnostic tool to discriminate alleles of avirulence genes and haplotypes of Phytophthora sojae using high-resolution melting analysis.

Effectors encoded by avirulence genes (Avr) interact with the Phytophthora sojae resistance gene (Rps) products to generate incompatible interactions. The virulence profile of P. sojae is rapidly evolving as a result of the large-scale deployment of Rps genes in soybean. For a successful exploitation of Rps genes, it is recommended that soybean growers use cultivars containing the Rps genes corresponding to Avr genes present in P. sojae populations present in their fields. Determination of the virulence profile of P. sojae isolates is critical for the selection of soybean cultivars. High-resolution melting curve (HRM) analysis is a powerful tool, first applied in medicine, for detecting mutations with potential applications in different biological fields. Here, we report the development of an HRM protocol, as an original approach to discriminate effectors, to differentiate P. sojae haplotypes for six Avr genes. An HRM assay was performed on 24 P. sojae isolates with different haplotypes collected from soybean fields across Canada. The results clearly confirmed that the HRM assay discriminated different virulence genotypes. Moreover, the HRM assay was able to differentiate multiple haplotypes representing small allelic variations. HRM-based prediction was validated by phenotyping assays. This HRM assay provides a unique, cost-effective and efficient tool to predict virulence pathotypes associated with six different Avr (1b, 1c, 1d, 1k, 3a and 6) genes from P. sojae, which can be applied in the deployment of appropriate Rps genes in soybean fields.

Simple Sequence Repeat Fingerprint Identification of Essential-Oil-Bearing Rosa rugosa via High-Resolution Melting (HRM) Analysis.

Oil-bearing Rosa rugosa are popular in the essential oil and perfume markets. The similar botanical characteristics between high-oil-yield or low-oil-yield cultivars are confusing and it is hard for farmers or breeders to identify the high-oil-yield cultivar by phenotype difference. High-resolution melting (HRM) analysis of simple sequence repeats (SSRs) can construct accurate DNA fingerprints quickly, which was shown to be effective for identification of closely related cultivars of R. rugosa. Optimization of HRM-SSR indicated that the 10 µL HRM reaction mixture containing 20 ng of genomic DNA of R. rugosa and 0.75 µL of 10 µmol/L of each primer with an annealing temperature of 64 °C was a robust SSR genotyping protocol. Using this protocol, 9 polymorphic SSR markers with 3-9 genotypes among the 19 R. rugosa cultivars were identified. The top three polymorphic makers SSR9, SSR12 and SSR19 constructed a fingerprint of all cultivars, and the rare insertion in the flanking sequences of the repeat motif of SSR19 generated three characteristic genotypes of three high-oil-yield cultivars. These results may be economical and practical for the identification of high-oil-yield R. rugosa and be helpful for the selection and breeding of oil-bearing roses.

A circulating cell-free DNA methylation signature for the detection of hepatocellular carcinoma.

To address the shortcomings of current hepatocellular carcinoma (HCC) surveillance tests, we set out to find HCC-specific methylation markers and develop a highly sensitive polymerase chain reaction (PCR)-based method to detect them in circulating cell-free DNA (cfDNA). The analysis of large methylome data revealed that Ring Finger Protein 135 (RNF135) and Lactate Dehydrogenase B (LDHB) are universally applicable HCC methylation markers with no discernible methylation level detected in any other tissue types. These markers were used to develop Methylation Sensitive High-Resolution Analysis (MS-HRM), and their diagnostic accuracy was tested using cfDNA from healthy, at-risk, and HCC patients. The combined MS-HRM RNF135 and LDHB analysis detected 57% of HCC, outperforming the alpha-fetoprotein (AFP) test's sensitivity of 45% at comparable specificity. Furthermore, when used with the AFP test, the methylation assay can detect 70% of HCC. Our findings suggest that the cfDNA methylation assay could be used for HCC liquid biopsy.

High-resolution melting analysis for simultaneous detection and discrimination between wild-type and vaccine strains of feline calicivirus.

High-resolution melting (HRM) analysis, a post-polymerase chain reaction (PCR) application in a single closed tube, is the straightforward method for simultaneous detection, genotyping, and mutation scanning, enabling more significant dynamic detection and sequencing-free turnaround time. This study aimed to establish a combined reverse-transcription quantitative PCR and HRM (RT-qPCR-HRM) assay for diagnosing and genotyping feline calicivirus (FCV). This developed method was validated with constructed FCV plasmids, clinical swab samples from living cats, fresh-frozen lung tissues from necropsied cats, and four available FCV vaccines. We performed RT-qPCR to amplify a 99-base pair sequence, targeting a segment between open reading frame (ORF) 1 and ORF2. Subsequently, the HRM assay was promptly applied using Rotor-Gene Q® Software. The results significantly revealed simultaneous detection and genetic discrimination between commercially available FCV vaccine strains, wild-type Thai FCV strains, and VS-FCV strains within a single PCR reaction. There was no cross-reactivity with other feline common viruses, including feline herpesvirus-1, feline coronavirus, feline leukemia virus, feline immunodeficiency virus, and feline morbillivirus. The detection limit of the assay was 6.18 × 101 copies/µl. This study, therefore, is the first demonstration of the uses and benefits of the RT-qPCR-HRM assay for FCV detection and strain differentiation in naturally infected cats.

Application of High-Resolution Melting and DNA Barcoding for Discrimination and Taxonomy Definition of Rocket Salad (Diplotaxis spp.) Species.

Nuclear and cytoplasmic DNA barcoding regions are useful for plant identification, breeding, and phylogenesis. In this study, the genetic diversity of 17 Diplotaxis species, was investigated with 5 barcode markers. The allelic variation was based on the sequences of chloroplast DNA markers including the spacer between trnL and trnF and tRNA-Phe gene (trnL-F), the rubisco (rbcl), the maturase K (matk), as well as the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA. A highly polymorphic marker (HRM500) derived from a comparison of cytoplasmic genome sequences in Brassicaceae, was also included. Subsequently, a real-time PCR method coupled with HRM analysis was implemented to better resolve taxonomic relationships and identify assays suitable for species identification. Integration of the five barcode regions revealed a grouping of the species according to the common chromosomal set number. Clusters including species with n = 11 (D. duveryrieriana or cretacea, D. tenuifolia, D. simplex and D. acris), n = 8 (D. ibicensis, D. brevisiliqua and D. ilorcitana), and n = 9 (D. brachycarpa, D. virgata, D. assurgens, and D. berthautii) chromosomes were identified. Both phylogenetic analysis and the genetic structure of the collection identified D. siifolia as the most distant species. Previous studies emphasized this species' extremely high glucosinolate content, particularly for glucobrassicin. High-resolution melting analysis showed specific curve patterns useful for the discrimination of the species, thus determining ITS1 as the best barcode for fingerprinting. Findings demonstrate that the approach used in this study is effective for taxa investigations and genetic diversity studies.

A high-resolution melting approach for the simultaneous differentiation of five human babesiosis-causing Babesia species.

BACKGROUND: Six species of apicomplexan parasites of the genus Babesia, namely B. microti, B. divergens, B. duncani, B. motasi, B. crassa-like and B. venatorum, are considered to be the primary causal agents of human babesiosis in endemic areas. These six species possess variable degrees of virulence for their primary hosts. Therefore, the accurate identification of these species is critical for the adoption of appropriate therapeutic strategies. METHODS: We developed a real-time PCR-high-resolution melting (qPCR-HRM) approach targeting 18S ribosomal RNA gene of five Babesia spp. based on melting temperature (Tm) and genotype confidence percentage values. This approach was then evaluated using 429 blood samples collected from patients with a history of tick bites, 120 DNA samples mixed with plasmids and 80 laboratory-infected animal samples. RESULTS: The sensitivity and specificity of the proposed qPCR-HRM method were 95% and 100%, respectively, and the detection limit was 1-100 copies of the plasmid with the cloned target gene. The detection level depended on the species of Babesia analyzed. The primers designed in this study ensured not only the high interspecific specificity of our proposed method but also a high versatility for different isolates from the same species worldwide. Additionally, the Tm obtained from the prepared plasmid standard is theoretically suitable for identifying isolates of all known sequences of the five Babesia species. CONCLUSIONS: The developed detection method provides a useful tool for the epidemiological investigation of human babesiosis and pre-transfusion screening.

Associations of multiple genetic variations with plasma levels of Von Willebrand Factor and clinical phenotype in Iranian patients with Von Willebrand disease type 1.

BACKGROUND: Genetic variations influence the Von Willebrand Factor plasma level and function. This study aims to evaluate the frequency and clinical phenotype effects of eight single nucleotide polymorphism candidates in four genes (VWF, STXBP5, CLEC4M, and ABO) in Iranian patients with VWD type 1. METHOD: The study recruited 50 patients with VWD type 1 and 100 healthy individuals. The demographic data from all participants were collected, and the High-Resolution Melting technique was used to determine the frequency of specific single nucleotide polymorphisms. Bleeding scores were also obtained from all patients to assess how these genetic variations might affect the severity of their bleeding symptoms. RESULTS: The study found notable variations in the occurrence of certain SNPs (rs7853989 and rs8176743 for ABO gene and rs1063856 and rs1063857 for VWF gene) between the control group and the patients. Additionally, the study discovered that two SNPs (rs868875 for CLEC4M gene and rs9390459 for STXBP5 gene) were significantly linked to the severity of bleeding, and two others (rs868875 for CLEC4M gene and rs8176746 for ABO gene) were associated with reduced levels of VWF antigen in the patients. CONCLUSION: According to this study, the above-selected SNPs can cause variations in VWF plasma levels in patients with VWD type 1. Furthermore, the effects of SNPs on bleeding phenotype prove the role of these SNPs in the severity of bleeding manifestations in patients.