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.

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.

Revealing of TLR-9 gene polymorphisms by qPCR HRM technique and their influence on TLR-9 serum level in acute myeloid leukemia patients: Case-control study.

Acute myeloid leukemia (AML) is one of the most common and fatal malignancies that affect adults, which can quickly become aggressive if left untreated, and leukemia cells invade the bone marrow. TLR-9 is an innate immune cell receptor sensitive to various PAMPs and encoded by the TLR-9 gene. As is often known, genetic polymorphisms in any gene can help the development of the disease, and these three polymorphisms, rs187084, rs5743836, and rs352140 of TLR-9, have been studied in many different cancer disorders. Therefore, this study aimed to discover the multiple forms of a TLR-9 gene in a sample of Iraqi AML patients. A total of 120 participants in a case-control study were enrolled in the current study. Using CBC, some hematological parameters were evaluated, and the serum level of TLR-9 was assessed using the ELISA technique. DNA was extracted directly from blood, and a high-resolution melting (HRM) analysis was then carried out. The results revealed a significant difference in some blood parameters among patients and healthy control, while WBC and lymphocytes were without an evident difference between the two groups of the current investigation. The serum concentration of TLR-9 showed an elevated level in patients (P value < 0.01). Nonetheless, this increase was not affected by the genotype patterns of polymorphisms. According to the P-value, there was a significant difference in wild genotypes of the three polymorphisms (rs187084, rs5743836, and rs352140). At the same time, the odds ratio revealed the association with the disease as a protective factor. In contrast, there was a significant difference in the heterozygous and mutant genotypes of TLR-9 polymorphisms, though the odds ratio confirmed the association with the AML as a risk factor. The results of rs352140 were compatible with H.W.E since there were no significant differences between the observed and expected values for either patients or healthy controls. In contrast, the result of rs5743836 was not consistent with the HWE. Furthermore, although it corresponds with the healthy one, the finding of rs187084 conflicted with H.W.E. in the patient group. In conclusion, High serum levels of TLR-9 in patients could act as biomarkers for AML. The TLR-9 gene polymorphisms (rs187084, rs5743836, and rs352140) have been linked to an increased risk of AML and may impact the disease progression in the Iraqi population.

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.

Characteristics of Candida albicans Derived From HIV-Positive Individuals With Oral Candidiasis: Genotyping, Phenotypic Variation, Antifungal Susceptibility, and Biofilm Formation.

BACKGROUND: Oral candidiasis (OC) is one of the most common mucosal infections in those afflicted with HIV/AIDS. This study aimed to provide detailed information on the phenotype, genotype, antifungal susceptibility, and biofilm formation ability of oral Candida albicans isolated from HIV-infected patients with OC. METHODS: A total of 25 C. albicans isolates were collected from oral lesions of HIV-infected patients referred to Behavioral Diseases Counseling Center affiliated with Ahvaz Jundishapur University of Medical Sciences, Iran. The antifungal susceptibility testing was done according to CLSI M27 guideline (fourth edition). The crystal violet method was used to evaluate the biofilm formation ability of isolates. Different phenotypes were identified on yeast extract-peptone-dextrose agar medium supplemented with phloxine B. Genotyping analysis of the isolates was performed using high-resolution melting (HRM) assays and ABC genotyping. RESULTS: The highest and lowest susceptibility of the C. albicans isolates was found for fluconazole 24 (96%) and ITC 18 (72%), respectively. Forty-eight percent of the isolates had high biofilm formation ability and exhibited gray cell type. The most common genotype was genotype B (52%). HRM analysis of HIS3, EF3, and CDC3 markers showed three, four, and five different groups, respectively. CONCLUSION: Investigating the phenotype, antifungal susceptibility and biofilm formation ability of the C. albicans isolates obtained from oral lesions of HIV-infected patients revealed that the dominant genotypes in the current research could cause more serious infections from the oral source. We recommend further research with a larger sample size to determine the molecular epidemiology of C. albicans among HIV patients in Iran.

High-resolution melting real-time polymerase chain reaction 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 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 to 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.

Identification and Differentiation of the Fusarium graminearum NX-2 Chemotype Using High-Resolution Melting (HRM).

Fusarium head blight causes significant yield losses in wheat and other cereals and contaminates grain products with trichothecene mycotoxins. F. graminearum isolates are classified into different chemotypes depending on the type of mycotoxin produced, including the type B trichothecenes 3-acetyl deoxynivalenol (3-ADON), 15-acetyl deoxynivalenol (15-ADON), nivalenol (NIV), and the recently identified type A trichothecene NX-2. Molecular tools to differentiate NX-2 producers from other chemotypes have remained relatively laborious and time consuming. In this study, we developed and validated a high-resolution melting (HRM) assay that can identify NX-2 producers quickly and cost-effectively. By analyzing TRI1 coding sequences from 183 geographically diverse isolates representing all four F. graminearum chemotypes, we selected a 75-base pair region containing four non-synonymous single nucleotide polymorphisms (SNPs) that are specific to the NX-2 genotypes. The amplicon generated two HRM profiles, one of which was specific for only NX-2. We confirmed that the assay is robust across qPCR platforms and unambiguously differentiates NX-2 from other chemotypes using a panel of 72 diverse isolates previously collected from North America. The HRM assay was also successful in identifying NX-2 producers directly from DNA extracted from infected wheat spikes with varying levels of disease severity and fungal DNA. The assay can detect as little as 0.01 ng of fungal DNA in a background of 50 ng of plant DNA. This new diagnostic assay can be used for high-throughput molecular detection of the NX-2 chemotype of F. graminearum from infected plant samples and culture collections, thus making it a valuable tool for surveys of contemporary and historical FHB pathogen populations.

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.

High-resolution melting analysis to authenticate deer-derived materials in processed products in China using a cytochrome oxidase I mini-barcode.

BACKGROUND: Deer-derived materials (antler, venison, fetus, penis, bone, tail, and others) are some of the most valuable traditional animal-based medicinal and food materials in China. In production, processing, and trade, the quality of deer products varies. The market is confusing, and counterfeit and shoddy products are common. There is an urgent need to establish an accurate identification method. RESULTS: Two pairs of primers suitable for identifying deer-derived medicinal materials were obtained by screening the cytochrome oxidase I (COI) sequences of 18 species from nine genera of the deer family. The two primers were used to identify the species and adulteration of 22 batches of commercially available deer-derived products with a mini-barcode combining high-resolution melting (HRM) technology and methodical investigation. Deer-derived materials (sika and red deer) were correctly identified by species using varying DNA amounts (1 to 500 ng). The two pairs of primers COI-1FR and COI-2FR yielded melting temperatures (Tm) of 80.55 to 81.00 °C and 82.00 to 82.50 °C for sika deer, and 81.00 to 82.00 °C and 81.40 to 82.00 °C for red deer. Twenty-two batches of commercially available samples were analyzed by HRM analysis and conventional amplification sequencing, and it was found that the species samples had an error rate of species labeling of 31.8%. Four batches of samples were identified as mixed (adulterated) in the HRM analysis. CONCLUSION: The combination of DNA mini-barcode with HRM analysis facilitated the accurate identification of species of deer-derived materials, especially the identification of samples in an adulterated mixed state. © 2024 Society of Chemical Industry.

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.

Leishmania donovani Transmission Cycle Associated with Human Infection, Phlebotomus alexandri Sand Flies, and Hare Blood Meals, Israel1.

Cutaneous leishmaniasis caused by Leishmania major or L. tropica and visceral leishmaniasis caused by L. infantum have been reported in Israel. We collected Phlebotomus spp. sand flies in the Negev desert of southern Israel to identify circulating Leishmania spp. Of 22,636 trapped sand flies, 80% were P. alexandri. We sequenced Leishmania-specific internal transcribed spacer 1 fragments and K26 genes. Of 5,019 Phlebotomus female sand flies, 2.5% were Leishmania DNA-positive; 92% of infections were L. donovani. Phylogenetic analyses showed separate clustering of L. donovani and L. infantum. P. alexandri flies positive for L. donovani harbored blood meals from European hares. Leishmania DNA isolated from a patient with cutaneous leishmaniasis who lived in the survey area was identical to L. donovani from P. alexandri flies. We report circulation of L. donovani, a cause of visceral leishmaniasis, in southern Israel. Prompt diagnosis and Leishmania spp. identification are critical to prevent leishmaniasis progression.

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.

High-Resolution Melting-Based Marker Development for Wheat Leaf Rust Resistance Gene Lr34.

Deploying adult plant resistance (APR) against rust diseases is an important breeding objective of most wheat-breeding programs. The gene Lr34 is an effective and widely deployed broad-spectrum APR gene in wheat against leaf rust fungus Puccinia triticina. Various molecular markers have been developed for Lr34, but they either require post-PCR handling processes or are not economical. Herein, we developed a high-resolution melting (HRM)-based diagnostic assay for Lr34 based on a 3-bp 'TTC' deletion in exon 11 of the resistant allele. The susceptible cultivar Thatcher (Tc) and the near-isogenic Thatcher line (RL6058) with Lr34 yielded distinct melting profiles and were differentiated with high reproducibility. For further validation, all three copies of Lr34 were cloned in plasmid vectors, and HRM analysis using individual and combination (equimolar mixture of three copies) homoeologs yielded distinct melting profiles. An additional layer of genotyping was provided by a LunaProbe assay. The allele-specific probes successfully distinguished the homoeologs but not Tc and RL6058. Furthermore, the practical deployment of the HRM assay was tested by running the marker on a set of breeding lines. When compared with a kompetitive allele-specific PCR (KASP) Lr34 assay, the HRM assay had similar genotyping results and was able to accurately differentiate the resistant and susceptible breeding lines. However, our HRM assay was unable to detect the heterozygote. To our knowledge, this is the first report of an HRM assay for genotyping a wheat rust resistance gene.

High-throughput identification of tuna (Thunnus spp.) larvae in the Gulf of Mexico using unlabelled-probe high-resolution melting analysis.

The genus Thunnus (family Scombridae) comprises eight species of tunas of which all but one are targeted by industrialized fisheries. Although intact individuals of these species can be distinguished by morphological characteristics, researchers and managers often rely on dressed, frozen, juvenile or larval fish samples, which often necessitates the identification of molecular species. Here the authors investigate short amplicon (SA) and unlabelled probe high-resolution melting analysis (UP-HRMA) as a low-cost, high-throughput molecular genotyping assay capable of distinguishing between albacore tuna (Thunnus alalunga), blackfin tuna (Thunnus atlanticus), bigeye tuna (Thunnus obesus), Atlantic bluefin tuna (Thunnus thynnus) and yellowfin tuna (Thunnus albacares) in the Gulf of Mexico. Although SA-HRMA of variable regions in the NADH dehydrogenase subunit 4 (ND4) and subunit 5 (ND5), and subunit 6 (ND6) of the mtDNA genome did yield some species-specific diagnostic melting curves (e.g., ND4 assay can reliably distinguish Atlantic bluefin tuna), genotype masking produced excessive variation in melting curves for reliable multi-species identification. To minimize the genotyping masking of SA-HRMA a 26 base pair long UP containing four SNPs was developed within a 133 bp segment of ND4. The UP-HRMA is able to reliably distinguish Gulf of Mexico species T. thynnus, T. obesus, T. albacares and T. atlanticus by UP melting temperature at 67, 62, 59 and 57°C, respectively. The developed UP-HRMA assay is a lower-cost, higher-throughput, alternative to previously published molecular assays for tuna identification that can be easily automated for large data sets, including ichthyological larval surveys, fisheries specimens lacking distinguishing morphological characteristics or detection of fraudulent trading of tuna species.

Two-Step Identification of N-, S-, R- and T-Cytoplasm Types in Onion Breeding Lines Using High-Resolution Melting (HRM)-Based Markers.

High-resolution melting (HRM) analysis is a powerful detection method for fast, high-throughput post-PCR analysis. A two-step HRM marker system was developed for identification of the N-, S-, R- and T-cytoplasms of onion. In the first step for the identification of N-, S- and R-cytoplasms, one forward primer was designed to the identical sequences of both cox1 and orf725 genes, and two reverse primers specific to the polymorphic sequences of cox1 and orf725 genes were used. For the second step, breeding lines with N-cytoplasm were evaluated with primers developed from the orfA501 sequence to distinguish between N- and T-cytoplasms. An amplicon with primers to the mitocondrial atp9 gene was used as an internal control. The two-step HRM marker system was tested using 246 onion plants. HRM analysis showed that the most common source of CMS, often used by Russian breeders, was S-cytoplasm; the rarest type of CMS was R-cytoplasm; and the proportion of T-cytoplasm among the analyzed breeding lines was 20.5%. The identification of the cytoplasm of a single plant by phenotype takes from 4 to 8 years. The HRM-based system enables quick and easy distinguishing of the four types of onion cytoplasm.

MS-HRM protocol: a simple and low-cost approach for technical validation of next-generation methylation sequencing data.

DNA methylation is a fundamental epigenetic process and have a critical role in many biological processes. The study of DNA methylation at a large scale of genomic levels is widely conducted by several techniques that are next-generation sequencing (NGS)-based methods. Methylome data revealed by DNA methylation next-generation sequencing (mNGS), should be always verified by another technique which they usually have a high cost. In this study, we offered a low-cost approach to corroborate the mNGS data. In this regard, mNGS was performed on 6 colorectal cancer (case group) and 6 healthy individual colon tissue (control group) samples. An R-script detected differentially methylated regions (DMRs), was further validated by high resolution melting (MS-HRM) analysis. After analyzing the data, the algorithm found 194 DMRs. Two locations with the highest level of methylation difference were verified by MS-HRM, which their results were in accordance with the mNGS. Therefore, in the present study, we suggested MS-HRM as a simple, accurate and low-cost method, useful for confirming methylation sequencing results.

Global DNA methylation profile at LINE-1 repeats and promoter methylation of genes involved in DNA damage response and repair pathways in human peripheral blood mononuclear cells in response to γ-radiation.

DNA methylation is an epigenetic mechanism, which plays an important role in gene regulation. The present study evaluated DNA methylation profile of LINE1 repeats and promoter methylation of DNA damage response (DDR) and DNA repair (DR) genes (PARP1, ATM, BRCA1, MLH1, XPC, RAD23B, APC, TNFα, DNMT3A, MRE11A, MGMT, CDKN2A, MTHFR) in human peripheral blood mononuclear cells (PBMCs) of healthy donors in response to γ-radiation. Methylation level was correlated with gene expression profile of selected DDR and DR genes (APC, MLH1, PARP1, MRE11A, TNFα, MGMT) to understand their role in gene regulation. Blood samples were collected from 15 random healthy donors, PBMCs were isolated, exposed to 0.1 Gy (low) and 2.0 Gy (high) doses of γ-radiation and proliferated for 48 h and 72 h. Genomic DNA and total RNA were isolated from irradiated PBMCs along with un-irradiated control. Methylation profile was determined from bisulphite converted DNA and amplified by methylation sensitive high resolution melting (MS-HRM) method. Total RNA was converted to cDNA and relative expression was analysed using real time quantitative-PCR. Our results revealed that at 0.1 Gy, MRE11A and TNFα showed significant (P < 0.05) increase in methylation at 72 h. At 2.0 Gy, significant increase (P < 0.05) in methylation profile was observed at LINE1, MRE11A, PARP1, BRCA1, DNMT3A and RAD23B at 48 h and 72 h. PARP1 showed significant positive correlation of methylation status with gene expression. In conclusion, low and high doses of γ-radiation have significant influence on DNA methylation status of LINE1, DDR and DR genes suggesting their potential role as epigenetic signatures in human PBMCs, which can be further explored in human populations.

Estimation of Lewis-negative alleles by high-resolution melting analysis of three tag SNPs of FUT3.

BACKGROUND AND OBJECTIVES: The expression of type 1 chain Lewis blood group antigens is regulated by secretor-type α(1,2)fucosyltransferase, encoded by FUT2, and Lewis α(1,3/1,4)fucosyltransferase, encoded by FUT3. Accumulating evidence has linked Lewis phenotypes or genotypes to various clinical conditions. Thus, in addition to FUT2, large-scale FUT3 genotyping is important. Because FUT3 has two paralogous genes (FUT5 and FUT6) with high DNA sequence similarity, we should select the polymerase chain reaction (PCR) primers carefully for FUT3 genotyping. Previously, we suggested that 13G>A (rs28362458), 59T>G (rs28362459) and 202T>C (rs812936) could be selected as tag single nucleotide polymorphisms (SNPs) for detection of Lewis-negative alleles (le). MATERIALS AND METHODS: In this study, three high-resolution melting (HRM) analyses for genotyping these SNPs were developed and applied for 140 Japanese, eight Ghanaians and four Sinhalese subjects. RESULTS: Each of three genotypes of 13G>A (G/G, G/A, A/A), 59T>G (T/T, T/G, G/G) and 202T>C (T/T, T/C, C/C) was discriminated clearly. Although we need to be careful in interpretation of results due to SNPs other than the 59T>G in the amplicon, the results of 59T>G genotyping were in full agreement with the results by a previous PCR-restriction fragment length polymorphism analysis in 140 Japanese. In addition, three heterozygotes of 202C substitution were identified, and no one having a 13A substitution was found in 140 Japanese. CONCLUSION: The present HRM analyses are useful and reliable methods for large-scale estimation of le alleles.

Molecular identification and anti-malarial drug resistance profile of Plasmodium falciparum from patients attending Kisoro Hospital, southwestern Uganda.

BACKGROUND: The evolution of malaria infection has necessitated the development of highly sensitive diagnostic assays, as well as the use of dried blood spots (DBS) as a potential source of deoxyribonucleic acid (DNA) yield for polymerase chain reaction (PCR) assays. This study identified the different Plasmodium species in malaria-positive patients, and the anti-malarial drug resistance profile for Plasmodium falciparum using DBS samples collected from patients attending Kisoro Hospital in Kisoro district, Southwestern Uganda. METHODS: The blood samples were prospectively collected from patients diagnosed with malaria to make DBS, which were then used to extract DNA for real-time PCR and high-resolution melting (HRM) analysis. Plasmodium species were identified by comparing the control and test samples using HRM-PCR derivative curves. Plasmodium falciparum chloroquine (CQ) resistance transporter (pfcrt) and kelch13 to screen the samples for anti-malarial resistance markers. The HRM-PCR derivative curve was used to present a summary distribution of the different Plasmodium species as well as the anti-malarial drug profile. RESULTS: Of the 152 participants sampled, 98 (64.5%) were females. The average age of the participants was 34.9 years (range: 2 months-81 years). There were 134 samples that showed PCR amplification, confirming the species as Plasmodium. Plasmodium falciparum (N = 122), Plasmodium malariae (N = 6), Plasmodium ovale (N = 4), and Plasmodium vivax (N = 2) were the various Plasmodium species and their proportions. The results showed that 87 (71.3%) of the samples were sensitive strains/wild type (CVMNK), 4 (3.3%) were resistant haplotypes (SVMNT), and 31 (25.4%) were resistant haplotypes (CVIET). Kelch13 C580Y mutation was not detected. CONCLUSION: The community served by Kisoro hospital has a high Plasmodium species burden, according to this study. Plasmodium falciparum was the dominant species, and it has shown that resistance to chloroquine is decreasing in the region. Based on this, molecular identification of Plasmodium species is critical for better clinical management. Besides, DBS is an appropriate medium for DNA preservation and storage for future epidemiological studies.

A new insight into the SNP genotyping using high-resolution melting method after the correlation analysis of the SNPs with WSSV-resistant traits.

Procambarus clarkii is an important freshwater cultured crayfish in China. With the gradual development of its aquaculture industry, research on white spot disease, which is harmful to healthy culture of P. clarkii, increases gradually. The prophenoloxidase (proPO) system is an important part of crayfish's innate immunity and plays a role in virus resistance. In this study, based on the early discovery of three SNP sites in the intron of proPO gene, the linkage disequilibrium and haplotype were analyzed for the SNPs, and it was found that there was a strong linkage disequilibrium relationship among them. Through the analysis on association between the haplotypes and genotype of each SNP site with the WSSV-resistant traits, the detection of the SNP_7081 genotype was considered as the most convenient and efficient way for WSSV-resistant group selection. Furtherly, the high-resolution melting curve (HRM), which is a rapid and economic genotyping method, was chosen to establish for SNP_7081 site genotyping. The 68 bp target fragment with 27.94% GC content was amplified and melting curve analysis were performed. However, the appearance of false negatives which led to unable automatically grouped although the melting curves of genotypes CC, C>T and T>C were obviously different, and could be treated as standard to manually genotype the samples with an accuracy rate of 97.61%. The low GC content which correlated with the Tm value, was confirmed as the reason for the false negatives by the assay about the recombinant plasmid PMD18-T-SNP_7081 constructed with 45.24% GC content. Eventually, the adaptor primers were used to increase the GC content of the target fragment, and a modified HRM method for genotyping SNP_7081 site that could group automatically was established, which could provide a new insight for the HRM method to genotype SNPs.