High-resolution Melting Analysis Research Articles

Establishing a Mouse Genotyping Core Facility Based on Automation, High-resolution Melting Analysis, and Purpose-developed MATLAB Applications for Sample Tracking, Data Analysis, and Report Generation

An in-house genotyping facility should aim to be more cost-effective than outsourced service and more reliable than genotyping performed by short-term employees or students of individual research groups. Reliable genotyping allows efficient and economical management of mice colonies and promotes accurate and reproducible research results. Here we provide a detailed description of our approach to establishing a genotyping core facility, relying on automated PCR assembly and high-resolution melting (HRM) analysis (first derivative). The workflow we devised was tightly managed by purpose-designed applications developed using MATLAB App Designer that allowed straightforward work planning, ensured sample tracking throughout the process, and provided a platform for reliable data analysis and generation of genotyping reports. We successfully transitioned PCR product analysis of more than 250 different target genes from standard gel electrophoresis to the more advanced HRM analysis. About 23% of the target genes required a redesign of primers to adapt to our protocol. The process was highly universal, and only 2% of the target genes required deviation from the standard PCR method to a more restricted protocol that reduces the amplification of nonspecific products. We currently run more than 1,000 PCR reactions weekly, of samples taken at weaning or experimental endpoint, and assemble a large variety of target genes in every PCR plate. We also showed that genotyping of blastocytes instead of embryos can serve as quality control of cryopreservation. Thus, our genotyping protocol promotes the 3Rs (Replacement, Reduction, and Refinement) principles. Our refined genotyping process facilitates cost-effective colony management, replaces tissue types as well as traditional methods with advanced ones, and provides reliable results in a timely manner. MATLAB codes and related data are available in supplementary materials and online.

Simultaneous detection and differentiation of classical Muscovy duck reovirus and goose-origin Muscovy duck reovirus by RT-qPCR assay with high-resolution melting analysis.

Classical Muscovy duck reovirus (C-MDRV) and goose-origin Muscovy duck reovirus (Go-MDRV) infections cause "Liver white-spots disease" in Muscovy duckling and gosling. It is difficult to differentiate the infections caused by C-MDRV and Go-MDRV using conventional serological methods. Specific primers were designed and synthesized according to σNS and λA nucleotide sequences of C-MDRV and Go-MDRV, respectively. The PCR amplified products were cloned into the pMD-18-T vector. The recombinant plasmid DNA was used to establish an SYBR Green І based duplex real-time PCR assay for the simultaneous detection and differentiation of C-MDRV and Go-MDRV using high-resolution melting (HRM) analysis. The specificity, sensitivity, and repeatability of the methodology were examined based on the optimization of the reaction system and amplification conditions. C-MDRV and Go-MDRV were identified by their distinctive melting temperatures with 84.50 ± 0.25°C for C-MDRV and 87.50 ± 0.20°C for Go-MDRV, respectively. The amplifications were specific, and other non-targeted waterfowl viruses employed in this study did not show normalized melting peaks. The intra- and inter-assay coefficients of variations were between 0.05 and 1.83%, demonstrating good repeatability. The detection limits of this assay were 51.4 copies·μl-1 for C-MDRV and 61.8 copies·μl-1 for Go-MDRV, respectively. A total of 45 clinical samples were tested by RT-qPCR, with positive rates of 15.56% for C-MDRV and 22.22% for Go-MDRV, without co-infections. These results suggest that this duplex RT-qPCR method is highly sensitive, specific, and reproducible. The HRM assay established in this study provides a powerful tool for the differential detection and epidemiological investigation of C-MDRV and Go-MDRV.

Establishing a Mouse Genotyping Core Facility Based on Automation, High-resolution Melting Analysis, and Purpose-developed MATLAB Applications for Sample Tracking, Data Analysis, and Report Generation.

An in-house genotyping facility should aim to be more cost-effective than outsourced service and more reliable than genotyping performed by short-term employees or students of individual research groups. Reliable genotyping allows efficient and economical management of mice colonies and promotes accurate and reproducible research results. Here we provide a detailed description of our approach to establishing a genotyping core facility, relying on automated PCR assembly and high-resolution melting (HRM) analysis (first derivative). The workflow we devised was tightly managed by purpose-designed applications developed using MATLAB App Designer that allowed straightforward work planning, ensured sample tracking throughout the process, and provided a platform for reliable data analysis and generation of genotyping reports. We successfully transitioned PCR product analysis of more than 250 different target genes from standard gel electrophoresis to the more advanced HRM analysis. About 23% of the target genes required a redesign of primers to adapt to our protocol. The process was highly universal, and only 2% of the target genes required deviation from the standard PCR method to a more restricted protocol that reduces the amplification of nonspecific products. We currently run more than 1,000 PCR reactions weekly, of samples taken at weaning or experimental endpoint, and assemble a large variety of target genes in every PCR plate. We also showed that genotyping of blastocytes instead of embryos can serve as quality control of cryopreservation. Thus, our genotyping protocol promotes the 3Rs (Replacement, Reduction, and Refinement) principles. Our refined genotyping process facilitates cost-effective colony management, replaces tissue types as well as traditional methods with advanced ones, and provides reliable results in a timely manner. MATLAB codes and related data are available in supplementary materials and online.

Assessing ST18 gene polymorphisms (rs17315309, rs2304365) in Iraqi patients with Pemphigus vulgaris.

Pemphigus vulgaris (PV) is a potentially fatal autoimmune disease characterized by blistering of the skin, mucous membranes, and oral cavity. Genetics are implicated in its etiology, with the ST18 gene identified as a potential risk factor for pemphigus in certain populations, suggesting its role as a novel molecular target for therapeutic intervention. This study aimed to detect single nucleotide polymorphisms (SNPs) rs17315309 A/G and rs2304365 C/G in the ST18 gene among Iraqi/Arabic patients with PV. A total of 90 Iraqi subjects participated in this study, including 45 patients diagnosed with PV and 45 healthy controls. SNP analysis was performed using High-Resolution Melt Analysis (HRMA) with Eva Green I Dye. For SNP rs17315309 A/G, the distribution of heterozygous genotypes showed highly significant differences between the patient and healthy groups (P = 0.005), with the mutant G-allele being significantly more prevalent in patients than in the healthy group (P = 0.001). In contrast, for SNP rs2304365 C/G, the distribution of heterozygous and mutant genotypes did not differ significantly between patients and healthy individuals (P = 0.8 and P = 0.3, respectively), with the mutant G-allele also showing no significant difference (P = 0.4). Our data indicate a significant association between PV and the rs17315309 A/G SNP in the ST18 gene among the Iraqi population of Arabic origin. However, no association was found between patients with PV and the rs2304365 C/G SNP in the same gene.

TaqMan® and HRM approaches for SNP genotyping in genetic traceability of musts and wines

The fight against fraud in the wine sector requires continuous improvements and validations of new technologies applicable to musts and wines. Starting from published data from the Vitis18kSNP array, a series of new specific single nucleotide polymorphism (SNP) markers have been identified for some important north-western Italian cultivars, such as Barbera, Dolcetto and Arneis (Vitis vinifera L.), used in the production of high-quality wines under Protected Denomination of Origin. A pair of new SNP markers for each grape variety were selected and validated using two real-time PCR techniques: TaqMan® genotyping assays and high-resolution melting analysis (HRM). The TaqMan® assay has proven to be more reliable and repeatable than HRM analysis because despite being an economical and versatile technique for the detection of different types of genomic mutations (SNPs, insertions or deletions), HRM has shown limitations in the presence of poor-quality DNA extracted from musts and wines. TaqMan® assays have successfully identified Barbera, Dolcetto and Arneis in their respective musts and experimental wines, and with good efficiency in commercial wines. Marked differences between genotypes were observed, varietal identification in Dolcetto-based musts/wines was more efficient than that in Arneis-based wines. Therefore, the TaqMan® assay has considerable potential for varietal identification in wines and the procedure described in the present work can be easily adapted to all wines with adequate setup of DNA extraction methods that should be adapted to different wines.

Quantitative DNA Methylation Analysis and Epigenotype-Phenotype Correlations in Taiwanese Patients with Silver-Russell Syndrome.

Background: Silver-Russell syndrome (SRS; OMIM #180860) is a clinically and genetically heterogeneous imprinting disorder characterized by prenatal and postnatal growth failure. The aim of this study was to identify the epigenotype-phenotype correlations in these patients using quantitative DNA methylation analysis. Methods: One hundred and eighty-three subjects clinically suspected of having SRS were referred for diagnostic testing by the methylation profiling of H19-associated imprinting center (IC) 1 and imprinted PEG1/MEST regions using methylation-specific high-resolution melting analysis and methylation quantification with the MassARRAY assay. Correlations between quantitative DNA methylation status and clinical manifestations of the subjects according to the Netchine-Harbison (N-H) clinical scoring system for SRS were analyzed. Results: Among the 183 subjects, 90 had a clinical diagnosis of SRS [N-H score ≥ 4 (maximum = 6)] and 93 had an SRS score < 4. Molecular lesions were detected in 41% (37/90) of the subjects with a clinical diagnosis of SRS, compared with 3% (3/93) of those with an N-H score < 4. The IC1 methylation level was negatively correlated with the N-H score. The molecular diagnosis rate was positively correlated with the N-H score. Thirty-one subjects had IC1 hypomethylation (IC1 methylation level <35% by the MassARRAY assay), seven had maternal uniparental disomy 7, and two had pathogenic copy number variants. Among the 90 subjects with an N-H score ≥ 4, the IC1 methylation level was significantly different between those with or without some clinical SRS features, including birth length ≤ 10th centile, relative macrocephaly at birth, normal cognitive development, body asymmetry, clinodactyly of the fifth finger, and genital abnormalities. Conclusions: This study confirmed the suitability of the N-H clinical scoring system as clinical diagnostic criteria for SRS. Quantitative DNA methylation analysis using the MassARRAY assay can improve the detection of epigenotype-phenotype correlations, further promoting better genetic counseling and multidisciplinary management for these patients.

DNA barcoding combined with high-resolution melting analysis to discriminate rhubarb species and its traditional Chinese patent medicines.

Introduction: Rhubarb is a frequently used and beneficial traditional Chinese medicine. Wild resources of these plants are constantly being depleted, meaning that rhubarb products have been subjected to an unparalleled level of adulteration. Consequentially, reliable technology is urgently required to verify the authenticity of rhubarb raw materials and commercial botanical drugs. Methods: In this study, the barcode-DNA high-resolution melting (Bar-HRM) method was applied to characterize 63 rhubarb samples (five Polygonaceae species: Rheum tanguticum, Rh. palmatum, Rh. officinale, Rumex japonicus and Ru. sp.) and distinguish the rhubarb contents of 24 traditional Chinese patent medicine (TCPM) samples. Three markers, namely ITS2, rbcL and psbA-trnH, were tested to assess the candidate DNA barcodes for their effectiveness in distinguishing rhubarb from its adulterants. A segment from ITS2 was selected as the most suitable mini-barcode to identify the botanical drug rhubarb in TCPMs. Then, rhubarbs and TCPM samples were subjected to HRM analysis based on the ITS2 barcode. Results: Among the tested barcoding loci, ITS2 displayed abundant sites of variation and was effective in identifying Polygonaceae species and their botanical origins. HRM analysis based on the ITS2 mini-barcode region successfully distinguished the authenticity of five Polygonaceae species and eight batches of TCPMs. Of the 18 TCPM samples, 66.7 % (12 samples) were identified as containing Rh. tanguticum or Rh. officinale. However, 33.3 % were shown to consist of adulterants. Conclusions: These results demonstrated that DNA barcoding combined with HRM is a specific, suitable and powerful approach for identifying rhubarb species and TCPMs, which is crucial to guaranteeing the security of medicinal plants being traded internationally.

Putative bloodmeal sources in Glossina austeni tsetse fly of Arabuko Sokoke National Reserve in Kenya.

Tsetse flies, the sole biological vectors of trypanosomiasis, are predominantly controlled using visual traps and targets baited with attractant lures. Formulation of the lures is informed by compositions of odors from vertebrate hosts preferred by specific tsetse species. However, there are no effective lures for Glossina austeni, a major vector of trypanosomiasis along eastern-coastal region of Africa. Formulation of the lure can be informed by knowledge of G. austeni, preferred vertebrate hosts. We thus sought to understand these hosts by assessment of putative bloodmeal sources of this tsetse fly in Arabuko Sokoke National Reserve where this species is naturally present. We sampled tsetse flies using NGU traps, isolated non-teneral G. austeni flies based on their feeding status, and identified vertebrate source of bloodmeals in their midgut contents using vertebrate 16S rRNA-PCR High-Resolution Melting analysis. We analyzed the relative vertebrate species frequencies in the bloodmeals using Fisher's exact tests. Overall, we trapped 122 flies, most of which (66.39%) were non-teneral, among which we successfully identified the vertebrate bloodmeals in 30 samples. Specifically, we detected putative suni antelope (Neotragus moschatus), harnessed bushbuck (Tragelaphus scriptus), buffalo (Syncerus caffer) and cattle (Bos taurus) derived bloodmeals. Putative suni antelope bloodmeals were significantly more frequent (63.22%), than those of the harnessed bushbuck (23.33%), buffalo (10.00%) or cattle (3.33%) (p < 0.05 Fisher's exact tests) among the samples analyzed. Suni antelope thus appears to predominate vertebrate bloodmeal source for G. austeni in the reserve, coincident with findings reported elsewhere, and is therefore a viable candidate for bioprospecting for G. austeni responsive attractants.

First report of cutaneous leishmaniasis caused by Leishmania donovani in Ethiopia

BackgroundLeishmaniasis is a common neglected tropical disease in Ethiopia. Visceral leishmaniasis (VL) caused by Leishmania donovani presents in the lowlands, while cutaneous leishmaniasis (CL) affects people living in the highlands. Although CL is described as being caused by Leishmania aethiopica, there is also evidence of L. tropica and L. major isolated from a patient, sand flies and potential reservoirs. Information on species causing CL in Ethiopia is patchy, and no nation-wide study has ever been done. Understanding which species are causing CL in Ethiopia can have important implications for patient management and disease prevention.MethodsWe analyzed stored routine samples and biobanked DNA isolates from previously conducted studies of CL patients from different centers in the north, center and south of Ethiopia. Species typing was performed using ITS-1 PCR with high-resolution melt (HRM) analysis, followed by HSP70 amplicon sequencing on a selection of the samples. Additionally, sociodemographic, clinical and laboratory data of patients were analyzed.ResultsOf the 226 CL samples collected, the Leishmania species could be determined for 105 (45.5%). Leishmania aethiopica was identified in 101 (96.2%) samples from across the country. In four samples originating from Amhara region, northwestern Ethiopia, L. donovani was identified by ITS-1 HRM PCR, of which two were confirmed with HSP70 sequences. While none of these four patients had symptoms of VL, two originated from known VL endemic areas.ConclusionsThe majority of CL was caused by L. aethiopica, but CL due to L. tropica and L. major cannot be ruled out. Our study is the first to our knowledge to demonstrate CL patients caused by L. donovani in Ethiopia. This should spark future research to investigate where, how and to which extent such transmission takes place, how it differs genetically from L. donovani causing VL and whether such patients can be diagnosed and treated successfully with the currently available tools and drugs.Graphical

10111-MPC-11 CLINICAL AND MOLECULAR NEUROPATHOLOGICAL FEATURES OF EPITHELIOID GLIOBLASTOMA

Abstract BACKGROUND Epithelioid Glioblastoma (E-GBM) has been identified as a pathological subtype of Glioblastoma. It is a rare glioblastoma and is mainly known in case reports, and the details of its clinical features and genetic characteristics are not clear. Here, we retrospecivly studied the results of clinical course and genetic features of E-GBM. METHODS Four patients who underwent surgery at our institution and were diagnosed with E-GBM were included in this study. Histological diagnosis was obtained by surgical excision in all cases. We extracted DNA from the resected tissues and analyzed glioma-related genes using various methods such as high resolution melting (HRM), MGMT methylation-specific HRM, digital PCR, and Multiple ligation-dependent probe amplification (MLPA) analysis. Moreover, we assayed methylation EPIC BeadChip arrays for each sample. RESULTS E-GBM tended to develop at a slightly younger age than other general glioblastoma. All patients underwent surgical resection followed by radiation therapy and concurrent temozolomide therapy. However, none of the patients were able to transition to temozolomide maintenance therapy due to disease progression, and the overall survival period was 8 to 19 weeks, indicating an extremely poor prognosis. Genetic analysis revealed both TERT promoter mutations and BRAFV600E mutation in all cases. In addition, IDH mutation was not found, and MGMT promoter methylation was unmethylated in all cases. EGFR amplification, CDKN2A/2B homozygous deletion and 1p/19q co-deletion were not found in any cases. Methylation-based classification did not reach E-GBM in all samples. CONCLUSION The simultaneous mutations of TERT promoter with BRAFV600E could serve as diagnostic molecular markers for the diagnosis of E-GBM. Standard therapy for glioblastoma was not expected to be effective in treating this tumor. It was considered necessary to introduce new therapeutic strategies such as early administration of BRAF and MEK inhibitors.

Rapid monitoring of SARS-CoV-2 variants of concern through high-resolution melt analysis

The current global pandemic of COVID-19 is characterized by waves of infection due to the emergence of new SARS-CoV-2 variants carrying mutations on the Spike (S) protein gene. Since autumn 2020 many Variants of Concern (VOC) have been reported: Alpha/B.1.1.7, Beta/B.1.351, Gamma/P.1, Delta/B.1.617.2, Omicron/B.1.1.529, and sublineages. Surveillance of genomic variants is currently based on whole-genome sequencing (WGS) of viral genomes on a random fraction of samples positive to molecular tests. WGS involves high costs, extended analysis time, specialized staff, and expensive instruments compared to a PCR-based test. To rapidly identify the VOCs in positive samples, six assays based on real-time PCR and high-resolution melting (HRM) were designed on the S gene and applied to 120 oro/nasopharyngeal swab samples collected from October 2020 to June 2022 (106 positive and 14 negative samples). Overall, the assays showed 100% specificity and sensitivity compared with commercial PCR tests for COVID-19. Moreover, 104 samples out of 106 (98.1%) were correctly identified as follows: 8 Wuhan (wild type), 12 Alpha, 23 Delta, 46 Omicron BA.1/BA.1.1, 15 Omicron BA.2/BA.4/BA.5. With our lab equipment, about 10 samples can be processed every 3 h at the cost of less than € 10 ($ 10.60) per sample, including RNA extraction. The implementation of this approach could help local epidemiological surveillance and clinical decision-making.

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.

Guardians of quality: advancing Castanea sativa traceability using DNA analysis from seed to processed food

Castanea sativa is one of the most appreciated species in Italy, due to timber and fruit quality; nuts are usually intended for fresh consumption, but also for the production of industrial foods. The demand for an efficient traceability system to identify Castanea species and cultivars along the supply chain, from the chestnut orchard to the processed product, is crucial to protect consumers and producers from frauds. Here we present a traceability protocol to identify the species of the Castanea genus and trace the cultivars of Castanea sativa, based on DNA markers (SSRs and SNPs) analyses. Microsatellites were applied to identify the cultivar in plant material (leaves, seed, and episperm) and processed food (creams, beverages, flour, and cookies). Although SSRs made it possible to trace cultivar starting from somatic tissues (e.g.: leaves), the presence of foreign paternal DNA from pollenizer plants in seed and processed food, interferes with the identification of the maternal one. To overcome this issue, we Illumina sequenced six C. sativa cultivars to highlight maternal-inherited markers. A set of SNP/INDEL markers in two chloroplastic regions were identified, able to effectively distinguish and trace C. sativa from C. mollissima and C. crenata. The High Resolution Melting (HRM) analysis was performed as a practical and efficient method for SNP/INDELvalidation and to develop a fast, reliable, and reproducible protocol for chestnut traceability in food.

Prevalence of BRCA1 and BRCA2 germline variants in an unselected pancreatic cancer patient cohort in Pakistan

BackgroundBRCA1 and BRCA2 (BRCA1/2) are the most frequently investigated genes among Caucasian pancreatic cancer patients, whereas limited reports are available among Asians. We aimed to investigate the prevalence of BRCA1/2 germline variants in Pakistani pancreatic cancer patients.MethodsOne hundred and fifty unselected and prospectively enrolled pancreatic cancer patients were comprehensively screened for BRCA1/2 germline variants using denaturing high-performance liquid chromatography and high-resolution melting analyses, followed by DNA sequencing of the variant fragments. The novel variants were analyzed for their pathogenic effect using in-silico tools. Potentially functional variants were further screened in 200 cancer-free controls.ResultsProtein truncating variant was detected in BRCA2 only, with a prevalence of 0.7% (1/150). A frameshift BRCA2 variant (p.Asp946Ilefs*14) was identified in a 71-year-old male patient of Pathan ethnicity, with a family history of abdominal cancer. Additionally, we found a novel variant in BRCA2 (p.Glu2650Gln), two previously reported variants in BRCA1 (p.Thr293Ser) and BRCA2 (p.Ile2296Leu) and a recurrent nonsense variant in BRCA2 (p.Lys3326Ter). These variants were classified as variants of uncertain significance (VUS). It is noteworthy that none of these VUS carriers had a family history of pancreatic or other cancers.ConclusionsIn this first study, BRCA1/2 pathogenic variant is identified with a low frequency in pancreatic cancer patients from Pakistan. Comprehensive multigene panel testing is recommended in the Pakistani pancreatic cancer patients to enhance genetic understanding in this population.

PATH-22. CLINICAL AND MOLECULAR NEUROPATHOLOGICAL FEATURES OF EPITHELIOID GLIOBLASTOMA

Abstract BACKGROUND Epithelioid Glioblastoma (E-GBM) has been identified as a pathological subtype of Glioblastoma. It is a rare glioblastoma and is mainly known in case reports, and the details of its clinical features and genetic characteristics are not clear. Here, we retrospecivly studied the results of clinical course and genetic features of E-GBM. METHODS Four patients who underwent surgery at our institution and were diagnosed with E-GBM were included in this study. Histological diagnosis was obtained by surgical excision in all cases. We extracted DNA from the resected tissues and analyzed glioma-related genes using various methods such as high resolution melting (HRM), MGMT methylation-specific HRM, digital PCR, and Multiple ligation-dependent probe amplification (MLPA) analysis. RESULTS E-GBM tended to develop at a slightly younger age than other general glioblastoma. All patients underwent surgical resection followed by radiation therapy and concurrent temozolomide therapy. However, none of the patients were able to transition to temozolomide maintenance therapy due to disease progression, and the overall survival period was 8 to 19 weeks, indicating an extremely poor prognosis. Genetic analysis revealed both TERT promoter mutations and BRAFV600E mutation in all cases. In addition, IDH mutation was not found, and MGMT promoter methylation was unmethylated in all cases. EGFR amplification, CDKN2A/2B homozygous deletion and 1p/19q co-deletion were not found in any cases. CONCLUSION The simultaneous mutations of TERT promoter with BRAFV600E could serve as diagnostic molecular markers for the diagnosis of E-GBM. Standard therapy for glioblastoma was not expected to be effective in treating this tumor. It was considered necessary to introduce new therapeutic strategies such as early administration of BRAF and MEK inhibitors.

Single-tube multiplex real-time PCR with EvaGreen and high-resolution melting analysis for diagnosis of α0-thalassemia--SEA,--THAI, and--CR type deletions.

Regions with a high prevalence of α-thalassemia (α-thal) require simple, rapid, and accurate tests for carrier screening and prenatal diagnosis. Diagnosis of multiple deletions in a single tube is necessary to clearly identify individuals with α0-thalassemia in the routine setting, especially in at-risk couples. Therefore, we aimed to develop a single-tube multiplex real-time PCR with EvaGreen and high-resolution melting (HRM) analysis for the identification of α0-thalassemia Southeast Asian (SEA), Thai and Chiang Rai (CR) type deletions. The results of the HRM analysis indicated that the amplified fragments from α0-thal--CR,--THAI,--SEA, and the wild-type α-globin gene had specific peak heights at mean melting temperature (Tm) values of 85.40°C, 86.50°C, 87.65°C, and 91.04°C, respectively. The frequencies of α0-thal--SEA,--THAI,--CR obtained from routine testing in 2,135 samples were 17.89%, 0.19% and 0.19%, respectively. This method would be useful for preventing Hb Bart's hydrops fetalis. Detection of multiple deletions in a single run is cost-effective, highly accurate and timesaving. This technique could enable wider α-thalassemia diagnosis in high prevalence areas and served as an example for thalassemia routine setting.

Distinguishing fanged frogs (Limnonectes) species (Amphibia: Anura: Dicroglossidae), from Thailand using high resolution melting analysis

Morphologically, species of fanged frogs (Limnonectes) are exceedingly similar, making it difficult to distinguish them within the complex. In Thailand, it has been difficult to distinguish between the sympatric species L. bannaensis and L. taylori, particularly among tadpoles, adolescents, and adult females. A precise identification contributes to a greater understanding of biodiversity, particularly for assessing distributions and population dynamics. Therefore, a novel approach is required. The objective of this study was to develop a high resolution melting analysis (HRM) for the rapid and accurate identification of six species of Limnonectes of the L. kuhlii complex found in Thailand, particularly the two sympatric fanged frogs. Here, HRM assays using 16S rRNA mitochondrial primers were designed and developed. There was as much as a 25.3% variation in the nucleotide sequence of the fragment amplified by HRM16S primers among the six species of Limnonectes. Prior to conducting an in vitro HRM, the DNA sequences were used in a simulation HRM, uMELT Quartz, to predict the melting curve for each species of Limnonectes. There were discrepancies between the predicted melting curves of each species generated by the programme. Consequently, in vitro HRM tests were conducted. The obtained melting curve and Tm values were consistent with those predicted, albeit with a slightly different Tm value and a more distinct melting curve. All evaluated species of Limnonectes could be easily distinguished from one another by comparing the melting curve shapes. The HRM assay was then used to confirm the species of 18 Limnonectes samples in comparison to the reference samples (confidence interval > 90%). In addition, the results of HRM were consistent with those of experts who used morphological analysis to identify species. The HRM was found to be useful, and therefore the method would also contribute to future ecological and systematic studies on the target species.

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.

Ticks (Acari: Ixodidae) infesting cattle in coastal Kenya harbor a diverse array of tick-borne pathogens

Ticks and the microbes they transmit have emerged in sub-Saharan Africa as a major threat to veterinary and public health. Although progress has been made in detecting and identifying tick-borne pathogens (TBPs) across vast agroecologies of Kenya, comprehensive information on tick species infesting cattle and their associated pathogens in coastal Kenya needs to be updated and expanded. Ticks infesting extensively grazed zebu cattle in 14 villages were sampled and identified based on morphology and molecular methods and tested for the presence of bacterial and protozoan TBPs using PCR with high-resolution melting analysis and gene sequencing. In total, 3,213 adult ticks were collected and identified as Rhipicephalus appendiculatus (15.8%), R. evertsi (12.8%), R. microplus (11.3%), R. pulchellus (0.1%), Amblyomma gemma (24.1%), A. variegatum (35.1%), Hyalomma rufipes (0.6%), and H. albiparmatum (0.2%). Ticks were infected with Rickettsia africae, Ehrlichia ruminantium, E. minasensis, Theileria velifera and T. parva. Coxiella sp. endosymbionts were detected in the Rhipicephalus and Amblyomma ticks. Co-infections with two and three different pathogens were identified in 6.9% (n = 95/1382) and 0.1% (n = 2/1382) of single tick samples, respectively, with the most common co-infection being R. africae and E. ruminantium (7.2%, CI: 4.6 – 10.6). All samples were negative for Coxiella burnetii, Anaplasma spp. and Babesia spp. Our study provides an overview of tick and tick-borne microbial diversities in coastal Kenya.

Multiplex Bar-HRM for differentiating Centella asiatica (L.) Urb. from possible substituent species

Global market demand for plant-derived products has increased significantly due to their extensive use in the treatment and prevention of various diseases. However, mislabeling of commercial herbal products poses a major safety concern and affects their efficacy. Differentiating species among Centella asiatica (L.) Urb., Hydrocotyle umbellata L., Bacopa monnieri L., and Bacopa caroliniana (Walter) B.L.Rob, which share morphological features and common local names is challenging. To address this issue, DNA barcoding coupled with high-resolution melting (Bar-HRM) analysis was performed using ITS, matK, and rbcL loci. Suitable primer sets were designed based on nucleotide diversity analysis from multiple alignments of each barcode. Our findings showed that multiplex Bar-HRM (mBar-HRM) using matK1 and rbcL1 successfully discriminated C. asiatica from the other species and enabled contamination detection of C. asiatica and species specificity of various medicinal plants. Analytical sensitivity determined the minimal DNA concentration to generate melting patterns at 0.01 ng per reaction. mBar-HRM was also tested for adulteration detection by mixing DNA from C. asiatica with H. umbellata or B. monnieri at more than 75%. Results demonstrated the potential of mBar-HRM as a reliable method for species authentication in C. asiatica-derived commercial products.