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INTRODUCTION: The C-C chemokine receptor type 5 (CCR5) is a major co-receptor for human immunodeficiency virus (HIV). Some individuals carry the CCR5 delta-32 genetic polymorphism. People with homozygous CCR5 delta-32 gene are nearly completely resistant to HIV-1 infection. High-resolution melting curve (HRM) analysis is a post-PCR technique utilized for identifying genetic variations in a quick, affordable, and closed-tube assay. The objective of this study was to develop an HRM assay for easy detection of delta-32 mutations. MATERIALS AND METHODS: DNA was extracted from peripheral blood mononuclear cells. HRM was performed to detect delta-32 mutation. The study investigated the impact of various factors, including annealing temperature, template concentration, touchdown PCR, additives, amplicon size, and program settings, on HRM Tm differentiation. RESULTS: It was expected that there would be a 4°C Tm difference between amplicons with and without delta-32 mutation, but the test showed a difference of only 2.3°C. In attempts to identify heterozygote delta-32 variants, a Tm difference of only 0.4°C could be achieved. Various modifications were applied, such as adjusting the template concentration, using touchdown PCR, and adding DMSO and glycerol. However, none of these changes helped to differentiate the Tm effectively, especially in delta-32 heterozygote samples. CONCLUSION: The HRM test identified four samples with heterozygote mutations in each HIV-infected (8.89%) and control (5.72%) groups. More importantly, this study showed that identifying the delta-32 mutation of the CCR5 gene using HRM assay is not as straightforward as previously suggested in some literature, and it requires special setup conditions.

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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.

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Leucemia Mieloide Aguda , Polimorfismo de Nucleótido Simple , Receptor Toll-Like 9 , Adulto , Femenino , Humanos , Masculino , Estudios de Casos y Controles , Predisposición Genética a la Enfermedad , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/sangre , Polimorfismo de Nucleótido Simple/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Toll-Like 9/genética

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Agarwood is a valuable traditional medicine and fragrance. The production process is a typical injury-induced defense response. Currently, there are approximately 22 known species in the genus Aquilaria Lam., all of which can produce agarwood, whereas there are only two legal species of traditional Chinese medicinal agarwood, Aquilaria sinensis (Lour.) Spreng. and Aquilaria agallocha (Lour.) Roxb. The Taiwan herbal Pharmacopoeia of China stipulates that the medicinal agarwood species are A. sinensis and its relatives in the same genus. Moreover, there are five species of agarwood available for clinical medicinal use in Japan, including A. agallocha and A. sinensis, which are often confused with each other or used in a mixed way in the trade process. Therefore, accurate identification of traditional Chinese medicinal agarwood species is important to ensure the authenticity of traditional medicines and to guide the safety of clinical medication. In this study, 59 specific single-nucleotide polymorphism loci were screened and obtained from the chloroplast genomes of 12 species of the genus Aquilaria Lam. We established an identification method for traditional Chinese medicinal agarwood using mini-barcoding combined with high-resolution melting (HRM) and designed and validated 10 pairs of primers from the psbM-trnD, psbA, rps16, petN, ndhE-psaC, rps4, atpE, ycf1, rps15-trnN, and matK regions. The amplification products were all less than 200 bp, with a high success rate of amplification. The method was applied to successfully identify traditional Chinese medicinal agarwood species from commercial agarwood samples. Overall, the sensitivity of this method was sufficient to detect 1% of adulterants in medicinal agarwood products, proving that mini-barcoding HRM is a powerful and flexible tool. This method can be used as a fast and effective high-throughput method for authenticity testing of traditional Chinese medicinal agarwood and its raw materials containing agarwood-containing proprietary Chinese medicines and is recommended for industrial applications.

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Blood culture, the gold-standard method for identifying pathogens in bloodstream infections, is time-consuming and demonstrates low sensitivity. These drawbacks are related to high mortality, especially among pediatric oncology patients presenting febrile neutropenia episodes. Here we describe two novel High-Resolution Melting assays designed for pathogen detection in bloodstream infections. The assays were initially evaluated using five sepsis-associated pathogens. Both assays demonstrated 100 % specificity, detected as low as 100 fg of bacterial DNA, and exhibited reproducibility. Clinical isolates from blood cultures were 100 % identified by both assays. Moreover, blind and direct identification of blood samples from pediatric cancer patients demonstrated sensitivities of 61.5 % and 69.2 % for "Primer Set 1" and "Primer Set 2", respectively. Our study highlights the potential of HRM-based assays as a rapid and efficient diagnostic approach for sepsis-related microorganisms. Further advancements could enhance their clinical utility for better management of febrile neutropenia episodes, especially in pediatric oncology patients.

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Neoplasias , Sensibilidad y Especificidad , Humanos , Niño , Neoplasias/complicaciones , Bacteriemia/diagnóstico , Bacteriemia/microbiología , Sepsis/microbiología , Sepsis/diagnóstico , Reproducibilidad de los Resultados , Técnicas de Diagnóstico Molecular/métodos , Bacterias/aislamiento & purificación , Bacterias/clasificación , Bacterias/genética , Preescolar , ADN Bacteriano/genética , Lactante

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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.

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Chestnut production is considered one of the most important economic resources of rural mountainous areas in Greece. Lately, producers report a steep rise in the incidence of brown rot disease caused by the fungus Gnomoniopsis smithogilvyi (Gnomoniaceae, Diaporthales), which results in severe chestnut rot. The pathogen is considered an emerging pathogen in many countries worldwide (Italy, France, Switzerland, Australia, New Zealand). This study aimed at (a) exploring the incidence of the brown rot disease in Vria (Regional Unit of Pieria, Region of Central Makedonia, Greece), (b) isolating and identifying the causal agent of the disease, (c) exploring the fungus presence at different phenological stages of the chestnut trees, and (d) implementing species-specific Bar- High Resolution Melting Analysis (HRM) for the early detection of G. smithogilvyi in chestnuts. G. smithogilvyi occurrence in chestnut tissues was more severe in June (59 %), nearly disappeared in July (19 %) and August (7 %) and increased again during harvesting time in September (57 %). This result could be attributed to a sum of different factors, including climate conditions. Moreover, it was demonstrated that G. smithogilvyi can be identified using a Bar-HRM analysis of chestnut tissues (buds, flowers and nuts). Results of this study clearly demonstrate that Bar-HRM can be used for the accurate, rapid and reliable identification of G. smithogilvyi universally on infected samples from different localities.

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Ascomicetos , Fagaceae , Flores , Enfermedades de las Plantas , Enfermedades de las Plantas/microbiología , Ascomicetos/aislamiento & purificación , Ascomicetos/genética , Ascomicetos/clasificación , Grecia , Flores/microbiología , Fagaceae/microbiología , Incidencia

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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.

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Metilación de ADN , Reacción en Cadena en Tiempo Real de la Polimerasa , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Desnaturalización de Ácido Nucleico , Semiconductores , Temperatura de Transición , Reproducibilidad de los Resultados

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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.

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Tipificación de Secuencias Multilocus , Humanos , Tipificación de Secuencias Multilocus/métodos , Temperatura de Transición , Mycobacterium/genética , Mycobacterium/clasificación , Mycobacterium/aislamiento & purificación , Proteínas Bacterianas/genética , Micobacterias no Tuberculosas/genética , Micobacterias no Tuberculosas/clasificación , Micobacterias no Tuberculosas/aislamiento & purificación , ADN Bacteriano/genética , Infecciones por Mycobacterium no Tuberculosas/microbiología , Infecciones por Mycobacterium no Tuberculosas/diagnóstico

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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.

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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.

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Metilación de ADN , Desnaturalización de Ácido Nucleico , Calibración , Humanos , Regiones Promotoras Genéticas , Metilasas de Modificación del ADN/genética , Proteínas Supresoras de Tumor/genética , Temperatura , Enzimas Reparadoras del ADN/genética , Islas de CpG , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ADN/normas , ADN/genética

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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.

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Antieméticos , Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias de la Mama , Ciclofosfamida , Náusea , Polimorfismo de Nucleótido Simple , Receptores de Neuroquinina-1 , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Persona de Mediana Edad , Antieméticos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Receptores de Neuroquinina-1/genética , Náusea/inducido químicamente , Náusea/genética , Ciclofosfamida/efectos adversos , Ciclofosfamida/uso terapéutico , Ciclofosfamida/administración & dosificación , Vómitos/inducido químicamente , Vómitos/genética , Antagonistas del Receptor de Neuroquinina-1/uso terapéutico , Adulto , Estudios de Asociación Genética , Haplotipos , Anciano , Docetaxel/uso terapéutico , Docetaxel/efectos adversos , Farmacogenética , Antraciclinas/efectos adversos , Antraciclinas/uso terapéutico , Genotipo

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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.

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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.

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ADN , Desnaturalización de Ácido Nucleico , Humanos , ADN/genética , ADN/química , Reacción en Cadena de la Polimerasa/métodos

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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.

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Escherichia coli , Leche , Reacción en Cadena en Tiempo Real de la Polimerasa , Leche/microbiología , Animales , Escherichia coli/genética , Reacción en Cadena de la Polimerasa Multiplex/métodos , Sensibilidad y Especificidad , Reproducibilidad de los Resultados

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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.

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Infecciones por Circoviridae , Circovirus , Enfermedades de las Aves de Corral , Animales , Pollos/genética , Reacción en Cadena de la Polimerasa/veterinaria , Circovirus/genética , Infecciones por Circoviridae/diagnóstico , Infecciones por Circoviridae/veterinaria , Infecciones por Circoviridae/epidemiología

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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.

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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.

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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Metabolómica , Cromatografía Líquida de Alta Presión , Espectrometría de Masas , Extractos Vegetales

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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.

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Cardiopatías Congénitas , Metilenotetrahidrofolato Reductasa (NADPH2) , Recién Nacido , Humanos , Niño , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Metilenotetrahidrofolato Reductasa (NADPH2)/metabolismo , Pakistán/epidemiología , Cardiopatías Congénitas/genética , Genotipo , Reacción en Cadena de la Polimerasa , Predisposición Genética a la Enfermedad , Estudios de Casos y Controles

RESUMEN

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.

Asunto(s)

Dermatoglifia del ADN , Repeticiones de Microsatélite , Masculino , Humanos , Femenino , Dermatoglifia del ADN/métodos , Reproducibilidad de los Resultados , ADN/análisis , Cromosomas Humanos Y

RESUMEN

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.

Asunto(s)

Phytophthora , Alelos , Haplotipos/genética , Phytophthora/genética , Patología Molecular , Genotipo , Enfermedades de las Plantas/genética , Resistencia a la Enfermedad/genética