One-shot high-resolution melting curve analysis for KRAS point-mutation discrimination on a digital microfluidics platform

Proposed high-accuracy one-shot melting curve analysis on a digital microfluidics platform, enabling KRAS gene discrimination with single-nucleotide mutations in 3 seconds.

Evaluation of the Multiplex Real-Time PCR DermaGenius® Assay for the Detection of Dermatophytes in Hair Samples from Senegal

For the successful treatment of dermatophytoses, especially tinea capitis, there is a need for accurate and rapid diagnostic methods. A lot of recent literature has focused on the detection of dermatophytes directly on sample material such as nails, hair and skin scrapings. Molecular tools offer the ability to rapidly diagnose dermatophytosis within 48 h. This study aimed to compare the results of a commercial real-time PCR (real-time PCR) assay DermaGenius®(DG) 2.0 complete multiplex kit with those of conventional diagnostic methods (direct microscopy and culture). A total of 129 hair samples were collected in Dakar (Senegal) from patients suspected of dermatophytosis. DG was applied for the molecular detection of Candida albicans, Trichophyton rubrum/soudanense, T. interdigitale, T. tonsurans, T. mentagrophytes, T. violaceum, Microsporum canis, M. audouinii, Epidermophyton floccosum, T. benhamiae and T. verrucosum. Dermatophytes species and C. albicans were differentiated by melting curve analysis. The sensitivity and specificity of the PCR assay were 89.3% and 75.3%, respectively. DG PCR was significantly more sensitive than culture (p < 0.001). DG PCR is fast and robust to contamination. In this paper, the main questions discussed were the replacement of culture by a broad-spectrum fungal real-time PCR and the implementation of DG PCR into a routine laboratory in Senegal.

Advanced Multiplex Loop Mediated Isothermal Amplification (mLAMP) Combined with Lateral Flow Detection (LFD) for Rapid Detection of Two Prevalent Malaria Species in India and Melting Curve Analysis

Isothermal techniques with lateral flow detection have emerged as a point of care (POC) technique for malaria, a major parasitic disease in tropical countries such as India. Plasmodium falciparum and Plasmodium vivax are the two most prevalent malaria species found in the country. An advanced multiplex loop-mediated isothermal amplification (mLAMP) combined with a lateral flow dipstick (LFD) technique was developed for the swift and accurate detection of P. falciparum and P. vivax, overcoming the challenges of the existing RDTs (rapid diagnostic tests). A single set of LAMP primers with a biotinylated backward inner primer (BIP primer) was used for DNA amplification of both malaria species in a single tube. The amplified DNA was hybridized with fluorescein isothiocyanate (FITC) and digoxigenin-labelled DNA probes, having a complemented sequence for the P. falciparum and P. vivax genomes, respectively. A colour band appeared on two separate LFDs for P. falciparum and P. vivax upon running the hybridized solution over them. In total, 39 clinical samples were collected from ICMR-NIMR, New Delhi. Melting curve analysis, with cross primers for both species, was used to ascertain specificity, and the sensitivity was equated with a polymerase chain reaction (PCR). The results were visualized on the LFD for both species within 60 min. We found 100% sensitivity and specificity, when compared with a traditional PCR. Melting curve analysis of mLAMP revealed the lowest detection limit of 0.15 pg/μL from sample genomic DNA. The mLAMP-LFD assays could be a potential point of care (POC) tool for early diagnosis in non-laboratory conditions, with the convenience of a reduced assay time and the simple interpretation of results.

High-Resolution Melting Curve Analysis to Predict Extended Blood Group Phenotypes among Thai Donors and Patients

<b><i>Background:</i></b> High-resolution melting (HRM) analysis is an alternative method for red cell genotyping. Differences in melting curves between homozygous and heterozygous genotypes can predict phenotypes in blood group systems based on single-nucleotide polymorphisms. This study aimed to implement HRM analysis to predict additional extended blood group phenotypes in Thai donor and patient populations. <b><i>Methods:</i></b> Blood samples obtained from 300 unrelated Thai blood donors and 23 patients with chronic transfusions were included. HRM analysis was developed and validated in genotyping of <i>KEL</i>*<i>01</i> and <i>KEL</i>*<i>02</i>, <i>JK</i>*<i>01</i> and <i>JK</i>*<i>02</i>, <i>FY</i>*<i>01</i>, <i>FY</i>*<i>02</i>, and <i>FY</i>*<i>02 N.01</i>, <i>DI</i>*<i>01</i> and <i>DI</i>*<i>02</i>, <i>GYPB</i>*<i>03</i> and <i>GYPB</i>*<i>04</i>, <i>RHCE</i>*<i>E</i> and <i>RHCE</i>*<i>e,</i> and <i>DO</i>*<i>01</i> and <i>DO</i>*<i>02.</i> Then genotyping results from HRM and polymerase chain reaction with sequence-specific primer (PCR-SSP) and phenotyping results were compared. <b><i>Results:</i></b> The validated genotyping results in known DNA controls by HRM analysis agreed with DNA sequencing. The genotyping results among 300 donors in 15 alleles by HRM analysis were in complete concordance with those obtained by serological testing and PCR-SSP. The sensitivity and specificity of the HRM assay were both 100%. Among patients, 13 had alloantibodies that possessed predicted antigen-negative phenotypes corresponding to those antibody specificities, and the highest probability of genotyped-matched donors was given to the remaining patients. <b><i>Conclusions:</i></b> We developed and implemented the HRM analysis assay for red cell genotyping to predict extended blood group antigens in Thai donor and patient populations. The data from this study may help inform about and support transfusion care of Thai patients to reduce the risk of alloimmunisation.

Methylation of FBN1, SPG20, ITF2, RUNX3, SNCA, MLH1, and SEPT9 genes in circulating cell-free DNA as biomarkers of colorectal cancer

BACKGROUND: Investigating aberrant tumor-specific methylation in plasma cell-free DNA provides a promising and noninvasive biomarker for cancer detection. OBJECTIVE: We aimed to investigate methylation status of some promoter regions in the plasma and tumor tissues to find biomarkers for early detection of colorectal cancer. METHODS: This case-control study on seventy colorectal cancer patients and fifty matched healthy controls used Methylation-Specific High-Resolution Melting Curve analysis to evaluate the methylation of the selected promoter regions in converted genomic tissue DNA and plasma cfDNA. RESULTS: The methylation levels in selected regions of SPG20 (+24375 to +24680, +24209 to +24399, and +23625 to +23883), SNCA (+807 to +1013, +7 to +162, and -180 to +7), FBN1 (+223 to +429, +1 to +245, and -18 to -175), ITF2 (+296 to +436 and -180 to +55), SEPT9 (-914412 to -91590 and -99083 to -92264), and MLH1 (-13 to +22) were significantly higher in tumor tissues compared with normal adjacent tissues. The methylation levels of FBN1, ITF2, SNCA, and SPG20 promoters were significantly higher in the patient’s plasma compared to patient’s normal tissue and plasma of healthy control subjects. FBN1, SPG20, and SEPT9 promoter methylation had a good diagnostic performance for discriminating CRC tissues from normal adjacent tissues (AUC > 0.8). A panel of SPG20, FBN1, and SEPT9 methylation had a higher diagnostic value than that of any single biomarker and other panels in tissue-based assay (AUC > 0.9). The methylation of FBN1(a) and SPG20(a) regions, as the closest region to the first coding sequence (CDS), had a good diagnostic performance in plasma cfDNA (AUC > 0.8) while a panel consisted of FBN1(a) and SPG20(a) regions showed excellent diagnostic performance for CRC detection in plasma cfDNA (AUC > 0.9). CONCLUSION: Methylation of FBN1(a) and SPG20(a) promoter regions in the plasma cfDNA can be an excellent simple, non-invasive blood-based test for early detection of CRC.

Optimasi Analisis Melting Curve untuk Skrining Cepat dan Sensitif Mutasi V1016G pada Aedes aegypti Resisten Sintetik Piretroid dengan Reaksi Rantai Polimerase Spesifik Alel

Detection of V1016G mutation is important for identifying the mechanism of synthetic pyrethroid resistance in Aedes aegypti population. The previous method has described an allele specific polymerase chain reaction (AS-PCR) using conventional PCR to detect the mutation. Although the method has great differentiating power and reproducibility, faster and more sensitive genotyping method is essential to accurately detect the mutation. This study evaluate the used of SYBR® Green real-time PCR and melting curve analysis (MCA) to identify the V1016G mutation. The collection of homozygous 1016G, heterozygous, and wild type (1016 V) mosquitoes DNA genome was extracted using genomic DNA mini kit. The SsoAdvanced™ Universal SYBR® Green Supermix was used to identify alleles by real-time PCR followed melting curve analysis of the amplicons. Melting curve analysis produced reproducible results for the loci tested. The melting temperature was reached at 78.5 oC for homozygous 1016G mosquito and at 86 oC for wild type mosquito. Meanwhile, the heterozigous mosquito revealed two peaks of melting temperature at both 78.5 oC and 86 oC. These easily interpretable and distinguishable melting curve results were consistent with AS-PCR results obtained for the same alleles. The described MCA application for screening V1016G mutation is fast and widely accessible also could be implemented under field conditions

Melting curve analysis to differentiate Rickettsia spp. and Rickettsia felis

Rickettsiosis, caused by Rickettsia species, is one of the old arthropod-borne illness that commonly found in humans and animals. One of the barriers to rickettsiosis control is the intricacy and time-consuming nature of rickettsiosis laboratory diagnosis. This study aimed to establish quantitative real-time PCR targeting the gltA gene for the DNA differentiation of Rickettsia spp. and Ricketsia felis. The collection of cat flea was extracted to acquire the DNA of Rickettsia. Primers were designed based on the analysis of Rickettsia gltA gene sequences. The confirmation of R. felis was performed by sequencing of PCR product. BLAST analysis was done to confirm the closest similarity of the sequences. Results of this study highlighted the melting temperature was reached at 78,5 °C for Rickettsia spp. and 76.5+0.5 °C for Rickettsia felis. The melting peak temperatures were significantly different between Rickettsia spp. and R. felis (p<0.05). The findings of this work are crucial in the development of powerful diagnostic procedures for detecting and distinguishing Rickettsia spp. and R. felis species.

Prognostic relevance of combined IDH1 and NPM1 mutations in the intermediate cytogenetic de novo acute myeloid leukemia

Despite the great advance in treatment, cytogenetically normal Acute myeloid leukemia (CN-AML) is still a challenging entity. The discovery of IDH1 mutation in AML together with the frequent co-mutations; NPM1 and FLT3-ITD throughs a new insight into the pathogenesis and outcome of CN-AML. Recently, there has been an increasing number of recurring mutations in other genes for which the forecasting effect is still required. Despite the large number of risk variables established, there are relatively few prognostic indicators that can help in treatment decisions in AML patients. This study aimed at recording the frequency of IDH1 and NPM1 mutations in newly diagnosed AML and, dual clinicopathological significance. IDH1 and NPM1 mutations were analyzed using High-Resolution Melting curve analysis PCR in 78 newly diagnosed AML patients; 30 pediatric and 48 adult AML patients. IDH1 mutation was detected in 6 out of the 48 adult AML cases (12.5%) and all of them had intermediate cytogenetic prognostic stratification. 5/6 mutant IDH1 patients showed NPM1 co-mutation (P-value= 0.008). Mutant IDH1 patients showed significant resistance to induction therapy (P-value <0.001) and even those who achieved complete remission were relapsed later. Within the intermediate cytogenetic group, the IDH1 mutated patients had short overall survival (HR 12.9, 95% CI (3.1- 53.45) and event-free survival (HR 15.7, 95% CI (2.99-82.72) and P-value <0.001). IDH1 mutation is closely linked to the intermediate cytogenetic stratified group and in particular old age patients and has a great impact on their survival.