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

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

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.

Optimization and development of high-resolution melting curve analysis (HRMA) assay for detection of New Delhi metallo-ß-lactamase (NDM) producing Pseudomonas aeruginosa.

New Delhi metallo-ß-lactamase-1 (NDM-1) producing Pseudomonas aeruginosa strain detection plays a vital role in confirming bacterial disease diagnosis and following the source of an outbreak for public health. However, the standard method for NDM-1 determination, which relies on the features of the colony of the bacteria cultured from the patient's specimen, is time-consuming and lacks accuracy and sensitivity. This study aimed to standardize a high-resolution melting curve analysis (HRMA) assay to detect NDM producing P. aeruginosa. For optimization and development of the HRMA method, a reference strain of P. aeruginosa was used. For evaluating the broad range PCR data, ABI Step One-Plus Manager Software version 3.2 and Precision Melt Analysis Software 3.02 (Applied Biosystems) were used. Based on the results, expected results were obtained for all tested strains, with high analytical sensitivity and specificity. Temperature melting analyses of the HRMA time PCR assays showed the Tm at 89.57 °C, 76.92 °C and 82.97 °C for N-1, N-2 and N-3 genes, respectively. Also, melting point temperatures of the bla VIM, bla SPM and bla SIM amplicons for isolates identified as MBL strains were 84.56 °C, 85.35 °C and 86.62 °C, respectively. The amplification results using negative control genomes as templates were negative, showing the specificity of the designed assays. Our study's data indicated that the sensitivity and specificity of the HRMA method are linked to the primer length and the fluorescent dye. We can further identify antibiotic resistance in NDMproducing P. aeruginosa by software analysis and melting curve analysis.

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

Background: 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. Methods: 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 KEL*01 and KEL*02, JK*01 and JK*02, FY*01, FY*02, and FY*02 N.01, DI*01 and DI*02, GYPB*03 and GYPB*04, RHCE*E and RHCE*e, and DO*01 and DO*02. Then genotyping results from HRM and polymerase chain reaction with sequence-specific primer (PCR-SSP) and phenotyping results were compared. Results: 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. Conclusions: 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.

Identification of the SARS-CoV-2 Delta variant C22995A using a high-resolution melting curve RT-FRET-PCR.

Knowledge of SARS-CoV-2 variants is essential for formulating effective control policies. Currently, variants are only identified in relatively small percentages of cases as the required genome sequencing is expensive, time-consuming, and not always available. In countries with facilities to sequence the SARS-CoV-2, the Delta variant currently predominates. Elsewhere, the prevalence of the Delta variant is unclear. To avoid the need for sequencing, we investigated a RT-FRET-PCR that could detect all SARS-CoV-2 strains and simultaneously identify the Delta variant. The established Delta RT-FRET-PCR was performed on reference SARS-CoV-2 strains, and human nasal swab samples positive for the Delta and non-Delta strains. The Delta RT-FRET-PCR established in this study detected as few as ten copies of the DNA target and 100 copies of RNA target per reaction. Melting points of products obtained with SARS-CoV-2 Delta variants (around 56.1°C) were consistently higher than products obtained with non-Delta strains (around 52.5°C). The Delta RT-FRET-PCR can be used to diagnose COVID-19 patients and simultaneously identify if they are infected with the Delta variant. The Delta RT-FRET-PCR can be performed with all major thermocycler brands meaning data on Delta variant can now be readily generated in diagnostic laboratories worldwide.

High-resolution melting curve FRET-PCR rapidly identifies SARS-CoV-2 mutations.

Reverse transcription fluorescence resonance energy transfer-polymerase chain reaction (FRET-PCRs) were designed against the two most common mutations in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) (A23403G in the spike protein; C14408T in the RNA-dependent RNA polymerase). Based on high-resolution melting curve analysis, the reverse transcription (RT) FRET-PCRs identified the mutations in american type culture collection control viruses, and feline and human clinical samples. All major makes of PCR machines can perform melting curve analysis and thus further specifically designed FRET-PCRs could enable active surveillance for mutations and variants in countries where genome sequencing is not readily available.

Development of a novel, high resolution melting analysis based genotyping method for Cryptosporidium parvum.

This study employed the post-real-time PCR application, high resolution melting (HRM) analysis, in order to differentiate between characterised clinical and reference Cryptosporidium parvum samples obtained from Cork University Hospital (Cork, Ireland) and the Cryptosporidium Reference Unit (Swansea, Wales). A sample set composed of 18 distinct C. parvum gp60-subtypes of the IIa gp60-subtype family (an allele family accounting for over 80% of all cryptosporidiosis cases in Ireland) was employed. HRM analysis-based interrogation of the gp60, MM5 and MS9-Mallon tandem repeat loci was found to completely differentiate between 10 of the 18 studied gp60-subtypes. The remaining eight gp60-subtypes were differentiated into three distinct groupings, with the designations within these groupings resolved to two to three potential gp60-subtypes. The current study aimed to develop a novel, reproducible, real-time PCR based multi-locus genotyping method to distinguish between C. parvum gp60-subtypes. These preliminary results support the further expansion of the multi-locus panel in order to increase the discriminatory capabilities of this novel method.

A novel genotyping method for Cryptosporidium hominis.

Cryptosporidiosis remains the leading protozoan induced cause of diarrhoea-associated mortality worldwide. Cryptosporidium hominis, the anthroponotically transmitted species within the Cryptosporidium genus, contributes significantly to the global burden of infection, accounting for the majority of clinical cases in many countries. This study applied high resolution melting analysis, a post-real-time PCR application, to the differentiation of six globally prevalent C. hominisgp60-subtypes. This novel method targeted three microsatellite, tandem repeat containing genetic markers, gp60, the genetic marker upon which current Cryptosporidium subtype nomenclature is based, MSB, and MSE, by which to differentiate between C. hominis isolates. This multi-locus approach successfully differentiated between all six C. hominisgp60-subtypes studied, some of which, such as IbA10G2, are known to exhibit global ubiquity. Thus, this method has the potential to be universally employed as a sensitive, cost effective and highly reproducible means to rapidly differentiate between C. hominisgp60-subtypes. Such a method would be of particular utility in epidemiological studies and outbreak scenarios, providing cost effective, clinically accessible alternative to DNA sequencing. The success of this preliminary study also supports further analysis of an expanded C. hominisgp60-subtype range and the potential expansion of the multi-locus panel in order to improve the discriminatory power of this approach.

Genotypic analysis of SLC4A1 A858D mutation in Indian population associated with distal renal tubular Acidosis (dRTA) coupled with hemolytic anemia.

INTRODUCTION: Although distinctive, distal renal tubular acidosis (dRTA) and Hereditary Spherocytosis (HS) shares a common protein, the anion exchanger1 (AE1) encoded by SLC4A1gene. In spite of this, the co-existence of dRTA and HS has rarely been observed. To date, 23 mutations have been identified in SLC4A1 gene causing both autosomal recessive (AR) and autosomal dominant (AD) forms of dRTA. METHODS: We have assessed the applicability of the High Resolution Melting curve (HRM) method for the detection of SLC4A1 (A858D) mutation in 12 Indian families having AR dRTA coupled with HS. The reliability of the HRM analysis was verified by comparing the results of the HRM method with those of conventional methods such as Polymerase Chain Reaction-Restriction Fragment-Length Polymorphism (PCR-RFLP) and Sanger sequencing thereby confirming the diagnosis. RESULTS: We here described the clinical, hematological and genetic data of 16 individuals from 12 families having AR dRTA coupled with HS. All patients carried homozygous SLC4A1 (A858D) mutation, whereas their family members had heterozygous A858D obtained by HRM analysis and confirmed by RFLP and Sanger sequencing. CONCLUSION: Our data indicates that a missense mutation of A858D in SLC4A1 gene is the most common cause of dRTA coupled with HS in the Indian population. HRM analysis can be used as a rapid screening method for common SLC4A1 mutations that cause AR dRTA in the Indian population.

Genotypic Distribution and a Potential Diagnostic Assay of Multidrug-Resistant Tuberculosis in Northern Thailand.

INTRODUCTION: Knowledge of the prevalence and distribution of multidrug-resistant tuberculosis (MDR-TB) genotypes in northern Thailand is still limited. An accurate, rapid, and cost-effective diagnostic of MDR-TB is crucial to improve treatment and control of increased MDR-TB. MATERIALS AND METHODS: The molecular diagnostic assays named "RIF-RD" and "INH-RD" were designed to detect rifampicin (RIF) and isoniazid (INH) resistance based on real-time PCR and high-resolution melting curve analysis. Applying the ∆Tm cutoff values, the RIF-RD and INH-RD were evaluated against the standard drug susceptibility testing (DST) using 107 and 103 clinical Mycobacterium tuberculosis (Mtb) isolates from northern Thailand. DNA sequence analysis of partial rpoB, katG, and inhA promoter of 73 Mtb isolates, which included 30 MDR-TB, was performed to elucidate the mutations involved with RIF and INH resistance. RESULTS: When compared with the phenotypic DST, RIF-RD targeting rpoB showed sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 83.9, 98.6, 96.9, and 92.0%, respectively. The multiplex reaction of the INH-RD targeted both katG and inhA promoter showed high sensitivity, specificity, PPV, and NPV of 97.1, 94.2, 89.2, and 98.5%, respectively. Six patterns of rpoB mutation, predominately at codons 531 (50%) and 526 (40%) along with a rare S522L (3.33%) and D516V (3.33%), were detected. A single pattern of katG mutation (S315T) (63.3%) and four patterns of inhA promoter mutation, predominately -15 (C>T), were found. Approximately, 17% of MDR-TB strains possessed double mutations within the katG and inhA promoter. CONCLUSION: Up to 86.7% and 96.7% of MDR-TB could be accurately detected by RIF-RD and INH-RD, emphasizing its usefulness as a low unit price assay for rapid screening of MDR-TB, with confirmation of INH resistance in low and middle-income countries. The MDR-TB genotypes provided will be beneficial for TB control and the development of drug-resistant TB diagnostic technology in the future.

A HRM Assay for Rapid Identification of Members of the Seedcorn Maggot Complex (Delia florilega and D. platura) (Diptera: Anthomyiidae) and Evidence of Variation in Temporal Patterns of Larval Occurrence.

The seedcorn maggot Delia platura (Meigen), and the bean seed maggot Delia florilega (Zetterstedt) can cause considerable feeding damage to a wide range of cultivated crops. The recent discovery of two distinct genetic lines of D. platura, each with a unique distribution pattern overlapping only in eastern Canada, suggests the presence of a new cryptic species for the group. The reliable identification of the three species/lines in the seedcorn maggot complex is crucial to our understanding of their distribution, phenology, and respective contribution to crop damage as well as to the development of specific integrated pest management approaches. As these taxa are morphologically indistinguishable in the immature stages, we developed a high-resolution melting PCR (HRM) assay using primers amplifying a variable 96-bp PCR product in the CO1 mitochondrial gene for rapid and economical identification of specimens. The three species/lines exhibited distinguishable melting profiles based on their different Tm values (between 0.4 and 0.9°C) and identification results based on HRM and DNA sequencing were congruent for all specimens in the validation data set (n = 100). We then used the new, highly sensitive HRM assay to identify survey specimens from the seedcorn maggot complex collected in Quebec, Canada, between 2017 and 2019. Progress curves developed to document the temporal occurrence patterns of each species/lines indicate differences between taxa, with the N-line (BOLD:AAA3453) of D. platura appearing approximately 17 d before D. florilega (BOLD:ACR4394) and the H-line (BOLD:AAG2511) of D. platura.

Prevalence and Molecular Typing of Colistin-Resistant Pseudomonas aeruginosa (CRPA) Among ß-Lactamase-Producing Isolates: A Study Based on High-Resolution Melting Curve Analysis Method.

BACKGROUND: The frequency and production of ß-lactamase enzymes may be different in colistin-resistant Pseudomonas aeruginosa (CRPA) strains compared to susceptible strains. The purpose of this study was to investigate the relationship between colistin resistance and ß-lactamase enzymes in different Sequence Types (ST) of P. aeruginosa. METHODS: A total of 101 P. aeruginosa isolates were collected from different samples. The antimicrobial susceptibilities of the bacterial isolates were examined by disk diffusion and MIC E-test methods. Also, real-time PCR and high-resolution melting curve analysis (HRMA) assay were performed to detect the resistance genes. RESULTS: Out of the 101 P. aeruginosa isolates, four isolates (3.96%) were resistant to colistin. Also, 39 isolates (38.61%) were considered as MDR, and eight isolates (7.92%) were considered as XDR. Further, 25 (24.75%) and 26 isolates (25.74%) were produced ESBL and carbapenemase enzymes, respectively. According to HRMA results, four isolates (3.96%) were positive for pmrA, three isolates (2.97%) were positive for mcr-1, 25 isolates (24.75%) were positive for blaTEM, 24 isolates (23.76%) were positive for blaSHV, 26 isolates (25.75%) were positive for blaKPC, and 23 isolates (22.77%) were positive for blaIMP genes. Furthermore, ST108 and ST250 showed the highest distribution in P. aeruginosa isolates. Also, ST217, ST1078, and ST3340 were reported as novel types in CRPA strains. CONCLUSION: Concerns about the prevalence of CRPA strains should be taken seriously. Also, our results showed that the mcr-1 gene plays a vital role in the distribution of ESBL and KPC-producing P. aeruginosa strains.

Co-harboring of mcr-1 and ß-lactamase genes in Pseudomonas aeruginosa by high-resolution melting curve analysis (HRMA): Molecular typing of superbug strains in bloodstream infections (BSI).

Background Colistin resistance in P. aeruginosa (CRPA) is due to the appearance of superbug strains. As this pathogen gains more transferrable resistance mechanisms and continues to adapt to acquire additional resistance mechanisms during antimicrobial therapy rapidly, we face the growing threat of CRPA in bloodstream infections (BSI). This study designed to evaluate the frequency of CRPA strains producing different ß-lactamases by the High-Resolution Melting Curve Analysis (HRMA) method in BSI and to characterize the different types by multilocus sequence typing (MLST). MATERIAL AND METHODS: Sixty-nine (69) P. aeruginosa isolates were collected from blood culture. MIC E-test methods examined the antimicrobial susceptibilities of the bacterial isolates. Detection of resistant strains performed by using HRMA assay. RESULTS: The strains resistant to amikacin (n = 11; 15.94%) and colistin (n = 10; 14.49%) were the least abundant and the gentamicin (n = 56; 82.6%) and ciprofloxacin (n = 67; 97.10%) resistant strains were the most frequent. Also, 39 isolates (56.52%) considered as multidrug-resistant (MDR), 20 isolates (28.98%) as extensively drug resistant (XDR), and 11 isolates (15.94%) as Pandrug Resistance (PDR). Further, 32 isolates (46.37%) considered as AmpC producer, and 28 isolates (40.57%) were considered an MBL producer. According to HRMA results, the blaSPM gene was detected in 19 isolates (27.53%), blaNDM gene in 11 isolates (15.94%), blaFOX gene in 31 isolates (44.92%), mcr-1 gene in 10 isolates (14.49%), blaACC and blaVIM genes in 27 isolates (39.13%), and blaTEM gene was reported in 20 isolates (28.98%). Furthermore, P. aeruginosa PASGNDM699, ST3340, and ST235 identified in 1.44%, 11.59% and 17.39% isolates, respectively. CONCLUSION: CRPA strains play an essential role in the spread of antibiotic resistance in BSI. Likewise, the HRMA method was sensitive and specific for the detection of superbugs. Moreover, MLST analysis of a diverse collection of P. aeruginosa from blood culture suggests that particular strains or clonal complexes are associated with antibiotic resistance profile.

Distribution of Class B and Class A ß-Lactamases in Clinical Strains of Pseudomonas aeruginosa: Comparison of Phenotypic Methods and High-Resolution Melting Analysis (HRMA) Assay.

BACKGROUND: There are various phenotypic methods for identifying class B and class A ß-lactamase enzymes in Pseudomonas aeruginosa. The purpose of this study was to compare the sensitivity and specificity of different phenotypic methods with HRMA assay to detect ß-lactamase-producing P. aeruginosa strains. METHODS: Eighty-eight of P. aeruginosa isolates were collected from different specimens. Conventional double-disk test (DDT) and EDTA-imipenem microbiological (EIM) were performed to detect ESBL and MBL-producing strains, respectively. Meanwhile, the Modified Hodge test and Carba-NP test were performed on all carbapenem-resistant strains. HRMA method and sensitivity and specificity of primers were determined based on the melt curve temperature range. In all comparisons, PCR was considered as the gold standard. RESULTS: Of the 402 isolates collected from different clinical specimens, 88 isolates of P. aeruginosa were identified. However, 43 strains were (48.88%) ESBL-producing, and 7 strains (7.95%) were MBL-producing. Also, using the Modified Hodge test and Carba-NP method, 11 (12.5%) and 19 (21.59%) strains were carbapenemase-producing, respectively. The results of the HRMA test revealed that genes coding for bla SHV, bla TEM, bla KPC, bla IMP, bla VIM, and bla GES were detected in 44.31%, 22.72%, 13.63%, 14.7%, 5.6%, and 2.27% of P. aeruginosa isolates. Nonetheless, for bla KPC and bla GES genes, sensitivity and specificity of the Carba-NP test were 90.47%, 94.87%, and 83.36%, 94.80%, respectively. However, sensitivity and specificity of MHT was 91.66%, 98.70%, and 77.77%, 96.42%, respectively. For bla SHV and bla TEM genes, sensitivity and specificity of DDT were 95.55%, 95.55%, and 86%, 83.50%, respectively. However, sensitivity and specificity of EMI were 77.77%, 97.59%, and 91.66%, 97.43% for bla VIM and bla IMP, respectively. CONCLUSION: The HRMA is a powerful, accurate, closed-tube, rapid method for detecting ß-lactamase genes in P. aeruginosa. The high sensitivity and specificity of this method, along with phenotypic tests, play a useful role in increasing the predictive value of clinical reports.

High-resolution melting curve analysis for infectious bronchitis virus strain differentiation.

BACKGROUND AND AIM: Belonging to the Coronaviridae family, avian infectious bronchitis virus (IBV) causes respiratory, reproductive, and renal diseases in poultry. Preventative measures lie mainly in vaccination, while the gold standard for IBV classification and differentiation is based on the sequence analysis of the spike 1 (S1) gene. In this study, we tested a new assay for IBV strain classification that is less expensive and requires reduced time and effort to perform. We carried out a quantitative real-time polymerase chain reaction followed by high-resolution melting (qRT-PCR/HRM) curve analysis. MATERIALS AND METHODS: In this study, qRT-PCR was conducted on a partial fragment S1 gene followed by a high resolution melting curve analysis (qRT-PCR/HRM) on 23 IBV-positive samples in Jordan. For this assay, we utilized the most common IBV vaccine strains (Mass and 4/91) as a reference in the HRM assay. To evaluate the discrimination power of the qRT-PCR/HRM, we did the sequencing of the partial S1 gene. RESULTS: It was shown that HRM was able to classify IBV samples into four clusters based on the degree of similarity between their melting points: The first cluster exhibited the highest similarity to the 4/91 strain, while the second was similar to the Mass-related IBV strain. Although the third cluster contained the highest number of samples, it displayed no similarity to any of the reference vaccine strains, and, after comparing them with the sequencing results, we found that the samples in the third cluster were similar to the variant II-like (IS-1494-06) IBV field strain. Finally, the fourth cluster comprised one unique sample that was found to belong to the Q1 IBV strain. CONCLUSION: Our developed qRT-PCR/HRM curve analysis was able to detect and rapidly identify novel and vaccine-related IBV strains as confirmed by S1 gene nucleotide sequences, making it a rapid and cost-effective tool.

Development of high-resolution melting curve analysis in rapid detection of vanA gene, Enterococcus faecalis, and Enterococcus faecium from clinical isolates.

BACKGROUND: High-resolution melting analysis (HRMA) is a novel molecular technique based on the real-time PCR that can be used to detect vancomycin resistance Enterococcus (VRE). The purpose of this study was to identify VRE species with HRMA in clinical isolates. RESULTS: Out of 49 Enterococcus isolates, 11 (22.44%) E. faecium isolates and 19 (38.77%) E. faecalis isolates were detected. Average melting temperatures for divIVA in E.faecalis, alanine racemase in E.faecium, and vanA in VRE strains were obtained as 79.9 ± 0.5 °C, 85.4 ± 0.5 °C, and 82.99 ± 0.5 °C, respectively. Furthermore, the data showed that the HRMA method was sensitive to detect 100 CFU/ml for the divIVA, alanine racemase, and vanA genes. Also, out of 49 Enterococcus spp., which were isolated by HRMA assay, 8 isolates (16.32%) of E. faecium and 18 isolates (36.73%) of E. faecalis were detected. The vanA gene was reported in 2 isolates (25%) of E. faecium and 9 isolates (50%) of E. faecalis. CONCLUSIONS: This study demonstrated that using the HRMA method, we can detect E. faecium, E. faecalis, and the vanA gene with high sensitivity and specificity.

Development and evaluation of high-resolution melting curve analysis for rapid detection and subtyping of Blastocystis and comparison the results with sequencing.

Blastocystis is a prevalent parasite that has a wide distribution. In order to design HRM real-time PCR, primers were selected from SSU rRNA gene to amplify specific fragment with different melting temperatures for each subtype of Blastocystis. Subsequently, HRM real-time PCR was performed and melting curve analysis was done by Rotor-Gene Q software. The results of HRM real-time PCR was then compared with sequence results of "barcoding region" of SSU rRNA gene of Blastocystis. To evaluate sensitivity of test, 10-fold serial dilutions of the parasite were prepared from ~ 106 to 1 parasite per mL of stool sample and were investigated by HRM real-time PCR. In order to determine specificity of method, HRM real-time PCR was done for some microorganisms and Blastocystis-negative stool samples. In silico analysis showed that all seventeen subtypes of Blastocystis were distinguish. In vitro analysis revealed that the test discriminated subtypes with specific melting temperatures.

One-Step ARMS-PCR for the Detection of SNPs-Using the Example of the PADI4 Gene.

In eukaryotes, cellular functions are tightly controlled by diverse post-translational modifications (PTMs) of proteins. One such PTM affecting many proteins is the deimination of arginine to citrulline. This process, called citrullination is catalyzed by a group of hydrolases called protein arginine deiminases (PADs), of which five isoforms have been identified. Hypercitrullination, as a result of increased PAD expression or activity, is associated with autoimmune diseases e.g., rheumatoid arthritis, lupus, Alzheimer's disease, ulcerative colitis, multiple sclerosis, and certain cancers. Three common single nucleotide polymorphisms (SNPs) in the PADI4 gene have been described, namely rs874881, rs11203366, and rs11203367, which are thought to affect PAD4 expression and activity. We here compared the suitability of four methods for the screening of SNPs in the PADI4 gene: (i) SYBR-green based real-time polymerase chain reaction followed by high resolution melting curve analysis (HRM-PCR); (ii) PCR followed by detection of restriction fragment length polymorphisms (PCR-RFLP); (iii) conventional tetra-primer amplification refractory mutation system PCR (ARMS-PCR); and (iv) real-time PCR based on the one-step ARMS-PCR. Of these, ARMS-PCR proved to be the most suitable method regarding handling, duration, and cost of experiments. Using the method with SYBR-green based real-time PCR reagents further diminished handling steps and thus potential sources of error.

Molecular detection of rifampin, isoniazid, and ofloxacin resistance in Iranian isolates of Mycobacterium tuberculosis by high-resolution melting analysis.

BACKGROUND: The emergence of drug resistance among Mycobacterium tuberculosis (MTB) strains is a serious health concern worldwide. The development of rapid molecular diagnostic methods in recent years has a significant impact on the early detection of resistance to major anti-TB drugs in MTB isolates, which helps in employing appropriate treatment regimen and prevents the spread of drug-resistant strains. This study was designed to evaluate the efficacy of real-time PCR and high-resolution melting (HRM) curve analysis for the determination of resistance to rifampin (RIF), isoniazid (INH), and ofloxacin (OFX) in MTB isolates and to investigate their resistance-related mutations. METHODS: HRM analysis was performed to screen 52 (32 drug-resistant and 20 fully susceptible) MTB clinical isolates for mutations in rpoB, katG, mab-inhA, and gyrA genes. The HRM results were then confirmed by DNA sequencing. RESULTS: In total, 32 phenotypically resistant isolates, comprising 18 RIF-, 16 INH-, and five OFX- resistant strains, were investigated. HRM analysis successfully identified 15 out of 18 mutations in rpoB, 14 out of 16 mutations in katG and mab-inhA, and four out of five mutations in gyrA conferring resistance to RIF, INH, and OFX, respectively. The obtained sensitivity and specificity, respectively, for HRM in comparison with phenotypic susceptibility testing were found to be 83.3% and 100% for RIF, 87.5% and 100% for INH, and 80% and 100% for OFX. In five resistant strains (12.8%), no mutation was detected by using HRM and DNA sequencing. CONCLUSION: HRM assay is a rapid, accurate, and cost-effective method possessing high sensitivity and specificity for the determination of antibiotic resistance among MTB clinical isolates and screening of their associated mutations. This method can generate results in a shorter period of time than taken by the phenotypic susceptibility testing and also allows for timely treatment and prevention of the emergence of possible MDR strains.

Detection of KIT mutations in core binding factor acute myeloid leukemia.

We have investigated the frequency and the effect of KIT mutations on the outcome of patients with CBF-AML. 69 patients (34 pediatrics and 35 adults) with CBF-AML were enrolled in the study. The frequency of KIT mutations was higher in adults compared to pediatrics (22.9% and 14.7%, p = 0.38) respectively. Leukocytosis ≥ 20 × 109 /L was significantly associated with pediatrics compared to adults. t(8;21)(q22;22) was significantly associated with thrombocytopenia in adults. We conclude that no significant difference is found between KIT mutated and unmutated CBF-AML in adults and pediatrics. Children with CBF-AML present with leukocytosis. t(8;21) is associated with thrombocytopenia.