The establishment of a high-resolution melting curve genotyping method for ABCG2*376T allele of Jr(a-) and its distribution frequency study / 中国输血杂志

【Objective】 To establish a high-throughput detection method for ABCG2*376T allele of Jr(a-), and apply it to the study of the frequency of this allele in the Chinese population. 【Methods】 The specific primers were designed and synthesized, the sample carrying homozygous ABCG2*376T alleles, obtained in the previous study, was used as the homozygous positive control, and the sample carrying heterozygous allele as the heterozygous positive control. The wild-type sample was used as a negative control, and a high-resolution melting curve(HRM) method for detecting this allele was established. The established method was used to screen DNA samples from blood donors in Guangzhou, and the samples carrying ABCG2*376T alleles were sequenced to confirm the accuracy of the HRM method. 【Results】 A HRM method, which can detect ABCG2*376T allele and accurately type homozygotes and heterozygotes at the same time, had been established successfully. Fifteen individuals with heterozygous alleles were screened out of 1 560 blood donors in Guangzhou, while none homozygous allele was detected. 【Conclusion】 The HRM method can be used to accurately screen and type ABCG2*376T allele. The frequency of this allele in Chinese population is about 0.48%(15/3120).

Analysis of Salivary Bacterial Community by Direct PCR and High Resolution Melting Curve and Its Forensic Applications / 法医学杂志

OBJECTIVES@#To develop a rapid test for salivary bacterial community based on direct PCR (dPCR) and high resolution melting (HRM) curve analysis, to evaluate its application value in forensic medicine.@*METHODS@#The salivary bacteria were collected by centrifugation and then resuspended in Tris-EDTA (TE) buffer, and directly used as the template for amplification and HRM curve analysis (dPCR-HRM) of the 16S rDNA V4 region. The genotype confidence percentage (GCP) of the HRM profiles compared with the reference profile was calculated. The template DNA was extracted by traditional kit and then PCR-HRM (namely kPCR-HRM) was used as reference to validate the feasibility of dPCR-HRM. The gradient dilution templates, population samples and simulated salivary stains were analyzed by dPCR-HRM to evaluate its sensitivity, typing ability and adaptability.@*RESULTS@#Using dPCR-HRM method, the HRM profiles of salivary bacterial community were obtained within 90 minutes. The GCP between dPCR-HRM and kPCR-HRM was greater than 95.85%. For general individuals, the HRM type of bacterial community could be determined with 0.29 nL saliva by dPCR-HRM. The 61 saliva samples could be divided into 10 types. The typing of salivary stains deposited within 8 h was the same as those of fresh saliva (GCP>90.83%).@*CONCLUSIONS@#dPCR-HRM technology can be used for rapid typing of salivary bacterial community, and has the advantage of low cost and simple operation.

Genetic study of a Chinese pedigree affected with pachyonychia congenita / 中华医学遗传学杂志

Objective@#To explore the genetic basis for a Chinese pedigree affected with pachyonychia congenita (PC).@*Methods@#With informed consent obtained, peripheral blood samples were taken from the pedigree. Genomic DNA was extracted with a phenol/chloroform method. Based on the clinical manifestation of the patients, candidate genes for PC were selected. Potential mutation was screened by PCR and Sanger sequencing. Suspected mutation was verified in other family members by PCR-high resolution melting (HRM) analysis. Haplotype analysis using microsatellite markers was also carried out to determine the founder of the mutation.@*Results@#A heterozygous c. 275A>G (Asn92Ser) mutation was discovered in exon 1 of the KRT17 gene in the proband. PCR-HRM analysis showed that all affected members were heterozygous carriers of the mutation. The same mutation was found in none of the unaffected members. Haplotype analysis and sequencing indicated the mother of the proband to be the founder.@*Conclusion@#The c. 275A>G (Asn92Ser) mutation of the KRT17 gene probably underlies the disease in this pedigree. Above finding has facilitated genetic counseling and prenatal diagnosis for this pedigree.

Identification of Gentiana rhodantha and Its Adulterants by DNA Barcoding Coupled with High Resolution Melting Analysis / 中国药学杂志

OBJECTIVE: To apply DNA barcoding coupled with high resolution melting analysis to distinguish Gentiana rhodantha from its adulterants. METHODS: The internal transcribed spacer 2 (ITS2) barcode was selected for HRM analysis to produce standard melting profile of the selected species. RESULTS: The ITS2 molecular regions coupled with HRM analysis can effectively differentiate five herbal species, including two Gentiana rhodantha and their four common adulterants. CONCLUSION: DNA barcoding coupled with HRM analysis is a accurate, reliable, rapid, cost-effective and robust tool, which may contribute to the quality control of traditional Chinese medicine in the natural health product industry.

Evaluation of High Resolution Melting Technique for Detection of JAK2-V617F Mutation in Formalin-Fixed Paraffin-Embedded Specimen from Myeloproliferative Neoplasm Cases

@#Introduction: Myeloproliferative neoplasm (MPN) is a group of myeloid disorders which leads to erythrocytosis, thrombocytosis and leucocytosis. MPN with BCR-ABL positive is chronic myeloid leukaemia (CML) while BCR-ABL negative MPN includes polycythaemia Vera (PV), essential thrombocytemia (ET) and primary myelofibrosis (PMF). One of the major criteria for diagnosis of BCR-ABL negative MPN is the presence of JAK2-V617F mutation which is positive in 95% of PV and around 60% of ET and MF. Beside peripheral blood specimen, formalin-fixed paraffin-embedded (FFPE) marrow specimen can be used for detection of this mutation. Unfortunately, FFPE produces low quality DNA that put a challenge for successful amplification of DNA. We aimed to evaluate the utility of High Resolution Melting (HRM) analysis for detection of JAK2-V617F mutation in FFPE specimen from MPN cases. Materials and Methods: This study is a descriptive crosssectional study. Forty FFPE marrow specimens were retrieved from the years 2014-2016. Bio-Rad Precision Melt Analysis software was used for analysis of HRM data. Allele-specific PCR was done for validation of results. Positive samples were subjected to Sanger sequencing. Results: JAK2-V617F mutation was positive in 13 out of 40 MPN cases. Level of agreement between HRM and AS-PCR was 97.5%. Conclusion: HRM is a rapid and powerful diagnostic assay which is suitable for detection of JAK2-V617F mutation in FFPE marrow specimen.

Real-time multiplex polymerase chain reaction with high-resolution melting-curve analysis for the diagnosis of enteric infections associated with diarrheagenic Escherichia coli

Introduction: Although diarrheagenic Escherichia coli (DEC) strains are important bacterial causative agents of diarrhoea, they are not routinely sought as stool pathogens in clinical laboratories as conventional microbiological testing are unable to distinguish between normal flora and pathogenic strains of E. coli. This study was undertaken to determine the prevalence of DEC pathotypes amongst children with and without diarrhoea and to detect specific virulent genes present in different DEC pathotypes, using real-time multiplex polymerase chain reaction (PCR) with high-resolution melting (HRM) technology. Materials and Methods: Stool samples were obtained from cases and controls. Using a set of conventional biochemical tests, E. coli strains were identified. Further, these isolates were subjected to multiplex PCR system for the detection of virulence genes of different pathotypes of DEC. Real-time multiplex PCR was performed for the detection of specific virulent genes of DEC pathotypes, using Rotor-Gene Q instrument (Qiagen) having High-resolution Melt analyser using Type-it HRM PCR kit (Qiagen) containing EvaGreen fluorescent intercalating dye. Results: In this study, we had successfully standardised two multiplex PCR assays which were found to be effective for direct detection of enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC) and enteroinvasive E. coli (EIEC). A total of 42 DEC strains were detected at an overall rate of 19.3% (n = 42), from the total 217 E. coli isolates recovered from the cases (n = 39, 17.9%) and control (n = 3, 3.8%) groups. Amongst the 42 DEC pathotypes (39 from cases and 3 from controls), EPEC (10%), EAEC (8.82%), ETEC (2.94%) and EIEC (1.18%) were found in children with diarrhoea (cases) and in children without diarrhoea (control) only EAEC (2.13%) and EPEC (4.26%) were detected. Age distribution, gender variation, seasonal variation and clinical features were also analysed Conclusion: This study helped evaluate the prevalence of DEC amongst children (<18 years of age) with and without diarrhoea using multiplex real-time PCR with HRM analysis.

High resolution melting real-time PCR detect and identify filarial parasites in domestic cats

Objective:To detect and identify filarial parasites in dried blood spots (DBS) collected from domestic cats using high resolution melting real-time PCR (HRM RT-PCR).Methods:A total of 208 DBS were collected from domestic cats in a brugian filariasis endemic areas in Surat Thani Province, southern Thailand. Microfilariae were found in 9 blood slides using Giemsa-stained thick blood film. The extracted DNA from blood spot volumes of 10 and 20 μL DBS with positive filarial parasites in cats were performed using HRM RT-PCR method. The primers were designed based on the partial mitochondrial 12S rRNA gene for identifying Brugia malayi, Brugia pahangi, Dirofilaria immitis. All purified samples were then detected.Results:Using different volumes of 10 μL and 20 μL DBS could easily distinguish filarial parasites and showed similar results. PCR amplicons of Brugia malayi, Brugia pahangi and Dirofilaria immitis were determined at melting peak (temperature) of 75.70 77.46 and 73.56 respectively. All 9 positive DBS samples showed positive Brugia pahangi and similar nucleotide sequences.Conclusions:This HRM RT-PCR method is able to diagnose, identify and discriminate filarial parasites collected from DBS, which is simple and inexpensive compared with other probe-based genotyping methods. Furthermore, this method is useful to survey, prevent and control filariasis.

High-resolution melting analysis in forensic research / 中国法医学杂志

High-resolution melting (HRM) analysis is a versatile method for variant scanning and genotyping. It involves amplification of the target in the presence of a saturation dye by the polymerase chain reaction (PCR). HRM analysis can be performed in one closed-tube, which does not require additional post-PCR separations and greatly reduces the possibility of contamination. HRM is faster, simpler, and less expensive than alternative approaches requiring labeled probes. Considering the many advantages of HRM analysis, many researchers have tried to apply this method to forensic research. This paper intends to summarize the principle, technical characteristics, limitations and application of HRM analysis in forensic science.

Association of ring-finger protein 3 (MKRN3) gene rs2239669 polymorphism with central precocious puberty susceptibility / 临床儿科杂志

Objectives To explore the association between the single nucleotide polymorphism (SNP) rs2239669 in makorin ring-finger protein 3 (MKRN3) gene and the susceptibility to central precocious puberty (CPP). Methods A case-control study including 246 children with CPP and 269 healthy children was performed.The genotype and MKRN3 expression levels of patients were analyzed by PCR-HRM and RT-PCR,respectively. Results SNP rs2239669 genotype (TT,TC,CC) and allele frequencies (T and C) were different between cases and controls,with higher CC genotype in CPP patients. Under recessive model (CC/TT+TC),CC genotype was higher in CPP group and associated with higher risk of CPP (95%CI:1.062-2.143,P=0.021). MKRN3 expression levels were different among patients with different genotypes,of which TT genotype had the highest level followed by TC and CC (0.376±0.094, 0.330±0.068, 0.250±0.072, P=0.041). Conclusions MKRN3 SNP rs2239669 was associated with increased risk of CPP, and patients with TT genotype had higher MKRN3 levels.

High resolution melting analysis for detection of ALDH 2 and ADH1B gene polymorphisms / 国际检验医学杂志

Objective To establish the system of high resolution melting for detection of aldehyde dehydro-genase 2(ALDH2) and alcohol dehydrogenase-1B (ADH1B) gene polymorphisms .Methods The short prim-ers were designed for ALDH2 and ADH1B gene .Different PCR products were analyzed using Eva Green dyes after amplification ,which were confirmed by Sanger sequencing .Results The genotypes of ALDH2 rs671 and ADH1B rs1229984 were successfully detected in the same procedure by HRM within 90min ,and the results were consistent with Sanger sequencing .Conclusion The assay of HRM is simple ,rapid ,cost-effective ,and re-liable for the detection of ALDH2 and ADH1B polymorphism and it is worthy to be popularized .

Performance evaluation of the automated SNPs analyzer for the detection of JAK2 V617F mutation / 中华检验医学杂志

Objective To test and evaluate the JAK2 gene V617F mutation in patients with myeloproliferative tumors based on i-densy IS-5320 platform according to ISO15189 accreditation requirements.Methods Instrument performance verification.Selected from December 2014 to February 2017, 20 cases of JAK2 V617F mutation positive peripheral blood samples from Huashan Hospital of the Shanghai FuDan University Medical College and 20 cases of peripheral blood samples with negative JAK2V617F mutation.The Realtime PCR with TaqMan MGB probe was selected as the control method to verify and evaluate the accuracy of testing JAK 2 V617F mutation on i-densy IS-5320 platform.Whole blood samples were used to evaluate the reproducibility , cross-contamination and anti-interference ability of this platform.The ability of mutation load was verified by detecting mixtures of human erythnoleukemia cells and colorectal cancer cell HCT116 with 12 different proportions.Results I-densy IS-5320 platform and TaqMan MGB probe real-time fluorescence quantitative PCR show the same result .The within-run reproducibility and between-run reproducibility are both 100%.There is no observed contamination .High bilirubin and high triglyceride blood samples have no obvious interference on mutation detection .The mutation ratio with a load as low as 0.25%could be tested stably by i-densy IS-5320 platform.The detecting peak of melting curve can reflect the ratio of JAK2 V617F mutation to some extent.Conclusions I-densy IS-5320 can detect the mutation of JAK2 V617F gene in the whole blood directly.It has high sensitivity, accuracy and stability, and is easy to operate, and also can reflect the mutation load of JAK2 V617F, which could meet the clinical requirements for the detection of mutations.

Rapid screening of c.166A>G in SLC4A1 gene by high-resolution melting analysis / 中华检验医学杂志

Objective To explore the feasibility of a high-resolution melting(HRM)method for rapid screening of SLC4A1 mutation.Methods Two hereditary spherocytosis(HS)with a c.166A >G heterozygous mutation of SLC4A1 confirmed by DNA sequencing and thirty healthy controls were selected for the study.The HRM primer was designed by Primer Premier 6.0 software.All of these samples were detected by LightCycler?480 and analyzed by HRM.Results The HRM analysis was able to detect the c.166A>G heterozygous mutation of SLC4A1 effectively, and the specificity and sensitivity were both 100%.Conclusions The HRM analysis was appropriate for the detection of c.166A>G in SLC4A1.It was an efficient,accurate and cost-effective molecular diagnosis method.

High-resolution melting-based TILLING of γ ray-induced mutations in rice / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics strategy for the high-throughput screening of induced mutations. γ radiation, which often induces both insertion/deletion (Indel) and point mutations, has been widely used in mutation induction and crop breeding. The present study aimed to develop a simple, high-throughput TILLING system for screening γ ray-induced mutations using high-resolution melting (HRM) analysis. Pooled rice (Oryza sativa) samples mixed at a 1:7 ratio of Indel mutant to wild-type DNA could be distinguished from the wild-type controls by HRM analysis. Thus, an HRM-TILLING system that analyzes pooled samples of four M2 plants is recommended for screening γ ray-induced mutants in rice. For demonstration, a γ ray-mutagenized M2 rice population (n=4560) was screened for mutations in two genes, OsLCT1 and SPDT, using this HRM-TILLING system. Mutations including one single nucleotide substitution (G→A) and one single nucleotide insertion (A) were identified in OsLCT1, and one trinucleotide (TTC) deletion was identified in SPDT. These mutants can be used in rice breeding and genetic studies, and the findings are of importance for the application of γ ray mutagenesis to the breeding of rice and other seed crops.

High resolution melting real-time PCR detect and identify filarial parasites in domestic cats

Objective: To detect and identify filarial parasites in dried blood spots (DBS) collected from domestic cats using high resolution melting real-time PCR (HRM RT-PCR). Methods: A total of 208 DBS were collected from domestic cats in a brugian filariasis endemic areas in Surat Thani Province, southern Thailand. Microfilariae were found in 9 blood slides using Giemsa-stained thick blood film. The extracted DNA from blood spot volumes of 10 and 20 μL DBS with positive filarial parasites in cats were performed using HRM RT-PCR method. The primers were designed based on the partial mitochondrial 12S rRNA gene for identifying Brugia malayi, Brugia pahangi, Dirofilaria immitis. All purified samples were then detected. Results: Using different volumes of 10 μL and 20 μL DBS could easily distinguish filarial parasites and showed similar results. PCR amplicons of Brugia malayi, Brugia pahangi and Dirofilaria immitis were determined at melting peak (temperature) of 75.70 77.46 and 73.56 respectively. All 9 positive DBS samples showed positive Brugia pahangi and similar nucleotide sequences. Conclusions: This HRM RT-PCR method is able to diagnose, identify and discriminate filarial parasites collected from DBS, which is simple and inexpensive compared with other probe-based genotyping methods. Furthermore, this method is useful to survey, prevent and control filariasis.

HLA-B*15:02 screening in epileptic patients using a high resolution melting-real time PCR (HRM-QPCR) method

Background: The HLA-B*15:02 polymorphism in epileptic patients is known to be associated with carbamazepine-induced Stevens-Johnson syndrome (SJS). The prevalence of HLA-B*15:02 polymorphism seemed to be ethnic-specific with a higher frequency of HLA-B*15:02 in Asian compared to the Europeans. This study was performed to determine the frequency of the HLA-B*15:02 polymorphism in epileptic patients at the Chancellor Tuanku Muhriz Hospital-UKM Medical Centre (HCTM-UKMMC) using high resolution melting-real time PCR (HRM-QPCR) method. Methods: We performed a fast and effective in-house high resolution melting-real time polymerase chain reaction method and compared it with the conventional multiplex-PCR method. The specificity and sensitivity of each test were also determined using DNA from saliva. Results: Using the conventional multiplex-PCR approach for screening, 25 out of 64 (39.1%) epileptic patients were positive for HLA-B*15:02. However, using the HRM-QPCR technique, 24/64 (37.5%) of the patients were positive. The one patient who tested positive by the multiplex-PCR but negative using the HRM-QPCR turned out to be negative by DNA sequencing. The HRM-QPCR and DNA sequencing showed 100% sensitivity and specificity. The multiplex-PCR showed 100% sensitivity and 98.4% specificity compared to both HRM-QPCR and DNA sequencing. The HRM-QPCR is also more cost-effective (<$16.40 USD/test) and less time-consuming when compared to the multiplex-PCR ($25.15 USD/test).Conclusion: Our result suggested that multiplex PCR, HRM-QPCR and Sanger sequencing can be used for detection of HLA-B*15:02. However, a qualitative method such as multiplex PCR should be confirmed with other quantitative methods such as HRM-QPCR and Sanger sequencing.

Rapid screening for Klinefelter syndrome with a simple high-resolution melting assay: A multicenter study / 亚洲男科学杂志(英文版)

Klinefelter syndrome (KS) is the set of symptoms that result from the presence of an extra X chromosome in males. Postnatal population-based KS screening will enable timely diagnosis of this common chromosomal disease, providing the opportunity for early intervention and therapy at the time point when they are most effective and may prevent later symptoms or complications. Therefore, through this study, we introduced a simple high-resolution melting (HRM) assay for KS screening and evaluated its clinical sensitivity and specificity in three medical centers using 1373 clinical blood samples. The HRM assay utilized a single primer pair to simultaneously amplify specific regions in zinc finger protein, X-linked (ZFX) and zinc finger protein, Y-linked (ZFY). In cases of KS, the ratios of ZFX/ZFY are altered compared to those in normal males. As a result, the specific melting profiles differ and can be differentiated during data analysis. This HRM assay displayed high analytical specificity over a wide range of template DNA amounts (5 ng-50 ng) and reproducibility, high resolution for detecting KS mosaicism, and high clinical sensitivity (100%) and specificity (98.1%). Moreover, the HRM assay was rapid (2 h per run), inexpensive (0.2 USD per sample), easy to perform and automatic, and compatible with both whole blood samples and dried blood spots. Therefore, this HRM assay is an ideal postnatal population-based KS screening tool that can be used for different age groups.

Rapid screening for Klinefelter syndrome with a simple high-resolution melting assay: a multicenter study / 亚洲男科学杂志(英文版)

Klinefelter syndrome (KS) is the set of symptoms that result from the presence of an extra X chromosome in males. Postnatal population-based KS screening will enable timely diagnosis of this common chromosomal disease, providing the opportunity for early intervention and therapy at the time point when they are most effective and may prevent later symptoms or complications. Therefore, through this study, we introduced a simple high-resolution melting (HRM) assay for KS screening and evaluated its clinical sensitivity and specificity in three medical centers using 1373 clinical blood samples. The HRM assay utilized a single primer pair to simultaneously amplify specific regions in zinc finger protein, X-linked (ZFX) and zinc finger protein, Y-linked (ZFY). In cases of KS, the ratios of ZFX/ZFY are altered compared to those in normal males. As a result, the specific melting profiles differ and can be differentiated during data analysis. This HRM assay displayed high analytical specificity over a wide range of template DNA amounts (5 ng-50 ng) and reproducibility, high resolution for detecting KS mosaicism, and high clinical sensitivity (100%) and specificity (98.1%). Moreover, the HRM assay was rapid (2 h per run), inexpensive (0.2 USD per sample), easy to perform and automatic, and compatible with both whole blood samples and dried blood spots. Therefore, this HRM assay is an ideal postnatal population-based KS screening tool that can be used for different age groups.

Mutaciones en JAK2, MPL y CALR en neoplasias mieloproliferativas: análisis de disociación de alta resolución / Mutations in JAK2, MPL and CALR in myeloproliferative neoplasms: High Resolution Melting analysis / Mutações em JAK2, MPL e CALR em neoplasias mieloproliferativas: análise de dissociação em alta resolução

La policitemia vera (PV), la trombocitemia esencial (TE) y la mielofibrosis idiopática (MI) constituyen las Neoplasias Mieloproliferativas cromosoma Filadelfia negativas (NMP Ph-neg). La mutación V617F en el exón 14 del gen JAK2 ha sido descripta en un 90% de los casos de PV y en un 50% de TE y MI. Recientemente, se identificaron mutaciones en el exón 10 del gen MPL y en el exón 9 del gen CALR, presentes en un 5 y 73% de pacientes con TE y MI sin mutaciones en JAK2, respectivamente. En el presente trabajo se estudió la detección de dichas mutaciones en 52 pacientes con NMP, mediante amplificaciones por PCR en Tiempo Real con posterior análisis por High Resolution Melting (HRM) y secuenciación. La mutación V617F en JAK2 fue registrada en un 83,3% de pacientes con PV y 42,8% con TE y MI. Un 6,25% y 56,25% de pacientes con TE y MI JAK2 negativos resultaron positivos para mutaciones en el exón 10 de gen del receptor de la trombopoyetina (MPL) y el exón 9 de gen de la calreticulina (CALR). El análisis por HRM puede ser considerado como herramienta diagnóstica eficaz para las NMP debido a su alta sensibilidad, bajo costo y tiempo de procesado, teniendo en cuenta el impacto clínico que podría tener en los pacientes la detección temprana de dichas mutaciones.

Polycythemia vera (PV), essential thrombocythemia (TE) and idiopathic myelofibrosis (MI) are Philadelphia chromosome-negative myeloproliferative neoplasms (MPN-Ph. Neg). The presence of the V617F mutation in exon 14 of the JAK2 gene has been described in 90% of cases of PV and 50% of MI and TE. Recently, mutations in exon 10 of the MPL gene and exon 9 of CALR gene have been identified, which are present in 5 to 73% of patients with TE and MI without mutations in JAK2, respectively. In this work, the detection of these mutations was studied in 52 patients with NMP, using real time PCR amplifications with subsequent High Resolution Melting (HRM) analysis and sequencing. A total of 83.3% of patients with PV and 42.8% with MI and TE were recorded as positive for the V617F mutation in JAK2. A total of 6.25% and 56.25% of the patients with MI and TE with non-mutated JAK2 were positive for mutations in MPL exon 10 and CALR exon 9. HRM analysis could be considered an effective diagnostic tool for NMP due to its high sensitivity, low cost and processing time, taking into account the clinical impact that early detection of such mutations could have on patients.

Policitemia vera (PV), trombocitemia essencial (TE) e mielofibrose idiopática (MI) constituem as Neoplasias Mieloproliferativas cromossomo Filadélfia negativas (NMP Ph-neg). A mutação V617F no exon 14 do gene JAK2 foi descrita em 90% dos casos de PV e em 50% de TE e MI. Recentemente, foram identificadas mutações no exon 10 do gene MPL e no exon 9 do gene CALR, presentes em 5 a 73% de pacientes com TE e MI sem mutações em JAK2, respectivamente. Neste trabalho foi estudada a detecção de tais mutações em 52 pacientes com NMP, usando amplificações por PCR em Tempo Real, com posterior análise por High Resolution Melting (HRM) e sequenciamento. 83,3% dos pacientes com PV e 42,8% com TE e MI foram positivos para a mutação V617F em JAK2. 6,25% e 56,25% de pacientes com TE e MI JAK2 negativo foram positivos para mutações no exon 10 de gene do receptor da trombopoietina (MPL) e o exon 9 de gene da calreticulina (CALR). A análise por HRM pode ser considerada como ferramenta de diagnóstico eficaz para as NMP, devido à sua alta sensibilidade, baixo custo e tempo de processamento, tendo em conta o impacto clínico que poderia ter a detecção precoce de tais mutações nos pacientes.

Evaluation of SYTO13 as fluorescent dye for high resolution melting based single nucleotide polymorphism genotyping / 中华检验医学杂志

Objective To evaluate the utility of fluorescent dye SYTO 13 for high -resolution melting ( HRM) detection in single nucleotide polymorphism ( SNP) genotyping and its clinical application . Methods This is a performance verification study .36 genotype defined samples were divided into three groups:SNP rs3125734 C>T (class Ⅰ SNP) ,rs255758 A>C (class ⅡSNP) and rs688C>T.These samples were used to evaluate SYTO 13′s SNP genotyping capability of class ⅠSNP, classⅡSNP, and two PCR products of different lengths (52 and 107 bp) covering the same SNP of rs688C>T.The commercial HRM dye of LCGreen Plus was used as the control .The genotyping capability is indicated by the Tm difference(ΔTm) between wild type and homozygous mutant genotypes .The Tm differences between wild genotype and homozygous mutant genotype were compared using the Independent Samples t test.Paired t test was used to evaluate genotyping capability of the two dyes .The clinical applicability is evaluated by synchronously performing PCR amplification and HRM analysis on thirty -five randomly selected DNA samples with known genotypes of the three SNPs .Results The SNPs of class Ⅰ and class Ⅱ can be genotyped directly and clearly with SYTO13 (ΔTmclas Ⅰ =0.36 ±0.05,tclas Ⅰ =14.827,Pclas Ⅰ =0.000;ΔTm clas Ⅱ =0.42 ±0.110,tclasⅡ =9.539,Pclas Ⅱ =0.000).The classⅠSNP genotyping results was better using SYTO13 (ΔTmSYTO13 =0.39 ±0.027), while the SNP genotyping for small amplicon did not discriminated clearly in this study .Long amplicons of class ⅠandⅡSNPs can be identified directly except for several samples which can be genotyped accurately after having performed reexamination .Conclusion SYTO13 can apply for HRM analysis of genotyping classⅠand ⅡSNPs with long amplicon and for clinical routine detection.

Rapid detection of isoniazid resistance in clinical Mycobacterium tuberculosis isolates by high-resolution melting curve analysis / 中国人兽共患病学报

We detected the isoniazid resistance in clinical Mycobacterium tuberculosis isolates by high-resolution melting (HRM) curve analysis and assessed the application value of the assay.The isoniazid resistance of 49 M.tuberculosis isolates preserved in laboratory was analyzed by the drug sensitivity test (traditional proportion method).Further analysis was made on the sequencing of the isoniazid resistance determining region in these test strains,and their mutation sites were screened.Specific primers used in the HRM curve analysis were designed based on the screened mutation sites,DNA mutations were assayed in the isoniazid-resistant gene determining region by the HRM curve analysis,and an assessment was made of the detection efficiency of the assay in isoniazid resistance in M.tuberculosis.Results of the drug sensitivity test (proportion method) showed that,of the 49 test strains,there were 20 isoniazid-resistant strains,29 isoniazid-sensitive strains.Results of the sequencing analysis showed that:1) KatG gene had four mutation patterns,i.e.,point mutations at site 234,at sites 234 and 315,at sites 234 and 463,and at sites 234,315 and 463;2) there were three mutations were detected in inhA gene,i.e.,mutations in inhA-8,-15 and-152.Analysis of gene mutation in drug-resistant strains found that of the 20 isoniazid-resistant strains,11 (55 ％) were mutated at codon 315 of KatG gene;6 (30％) were mutated in inhA-15 (4/20),-8 (1/20) and-153 (1/20) of inhA gene;two (10％) were mutated at codon 315 of KatG gene and in inhA-15;in one strain (5％),no mutation was detected in KatG and inhA genes.Through the gene mutation detection,the sensitivity and specificity of isoniazid resistance in M.tuberculosis were 95 ％ and 100 ％,respectively.Results of HRM curve analysis of drug-resistance gene mutations in test strains showed gene mutations were present in 18 strains and absent in 24 ones;referring to DNA sequencing results,the sensitivity and specificity of the assay were 94.7％ and 80％,respectively.Judged by mutations as drug-resistance via the HRM curve analysis,19 resistant and 24 sensitive strains were tested.With the drug sensitivity test results by the proportion method as controls,the sensitivity and specificity of the assay were 95 ％ and 82.76 ％,respectively.Use of the HRM curve in the detection of resistance of M.tuberculosis to isoniazid is characterized by good sensitivity and short time consuming,and has certain value in the rapid diagnosis of isoniazid-resistant tuberculosis.