Probe Melting Curve Analysis Research Articles

Screening results and mutation frequency analysis of G6PD deficiency in 1,291,274 newborns in Huizhou, China: a twenty-year experience.

This study aimed to investigate the incidence rate and spectrum of gene mutations of Glucose-6-phosphate dehydrogenase (G6PD) deficiency in the Huizhou city of southern China to provide a scientific basis for disease prevention and control in the area. From March 2003 to December 2022, newborn screening for G6PD enzyme activity was carried out in Huizhou city using the fluorescence quantitative method. Infants who tested positive during the initial screening were diagnosed using the nitroblue tetrazolium ratio method, while a subset of infants received further gene mutation analysis using the multicolor probe melting curve analysis method. A total of 1,291,274 newborns were screened and the screening rate has increased from 20.39% to almost 100%. In the 20-year period, 57,217 (4.43%) infants testing positive during the initial screening. Out of these infants, 49,779 (87%) were recalled for confirmatory testing. G6PD deficiency was confirmed in 39,261 of the recalled infants, indicating a positive predictive value of 78.87%. The estimated incidence rate of G6PD deficiency in the region was 3.49%, which was significantly higher than the average incidence rate of 2.1% in southern China. On the other hand, seven pathogenic G6PD variants were identified in the analysis of the 99 diagnosed infants with the most common being c.1388 G > A (48.5%), followed by c.95 A > G (19.2%), c.1376 G > T (15.2%), c.871 G > A (9.1%), c.1360 C > T (3.0%), c.392 G > T (3.0%), and c.487 G > A (1.0%). The incidence of G6PD deficiency in newborns in the Huizhou city was higher than the southern China average level, while the types and frequencies of gene mutations were found to vary slightly from other regions. Our findings suggested that free government screening and nearby diagnosis strategies could reduce the incidence of G6PD deficiency in the area.

A Novel Molecular Method for Simultaneous Identification of Vibrio parahaemolyticus 57 K-Serogroups Using Probe Melting Curve Analysis.

The serotyping of Vibrio parahaemolyticus, which is crucial to the surveillance and detection of outbreaks of vibriosis infection, has been widely used in many countries. In this study, we developed a molecular assay, named multiplex ligation reaction based on probe melting curve analysis (MLMA), for simultaneous identification of V. parahaemolyticus 57 K-serogroups. Based on the previous genomes of 418 strains including 39 K-serogroups and the 18 K-serogroups sequences from public databases, we obtained 57 K-serogroups specific gene sequences for designing primers and probes. The developed MLMA assay for identifying the V. parahaemolyticus 57 K-serogroups showed high reproducibility, with the intra- and inter-assay standard deviations and coefficients of variation of no more than 1°C and 1%, respectively. The limit of detection for all gene targets ranged from 0.1 to 1.0 ng/µl. We validated the MLMA assay with a double-blind test identifying 595 V. parahaemolyticus isolates using conventional serotyping methods for comparison. The results showed the kappa value between the MLMA assay and the traditional serological method was 0.936 and that there was a 96.97% consistency rate with conventional serotyping methods for all detected isolates. Additionally, five rare K-serogroups were identified using the MLMA assay, as well as 18 strains that could not be identified using the traditional serotyping method. Thus, the MLMA assay provides a rapid, robust, and promising tool for the molecular serotyping of V. parahaemolyticus K-serogroups and has the potential application to the detection of outbreaks and surveillance of V. parahaemolyticus infection.

Multiplex ligation reaction based on probe melting curve analysis: a pragmatic approach for the identification of 30 common Salmonella serovars

BackgroundWhile Salmonella serotyping is of paramount importance for the disease intervention of salmonellosis, a fast and easy-to-operate molecular serotyping solution remains elusive. We have developed a multiplex ligation reaction based on probe melting curve analysis (MLMA) for the identification of 30 common Salmonella serovars.MethodsSerovar-specific primers and probes were designed based on a comparison of gene targets (wzx and wzy encoding for somatic antigen biosynthesis; fliC and fljB for flagellar antigens) from 5868 Salmonella genomes. The ssaR gene, a type III secretion system component, was included for the confirmation of Salmonella.ResultsAll gene targets were detected and gave expected Tm values during assay evaluation. Cross reactions were not demonstrated between the 30 serovars (n = 211), or with an additional 120 serovars (n = 120) and other Enterobacteriaceae (n = 3). The limit of identification for all targets ranged from using 1.2 ng/μL to 1.56 ng/μL of DNA. The intra- and inter-assay standard deviations and the coefficients of variation were no more than 0.5 °C and less than 1% respectively, indicating high reproducibility. From consecutive outpatient stool samples (n = 3590) collected over a 10-month period at 11 sentinel hospitals in Shenzhen, China, we conducted a multicenter study using the traditional Salmonella identification workflow and the MLMA assay workflow in parallel. From Salmonella isolates (n = 496, 13.8%) derived by both workflows, total agreement (kappa = 1.0) between the MLMA assay and conventional serotyping was demonstrated.ConclusionsWith an assay time of 2.5 h, this simple assay has shown promising potential to provide rapid and high-throughput identification of Salmonella serovars for clinical and public health laboratories to facilitate timely surveillance of salmonellosis.

Simultaneous Identification of Clinically Common Vibrio parahaemolyticus Serotypes Using Probe Melting Curve Analysis

The dynamic nature of Vibrio parahaemolyticus epidemiology has presented a unique challenge for disease intervention strategies. Despite the continued rise of disease incidence and outbreaks of vibriosis, as well as the global emergence of pandemic clones and serovariants with enhanced virulence, there is a paucity of molecular methods for the serotyping of V. parahaemolyticus strains to improve disease surveillance and outbreak investigations. We describe the development of a multiplex ligation reaction based on probe melting curve analysis (MLMA) for the simultaneous identification of 11 clinically most common V. parahaemolyticus serotypes spanning a 10-year period. Through extensive sequence analyses using 418 genomes, specific primers and probes were designed for a total of 22 antigen gene targets for the O- and K- serogroups. Additionally, the toxR gene was incorporated into the assay for the confirmation of V. parahaemolyticus. All gene targets were detected by the assay and gave expected Tm values, without any cross reactions between the 11 clinically common serotypes or with 38 other serotypes. The limit of identification for all gene targets ranged from 0.1 to 1 ng/μL. The intra- and inter-assay standard deviations and the coefficients of variation were no more than 1°C and <1% respectively, indicating a highly reproducible assay. A multicenter double-blind clinical study was conducted using the traditional V. parahaemolyticus identification workflow and the MLMA assay workflow in parallel. From consecutive diarrheal stool specimens (n = 6118) collected over a year at 10 sentinel hospitals, a total of 153 V. parahaemolyticus isolates (2.5%) were identified by both workflows. A total agreement (kappa = 1.0) between the serotypes identified by the MLMA assay and conventional serological method was demonstrated. This is the first molecular assay to simultaneously identify multiple clinically important V. parahaemolyticus serotypes, which satisfies the acute need for a practical, rapid and robust identification of V. parahaemolyticus serotypes to facilitate the timely detection of vibriosis outbreaks and surveillance.

Evaluation of Real-Time PCR Coupled With Multiplex Probe Melting Curve Analysis for Pathogen Detection in Patients With Suspected Bloodstream Infections.

Background: This study aimed to evaluate real-time polymerase chain reaction coupled with multiplex probe melting curve analysis (PCR-MCA) for pathogen detection in patients with suspected bloodstream infections (BSIs).Methods: A PCR-MCA assay was developed for simultaneous identification of 28 kinds of the most common pathogens and two resistance genes within a few hours. The diagnostic performance of the PCR-MCA assay was determined and compared to the results of blood culture.Results: A total of 2,844 consecutive new episodes of suspected BSIs in 2,763 patients were included in this study. There were 269 episodes of pathogens identified by blood culture. For all the pathogens tested, the PCR-MCA assay exhibited a sensitivity of 88.8% (239/269), specificity of 100% (2,575/2,575), and agreement of 98.9% (2,814/2,844). For the pathogens on the PCR-MCA list, the PCR-MCA results had a sensitivity of 99.2% (239/241), specificity of 100% (2,575/2,575), and agreement of 99.9% (2,814/2,816) compared with the results of blood culture. For seven samples with multiple pathogens identified simultaneously during one blood culture investigation, the PCR-MCA assay verified the results of the blood culture, with an agreement rate of 100% for each.Conclusion: The PCR-MCA assay could discover 88.8% of the pathogens in clinical practice, showing excellent diagnostic performance vs. that of blood culture for pathogen detection in patients with suspected BSIs, and would contribute to rapid diagnosis and correct antibiotic administration.

Detection of IDH1 and IDH2 Mutation in Formalin-fixed Paraffin-embedded Gliomas Using Allele-specific COLD-PCR and Probe Melting Curve Analysis

Many tumors, especially gliomas, contain an isocitrate dehydrogenase (IDH) mutation that can be used for clinical diagnosis and prognosis. Our study aimed to develop a new reliable detection assay for IDH1 and IDH2 mutations for clinical diagnosis based on the allele-specific (AS) coamplification with lower denaturing-polymerase chain reaction (COLD-PCR) and probe melting curve analysis (PMCA). The method includes 3 elements allowing for the sensitive detection of low-abundance mutations: (1) PCR amplification of the target fragments with AS primers; (2) COLD-PCR; and (3) PMCA for differentiating the different mutations after amplification. We conducted a blinded study with 45 paraffin-embedded gliomas specimens and 13 fresh specimens screened for IDH mutations using Sanger sequencing. Concordance between the results of our AS-COLD-PCR/PMCA assay and Sanger sequencing was 100%. Our assay appeared to be superior to direct sequencing with a much higher sensitivity of 0.4% mutations. In summary, our assay is a cost-effective, convenient, and sensitive method for detecting IDH mutations and could be applied in the clinical setting to assess small brain biopsies.

A novel method of multiple nucleic acid detection: Real-time RT-PCR coupled with probe-melting curve analysis

A novel method, real-time reverse transcription PCR (real-time RT-PCR) coupled with probe-melting curve analysis, has been established to detect two kinds of samples within one fluorescence channel. Besides a conventional TaqMan probe, this method employs another specially designed melting-probe with a 5′ terminus modification which meets the same label with the same fluorescent group. By using an asymmetric PCR method, the melting-probe is able to detect an extra sample in the melting stage effectively while it almost has little influence on the amplification detection. Thus, this method allows the availability of united employment of both amplification stage and melting stage for detecting samples in one reaction. The further demonstration by simultaneous detection of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in one channel as a model system is presented in this essay. The sensitivity of detection by real-time RT-PCR coupled with probe-melting analysis was proved to be equal to that detected by conventional real-time RT-PCR. Because real-time RT-PCR coupled with probe-melting analysis can double the detection throughputs within one fluorescence channel, it is expected to be a good solution for the problem of low-throughput in current real-time PCR.

A portable microfluidic platform for rapid molecular diagnostic testing of patients with myeloproliferative neoplasms

The ability to simultaneously detect JAK2 V617F and MPL W515K/L mutations would substantially improve the early diagnosis of myeloproliferative neoplasms (MPNs) and decrease the risk of arterial thrombosis. The goal of this study is to achieve a point of care testing platform for simultaneous analysis of major genetic alterations in MPN. Here, we report a microfluidic platform including a glass capillary containing polypropylene matrix that extracts genomic DNA from a drop of whole blood, a microchip for simultaneous multi-gene mutation screening, and a handheld battery-powered heating device. The µmLchip system was successfully used for point-of-care identification of the JAK2 V617F and MPL W515K/L mutations. The µmLchip assays were then validated by mutation analysis with samples from 100 MPN patients who had previously been analyzed via unlabeled probe melting curve analysis or real-time PCR. The results from the µmLchip were in perfect agreement with those from the other methods, except for one discrepant result that was negative in the unlabeled probe melting curve analysis but positive in the µmLchip. After T-A cloning, sequences of cloned PCR products revealed JAK2 V617F mutation in the sample. The portable microfluidic platform may be very attractive in developing point-of-care diagnostics for MPL W515K/L and JAK2 V617F mutations.

Simultaneous Identification of Ten Bacterial Pathogens Using the Multiplex Ligation Reaction Based on the Probe Melting Curve Analysis

Pathogenic Vibrio spp., Aeromonas spp. and Plesiomonas shigelloides are associated with human gastroenteritis and wound infections, as well as fish diseases. The comprehensive and accurate identification of these pathogens is crucial for the current public health. The present study describes the development of a multiplex assay for the simultaneous identification of ten bacterial pathogens in a single reaction by using a multiplex ligation reaction based on probe melting curve analysis (MLMA). The specificity for target genes was 100%, as assessed with a panel of 67 bacterial pathogens, which indicated no cross-reactions. The detection limit of this assay ranged from 0.8 × 107 CFU/mL to 1.5 × 108 CFU/mL at the pure bacterial culture level and from 0.1 ng to 1.0 ng at the DNA level. The MLMA assay was used to detect ten species of pathogens in 269 clinical and seafood samples, and for further validation, the results were compared with the conventional culture method. The results indicated greater than 90% sensitivity and 100% specificity for each bacterial pathogen tested, and the kappa correlation for all the pathogens ranged from 0.95 to 1.00. Overall, this assay is well suited for public health laboratories for its high throughput, accuracy, and low cost.

Rapid and simultaneous detection of common aneuploidies by quadruplex real-time polymerase chain reaction combined with melting curve analysis.

BackgroundDuring the prenatal period, the number variation of chromosomes 13, 18, 21, X and Y accounts for more than 80% of the clinically significant chromosomal abnormalities diagnosed. Rapid tests for prenatal diagnosis of these abnormalities can improve pregnancy management and alleviate parental anxiety. Here, we present a molecular alternative method for detecting common aneuploidies.MethodsThis method is based on co-amplification of segmental duplications located on two different chromosomes using a single pair of primers. Segmental duplications have a high degree of sequence identity, but have single-nucleotide differences in some regions. These sequence differences can be quantified using melting curve analysis of dual-labeled probes to estimate the relative dosages of different chromosomes. We designed two quadruplex real-time PCR assays to detect aneuploidies of chromosomes 13, 18, 21, X and Y.ResultsWe examined 75 aneuploid DNA samples and 56 unaffected DNA control samples using these two assays and correctly identified all samples. Four cases of unbalanced translocation were also accurately detected. The observed averaged ratio for each chromosomal disorder was similar to the theoretically expected value.ConclusionsOur real-time assay is a robust, rapid, and easy to conduct technique for prenatal diagnosis of common aneuploidies, representing a competitive alternative for use in diagnostic laboratories.

The application of multicolor probe melting curve analysis for the prenatal diagnosis of β thalassemia

Objective To establish a method of multicolor melting curve analysis for the prenatal diagnosis of β thalassemia. Methods Methodology establishment. A total of 95 cases, including 9 fetal villi samples(10-13 weeks)and 86 amniotic fluid samples(18-24 weeks)were collected by Center for Prenatal Diagnosis of Shenzhen Maternity and Child Healthcare Hospital between January 2014 and December 2014. A double-blind test was done to detect the mutations of beta globin gene by means of reverse dot (RDB) blot and multicolor melting curve analysis(MMCA). The consistency of the two methods is compared. Results The results of 93 cases detected by MMCA and RDB are completely consistent. The results of the 2 cases detected by MMCA after correction are the same as the results detected by RDB. Finally, the coincidence rate of the result was 100%. Conclusion MMCA can be applied to the prenatal diagnosis of β thalassemia as an effective supplement to RDB.(Chin J Lab Med, 2016, 39: 192-196) Key words: beta-Thalassemia; Prenatal diagnosis; Reverse transcriptase polymerase chain reaction; Nucleic Acid Hybridization

New real-time-PCR method to identify single point mutations in hepatitis C virus.

AIMTo develop a fast, low-cost diagnostic strategy to identify single point mutations in highly variable genomes such as hepatitis C virus (HCV).METHODSIn patients with HCV infection, resistance-associated amino acid substitutions within the viral quasispecies prior to therapy can confer decreased susceptibility to direct-acting antiviral agents and lead to treatment failure and virological relapse. One such naturally occurring mutation is the Q80K substitution in the HCV-NS3 protease gene, which confers resistance to PI inhibitors, particularly simeprevir. Low-cost, highly sensitive techniques enabling routine detection of these single point mutations would be useful to identify patients at a risk of treatment failure. LightCycler methods, based on real-time PCR with sequence-specific probe hybridization, have been implemented in most diagnostic laboratories. However, this technique cannot identify single point mutations in highly variable genetic environments, such as the HCV genome. To circumvent this problem, we developed a new method to homogenize all nucleotides present in a region except the point mutation of interest.RESULTSUsing nucleotide-specific probes Q, K, and R substitutions at position 80 were clearly identified at a sensitivity of 10% (mutations present at a frequency of at least 10% were detected). The technique was successfully applied to identify the Q80K substitution in 240 HCV G1 serum samples, with performance comparable to that of direct Sanger sequencing, the current standard procedure for this purpose. The new method was then validated in a Catalonian population of 202 HCV G1-infected individuals. Q80K was detected in 14.6% of G1a patients and 0% of G1b in our setting.CONCLUSIONA fast, low-cost diagnostic strategy based on real-time PCR and fluorescence resonance energy transfer probe melting curve analysis has been successfully developed to identify single point mutations in highly variable genomes such as hepatitis C virus. This technique can be adapted to detect any single point mutation in highly variable genomes.

Toward point-of-care testing for JAK2 V617F mutation on a microchip

Molecular genetics now plays a crucial role in diagnosis, the identification of prognostic markers, and monitoring of hematological malignancies. Demonstration of acquired changes such as the JAK2 V617F mutation within myeloproliferative neoplasms (MPN) has quickly moved from a research setting to the diagnostic laboratory. Microfluidics-based assays can reduce the assay time and sample/reagent consumption and enhance the reaction efficiency; however, no current assay has integrated isothermal amplification for point-of-care MPN JAK2 V617F mutation testing with a microchip. In this report, an integrated microchip that performs the whole human blood genomic DNA extraction, loop-mediated isothermal nucleic acid amplification (LAMP) and visual detection for point-of-care genetic mutation testing is demonstrated. This method was validated on DNA from cell lines as well as on whole blood from patients with MPN. The results were compared with those obtained by unlabeled probe melting curve analysis. This chip enjoys a high accuracy, operability, and cost/time efficiency within 1h. All these benefits provide the chip with a potency toward a point-of-care genetic analysis. All samples identified as positive by unlabeled probe melting curve analysis (n=27) proved positive when tested by microchip assay. None of the 30 negative controls gave false positive results. In addition, a patient with polycythemia vera diagnosed as being JAK2 V617F-negative by unlabeled probe melting curve analysis was found to be positive by the microchip. This microchip would possibly be very attractive in developing a point-of-care platform for quick preliminary diagnosis of MPN or other severe illness in resource-limited settings.

Practical Prediction of Ten Common Streptococcus pneumoniae Serotypes/Serogroups in One PCR Reaction by Multiplex Ligation-Dependent Probe Amplification and Melting Curve (MLPA-MC) Assay in Shenzhen, China.

Background Streptococcus pneumoniae has more than 95 distinct serotypes described to date. However, only certain serotypes are more likely to cause pneumococcal diseases. Thus serotype surveillance is important for vaccine formula design as well as in post-vaccine serotype shift monitor. The goal of this study was to develop a practical screening assay for ten Shenzhen China common pneumococcal serotypes/serogroups in one molecular reaction.MethodsA molecular assay, based on multiplex ligation-dependent probe amplification (MLPA) and melting curve (MC) analysis, was developed in an integrated approach (MLPA-MC) for the detection of ten capsular serotypes/serogroups 4, 6 (6A/6B/6C/6D), 9V/9A, 14, 15F/15A, 15B/15C, 18 (18F/18A/18B/18C), 19F, 19A and 23F. We designed serotype/serogroup-specific MLPA probes and fluorescent detection probes to discriminate the different serotypes/serogroups in one molecular reaction. The three steps of MLPA-MC assay are continuous reactions in one well detected by LightCycler 480. A total of 210 S. pneumoniae isolates from our local Maternity and Child Health Hospital were randomly chosen to evaluate the assay against published multiplex PCR assays.ResultsOur results showed that 198 (94.3%) of S. pneumoniae isolates were type-able by our assays and the results were in complete concordance with the published multiplex PCRs. Using the MLPA-MC assay, 96 S. pneumoniae isolates could be typed within 3 hours with limited hands-on time. This serotype/serogroup-screening assay can be easily modified or extended by modification of the serotype/serogroup-specific MLPA probes combinations according to the needs of different laboratories.ConclusionsWe recommend use of this assay as a starting point for screening serotype/serogroup frequencies. There is a need for this assay to be combined with other molecular typing assays, like published serotype specific PCRs, or even the Quellung reaction for serotype confirmation.

212P - BRAF V600E: Prognostic Marker in Colorectal Cancer

ABSTRACT Activation of BRAF oncogene has been implicated in colorectal carcinogenesis. BRAF protein is a serin/threonine kinase, component of a conserved signaling pathway that regulates cellular responses to exstracellular signals. In human cancer, it is commonly activated by somatic hotspot mutation at nucleotide 1799 of the BRAF gene which leads to a single aminoacid substitution Val600Glu (V600E). The aim of this study was to compare clinical and pathological phenotype of colorectal cancers with incidence of BRAF gene mutation. Clinical and pathological characteristics such as age, sex, tumor size, histologic grade, Dukes' stage, angioinvasion and tumor position were collected for each patient. Sections from 113 formalin-fixed paraffin-embedded (FFPE) tumor samples were evaluated for the BRAF V600E mutation using LightCycler PCR with allele-specific fluorescent probe melting curve analysis. Differences in clinical and pathological variables between the groups of patients with and without BRAF mutation were analyzed using Fisher exact test. Our results show that BRAF gene mutation was detected in 8.8% (10/113) samples. Statistical analysis revealed a significant association between the BRAF mutation and Dukes' stage (p = 0.04) where all mutations were found in tumors classified as Dukes'C (10/10). Incidence of mutation was higher in males, patients older than 60 years, tumors bigger than 5 cm, tumors with angioinvasion and poor differentiated tumors, but no significant association was found. All BRAF gene mutations were detected in colon cancers (p = 0.01). We can conclude that incidence of BRAF gene mutation was higher in tumors with poor prognostic markers which have been associated with poor tumor prognosis and progression of disease. Disclosure All authors have declared no conflicts of interest.

Comparison between probe melting curve analysis based on real-time fluorescent PCR and reverse dot blot assay for gene and prenatal diagnosis of β-thalassemia

Objective To evaluate the reliability of the probe melting curve analysis (PMCA) based on real-time fluorescent PCR assay for the genetic diagnosis and prenatal diagnosis of β-thalassemia. Methods A total of 135 cases of peripheral blood samples were collected from Zhuhai Municipal Maternity and Child Healthcare Hospital between 2008 and 2010.All the samples were performed preliminary diagnosis according to the hematological data.Of these, 119 cases were diagnosed as β-thalassemia trait, 4 cases were diagnosed as severe thalassemia and 12 cases were normal.In addition, 44 cases of amniotic fluid and 8 cases of cord blood with high-risk for severe β-thalassemia were also collected.The diagnostic reliability of the PMCA assay and reverse dot blot assay was evaluated on 187 above-mentioned cases by direct DNA sequencing analysis in a double-blind study. Results The genotypes of 185 cases were detected accurately based on the PMCA assay except for two cases:one heterozygote with Ini(ATG>AGG) was omitted and another heterozygote couldn′t be distinguished between CD43(G>T) and CD37(G>A).For the RDB assay, only one heterozygote with CD71-72(+T) was not detected accurately in the above-mentioned cases.Compared with the DNA sequencing analysis, the sensitivity, specificity, negative predictive value, positive predictive value and diagnostic efficiency of the PMCA assay were 98.75%, 100.00%, 93.10%, 100.00% and 98.93%, respectively.The corresponding value of the RDB assay were 99.38%, 100.00%, 96.42%, 100.00% and 99.47%, respectively.There were no significant between-group differences in the diagnostic efficiency of the two assays (P>0.05).The results of prenatal diagnosis were in complete concordance with the follow up results, after the birth or induced labour of the fetuses. Conclusions The PMCA assay can be used as an alternative and verified method of RDB assay for the genetic diagnosis and prenatal diagnosis of β-thalassemia.(Chin J Lab Med, 2012，35：413-417) Key words: beta-Thalassemia; Polymerase chain reaction; Melting curve analysis; Nucleic acid hybridization; Prenatal diagnosis

Development and Inter-Laboratory Validation of Unlabeled Probe Melting Curve Analysis for Detection of JAK2 V617F Mutation in Polycythemia Vera

BackgroundJAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described.Methodology/Principal FindingsAsymmetric PCR for detection of JAK2 V617F with a 3′-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex) in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type) covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments.ConclusionsWith a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.

Abstract A23: Rare allele enrichment by DNA melting analysis

Abstract Since the sequencing of the human genome and the development of high throughput genotyping techniques, many genes have been identified that are associated with a variety of diseases through their SNPs and mutations. For example, in lung cancer, mutations in the gene encoding epidermal growth factor reporter (EGFR) can confer either positive therapeutic response or drug resistance to tyrosine kinase inhibitors. Additionally, mutations in the BRAF gene are strongly associated with cancerous thyroid nodules. In the early and post-treatment stages of cancer or in prenatal diagnostics, the proportion of the mutant allele is typically low within a background of wild-type. Even after amplification by standard PCR, the low levels of the minority allele that are present in tumor tissue or in plasma circulating DNA are not sufficient to permit genotyping or sequencing. Mutation detection plays a key role in several areas of medicine including diagnosis, treatment and prognosis. In cancer-related gene mutation diagnostics, low-level (&amp;lt;10%) mutations cannot be sequenced by standard PCR or genotyping techniques. We have developed methods of rare allele enrichment and melting curve detection using Snapback primers and unlabeled probes. A snapback primer is a tail at the 5′ end of a primer that is complementary to its own extension product. When intramolecular hybridization is favored over intermolecular pairing, a hairpin is formed whose stem melting behavior depends on sequence. Rapid cycle PCR with a short extension time (0 sec) and an extension temperature lower than the Tm of the hairpin of the wild-type, perfectly matched allele obstructs primer extension while mismatched alleles denature, thus providing the competitive advantage and preferential amplification of minority alleles. Minority alleles down to 0.1% (1:1000) can be detected. Even greater detection sensitivity can be achieved using allele specific PCR, probe blocking and melting analysis. Unlabeled probes, dual hybridization probes, or molecular beacons can be used. Allele-specific competitive blocker PCR (ACB-PCR) was modified by using asymmetric PCR and melting analysis to increase the sensitivity. The probe serves as both a blocker and produces the probe melting curve to implement detection. Mutation detection by probe melting curve analysis is more reliable than PCR quantification because only the specifically amplified allele produces the melting curve associated with the target. Quantitative PCR with dsDNA dyes cannot distinguish the intended PCR product from primer dimers and other non-specific PCR products. In contrast, when using probe melting curves to detect mutations, the melting temperature and overall melting profile of probe melting curves specifically identify the known mutant target. Additionally, based upon probe melting temperatures of wild-type and mutant hybrids, a precise annealing temperature between these Tms guarantees that the PCR selectively amplifies the minority allele. Low allele specific primer concentrations (asymmetric PCR) further increase the sensitivity and increase the probe signal allowing a sensitivity of 0.001% (1:105) mutant to wild-type genomic DNA, or just a single copy in a 500 ng genomic DNA. Methods that do not require labeled probes (snapback primer and unlabeled probes) can be used to selectively amplify minority alleles by methods that are rapid (less than 20 minutes), easy to design, inexpensive and reliable. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A23.

Rapid Detection and Molecular Differentiation of Toxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans Strains by LightCycler PCR

The systemic symptoms of diphtheria are caused by the tox-encoded diphtheria toxin (DT) which is produced by toxigenic Corynebacterium spp. Besides the classical agent C. diphtheriae, the zoonotic pathogen C. ulcerans has increasingly been reported as an emerging pathogen for diphtheria. The reliable detection of toxigenic Corynebacterium spp. is of substantial importance for both diphtheria surveillance in the public health sector and the clinical workup of a patient with diphtherialike symptoms. Since the respective tox genes of C. diphtheriae and C. ulcerans differ from each other in both DNA and amino acid sequence, both tox genes should be covered by novel real-time PCR methods. We describe the development and validation of a LightCycler PCR assay which reliably recognizes tox genes from both C. diphtheriae and C. ulcerans and differentiates the respective target genes by fluorescence resonance energy transfer (FRET) hybridization probe melting curve analysis.

5′-MGB Probes Allow Rapid Identification of Methanogens and Sulfate Reducers in Cold Marine Sediments by Real-Time PCR and Melting Curve Analysis

The analysis of microorganism communities in uncultured environmental samples requires laborious and cumbersome techniques such as denaturing gradient gel electrophoresis of amplicons generated with 16S rRNA generic primers with subsequent fragment sequencing. We have developed a simple method for genus identification of methanogen archaea and sulfate-reducing bacteria based on a real-time PCR hybridization probe melting curve analysis. The method takes advantage of a recent explosion of microorganism sequencing data conveniently packaged in the Ribosomal Database Project. Specificity of detection is based on a genus-specific real-time PCR fluorescent 5'-MGB-probe melt. As the probes are designed to have destabilizing mismatches with undesired genera, only samples with a proper melting temperature are called positive.