Probe-based Fluorescence Melting Curve Analysis Research Articles

Fluorescence Melting Curve Analysis for Concurrent Genotyping of Three Tag SNPs in FUT3.

The synthesis of Lewis blood group antigens is governed by two fucosyltransferase genes, FUT2 and FUT3. Evidence is accumulating to suggest that functional polymorphisms of FUT2 and FUT3 are associated with a variety of clinical conditions. Fluorescence melting curve analysis (FMCA), using three different dual-labeled probes for concurrent genotyping of three single nucleotide polymorphisms (SNPs) of FUT3, c.59T>G, c.314C>T, and c.484G>A for Lewis-negative allele inference, was developed and validated using Ghanaian and Caucasian subjects. Although two other SNPs, c.55G>A, and c.61C>T, are located in the probe sequence for c.59T>G, it seems feasible to detect these two SNPs along with c.59T>G. The results obtained by probe-based FMCA were in perfect accordance with those obtained by Sanger sequencing for 106 Ghanaians and 100 Caucasians. The present method is useful and reliable for estimating Lewis-negative alleles on a relatively large scale.

Retrospective analysis of a ten-year screening project for G6PD deficiency in neonates in Hainan Province

Objective To investigate the prevalence of glucose-6-posphate dehydrogenase (G6PD) deficiency and its gene mutations among neonates in Hainan Province. Methods The G6PD activity of dried blood spots of 914 520 neonates born from 2007 to 2016 was screened by fluorescence spot test in Hainan Province. The G6PD/6-glucose phosphate dehydrogenase (6GPD) ratio method was used to confirm the diagnosis of suspected specimens, and 3 012 of year 2016 dried blood spots of neonates with G6PD deficiency were genotyped using the multicolor probe-based fluorescence melting curve analysis. Results From 2007 to 2016, 36 314 positive cases were screened in 914 520 neonates. A total of 26 370 cases of G6PD deficiency were diagnosed with an incidence rate of 2.88%(26 370/914 520) in Hainan Province. The incidences of G6PD deficiency were 2.80%(21 688/774 555) in ethnic Han population, 3.45% (4 292/124 419) in ethnic Li population, 3.31%(212/6 401) in ethnic Miao population and 1.95%(178/9 145) in other ethnic groups. There were significant differences in the incidence of G6PD deficiency in ethnic Han population and ethnic Li population(χ2=161.261, P=0.000), ethnic Miao population(χ2=6.104, P=0.013) and other ethnic groups(χ2=24.283, P=0.000). A total of 13 mutation types were detected by gene detection in 3 012 confirmed cases of G6PD deficiency, of which c.1376 G>T, c.1388 G>A, c.95 A>G and c.1024 C>T mutations and related combinations accounted for approximately 91.74%. Two mutations outside 16 genotypes, c.86 C>T and c.1311 C>T, were found by gene sequencing. Conclusions The incidence of G6PD deficiency among newborns in Hainan Province is high, and there are ethnic and regional differences. The dominant genetic mutations in Hainan Province are c.1376 G>T, c.1388 G>A, c.95 A>G and c.1024 C>T. Key words: Glucosephosphate dehydrogenase deficiency; Incidence; Retrospective studies; Neonatal screening

Simultaneous Detection of Multiple Pathogenic Targets with Stem-Tagged Primer Sets.

Simultaneous multiple gene detection is indispensable for the detection of various genes in a small sample obtained by an invasive method. A typical detection method is probe-based fluorescence melting curve analysis by means of real-time PCR. It is very limited because, for each target, a probe sequence with at least a different Tm must be designed. To overcome this limitation, we developed a simultaneous multiple gene detection method based on a giant amplicon molecular beacon. PCR was performed by attaching stem sequences with different Tm values to each primer set, and the melting Tm was measured by hybridizing the stem sequences at both ends of the amplified amplicon; this generated well-separated Tm signals. The important point here is that the stem sequence that produces the Tm signal is an arbitrarily selectable sequence unrelated to the target gene. Because it is arbitrarily selectable, the desired Tm can be freely adjusted. As a result, we succeeded in the simultaneous detection of four samples with the use of only one fluorophore. Theoretically, a combination of five fluorophores could detect more than 20 multiple genes simultaneously.

Establishment of Coamplification at Lower Denaturation Temperature PCR/Fluorescence Melting Curve Analysis for Quantitative Detection of Hepatitis B Virus DNA, Genotype, and Reverse Transcriptase Mutation and Its Application in Diagnosis of Chronic Hepatitis B

Dynamic and real-time hepatitis B virus (HBV) DNA, genotype, and reverse transcriptase mutation analysis plays an important role in diagnosing and monitoring chronic hepatitis B (CHB) and in assessing the therapeutic response. We established a highly sensitive coamplification at lower denaturation temperature PCR (COLD-PCR) coupled with probe-based fluorescence melting curve analysis (FMCA) for precision diagnosis of CHB patients. The imprecision with %CV and detection limit of HBV DNA detected by COLD-PCR/FMCA were 2.58% to 4.42% and 500 IU/mL, respectively. For mutation, the imprecision and detection limit were 3.35% to 6.49% and 1%, respectively. Compared with Sanger sequencing, the coincidence rates of genotype and mutation were 96.0% and 82.5%, respectively, whereas the inconsistent data resulted from a low proportion (<20%) of mixed genotypes or mixed mutations. The mutation ratio in HBV infection patients was as follows: hepatitis B e antigen (HBeAg)-positive infection (0/0.0%)<HBeAg-negative infection (16/4.5%)<HBeAg-positive hepatitis (30/5.5%)<HBeAg-negative hepatitis (36/6.5%). In patients with entecavir therapy, the proportion of mutation at baseline or week 4 in virologic response (VR) group was <4%, whereas in the partial VR group, it was mostly≥4%. COLD-PCR/FMCA provides a novel tool with high sensitivity, convenience, and practicability for the simultaneous quantification of HBV DNA, genotype, and mutation. It might be used for distinguishing the different phases of HBV infection and predicting VR of CHB patients.

Application of Duplex Fluorescence Melting Curve Analysis (FMCA) to Identify Canine Parvovirus Type 2 Variants.

Canine parvovirus (CPV-2) is an enteric virus causing morbidity and mortality in dogs worldwide. Since CPV-2 emerged as canine pathogen, the original CPV-2 strain has constantly evolved, and its primary variants (CPV-2a, CPV-2b, and CPV-2c) co-circulate to varying extents in canine populations worldwide. Thus, rapid and accurate laboratory diagnoses of CPV-2 variants are crucial to monitor CPV-2 evolution. Conventional methods for CPV-2 genotyping are laborious, time consuming, and determining the genotype of a CPV-2 variant often requires two or more reaction tubes. The present study developed a probe-based fluorescence melting curve analysis (FMCA) for genotyping six different CPV-2 variants (original CPV-2, CPV-2a, CPV-2b, CPV-2c, and vaccine strains of CPVpf and CPVint) in a single reaction tube using only two TaqMan probes. One of the TaqMan probes (FAM labeled) was designed to perfectly match with the target sequence of CPV-2a, this probe allows a 1-bp mismatched hybridization with the CPV-2b VP2 gene region (A4062G), and a 2-bp mismatched hybridization for CPV-2c (A4062G and T4064A); Another TaqMan probe (HEX labeled) was produced to perfectly match with the target sequence of original CPV-2, this probe enables 1-bp mismatched hybridization with the other CPV-2 variants (A3045T). Using the two TaqMan probes, all six CPV-2 variants were readily distinguished by their respective melting temperature values in a single reaction tube. The detection limits of this assay were 1–10 copies per reaction for six CPV-2 construction plasmids and no cross reactions were observed with several other common canine viruses. In this assay, co-infected samples were also directly identified via probe-based FMCA without using a mixing control; only a pure control is required. The clinical evaluation of this assay was demonstrated by analyzing 83 clinical fecal samples, among which 41 (49.39%), 8 (9.63%), and 14 (16.87%) samples were found to be positive for CPV-2a, CPV-2b, and CPV-2c, respectively. The concordance rate between probe-based FMCA and Sanger sequencing was 100%. Thus, the duplex FMCA is effective, rapid, simple, high-throughput, and straightforward for genotyping CPV-2 variants, and is useful to effectively diagnose and monitor CPV-2 epidemiology.

Peptide Nucleic Acid Probe-Based Analysis as a New Detection Method for Clarithromycin Resistance in Helicobacter pylori.

Background/AimsHelicobacter pylori eradication rates are decreasing because of increases in clarithromycin resistance. Thus, finding an easy and accurate method of detecting clarithromycin resistance is important.MethodsWe evaluated 70 H. pylori isolates from Korean patients. Dual-labeled peptide nucleic acid (PNA) probes were designed to detect resistance associated with point mutations in 23S ribosomal ribonucleic acid gene domain V (A2142G, A2143G, and T2182C). Data were analyzed by probe-based fluorescence melting curve analysis based on probe-target dissociation temperatures and compared with Sanger sequencing.ResultsAmong 70 H. pylori isolates, 0, 16, and 58 isolates contained A2142G, A2143G, and T2182C mutations, respectively. PNA probe-based analysis exhibited 100.0% positive predictive values for A2142G and A2143G and a 98.3% positive predictive value for T2182C. PNA probe-based analysis results correlated with 98.6% of Sanger sequencing results (κ-value=0.990; standard error, 0.010).ConclusionsH. pylori clarithromycin resistance can be easily and accurately assessed by dual-labeled PNA probe-based melting curve analysis if probes are used based on the appropriate resistance-related mutations. This method is fast, simple, accurate, and adaptable for clinical samples. It may help clinicians choose a precise eradication regimen.

Peptide nucleic acid probe-based fluorescence melting curve analysis for rapid screening of common JAK2, MPL, and CALR mutations

BackgroundWe developed and evaluated the feasibility of peptide nucleic acid (PNA)-based fluorescence melting curve analysis (FMCA) to detect common mutations in myeloproliferative neoplasms (MPNs). MethodsWe have set up two separate reactions of PNA-based FMCA: JAK2 V617F &CALR p.Leu367fs*46 (set A) and MPL W515L/K &CALR p.Lys385fs*47 (set B). Clinical usefulness was validated with allele-specific real-time PCR, fragment analysis, Sanger sequencing in 57 BCR-ABL1-negative MPNs. ResultsThe limit of detection (LOD) of PNA-based FMCA was approximately 10% for each mutation and interference reactions using mixtures of different mutations were not observed. Non-specific amplification was not observed in normal control. PNA-based FMCA was able to detect all JAK2 V617F (n=20), CALR p.Leu367fs*46 (n=10) and p.Lys385fs*47 (n=8). Three of six MPL mutations were detected except three samples with low mutant concentration in out of LOD. JAK2 exon 12 mutations (n=7) were negative without influencing V617F results. Among six variant CALR exon 9 mutations, two were detected by this method owing to invading of probe binding site. ConclusionsPNA-based FMCA for detecting common JAK2, MPL, and CALR mutations is a rapid, simple, and sensitive technique in BCR-ABL1-negative MPNs with >10% mutant allele at the time of initial diagnosis.

Easy method for discriminating the origins of manila clam Ruditapes philippinarum with a dual-labelled PNA-probe-based melting curve analysis

Probe-based fluorescence melting curve analysis is a powerful tool used to detect mutations, based on the melting temperature from the thermal denaturation of the probe-target hybrid. PNA (Peptide nucleic acid) probes have several advantages: easy to design and modify and do not give false negative results. In this study, we developed and applied a MeltingArray method using PNA-probe-based fluorescence melting curve analysis to quickly determine the origins of manila clams. Five PNA probes were designed to discriminate between imported (from Dalian, China) and domestic (Korea) manila clams and positioned at KRph27135, KRph29263 and KRph37987. The KRph 37987-159C PNA probe had the highest discrimination ability. The KRph37987-159C probe had perfect match binding with the domestic manila clams at 80.8-100%, and mismatch binding with the imported manila clams excluding heterozygous samples at 66.7-76.8%. Moreover, domestic manila clams were discriminated by the PNA probes KRph29263-143G and KRph29263-205C at a ratio of 96.8% and 100%, and the imported manila clams were discriminated 100% and 98.5%, respectively. Here, we report that domestic and imported manila clams can be distinguished by the MeltingArray system and that it is a more rapid, simple and accurate method than that of sanger sequencing. The dual-labeled PNA probes offer the advantage of improved flexibility in probe design, which could be used in future applications for genotyping a wide range of subjects.

Fluorescence melting curve analysis using self-quenching dual-labeled peptide nucleic acid probes for simultaneously identifying multiple DNA sequences

Previous fluorescence melting curve analysis (FMCA) used intercalating dyes, and this method has restricted application. Therefore, FMCA methods such as probe-based FMCA and molecular beacons were studied. However, the usual dual-labeled probes do not possess adequate fluorescence quenching ability and sufficient specificity, and molecular beacons with the necessary stem structures are hard to design. Therefore, we have developed a peptide nucleic acid (PNA)-based FMCA method. PNA oligonucleotide can have a much higher melting temperature (Tm) value than DNA. Therefore, short PNA probes can have adequate Tm values for FMCA, and short probes can have higher specificity and accuracy in FMCA. Moreover, dual-labeled PNA probes have self-quenching ability via single-strand base stacking, which makes PNA more favorable. In addition, this method can facilitate simultaneous identification of multiple DNA templates. In conventional real-time polymerase chain reaction (PCR), one fluorescence channel can identify only one DNA template. However, this method uses two fluorescence channels to detect three types of DNA. Experiments were performed with one to three different DNA sequences mixed in a single tube. This method can be used to identify multiple DNA sequences in a single tube with high specificity and high clarity.

Abstract 1518: A new multiplex genotyping assay using PNA clamping-assisted fluorescence melting curve analysis

Abstract Recent studies on molecular mechanisms of anti-EGFR therapies revealed importance of mutation screening and genotyping of multiple target oncogenes. For example, KRAS mutations have been associated with poor responsiveness to anti-EGFR therapies. Determining the KRAS status of a newly diagnosed cancer patient prior to initiating anti-EGFR therapy would be required in selecting effective therapeutics among tyrosine kinase inhibitor and monoclonal antibody therapy. Peptide nucleic acid (PNA) is an artificially synthesized polymer. The negatively charged deoxyribose phosphate backbone of DNA is replaced by an electroneutral peptide-like backbone consisting of N-(2-aminoethyl) glycine units directly linked to the nucleotide bases. Complementary PNA/DNA complex has stronger binding affinity than complementary DNA/DNA complex. A PNA probe has higher specificity and mismatch discrimination towards its target DNA than a DNA probe. Furthermore, it shows a remarkable biological stability to enzymatic degradation. PNA is not recognized by DNA polymerase so it cannot be used as a primer. PNAClamp™ technique is the PNA-based PCR clamping that selectively amplifies only the target (mutated) DNA sequence as a minor portion in the mixture with the major non-target (wild type) DNA sequences. Therefore, PNAClamp technique provides better limit of detection than conventional PCR method. PANA S-Melting™ (PNA probe-based fluorescence melting curve analysis) based on change in fluorescent signal as a sample thermally denatured (melting temperature shift). This is a powerful tool for mutation detection (screening and genotyping) by a single probe. It can distinguish multiple sequence variants adjacent to each other on a small target region. We developed a new technique, which combines two techniques; PNAClamp and PNA probe-based fluorescence melting curve analysis. This new PNAClamp-assisted melting curve method can discriminate a sequence variant from a wild-type sequence with a relatively large Tm difference. Therefore, it could easily detect presence of sequence variation without an additional data analysis process. This technique can be performed in any real-time PCR machine, which has melting curve analysis function. We tested a new multiplex genotyping assay of KRAS gene mutation using this combined method in lung cancer cell line samples. This new assay could detect a total of seven sequence variants (6 genotypes of codon 12 and 1 genotype of codon 13) in a single closed reaction tube using three different fluorescent dyes. The results showed that 0.5 % mutations were detected using this multiplexing assay system. This new multiplex genotyping method offers a cost-effective and highly sensitive mutation genotyping method that can be used in various clinical areas including personalized medicine. Citation Format: Yongtae Kim, Jin W. Kim, Sung-Kee Kim, Goon Ho Joe. A new multiplex genotyping assay using PNA clamping-assisted fluorescence melting curve analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1518. doi:10.1158/1538-7445.AM2014-1518

Multiplex Fluorescence Melting Curve Analysis for Mutation Detection with Dual-Labeled, Self-Quenched Probes

Probe-based fluorescence melting curve analysis (FMCA) is a powerful tool for mutation detection based on melting temperature generated by thermal denaturation of the probe-target hybrid. Nevertheless, the color multiplexing, probe design, and cross-platform compatibility remain to be limited by using existing probe chemistries. We hereby explored two dual-labeled, self-quenched probes, TaqMan and shared-stem molecular beacons, in their ability to conduct FMCA. Both probes could be directly used for FMCA and readily integrated with closed-tube amplicon hybridization under asymmetric PCR conditions. Improved flexibility of FMCA by using these probes was illustrated in three representative applications of FMCA: mutation scanning, mutation identification and mutation genotyping, all of which achieved improved color-multiplexing with easy probe design and versatile probe combination and all were validated with a large number of real clinical samples. The universal cross-platform compatibility of these probes-based FMCA was also demonstrated by a 4-color mutation genotyping assay performed on five different real-time PCR instruments. The dual-labeled, self-quenched probes offered unprecedented combined advantage of enhanced multiplexing, improved flexibility in probe design, and expanded cross-platform compatibility, which would substantially improve FMCA in mutation detection of various applications.