Fluorescence Melting Curve Analysis Research Articles

A prospective phase 2 study of expeditious EGFR genotyping and immediate therapeutic initiation through extracellular vesicles (EV)-based bronchoalveolar lavage fluid (BALF) liquid biopsy in advanced NSCLC patients.

In our previous study, epidermal growth factor receptor (EGFR) genotyping using extracellular vesicles (EV)-derived DNA isolated from bronchoalveolar lavage fluid (BALF) was proven to be highly concordant with conventional tissue-based genotyping and its turn-around-time (TAT) was only 1-2 days. On this background, we prospectively validated the performance of EV-based BALF liquid biopsy for EGFR genotyping in the real practice of advanced non-small cell lung cancer (NSCLC) patients. After screening 120 newly diagnosed stage III-IV NSCLC patients, 51 cases were detected as EGFR-mutated by EV-based BALF EGFR genotyping and 40 patients were enrolled for gefitinib treatment. BALF EV were isolated by ultracentrifuge method and EGFR genotyping was performed with PCR-based PNA-clamping assisted fluorescence melting curve analysis. The objective response rate, progression-free survival (PFS), TAT, time to treatment initiation (TTI), and concordance rate were analyzed with clinical parameters. There was only one false positive case among the 120 screened patients and the overall concordance rate between tissue biopsy and EV-based BALF liquid biopsy was 99.2% including the subtype of EGFR mutations. TAT for EV-based BALF EGFR genotyping was 1.9±1.1 days, while tissue-based TAT was 12.1±7.2 days (P<0.001). EGFR genotyping was determined even before obtaining histopathologic report in most cases. TTI in BALF EGFR genotyping was faster than tissue genotyping (7.8±6.5 vs. 13.8±12.9 days). Therapeutic outcomes of response rate and PFS were almost similar to tissue-based results. We demonstrated, for the first time, that EV-based BALF liquid biopsy should be an excellent platform for expeditious EGFR genotyping and rapid therapeutic intervention even before obtaining the result of histopathology in advanced NSCLC patients.

Detection of c.375A&gt;G, c.385A&gt;T, c.571C&gt;T, and sedel2 of FUT2 via Real-Time PCR in a Single Tube

α(1,2)fucosyltransferase (Se enzyme) encoded by FUT2 is involved in the secretor status of ABH(O) blood group antigens. The sedel2 allele is one of the non-functional FUT2 (se) alleles in which 9.3 kb, containing the entire coding region of FUT2, is deleted by Alu-mediated nonhomologous recombination. In addition to this allele, three SNPs of FUT2, c.375A&gt;G, c.385A&gt;T, and c.571C&gt;T, appear to be prevalent in certain Oceanian populations such as Polynesians. Recently, we developed an endpoint genotyping assay to determine sedel2 zygosity, using a FAM-labeled probe for detection of the sedel2 allele and a VIC-labeled probe for the detection of FUT2. In this study, instead of the VIC probe, a HEX-labeled probe covering both c.375A&gt;G and c.385A&gt;T and a Cy5-labeled probe covering c.571C&gt;T were added to the sedel2 allele assay mixture to allow for the simultaneous detection of these four variations via endpoint genotyping for sedel2 zygosity and fluorescence melting curve analysis for c.375A&gt;G, c.385A&gt;T, and c.571C&gt;T genotyping. The results obtained from 24 Samoan subjects using this method were identical to those obtained using previous methods. Therefore, it appears that the present method can accurately determine these four variations simultaneously.

Estimation of Lewis Blood Group Status by Fluorescence Melting Curve Analysis in Simultaneous Genotyping of c.385A&gt;T and Fusion Gene in FUT2 and c.59T&gt;G and c.314C&gt;T in FUT3

Lewis blood group status is determined by two fucosyltransferase activities: those of FUT2-encoded fucosyltransferase (Se enzyme) and FUT3-encoded fucosyltransferase (Le enzyme). In Japanese populations, c.385A>T in FUT2 and a fusion gene between FUT2 and its pseudogene SEC1P are the cause of most Se enzyme-deficient alleles (Sew and sefus), and c.59T>G and c.314C>T in FUT3 are tag SNPs for almost all nonfunctional FUT3 alleles (le59, le59,508, le59,1067, and le202,314). In this study, we first conducted a single-probe fluorescence melting curve analysis (FMCA) to determine c.385A>T and sefus using a pair of primers that collectively amplify FUT2, sefus, and SEC1P. Then, to estimate Lewis blood group status, a triplex FMCA was performed with a c.385A>T and sefus assay system by adding primers and probes to detect c.59T>G and c.314C>T in FUT3. We also validated these methods by analyzing the genotypes of 96 selected Japanese people whose FUT2 and FUT3 genotypes were already determined. The single-probe FMCA was able to identify six genotype combinations: 385A/A, 385T/T, sefus/sefus, 385A/T, 385A/sefus, and 385T/sefus. In addition, the triplex FMCA successfully identified both FUT2 and FUT3 genotypes, although the resolutions of the analysis of c.385A>T and sefus were somewhat reduced compared to that of the analysis of FUT2 alone. The estimation of the secretor status and Lewis blood group status using the form of FMCA used in this study may be useful for large-scale association studies in Japanese populations.

Molecular Diagnosis of Hepatitis C Viruses; Technologies and Their Clinical Applications

Hepatitis-C is one of the most common viral diseases caused by hepatitis C virus (HCV). It is responsible for millions of deaths each year in the developing world. The common dissemination paths of HCV include the use of contaminated water and transfusion of infected blood. Control of this virus has become a challenge for scientists and health professionals due to its versatility and adaptability in different host environments. Along with other problems, lack of efficient diagnosis, quantification and genotyping of viral strains are the major hindrances in a management of this notorious epidemic. The knowledge of HCV genotype and an amount of virus in patient’s blood are pre-requisites to determine the duration and method of treatment. In this review, we discuss the implications of HCV molecular diagnostic methods and their clinical applications. We conclude that while, several commercial and home-brewed methods are available for this purpose, and there is a visible vacuum for cost effective, robust, sensitive assays that can detect multiple viral genotypes in a single reaction. We are of the view that the level of sensitivity offered by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) technique is unequivocal as compared to other techniques. Therefore, researchers may explore further possibilities using this technique in the management of HCV.&#x0D; Key words: Hepatitis C virus, Genotyping, Mixed infection, Fluorescence melting curve analysis, Viral Load, Quantification

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&gt;G, c.314C&gt;T, and c.484G&gt;A for Lewis-negative allele inference, was developed and validated using Ghanaian and Caucasian subjects. Although two other SNPs, c.55G&gt;A, and c.61C&gt;T, are located in the probe sequence for c.59T&gt;G, it seems feasible to detect these two SNPs along with c.59T&gt;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.

Landscape of EGFR mutations in lung adenocarcinoma: a single institute experience with comparison of PANAMutyper testing and targeted next-generation sequencing.

BackgroundActivating mutations in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) are predictive biomarkers for response to EGFR–tyrosine kinase inhibitor (TKI) therapy in lung adenocarcinoma (LUAD). Here, we characterized the clinicopathologic features associated with EGFR mutations via peptide nucleic acid clamping-assisted fluorescence melting curve analysis (PANAMutyper) and evaluated the feasibility of targeted deep sequencing for detecting the mutations.MethodsWe examined EGFR mutations in exons 18 through 21 for 2,088 LUADs from July 2017 to April 2020 using PANAMutyper. Of these, we performed targeted deep sequencing in 73 patients and evaluated EGFR-mutation status and TKI clinical response.ResultsEGFR mutation was identified in 55.7% of LUADs by PANAMutyper, with mutation rates higher in females (69.3%) and never smokers (67.1%) and highest in the age range of 50 to 59 years (64.9%). For the 73 patients evaluated using both methods, next-generation sequencing (NGS) identified EGFR mutation–positive results in 14 of 61 patients (23.0%) who were EGFR-negative according to PANAMutyper testing. Of the 10 patients reportedly harboring a sensitizing mutation according to NGS, seven received TKI treatment, with all showing partial response or stable disease. In the 12 PANAMutyper-positive cases, NGS identified two additional mutations in exon 18, whereas a discordant negative result was observed in two cases.ConclusionsAlthough PANAMutyper identified high frequencies of EGFR mutations, targeted deep sequencing revealed additional uncommon EGFR mutations. These findings suggested that appropriate use of NGS may benefit LUAD patients with otherwise negative screening test results.

Real-Time Polymerase Chain Reaction for the Detection of Helicobacter pylori and Clarithromycin Resistance.

Real-time polymerase chain reaction (RT-PCR) is a fast and simple method for the simultaneous detection of clarithromycin (CLR) resistance and Helicobacter pylori. We evaluated the effectiveness of RT-PCR compared to that of the rapid urease test (RUT) and assessed its value in verifying CLR resistance. A total of 70 specimens with confirmed H. pylori infection in culture were enrolled and analyzed in this prospective study. All specimens were subjected to RT-PCR assay using fluorescence melting peak signals to detect H. pylori infection and CLR resistances caused by either A2142G or A2143G mutations in the 23S ribosomal RNA gene (23S rRNA). The results were compared to those of RUT and antimicrobial susceptibility culturing tests to investigate the efficacy of RT-PCR. Among the 70 specimens analyzed, the positivity rate was 97.1% (68/70) with RT-PCR and 82.9% (58/70) with RUT. CLR resistance (minimum inhibitory concentration >1.0 μg/mL) was confirmed in 18.6% (13/70), and fluorescence melting curve analysis showed that 84.6% (11/13) had point mutations in 23S rRNA. Ten specimens had only A2143G mutation, and one specimen contained both A2142G and A2143G mutations. RT-PCR assay was found to be more efficient than RUT in detecting H. pylori infection and could effectively verify CLR resistance compared to the antimicrobial susceptibility culturing test. Considering the high sensitivity and accessibility of RT-PCR method, it could be used to easily detect CLR-resistant H. pylori, thus helping clinicians select suitable treatment regimen and improve the eradication rate.

Extracellular Vesicle-Based Bronchoalveolar Lavage Fluid Liquid Biopsy for EGFR Mutation Testing in Advanced Non-Squamous NSCLC.

Simple SummaryTissue biopsy is the gold standard for molecular genotyping in lung cancer. However, obtaining tumor tissue is challenging due to its invasiveness, inadequate amount of tissue, or complications. To overcome the limitations of tissue biopsy, plasma liquid biopsy using cfDNA has been investigated extensively; however, its low sensitivity limits the clinical application. Therefore, we used the tumor-specific DNA of extracellular vesicles (EVs) in bronchoalveolar lavage fluid (BALF) as DNA source for EGFR genotyping. As a result, we demonstrated that EV-based BALF EGFR testing in advanced lung NSCLC is a highly accurate rapid method overcoming low sensitivity of plasma cfDNA-based EGFR genotyping. It can be used as an adjuvant or alternative method for lung biopsy in cases where obtaining an adequate amount of tissue is difficult.To overcome the limitations of the tissue biopsy and plasma cfDNA liquid biopsy, we performed the EV-based BALF liquid biopsy of 224 newly diagnosed stage III-IV NSCLC patients and compared it with tissue genotyping and 110 plasma liquid biopsies. Isolation of EVs from BALF was performed by ultracentrifugation. EGFR genotyping was performed through peptide nucleic acid clamping-assisted fluorescence melting curve analysis. Compared with tissue-based genotyping, BALF liquid biopsy demonstrated a sensitivity, specificity, and concordance rates of 97.8%, 96.9%, and 97.7%, respectively. The performance of BALF liquid biopsy was almost identical to that of standard tissue-based genotyping. In contrast, plasma cfDNA-based liquid biopsy (n = 110) demonstrated sensitivity, specificity, and concordance rates of 48.5%, 86.3%, and 63.6%, respectively. The mean turn-around time of BALF liquid biopsy was significantly shorter (2.6 days) than that of tissue-based genotyping (13.9 days; p < 0.001). Therefore, the use of EV-based BALF shortens the time for confirmation of EGFR mutation status for starting EGFR-TKI treatment and can hence potentially improve clinical outcomes. As a result, we suggest that EV-based BALF EGFR testing in advanced lung NSCLC is a highly accurate rapid method and can be used as an alternative method for lung tissue biopsy.

Detection of five common variants of ABO gene by a triplex probe-based fluorescence-melting-curve-analysis

Current studies have suggested that the ABO blood group system is associated with several clinical conditions. For large-scale genotyping of ABO alleles, we developed a triplex fluorescence melting curve analysis (FMCA) to determine five single nucleotide variants (SNVs), c.261delG, c.796C>A, c.802G>A and c.803G>C and c.1061delC, responsible for common ABO phenotypes using dual-labeled self-quenched (TaqMan) probes in a single tube. We accurately determined c.796C>A, c.802G>A, and c.803G>C genotypes using a FAM-labeled probe, c.261delG using a CAL Fluor Orange 560- labeled probe, and c.1061delC using a Cy5-labeled probe. The present genotyping results of five SNVs in 214 subjects of the 1000 Genomes Project were in full agreement with those of the database sequence. The predicted ABO phenotypes using combinations of these five SNVs by this method in 288 Japanese subjects were in complete agreement with those by hemagglutination assay, although we did not find any A2 (alleles containing c.1061delC) or O.02 (alleles containing c.802G>A) alleles. The present triplex probe-based FMCA is a valid and credible method for a considerably accurate large-scale determination of ABO allele genotypes and estimation of phenotypes.

Simultaneous genotyping of three major Se enzyme inactivating SNPs of FUT2 based on a triplex probe-based fluorescence melting-curve analysis

BackgroundThe ABO(H) secretor status is controlled by FUT2-encoded α(1,2)fucosyltransferase (Se enzyme) activity. Three SNPs of FUT2, 302C>T (rs200157007), 385A>T (rs1047781), and 428G>A (rs601338), cause three major variants of nonsecretor (se) or weak-secretor (Sew) alleles. Evidence has been accumulating that suggests the secretor status is associated with various conditions including infectious diseases but a robust multiplex method for assaying relatively large-scale samples to determine the genotype of these three SNPs simultaneously has not been developed yet. MethodsBy combined usage of two Eprobes and a dual-labeled fluorescence probe, we developed a real-time PCR, followed by triplex probe-based fluorescent melting-curve analysis (FMCA) for genotyping of 302C>T, 385A>T, and 428G>A of FUT2 in a single tube. ResultsThree genotypes of each of three variants of FUT2 were accurately determined by the triplex probe-based FMCA. We then validated this method using genomic DNA samples of 47 Bangladeshis, and the results obtained by using this method were fully concordant with those by previous Sanger sequencing. ConclusionsSince the present single triplex probe-based FMCA is robust, fast, and cost-effective, we are able to effectively estimate the secretor status of subjects on a large scale in many populations around the world.

Methodological Research on Rapid Detection and Genotyping of NPM1 Gene Mutations in Leukemia

To establish a method for rapid detection and typing of NPM1 mutations in acute myeloid leukemia (AML) by fluorescence melting curve analysis technology. A pair of primers and a fluorescent single-stranded probe (molecule beacon) were designed for the mutant genes mutA, mutB, mutD in exon 12 of nucleopsin (NPM1) and wild type. With a real-time qPCR, the A, B, and D gene mutations of NPM1 were detected and typed by different-melting curve peak value of the probe through RT-PCR. This method could detected the mutations of A, B, and D in NPM1 effectively with a sensitivity of 1%. Furthermore, 62 AML clinical samples were evaluated by the method. In the results, the detection rate and typing of NPM1 mutations were consistent with the sequencing results of clinical samples. There are three features in the method of fluorescence melting curve analysis: stable PCR system, easy to operate, and the easily distinguishable results. The method might meet the demand for rapid typing of NPM1 gene mutation in early diagnosis or concomitant diagnosis of AML.

High Risk Janus Kinase 2 V617F Allele Burden in a Seven-Year Cohort of Patients with Myeloproliferative Neoplasms.

JAK2 V617F is the most common somatic mutation associated with the classical Philadelphia (Ph) chromosome negative myeloproliferative neoplasms (MPNs), which include essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). JAK2 V617F allele burden may be used for establishing the diagnosis, determining prognosis, and monitoring progression in these diseases. Limited data is available regarding the epidemiology of MPNs in Africa, and there is paucity of data on demographic, laboratory, and clinical features of MPNs in South Africa. This study determined the JAK2 V617F allele burden in a seven-year retrospective cohort of patients diagnosed with MPNs and described the characteristics of these diseases in a South African setting. A laboratory database search was performed to identify patients diagnosed with ET, PV or PMF and a positive JAK2 V617F mutation, diagnosed qualitatively on Fluorescence Resonance Energy Transfer (FRET) real-time PCR and melting curve analysis. The allele burden for these patients was measured on archived residual DNA samples using a quantitative allele specific amplification (QUASA) assay. Demographic data and relevant laboratory results at presentation were analyzed. The search identified 87 patients who tested positive for JAK2 V617F mutation and fulfilled the diagnostic criteria for ET, PV or PMF from 2012 to 2018. Median age at diagnosis was 64 years with a male: female ratio of 1.2:1. ET, PV and PMF accounted for 11.5%, 44.8%, and 43.7% of the MPNs, respectively. Median allele burden for ET, PV, and PMF was 24.9%, 71.1%, and 55.8%, respectively. Allele burden was significantly lower in ET compared to PV (p = 0.0003) and PMF (p = 0.0023) and correlated with leukocytosis, neutrophilia, eosinophilia, and low erythrocyte mean cell volume (p < 0.05). JAK2 V617F-positive MPNs occurred predominantly in older patients with approximately equal gender ratio. ET was the least common MPN and there was a higher proportion of PMF cases than described in studies in Europe and America. Allele burden was also relatively high for all three subtypes of MPNs when compared to other published data, which may predispose to poorer prognosis.

Association of Nicotine Use Disorder with Neurexin 3 Gene Polymorphisms.

In this study, we aimed to investigate the Neurexin 3 gene (NRXN3) polymorphisms in the rs 221473, rs 221497, rs1004212 and rs11624704 regions in relation to nicotine use disorder (NUD) in the Turkish population. Power analysis indicated that the NUD group and the control group of this study should each comprise 200 participants in the 18-65 year age range. The NUD group consisted of individuals without a psychiatric first axis disorder except for NUD, mental retardation, past head trauma or a neurological disorder, who had smoked minimally10 cigarettes per day for at least 1 year. The control group included individuals without a serious chronic physical illness, a previous psychiatric disorder or mental retardation and who responded "no" to the question "have you ever smoked?" A sociodemographic questionnaire and the Fageström nicotine dependence scale (FNDS) for the NUD group were utilized. Venous blood samples of all participants were taken into tubes containing EDTA (ethylene daimine tetra acetic acid) for DNA extraction. Duplex fluorescence melting curve analysis was used for genotype detection and differentiation. The individuals carrying the AC allele and the AG allele at the rs11624704 and the rs1004212 regions respectively had a high risk of being addicted to cigarettes. This is first study investigating the relationship of the NRXN3 gene and nicotine addiction in the Turkish population. It was observed that the risk of NUD in the Turkish population may be related to the Neurexin gene.

Analysis of EGFR mutation status in malignant pleural effusion and plasma from patients with advanced lung adenocarcinoma.

Cell-free DNA (cfDNA) is emerging as a surrogate sample type for mutation analyses. We investigated the suitability of malignant pleural effusion (MPE) and plasma as a biomaterial for analyzing epidermal growth factor receptor (EGFR) mutation by peptide nucleic acid (PNA) clamping-assisted fluorescence melting curve (PANAMutyper™) analysis. Matched tissue, MPE cell block (MPE-CB), MPE supernatant, and plasma samples were collected from patients with advanced lung adenocarcinoma who had a MPE at the time of diagnosis. EGFR mutation was assessed by PANAMutyper™. Mutation analyses in matched tumor tissues, MPE-CB, MPE supernatant, and/or plasma samples were available for 67 patients. In comparison with tumor tissue and MPE-CB, MPE supernatant exhibited 84.4% sensitivity, 97.1% specificity, 96.4% positive predictive value (PPV), and 87.2% negative predictive value (NPV). In the same comparison, plasma exhibited 70.6% sensitivity, 100.0% specificity, 100.0% PPV, and 73.7% NPV. When sorted by mutation type, MPE supernatant had better sensitivity than plasma for the detection of two major EGFR mutations: 93.8% vs. 75.0% for exon 19 deletion and 73.3% vs. 60.0% for L858R. In this cohort of patients with MPEs, MPE supernatant demonstrated superior diagnostic performance compared with plasma using a PNA-based real-time PCR method.

Detection of Eight Respiratory Bacterial Pathogens Based on Multiplex Real-Time PCR with Fluorescence Melting Curve Analysis.

Background and Objective. Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis are primary respiratory bacterial pathogens contributing to morbidity and mortality in developing countries. This study evaluated the diagnostic performance of multiplex real-time PCR with fluorescence melting curve analysis (MCA) assay, which was used to detect eight respiratory bacterial pathogens simultaneously. Methods A total of 157 sputum specimens were examined by multiplex real-time with fluorescence MCA, and the results were compared with the conventional culture method. Results Multiplex real-time PCR with fluorescence MCA specifically detected and differentiated eight respiratory bacterial pathogens by different melting curve peaks for each amplification product within 2 hours and exhibited high repeatability. The limit of detection ranged from 64 to 102 CFU/mL in the multiplex PCR system. Multiplex real-time PCR with fluorescence MCA showed a sensitivity greater than 80% and a 100% specificity for each pathogen. The kappa correlation of eight bacteria ranged from 0.89 to 1.00, and the coefficient of variation ranged from 0.05% to 0.80%. Conclusions Multiplex real-time PCR with fluorescence MCA assay is a sensitive, specific, high-throughput, and cost-effective method to detect multiple bacterial pathogens simultaneously.

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.

Correlation of epidermal growth factor receptor mutation status in plasma and tissue samples of patients with non-small cell lung cancer.

Somatic mutations of the gene encoding epidermal growth factor receptor (EGFR) are detected in approximately 30%-50% of patients with non-small cell lung cancers (NSCLC), so detection of EGFR mutation is the pivotal step of treatment in patients with advanced NSCLC. However, difficulty in obtaining sufficient tissue and bias from the heterogeneity of the tumor samples are the major obstacles. Although analyzing EGFR with circulating tumor DNA (ctDNA) in plasma is a breakthrough, accuracy is the problem in variable methods. Peptide nucleic acid (PNA) clamping-assisted fluorescence melting curve analysis (PANAMutyper®) is a novel and highly sensitive method of detecting EGFR mutation in tumor tissues. This study was designed to evaluate PANAMutyper® for detecting EGFR mutation with ctDNA of patients with lung cancer. EGFR mutation status detected by PNA clamp with tissue samples and by PANAMutyper® with ctDNA was compared. Tissue biopsy was done in 158 patients with lung tumor, in which 23 cases were excluded and 135 cases were enrolled. EGFR mutation rate was 23.0% (31/135) in overall patients. All the plasma samples of the cases with mutant EGFR in tissue samples were verified by an already known highly sensitive method of droplet digital polymerase chain reaction (ddPCR). The concordance rate of tissue and plasma samples was 91.9% (124/135). The sensitivity, specificity, negative predictive value, and positive predictive value were 64.5%, 100%, 90.4%, and 100%, respectively, according to the tissue samples as a standard. PANAMutyper® method was not inferior to ddPCR for the detection of EGFR mutation including T790M with ctDNA. These results suggest that the detection of EGFR mutation status using ctDNA in plasma by PANAMutyper® is a feasible test prior to tissue biopsy.

A novel protocol to isolate, detect and differentiate taeniid eggs in leafy greens and berries using real-time PCR with melting curve analysis

BackgroundZoonotic taeniid cestodes are amongst the most important food-borne parasites affecting human health worldwide. Contamination of fresh produce with the eggs of Echinococcus granulosus (s.l.), Echinococcus multilocularis, and some Taenia species pose a potential food safety risk. However, very few studies have attempted to investigate the potential contamination of fresh produce with taeniid eggs and the available methods are not standardized for this purpose. Established protocols do exist for testing leafy greens and berries for contamination with protozoan parasites and are used in national surveillance programmes. This methodology could be suitable for the detection of taeniids. The objective of this project was to develop and standardize a sensitive and reliable method to detect contamination of leafy greens and berries with eggs of zoonotic taeniids and to differentiate between E. multilocularis, E. granulosus (s.l.) and Taenia spp.MethodsWe compared the efficacy of different wash solutions to remove Taenia spp. eggs from spiked produce, assessed two DNA extraction kits for their performance on Taenia spp. eggs, and adapted a published conventional multiplex PCR into a real-time PCR with fluorescence melting curve analysis (MCA) that was optimized for use on produce washes. Analytical specificity of this protocol was assessed using non-spiked produce washes as well as a variety of other potentially contaminating parasites.ResultsThe protocol as established in this study had an analytical sensitivity of detecting five eggs per spiked sample for both romaine lettuce and strawberries. Unequivocal identification of E. multilocularis, E. granulosus (s.l.) and Taenia spp. was possible through MCA. Amplicon sequencing allowed identification of Taenia to the species level. The real-time PCR also amplified DNA from Dicrocoelium sp., but with a clearly discernable melting curve profile.ConclusionThe new protocol for screening produce for taeniid contamination was highly sensitive. Melting curve analysis and the possibility of amplicon sequencing made this assay very specific. Once further validated, this method could be employed for surveillance of produce for contamination with taeniid parasites to assess potential risks for consumers.

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.