A highly sensitive and specific real-time quantitative PCR for BRAF V600E/K mutation screening.
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ABSTRACT:
SUBMITTER: Lung J
PROVIDER: S-EPMC7547094 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The BRAF V600E mutation is an important oncological target in certain central nervous system (CNS) tumors, for which a possible application of BRAF-targeted therapy grows continuously. In the present study, we aim to determine the prevalence of BRAF V600E mutations in a series of ganglioglioma (GG) and pilocytic astrocytoma (PA) cases. Simultaneously, we decided to verify whether the combination of fully automated tests-BRAF-VE1 immunohistochemistry (IHC) and Idylla BRAF mutation assay-may be useful to accurately predict it in the case of specified CNS tumors. The study included 49 formalin-fixed, paraffin-embedded tissues, of which 15 were GG and 34 PA. Immunohistochemistry with anti-BRAF V600E (VE1) antibody was performed on tissue sections using the VentanaBenchMark ULTRA platform. All positive or equivocal cases on IHC and selected negative ones were further assessed using the Idylla BRAF mutation assay coupled with the Idylla platform. The BRAF-VE1 IHC was positive in 6 (6/49; 12.3%) and negative in 39 samples (39/49; 79.6%). The interpretation of immunostaining results was complicated in 4 cases, of which 1 tested positive for the Idylla BRAF mutation assay. Therefore, the overall positivity rate was 14.3%. This included 2 cases of GG and 5 cases of PA. Our study found that BRAF V600E mutations are moderately frequent in PA and GG and that for these tumor entities, IHC VE1 is suitable for screening purposes, but all negative, equivocal, and weak positive cases should be further tested with molecular biology techniques, of which the Idylla system seems to be a promising tool.
| S-EPMC7448243 | biostudies-literature
Project description:BackgroundHigh frequencies of the BRAF V600E mutation have been reported in pleomorphic xanthoastrocytoma (PXA). Recently, a BRAF V600E mutation-specific antibody has been developed and validated. We evaluated the immunohistochemical (IHC) detection of BRAF V600E mutation in PXA by comparing to gold standard molecular analysis and investigating the interobserver variability of the IHC scoring. We performed BRAF V600E IHC in 46 cases, of which 37 (80%) cases had sufficient tumor tissue for molecular analysis. IHC detection was performed using monoclonal mouse antibody VE1 (Spring Bioscience). IHC slides were scored independently by four reviewers blind to molecular data, including a primary (gold standard) and three additional reviewers. BRAF V600E mutation status was assessed by allele-specific polymerase chain reaction (PCR) with fragment analysis.ResultsAll 46 cases showed interpretable BRAF V600E IHC results: 27 (59%) were positive (strong cytoplasmic staining), 19 (41%) were negative (6 of these cases with focal/diffuse weak cytoplasmic staining, interpreted as nonspecific by the primary reviewer). By molecular analysis, all 37 cases that could be tested had evaluable results: 22 (59%) cases were positive for BRAF V600E mutation and were scored as "IHC-positive", and 15 (41%) were negative (including 11 cases scored as "IHC-negative" and 4 cases scored as negative with minimal nonspecific staining). IHC detection of BRAF V600E mutant protein was congruent in all 37 cases that were successfully evaluated by molecular testing (sensitivity and specificity of 100%). Agreement for IHC scoring among the 4 reviewers was almost perfect (kappa 0.92) when cases were scored as "positive/negative" and substantial (kappa 0.78) when minimal nonspecific staining was taken into account.ConclusionsWe conclude that detection of BRAF V600E mutation by immunohistochemistry is highly sensitive and specific. BRAF V600E IHC interpretation is usually straightforward, but awareness of possible nonspecific staining is necessary and training is recommended. It is a practical rapid method that may avoid the need of labor-intensive molecular testing and may be most valuable in small biopsies unsuitable for molecular analysis.
| S-EPMC3893490 | biostudies-literature
Project description:The majority of congenital cytomegalovirus (cCMV) infections are asymptomatic at birth and therefore not diagnosed. Approximately 10-15% of these infants develop late-onset hearing loss and other developmental disorders. Implementation of a universal screening approach at birth may allow early initiation of symptomatic interventions due to a closer follow-up of infants at risk and offers the opportunity to consider treatment of late-onset disease. Real-time PCR assays for the detection of CMV DNA in buccal swab samples demonstrated feasibility and good clinical sensitivity in comparison to a rapid culture screening assay. Because most cCMV infections remain asymptomatic, a universal screening assay that stratifies CMV infected infants according to low and high risk of late-onset cCMV disease could limit the parental anxiety and reduce follow-up costs. We therefore developed and characterized a screening algorithm based on a highly-sensitive quantitative real-time PCR assay that is compatible with centralized testing of samples from universal screening and allows to determine CMV DNA load of saliva samples either as International Units (IU)/ml saliva or IU/105 cell equivalents. 18 of 34 saliva samples of newborns that tested positively by the screening algorithm were confirmed by detection of CMV DNA in blood and/or urine samples obtained during the first weeks of life. All screening samples that could not be confirmed had viral loads of <2.3x105 IU/ml saliva (median: 6.8x103) or 1.3x105 IU/105 cell equivalents (median: 4.0x102). The viral load of screening samples with confirmed cCMV infection ranged from 7.5x102 to 8.2x109 IU/ml saliva (median: 9.3x107) or 1.5x102 to 5.6x1010 IU/105 cell equivalents (median: 3.5x106). Clinical follow-up of these newborns with confirmed cCMV infection should reveal whether the risk of late-onset cCMV disease correlates with CMV DNA load in early life saliva samples and whether a cut-off can be defined identifying cCMV infected infants with or without risk for late-onset cCMV disease.
| S-EPMC6952102 | biostudies-literature
Project description:Mutations in the Janus kinase 2 (JAK2) gene have become an important identifier for the Philadelphia-chromosome negative chronic myeloproliferative neoplasms. In contrast to the JAK2V617F mutation, the large number of JAK2 exon 12 mutations has challenged the development of quantitative assays. We present a highly sensitive real-time quantitative PCR assay for determination of the mutant allele burden of JAK2 exon 12 mutations. In combination with high resolution melting analysis and sequencing the assay identified six patients carrying previously described JAK2 exon 12 mutations and one novel mutation. Two patients were homozygous with a high mutant allele burden, whereas one of the heterozygous patients had a very low mutant allele burden. The allele burden in the peripheral blood resembled that of the bone marrow, except for the patient with low allele burden. Myeloid and lymphoid cell populations were isolated by cell sorting and quantitative PCR revealed similar mutant allele burdens in CD16+ granulocytes and peripheral blood. The mutations were also detected in B-lymphocytes in half of the patients at a low allele burden. In conclusion, our highly sensitive assay provides an important tool for quantitative monitoring of the mutant allele burden and accordingly also for determining the impact of treatment with interferon-?-2, shown to induce molecular remission in JAK2V617F-positive patients, which may be a future treatment option for JAK2 exon 12-positive patients as well.
| S-EPMC3293922 | biostudies-literature
Project description:We have developed an assay for the detection of Norwalk-like viruses (NLVs) based on reverse transcription-PCR (RT-PCR) that is highly sensitive to a broad range of NLVs. We isolated virus from 71 NLV-positive stool specimens from 37 outbreaks of nonbacterial acute gastroenteritis and sequenced the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the NLV genome. The data were subjected to multiple-sequence alignment analysis and similarity plot analysis. We used the most conserved sequences that react with diverse NLVs to design primers and TaqMan probes for the respective genogroups of NLV, GI and GII, for use in a real-time quantitative RT-PCR assay. Our method detected NLV in 99% (80 of 81) of the stool specimens that were positive by electron microscopy, a better detection rate than with the two available RT-PCR methods. Furthermore, our new method also detected NLV in 20 of 28 stool specimens from the same NLV-related outbreaks that were negative for virus by electron microscopy. Our new assay is free from carryover DNA contamination and detects low copy numbers of NLV RNA. It can be used as a routine assay for diagnosis as well as for elucidation of the epidemiology of NLV infections.
| S-EPMC153860 | biostudies-literature
Project description:Brucellosis is a zoonotic disease caused by Brucella spp. and is a major threat to human and livestock health. The accurate and rapid detection of Brucella DNA is essential for the diagnosis and treatment of this disease. However, traditional diagnostic methods, such as culture and serological tests, have limitations in terms of sensitivity, specificity, and time consumption. To address these challenges, we developed a highly sensitive one-tube nested quantitative real-time PCR (qPCR) method to detect Brucella DNA using a closed-tube approach with two primers and two probes that sequentially react with encoding a Brucella outer membrane protein. The analytical sensitivity of this one-tube nested qPCR approach was 100 fg/μL, which is 100-fold higher than that of a conventional qPCR. Intra-batch and inter-batch replicates showed low coefficients of variation, both less than 5%. The study included 250 clinical samples, showing that this one-tube nested qPCR method has a specificity of 100% and a sensitivity of 98.6%, exceeding the sensitivity of conventional qPCR (84.1%) at a cycle threshold (CT) cutoff of 38. More importantly, the one-tube nested qPCR reduced the CT values by an average of 6.4 compared to conventional qPCR, significantly improving the detection rate of low-load samples (CT > 35). In conclusion, the bcsp31 one-tube nested qPCR method is a promising tool for the detection of brucellosis, owing to its high sensitivity and operational simplicity. This study highlights the potential of qPCR-based methods to improve the accuracy and speed of brucellosis diagnoses. IMPORTANCE This study developed a highly sensitive and efficient method for the detection of brucellosis by introducing a one-tube nested quantitative real-time PCR (qPCR) approach, representing a remarkable advance in the field. The method demonstrated an impressive analytical sensitivity of 100 fg/μL, surpassing conventional qPCR and enabling the detection of even low levels of Brucella DNA. In addition, the study's comprehensive evaluation of 250 clinical samples revealed a specificity of 100% and a sensitivity of 98.6%, underscoring its reliability and accuracy. Most importantly, the new method significantly improved the detection rate of low-burden samples, reducing cycle threshold values by an average of 6.4. These results underscore the immense potential of this approach to facilitate rapid and accurate brucellosis diagnosis, which is critical for effective disease management and control.
| S-EPMC10714840 | biostudies-literature
Project description:Coronavirus disease 2019 (COVID-19) has become a public health emergency. The reverse transcriptase real-time quantitative PCR (qRT-PCR) test is currently considered as the gold standard in the laboratory for the etiological detection of COVID-19. However, qRT-PCR results could be false-negative due to the inadequate sensitivity of qRT-PCR. In this study, we have developed and evaluated a novel one-step single-tube nested quantitative real-time PCR (OSN-qRT-PCR) assay for the highly sensitive detection of SARS-CoV-2 targeting the ORF1ab and N genes. The sensitivity of the OSN-qRT-PCR assay was 1 copy/reaction and 10-fold higher than that of the commercial qRT-PCR kit (10 copies/reaction). The clinical performance of the OSN-qRT-PCR assay was evaluated using 181 clinical samples. Among them, 14 qRT-PCR-negative samples (7 had no repetitive results and 7 had no cycle threshold (CT) values) were detected by OSN-qRT-PCR. Moreover, the 7 qRT-PCR-positives in the qRT-PCR gray zone (CT values of ORF1ab ranged from 37.48 to 39.07, and CT values of N ranged from 37.34 to 38.75) were out of the gray zone and thus were deemed to be positive by OSN-qRT-PCR, indicating that the positivity of these samples is confirmative. Compared to the qRT-PCR kit, the OSN-qRT-PCR assay revealed higher sensitivity and specificity, showing better suitability to clinical applications for the detection of SARS-CoV-2 in patients with low viral load.
| S-EPMC7299395 | biostudies-literature
Project description:Screening for theranostic biomarkers is mandatory for the therapeutic management of cutaneous melanoma. BRAF and NRAS genes must be tested in routine clinical practice. The methods used to identify these alterations must be sensitive to detect mutant alleles in a background of wild type alleles, and specific to identify the correct mutation. They should not require too much material, since in some cases the available samples are small biopsies. Finally, they should also be quick enough to allow a rapid therapeutic management of patients. Sixty five consecutive formalin-fixed paraffin-embedded (FFPE) melanoma samples were prospectively tested for BRAF mutations with the VE1 (anti-BRAF V600E) antibody and for both BRAF and NRAS mutations with the Idylla NRAS-BRAF-EGFR S492R Mutation Assay cartridges. Results were compared to our routine laboratory practice, allele specific amplification and/or Sanger sequencing and discordant cases confirmed by digital PCR. Excluding discordant by-design-mutations, system failures and DNA quantity or quality failures, BRAF IHC demonstrated an overall concordance of 89% for BRAF V600E mutation detection, the Idylla system gave a concordance of 100% for BRAF mutation detection and of 92.1% for NRAS mutation detection when compared to our reference. When discrepancies were observed, all routine results were confirmed by digital PCR. Finally, BRAF IHC positive predictive value (PPV) was of 82% and negative predictive value (NPV) of 92%. The Idylla cartridges showed a PPV and NPV of both 100% for BRAF mutation detection and a PPV and NPV of 100% and 87% respectively, for NRAS mutation detection. In conclusion, BRAF V600E immunohistochemistry is efficient for detecting the V600E mutation, but negative cases should be further evaluated by molecular approaches for other BRAF mutations. Since 3 NRAS mutations have not been detected by the Idylla NRAS-BRAF-EGFR S492R Mutation Assay, these cartridges should not be used as a substitute for traditional molecular methods in the conventional patient therapeutic care process without the expertise needed to have a critical view of the produced results.
| S-EPMC6695223 | biostudies-literature
Project description:BACKGROUND: Coxiella burnetii, the bacterium causing Q fever, is an obligate intracellular biosafety level 3 agent. Detection and quantification of these bacteria with conventional methods is time consuming and dangerous. During the last years, several PCR based diagnostic assays were developed to detect C. burnetii DNA in cell cultures and clinical samples. We developed and evaluated TaqMan-based real-time PCR assays that targeted the singular icd (isocitrate dehydrogenase) gene and the transposase of the IS1111a element present in multiple copies in the C. burnetii genome. RESULTS: To evaluate the precision of the icd and IS1111 real-time PCR assays, we performed different PCR runs with independent DNA dilutions of the C. burnetii Nine Mile RSA493 strain. The results showed very low variability, indicating efficient reproducibility of both assays. Using probit analysis, we determined that the minimal number of genome equivalents per reaction that could be detected with a 95% probability was 10 for the icd marker and 6.5 for the IS marker. Plasmid standards with cloned icd and IS1111 fragments were used to establish standard curves which were linear over a range from 10 to 10(7) starting plasmid copy numbers. We were able to quantify cell numbers of a diluted, heat-inactivated Coxiella isolate with a detection limit of 17 C. burnetii particles per reaction. Real-time PCR targeting both markers was performed with DNA of 75 different C. burnetii isolates originating from all over the world. Using this approach, the number of IS1111 elements in the genome of the Nine Mile strain was determined to be 23, close to 20, the number revealed by genome sequencing. In other isolates, the number of IS1111 elements varied widely (between seven and 110) and seemed to be very high in some isolates. CONCLUSION: We validated TaqMan-based real-time PCR assays targeting the icd and IS1111 markers of C. burnetii. The assays were shown to be specific, highly sensitive and efficiently reproducible. Cell numbers in dilutions of a C. burnetii isolate were reliably quantified. PCR quantification suggested a high variability of the number of IS1111 elements in different C. burnetii isolates, which may be useful for further phylogenetic studies.
| S-EPMC1360083 | biostudies-literature
Project description:Partial nucleotide sequences (459 bp) of the groEL gene (encoding the 60-kDa heat shock protein, HSP60) from 23 contemporary isolates of Orientia tsutsugamushi isolated from patients with acute scrub typhus in Thailand were compared with 16 reference strain sequences to evaluate the potential of groEL as a conserved and representative target for molecular diagnostics.. Overall nucleotide identity within all available O. tsutsugamushi isolates (n = 39) was 98.8% (range: 95.0-100), reflecting a high degree of conservation; nucleotide identities were 67.5% and 65.6%, respectively, when typhus and spotted fever group rickettsiae were included.. A highly sensitive and quantitative real-time PCR assay was designed and evaluated using 61 samples, including buffy coats from patients in Thailand and Laos. Reliable and accurate quantitation of bacterial loads allows further investigation of other diagnostic methods and may lead to an improved understanding of the pathophysiology of acute scrub typhus, an important but under-recognized disease.
| S-EPMC3429864 | biostudies-literature