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Identification and validation of biomarkers of IgV(H) mutation status in chronic lymphocytic leukemia using microfluidics quantitative real-time polymerase chain reaction technology.

ABSTRACT: To develop a model incorporating relevant prognostic biomarkers for untreated chronic lymphocytic leukemia patients, we re-analyzed the raw data from four published gene expression profiling studies. We selected 88 candidate biomarkers linked to immunoglobulin heavy-chain variable region gene (IgV(H)) mutation status and produced a reliable and reproducible microfluidics quantitative real-time polymerase chain reaction array. We applied this array to a training set of 29 purified samples from previously untreated patients. In an unsupervised analysis, the samples clustered into two groups. Using a cutoff point of 2% homology to the germline IgV(H) sequence, one group contained all 14 IgV(H)-unmutated samples; the other contained all 15 mutated samples. We confirmed the differential expression of 37 of the candidate biomarkers using two-sample t-tests. Next, we constructed 16 different models to predict IgV(H) mutation status and evaluated their performance on an independent test set of 20 new samples. Nine models correctly classified 11 of 11 IgV(H)-mutated cases and eight of nine IgV(H)-unmutated cases, with some models using three to seven genes. Thus, we can classify cases with 95% accuracy based on the expression of as few as three genes.

SUBMITTER: Abruzzo LV

PROVIDER: S-EPMC1975107 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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Identification and validation of biomarkers of IgV(H) mutation status in chronic lymphocytic leukemia using microfluidics quantitative real-time polymerase chain reaction technology.

Abruzzo Lynne V LV Barron Lynn L LL Anderson Keith K Newman Rachel J RJ Wierda William G WG O'brien Susan S Ferrajoli Alessandra A Luthra Madan M Talwalkar Sameer S Luthra Rajyalakshmi R Jones Dan D Keating Michael J MJ Coombes Kevin R KR

The Journal of molecular diagnostics : JMD 20070809 4

To develop a model incorporating relevant prognostic biomarkers for untreated chronic lymphocytic leukemia patients, we re-analyzed the raw data from four published gene expression profiling studies. We selected 88 candidate biomarkers linked to immunoglobulin heavy-chain variable region gene (IgV(H)) mutation status and produced a reliable and reproducible microfluidics quantitative real-time polymerase chain reaction array. We applied this array to a training set of 29 purified samples from pr ...[more]

PMID: 17690214

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Project description:BackgroundHelicobacter pylori (H. pylori) infection is a serious human health threat. The empiric H. pylori treatment paradigm guided by traditional testing technologies has led to antibiotic resistance. Here, we improved the qPCR method to provide technical support for precision H. pylori diagnosis and treatment.MethodsTwo pairs of primers and probes targeting the glmM gene were designed to detect H. pylori, and a multiplex qPCR method was established for virulence factor detection. Then, a rapid urease test (RUT), culturing and qPCR were performed on 141 specimens collected from Xinqiao Hospital of China in 2017 to evaluate the qPCR detection capability. Finally, the H. pylori infectious amount and virulence genes were detected by qPCR.Results1. The improved qPCR method which used two pairs of primers had a higher detection rate (100%) and better accuracy (p = 0.000), compared with the qPCR using a pair of primers. It also had better consistency with the bacterial culture than with RUT (Kappa =0.440, p < 0.001). 2. The H. pylori infectious amount was significantly positively associated with gastritis in corpus (p = 0.003) and gastric erosion (p = 0.043). The H. pylori infectious amount in gastric precancerous patients was significantly lower than that in H. pylori-positive patients (p < 0.05), and the infectious H. pylori-vacA s1+ amount was significantly greater than that of H. pylori-vacA s1- (p < 0.05). 3. The vacA s1 frequency was significantly higher than that of vacA m1/cagA+/babA2+ in chronic superficial gastritis (p = 0.000), peptic ulcer (p = 0.037) and gastric erosion (p = 0.009). The H. pylori-vacA+/cagA+/babA2+ frequency showed a significant positive correlation (p < 0.05).ConclusionsThe H. pylori infectious amount and presence of H. pylori virulence factors showed complex correlations with gastric disease occurrence and development. The improved qPCR with good detection performance can be used for quantitative H. pylori detection and testing for the virulence genes vacA s1, vacA m1, cagA and babA2 simultaneously. These findings will provide valuable information for disease diagnosis and treatment.

Project description:BACKGROUND AND OBJECTIVES:The variable response of hepatitis C virus (HCV) genotypes towards anti-viral treatment requires prior information on the genotype status before planning a therapeutic strategy. Although assays for typing or subtyping of HCV are available, however, a fast and reliable assay system is still needed. The present study was planned to develop a single-step multiplex quantitative real time polymerase chain reaction (qPCR) assay to determine HCV genotypes in patients' sera. METHODS:The conserved sequences from 5' UTR, core and NS5b regions of HCV genome were used to design primers and hydrolysis probes labeled with fluorophores. Starting with the standardization of singleplex (qPCR) for each individual HCV-genotype, the experimental conditions were finally optimized for the development of multiplex assay. The sensitivity and specificity were assessed both for singleplex and multiplex assays. Using the template concentration of 102 copies per microliter, the value of quantification cycle (Cq) and the limit of detection (LOD) were also compared for both singleplex and multiplex assays. Similarly, the merit of multiplex assay was also compared with sequence analysis and restriction fragment length polymorphism (RFLP) techniques used for HCV genotyping. In order to find the application of multiplex qPCR assay, it was used for genotyping in a panel of 98 sera positive for HCV RNA after screening a total number of 239 patients with various liver diseases. RESULTS:The results demonstrated the presence of genotype 1 in 26 of 98 (26.53%) sera, genotype 3 in 65 (66.32%) and genotype 4 in 2 (2.04%) sera samples, respectively. One sample showed mixed infection of genotype 1 and 3. Five samples could not show the presence of any genotype. Genotypes 2, 5 and 6 could not be detected in these sera samples. The analysis of sera by singleplex and RFLP indicated the results of multiplex to be comparable with singleplex and with clear merit of multiplex over RFLP. In addition, the results of multiplex assay were also found to be comparable with those from sequence analysis. The sensitivity, specificity, Cq values and LOD values were compared and found to be closely associated both for singleplex and multiplex assays. CONCLUSION:The multiplex qPCR assay was found to be a fast, specific and sensitive method that can be used as a technique of choice for HCV genotyping in all routine laboratories.

Dataset Information

Identification and validation of biomarkers of IgV(H) mutation status in chronic lymphocytic leukemia using microfluidics quantitative real-time polymerase chain reaction technology.

Publications

Identification and validation of biomarkers of IgV(H) mutation status in chronic lymphocytic leukemia using microfluidics quantitative real-time polymerase chain reaction technology.

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