RESUMO
BACKGROUND: The detection of somatic mutations in primary tumors is critical for the understanding of cancer evolution and targeting therapy. Multiple technologies have been developed to enable the detection of such mutations. Next generation sequencing (NGS) is a new platform that is gradually becoming the technology of choice for genotyping cancer samples, owing to its ability to simultaneously interrogate many genomic loci at massively high efficiency and increasingly lower cost. However, multiple barriers still exist for its broader adoption in clinical research practice, such as fragmented workflow and complex bioinformatics analysis and interpretation. METHODS: We performed validation of the QIAGEN GeneReader NGS System using the QIAact Actionable Insights Tumor Panel, focusing on clinically meaningful mutations by using DNA extracted from formalin-fixed paraffin-embedded (FFPE) colorectal tissue with known KRAS mutations. The performance of the GeneReader was evaluated and compared to data generated from alternative technologies (PCR and pyrosequencing) as well as an alternative NGS platform. The results were further confirmed with Sanger sequencing. RESULTS: The data generated from the GeneReader achieved 100% concordance with reference technologies. Furthermore, the GeneReader workflow provides a truly integrated workflow, eliminating artifacts resulting from routine sample preparation; and providing up-to-date interpretation of test results. CONCLUSION: The GeneReader NGS system offers an effective and efficient method to identify somatic (KRAS) cancer mutations.
Assuntos
Análise Mutacional de DNA , Proteínas Proto-Oncogênicas p21(ras)/genética , Neoplasias Colorretais/genética , Fixadores/química , Formaldeído/química , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Inclusão em Parafina , Reação em Cadeia da PolimeraseRESUMO
Hybrid Capture 2 (HC2) Human Papillomavirus (HPV) DNA Test® is FDA approved and is a proven aid in detecting HPV infections of the cervix and as an aid in diagnosing, with cytology, cervical disease. A prospective feasibility study was conducted to determine if HC2 testing has utility when screening for high-grade anal dysplasia (AIN2+). We enrolled 298 patients (45% HIV+) who had AIN2+ screening with cytology, histology and HC2 testing for two specimens: a swab into liquid-based cytology medium and either a swab or a brush collection in specimen transport medium (STM). High-resolution anoscopy was performed on all patients with biopsy of AIN2+ suspicious lesions. Cytology was benign (42%), atypical squamous cells of undetermined significance (30%), low-grade squamous intraepithelial lesion (18%), high-grade squamous intraepithelial lesion (1%), ASCUS possibly high-grade dysplasia (1.7%) and nondiagnostic (7%) and 36% had AIN2+ histology. Sensitivity and specificity for predicting AIN2+ histology for any abnormal cytology were 77 and 52%, whereas HC2 sensitivity and specificity were 91 and 40% (p = 0.005 for sensitivity), respectively. There was no significant difference in HC2 sensitivity or specificity between brush and swab or STM and residual cells from cytology. AIN2+ was found in 20% of patients with benign cytology. Only nine AIN2+ specimens were HC2-. This prospective study indicates that HC2 may be useful when screening for anal dysplasia; however, a larger study is recommended.
Assuntos
Doenças do Ânus/diagnóstico , Infecções por Papillomavirus/diagnóstico , Kit de Reagentes para Diagnóstico , Adulto , Idoso , Doenças do Ânus/patologia , Doenças do Ânus/virologia , Feminino , Soropositividade para HIV , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Sensibilidade e Especificidade , Adulto JovemRESUMO
We have developed a new research assay that combines sequence-specific sample preparation and isothermal amplification for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae infections. The assay targets both the omp gene and the cryptic plasmid of C. trachomatis and the multicopy opa gene of N. gonorrhoeae, which are amplified and detected in a single reaction. We evaluated the ability of the assay to detect C. trachomatis and N. gonorrhoeae infections in first-catch urine, swab, and liquid-based cytology samples. Total agreement between the new assay and APTIMA Combo 2 varied between 95.3% and 100%, depending on the sample type and target detected. Total agreement between the new assay and BD ProbeTec varied between 96.7% and 100%, depending on the sample type and target detected. The assay has a simple work flow, and endpoint results can be achieved in 3 h, including sample preparation. The assay described here was evaluated for research use and was compared to commercially available assays.
Assuntos
Colo do Útero/microbiologia , Gonorreia/diagnóstico , Linfogranuloma Venéreo/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Urina/microbiologia , Vagina/microbiologia , Proteínas da Membrana Bacteriana Externa/genética , Técnicas Bacteriológicas/métodos , Chlamydia trachomatis/isolamento & purificação , DNA Bacteriano/genética , Feminino , Gonorreia/microbiologia , Humanos , Linfogranuloma Venéreo/microbiologia , Masculino , Neisseria gonorrhoeae/isolamento & purificação , PlasmídeosRESUMO
Point-of-care (PoC) testing followed by personalized efficient therapy of infectious diseases may result in a considerable reduction of associated health care costs. Lab-on-a-chip (LoC) systems represent a potentially high efficient class of PoC tools. Here, we present a LoC system for automated pathogen analysis of respiratory viruses from nasopharyngeal specimens. The device prepares total nucleic acids from extracted swab samples using magnetic silica beads. After reverse transcription the co-purified viral RNA is amplified in accordance with the QIAplex multiplex PCR technology. Hybridized to corresponding QIAGEN LiquiChip beads and labelled with streptavidin R-phycoerythrin, the amplified target sequences are finally detected using a QIAGEN LiquiChip200 workstation. All chemicals needed are either stored freeze-dried on the disposable chip or are provided in liquid form in a reagent cartridge for up to 24 runs. Magnetic stir bars for mixing as well as turning valves with metering structures are integrated into the injection-moulded disposable chip. The core of the controlling instrument is a rotating heating bar construction providing fixed temperatures for fast cycling. PCR times of about half an hour (for 30 cycles) could be achieved for 120 µl reactions, making this system the fastest currently available high-volume PCR chip. The functionality of the system was shown by comparing automatically processed nasopharyngeal samples to ones processed manually according to the QIAGEN "ResPlex™ II Panel v2.0" respiratory virus detection kit. A prototype of the present instrument revealed slightly weaker signal intensities with a similar sensitivity in comparison to the commercially available kit and automated nucleic acid preparation devices, even without protocol optimization.
Assuntos
Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Nasofaringe/virologia , Infecções Respiratórias/diagnóstico , Técnicas de Laboratório Clínico , Humanos , Dispositivos Lab-On-A-Chip/economia , Técnicas Analíticas Microfluídicas/métodos , Ficoeritrina/química , Sistemas Automatizados de Assistência Junto ao Leito , Infecções Respiratórias/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Estreptavidina/química , Vírus/isolamento & purificaçãoRESUMO
The hc2 human papillomavirus DNA test (HC2) is effective when screening women for cervical dysplasia, and it might be effective in the screening for anal dysplasia. Differences between the anal and the cervical canals could affect the test performance. This prospective study (n=292) measured the HC2 signal and in agreement with a histologic endpoint of high-grade dysplasia for anal specimens collected in various ways. Sensitivities were 91%, 85%, and 62% for specimens collected in a sample transport medium and a liquid-based cytology medium processed by Gyn or Non-Gyn protocol, respectively. HC2 sensitivity and specificity to predict high-grade anal dysplasia were similar for brush or swab specimen collections, but HC2 signal was 6 times higher with the brush. Specificity and sensitivity were similar whether the sample was collected first or after a cytology sample for brush or swab, but swab specimens at the second collection had an HC2 signal (mean) 48% lower than that of the first collection, and the swab cellularity was lower. The presence of maximum stool decreased the HC2 signal in anal swab specimens. Consensus polymerase chain reaction (PCR) confirmed that the 13 human papillomavirus probe types in HC2 were optimal for performance. HC2 could potentially be further investigated for use in screening anal dysplasia. A larger prospective study is indicated.
Assuntos
Neoplasias do Ânus/diagnóstico , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Lesões Pré-Cancerosas/diagnóstico , Adulto , Idoso , Canal Anal/patologia , Canal Anal/virologia , Sondas de DNA de HPV , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Hibridização de Ácido Nucleico/métodos , Papillomaviridae/genética , Sensibilidade e Especificidade , Manejo de EspécimesRESUMO
Thermophilic helicase dependent amplification (tHDA), which employs helicase to unwind double-stranded DNA at constant temperature, is a relatively new isothermal nucleic acid amplification technology. In this study, the development and optimization of a 4-plex tHDA assay for detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are described. tHDA is combined with sequence-specific sample preparation on magnetic beads and homogeneous endpoint fluorescence detection using dual-labeled probes. This 4-plex tHDA assay was applied to the detection of 2 genes on CT and a multicopy gene on NG in the presence of an internal control. The assay showed high analytical sensitivity and specificity of simultaneous CT/NG detection and is compatible with a wide variety of sample types and media. The isothermal reaction conditions and homogeneous endpoint detection utilized in this assay are well suited for laboratory automation and high-throughput screening applications as well as for point-of-care testing.