Clinical Validation of a New Triplex Real-Time Polymerase Chain Reaction Assay for the Detection and Discrimination of Herpes simplex Virus Types 1 and.

Slides:

Advertisements

Similar presentations

Measurement of Relative Copy Number of CDKN2A/ARF and CDKN2B in Bladder Cancer by Real-Time Quantitative PCR and Multiplex Ligation-Dependent Probe Amplification.

Advertisements

Clinical Laboratory Analysis of Immunoglobulin Heavy Chain Variable Region Genes for Chronic Lymphocytic Leukemia Prognosis Philippe Szankasi, David.

Alexander A. Morley, Sue Latham, Michael J. Brisco, Pamela J

Direct DNA Amplification from Crude Clinical Samples Using a PCR Enhancer Cocktail and Novel Mutants of Taq Zhian Zhang, Milko B. Kermekchiev, Wayne.

Pre-Clinical Validation of a Novel, Highly Sensitive Assay to Detect PML-RARα mRNA Using Real-Time Reverse-Transcription Polymerase Chain Reaction James.

Evaluation of the Epstein-Barr Virus R-Gene Quantification Kit in Whole Blood with Different Extraction Methods and PCR Platforms Samira Fafi-Kremer,

Locked Nucleic Acids Can Enhance the Analytical Performance of Quantitative Methylation-Specific Polymerase Chain Reaction Karen S. Gustafson The Journal.

Direct DNA Amplification from Crude Clinical Samples Using a PCR Enhancer Cocktail and Novel Mutants of Taq Zhian Zhang, Milko B. Kermekchiev, Wayne.

Real-Time Polymerase Chain Reaction for Detecting Bacterial DNA Directly from Blood of Neonates Being Evaluated for Sepsis Jeanne A. Jordan, Mary Beth.

Optimization of a Relative Telomere Length Assay by Monochromatic Multiplex Real- Time Quantitative PCR on the LightCycler 480 Anthony Y.Y. Hsieh, Sara.

Molecular Assays for Quantitative and Qualitative Detection of Influenza Virus and Oseltamivir Resistance Mutations Erhard van der Vries, Jeer Anber,

Development and Evaluation of a SYBR Green–Based Real-Time Multiplex RT-PCR Assay for Simultaneous Detection and Serotyping of Dengue and Chikungunya.

MethySYBR, a Novel Quantitative PCR Assay for the Dual Analysis of DNA Methylation and CpG Methylation Density Pang-Kuo Lo, Hanano Watanabe, Pi-Chun.

High-Fidelity DNA Polymerase Enhances the Sensitivity of a Peptide Nucleic Acid Clamp PCR Assay for K-ras Mutations Bjørnar Gilje, Reino Heikkilä, Satu.

High Quality Assessment of DNA Methylation in Archival Tissues from Colorectal Cancer Patients Using Quantitative High-Resolution Melting Analysis Marija.

Clinical Validation of a New Triplex Real-Time Polymerase Chain Reaction Assay for the Detection and Discrimination of Herpes simplex Virus Types 1 and.

Establishment and Study of Different Real-Time Polymerase Chain Reaction Assays for the Quantification of Cells with Deletions of Chromosome 7 Elia Mattarucchi,

Detection of Cytomegalovirus in Whole Blood Using Three Different Real-Time PCR Chemistries Erica Vincent, Zhengming Gu, Markus Morgenstern, Candace.

Diagnostic Testing for Pandemic Influenza in Singapore

Michael T. Seipp, Jacob D. Durtschi, Michael A

Limitations and Practical Procedure in BclII-Ig Heavy Chain Gene Rearrangement Real- Time Quantitative Polymerase Chain Reaction Barbara Dessars, Pierre.

Single Nucleotide Polymorphism-Based System Improves the Applicability of Quantitative PCR for Chimerism Monitoring Egle Gineikiene, Mindaugas Stoskus,

Association of Clinical Status of Follicular Lymphoma Patients after Autologous Stem Cell Transplant and Quantitative Assessment of Lymphoma in Blood.

The Effect of Primer-Template Mismatches on the Detection and Quantification of Nucleic Acids Using the 5′ Nuclease Assay Ralph Stadhouders, Suzan D.

Clinical Relevance of Sensitive and Quantitative STAT3 Mutation Analysis Using Next- Generation Sequencing in T-Cell Large Granular Lymphocytic Leukemia

Evaluation of the Cepheid GeneXpert BCR-ABL Assay

Detection of Central Nervous System Leukemia in Children with Acute Lymphoblastic Leukemia by Real-Time Polymerase Chain Reaction Sharon R. Pine, Changhong.

Cornelis J. J. Huijsmans, Jeroen Poodt, Paul H. M. Savelkoul, Mirjam H

Protocol for the Use of Polymerase Chain Reaction in the Detection of Intraocular Large B-Cell Lymphoma in Ocular Samples Aires Lobo, Narciss Okhravi,

Performance Assessment of Eight High-Throughput PCR Assays for Viral Load Quantitation of Oncogenic HPV Types Roberto Flores-Munguia, Erin Siegel, Walter.

A Fast Real-Time Polymerase Chain Reaction Method for Sensitive and Specific Detection of the Neisseria gonorrhoeae porA Pseudogene Stig Ove Hjelmevoll,

Evaluation of Pre-Analytical Variables in the Quantification of Dengue Virus by Real- Time Polymerase Chain Reaction Azlinda Anwar, Guoqiang Wan, Kaw-Bing.

Real-Time Polymerase Chain Reaction for Detecting Bacterial DNA Directly from Blood of Neonates Being Evaluated for Sepsis Jeanne A. Jordan, Mary Beth.

Multiplexed Detection of Anthrax-Related Toxin Genes

Quantitative Determination of JAK2 V617F by TaqMan

Clinical Laboratory Analysis of Immunoglobulin Heavy Chain Variable Region Genes for Chronic Lymphocytic Leukemia Prognosis Philippe Szankasi, David.

Use of Single Nucleotide Polymorphisms (SNP) and Real-Time Polymerase Chain Reaction for Bone Marrow Engraftment Analysis Dwight H. Oliver, Richard E.

Validation and Implementation of a Custom Next-Generation Sequencing Clinical Assay for Hematologic Malignancies Michael J. Kluk, R. Coleman Lindsley,

Real-Time Polymerase Chain Reaction Detection of Herpes Simplex Virus in Cerebrospinal Fluid and Cost Savings from Earlier Hospital Discharge Kenneth.

Christine Formisano-Tréziny, Marina de San Feliciano, Jean Gabert

Critical Evaluation of Real-Time Reverse Transcriptase-Polymerase Chain Reaction for the Quantitative Detection of Cytokeratin 20 mRNA in Colorectal Cancer.

Olivier Gruselle, Thierry Coche, Jamila Louahed

Comparative Evaluation of Four Real-Time PCR Methods for the Quantitative Detection of Epstein-Barr Virus from Whole Blood Specimens Daelynn Buelow,

Rapid and Inexpensive Detection of α1-Antitrypsin Deficiency-Related Alleles S and Z by a Real-Time Polymerase Chain Reaction Suitable for a Large-Scale.

S. Hussain Askree, Shika Dharamrup, Lawrence N. Hjelm, Bradford Coffee

Development of a Diagnostic Microarray Assay to Assess the Risk of Recurrence of Prostate Cancer Based on PITX2 DNA Methylation Philipp Schatz, Dimo.

Development of a Quantitative Real-Time Polymerase Chain Reaction Assay for the Detection of the JAK2 V617F Mutation Elizabeth C. Wolstencroft, Katy.

Development and Laboratory Evaluation of a Real-Time PCR Assay for Detecting Viruses and Bacteria of Relevance for Community-Acquired Pneumonia Alicia.

Detection and Discrimination between Deletional and Non-Deletional Prader-Willi and Angelman Syndromes by Methylation-Specific PCR and Quantitative Melting.

Molecular Monitoring of Chronic Myelogenous Leukemia

Detection of Common Disease-Causing Mutations in Mitochondrial DNA (Mitochondrial Encephalomyopathy, Lactic Acidosis with Stroke-Like Episodes MTTL

Design and Evaluation of a Real-Time PCR Assay for Quantification of JAK2 V617F and Wild-Type JAK2 Transcript Levels in the Clinical Laboratory Jason.

Polymerase Chain Reaction Detection of Kaposi's Sarcoma-Associated Herpesvirus- Optimized Protocols and Their Application to Myeloma Langxing Pan, Laura.

Diagnosis of Human Congenital Cytomegalovirus Infection by Amplification of Viral DNA from Dried Blood Spots on Perinatal Cards Lori Scanga, Shu Chaing,

Rapid and Sensitive Real-Time Polymerase Chain Reaction Method for Detection and Quantification of 3243A>G Mitochondrial Point Mutation Rinki Singh,

Low Incidence of Minor BRAF V600 Mutation-Positive Subclones in Primary and Metastatic Melanoma Determined by Sensitive and Quantitative Real-Time PCR

Evaluation of the Abbott Investigational Use Only RealTime HIV-1 Assay and Comparison to the Roche Amplicor HIV-1 Monitor Test, Version 1.5 Michael T.

Volume 6, Issue 1, Pages (July 2002)

Amplification Refractory Mutation System, a Highly Sensitive and Simple Polymerase Chain Reaction Assay, for the Detection of JAK2 V617F Mutation in Chronic.

Cecily P. Vaughn, Elaine Lyon, Wade S. Samowitz

Optimized Allele-Specific Real-Time PCR Assays for the Detection of Common Mutations in KRAS and BRAF Alois H. Lang, Heinz Drexel, Simone Geller-Rhomberg,

Statistical Treatment of Fluorescence in Situ Hybridization Validation Data to Generate Normal Reference Ranges Using Excel Functions Allison L. Ciolino,

Development and Comparison of a Rapid Isothermal Nucleic Acid Amplification Test for Typing of Herpes Simplex Virus Types 1 and 2 on a Portable Fluorescence.

Custom Design of a GeXP Multiplexed Assay Used to Assess Expression Profiles of Inflammatory Gene Targets in Normal Colon, Polyp, and Tumor Tissue Janice.

Visual Format for Detection of Mycobacterium tuberculosis and M

Validation of Roche LightCycler Epstein-Barr Virus Quantification Reagents in a Clinical Laboratory Setting Margaret L. Gulley, Hongxin Fan, Sandra H.

Improved Detection of the KIT D816V Mutation in Patients with Systemic Mastocytosis Using a Quantitative and Highly Sensitive Real-Time qPCR Assay Thomas.

Monique A. Johnson, Marvin J. Yoshitomi, C. Sue Richards

Analytical Validation of a Highly Sensitive, Multiplexed Chronic Myeloid Leukemia Monitoring System Targeting BCR-ABL1 RNA Justin T. Brown, Ion J. Beldorth,

Real-Time Polymerase Chain Reaction Detection of Herpes Simplex Virus in Cerebrospinal Fluid and Cost Savings from Earlier Hospital Discharge Kenneth.

Presentation transcript:

Clinical Validation of a New Triplex Real-Time Polymerase Chain Reaction Assay for the Detection and Discrimination of Herpes simplex Virus Types 1 and 2 Heide Reil, Ariane Bartlime, Jana Drerup, Thomas Grewing, Klaus Korn The Journal of Molecular Diagnostics Volume 10, Issue 4, Pages 361-367 (July 2008) DOI: 10.2353/jmoldx.2008.070104 Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Accuracy of HSV quantitation. Specimens from three different quality proficiency panels (see Materials and Methods) were extracted using the MagNA Pure instrument (Roche). DNA extracts were subjected to the triplex real-time PCR on the ABI Prism 7000 instrument. A: Values from HSV-1 and HSV-2 quantitation are plotted against the expected reference values of each sample. B: Target values and results from real-time PCR are presented as columns; results from nested PCR are shown as − or + below. The Journal of Molecular Diagnostics 2008 10, 361-367DOI: (10.2353/jmoldx.2008.070104) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Probit analysis HSV-1/HSV-2. Dilution series have been set up for each HSV-1 and HSV-2 plasmid standard DNA, which was quantified by absorption spectroscopy. Different copy numbers ranging from 25.7 to 0.008 for HSV-1 and from 35.3 to 0.012 HSV copy equivalents/μl for HSV-2 were quantified in eight replicates on three different days on the ABI Prism 7000 instrument. The sensitivity was determined as 0.47 HSV-2 copies/μl, equivalent to 9.4 copies/reaction and 0.9 HSV-1 copies/μl, equivalent to 18 copies/reaction, defined as 95% cut-off value, by probit analysis using PriProbit version 1.63 software. The Journal of Molecular Diagnostics 2008 10, 361-367DOI: (10.2353/jmoldx.2008.070104) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Ct value distribution of HSV-positive clinical samples. A: Ct values achieved in triplex real-time PCR for CSF, BAL, and materials other than CSF and BAL (Table 1) positive for HSV-DNA are shown. B: Ct values of HSV and the Internal Control in all HSV-positive PCR reactions were plotted against each other. The dotted line depicts the HSV Ct value (Ct 22, corresponding to approximately 5 × 107 HSV copies/ml), where PCR competition between the amplification of HSV and the Internal Control begins to occur. When PCR competition is not relevant, the Ct value of the Internal Control vary between Ct 25 and 30 (arrows). The Journal of Molecular Diagnostics 2008 10, 361-367DOI: (10.2353/jmoldx.2008.070104) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 HSV-1 standard curve. Illustrated is a HSV-1 standard curve that was generated with five standards ranging from 5 × 103 to 5 × 106 HSV-1 copies/ml provided by the manufacturer. Data are mean values of five different runs performed on five different days. Standard deviation as indicated by error bars. The Journal of Molecular Diagnostics 2008 10, 361-367DOI: (10.2353/jmoldx.2008.070104) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 Interassay precision. HSV-1 and HSV-2 virus stocks were mixed, stored in aliquots at −20°C, and used as a control for each PCR run. Ct values of all three reporters (FAM for HSV-1; NED for HSV-2; and VIC for the Internal Control, IC) from 26 individual runs are documented. The mean Ct values were 28.5 ± 0.78 for HSV-1, 26.5 ± 0.65 for HSV-2, and 25.5 ± 0.45 for IC, corresponding to a mean coefficient of variation of 2.7%, 2.5%, and 1.8% respectively. The Journal of Molecular Diagnostics 2008 10, 361-367DOI: (10.2353/jmoldx.2008.070104) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions