Multiplex Detection of Ehrlichia and Anaplasma Species Pathogens in Peripheral Blood by Real-Time Reverse Transcriptase-Polymerase Chain Reaction Kamesh.

Slides:

Advertisements

Similar presentations

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

Advertisements

Performance Characteristics of a Quantitative, Homogeneous TaqMan RT-PCR Test for HCV RNA Joerg Kleiber, Thomas Walter, Gerd Haberhausen, Sue Tsang,

Effectiveness of Capillary Electrophoresis Using Fluorescent-Labeled Primers in Detecting T-Cell Receptor γ Gene Rearrangements Timothy C. Greiner, Ronald.

A Highly Sensitive, Multiplex Broad-Spectrum PCR-DNA-Enzyme Immunoassay and Reverse Hybridization Assay for Rapid Detection and Identification of Chlamydia.

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

One-Step Ligation on RNA Amplification for the Detection of Point Mutations Lei Zhang, Jingjing Wang, Mia Coetzer, Stephanie Angione, Rami Kantor, Anubhav.

RT-PCR Analysis of RNA Extracted from Bouin-Fixed and Paraffin-Embedded Lymphoid Tissues Annunziata Gloghini, Barbara Canal, Ulf Klein, Luigino Dal Maso,

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

Rapid Detection of the Epidermal Growth Factor Receptor Mutation in Non-Small-Cell Lung Cancer for Analysis of Acquired Resistance Using Molecular Beacons

Rapid detection of resistance in Mycobacterium tuberculosis: a review discussing molecular approaches D. García de Viedma Clinical Microbiology and.

Dual detection of Legionella pneumophila and Legionella species by real-time PCR targeting the 23S-5S rRNA gene spacer region G. Yang, R. Benson, T.

Detection and Species Identification of Malaria Parasites by Isothermal tHDA Amplification Directly from Human Blood without Sample Preparation Ying.

Genotyping of Chimerical BCR-ABL1 RNA in Chronic Myeloid Leukemia by Integrated DNA Chip Jong-Hun Kang, Hyun-Gyung Goh, Sang-Ho Chae, Sung-Yong Kim,

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

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

A Novel Technology for Multiplex Gene Expression Analysis Directly from Whole Blood Samples Stabilized at Ambient Temperature Using an RNA-Stabilizing.

Suppression of Wild-Type Amplification by Selectivity Enhancing Agents in PCR Assays that Utilize SuperSelective Primers for the Detection of Rare Somatic.

Detection of HIV-1 Minority Variants Containing the K103N Drug-Resistance Mutation Using a Simple Method to Amplify RNA Targets (SMART) Kenneth Morabito,

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

Comparison of BIOMED-2 Versus Laboratory-Developed Polymerase Chain Reaction Assays for Detecting T-Cell Receptor-γ Gene Rearrangements Keyur P. Patel,

Β-Glucuronidase Is an Optimal Normalization Control Gene for Molecular Monitoring of Chronic Myelogenous Leukemia Joong Won Lee, Qiaofang Chen, Daniel.

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.

B-Cell Clonality Determination Using an Immunoglobulin κ Light Chain Polymerase Chain Reaction Method Reetesh K. Pai, Artemis E. Chakerian, John M. Binder,

Multi-Probe Real-Time PCR Identification of Common Mycobacterium Species in Blood Culture Broth Suporn Foongladda, Suporn Pholwat, Boonchuay Eampokalap,

Philippe Szankasi, Mohamed Jama, David W. Bahler

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

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

Homogeneous Polymerase Chain Reaction Nucleobase Quenching Assay to Detect the 1-kbp Deletion in CLN3 That Causes Batten Disease Paul G. Rothberg, Denia.

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

Novel Molecular Method for Detection of Bovine-Specific Central Nervous System Tissues as Bovine Spongiform Encephalopathy Risk Material in Meat and Meat.

A Pyrosequencing-Based Assay for the Rapid Detection of IDH1 Mutations in Clinical Samples Prashanth Setty, Jennifer Hammes, Thomas Rothämel, Valentina.

Quantitative mRNA Expression Analysis from Formalin-Fixed, Paraffin-Embedded Tissues Using 5′ Nuclease Quantitative Reverse Transcription-Polymerase Chain.

Characterization of the Different BCR-ABL Transcripts with a Single Multiplex RT-PCR Jacques Chasseriau, Jérôme Rivet, Frédéric Bilan, Jean-Claude Chomel,

DNA Diagnostics by Surface-Bound Melt-Curve Reactions

Single Monochrome Real-Time RT-PCR Assay for Identification, Quantification, and Breakpoint Cluster Region Determination of t(9;22) Transcripts Marina.

Single-Run, Parallel Detection of DNA from Three Pneumonia-Producing Bacteria by Real-Time Polymerase Chain Reaction Reinhard B. Raggam, Eva Leitner,

Molecular Typing of West Nile Virus, Dengue, and St

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

Getting Things Backwards to Prevent Primer Dimers

Specific Detection of Cytokeratin 20-Positive Cells in Blood of Colorectal and Breast Cancer Patients by a High Sensitivity Real-Time Reverse Transcriptase-Polymerase.

A Consensus on Fungal Polymerase Chain Reaction Diagnosis?

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

SeqSharp The Journal of Molecular Diagnostics

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

Development of a Novel One-Tube Isothermal Reverse Transcription Thermophilic Helicase-Dependent Amplification Platform for Rapid RNA Detection James.

Sensitive Detection of Deletions of One or More Exons in the Neurofibromatosis Type 2 (NF2) Gene by Multiplexed Gene Dosage Polymerase Chain Reaction

Performance Characteristics of a Quantitative, Homogeneous TaqMan RT-PCR Test for HCV RNA Joerg Kleiber, Thomas Walter, Gerd Haberhausen, Sue Tsang,

Benjamin P. Song, Surbhi Jain, Selena Y. Lin, Quan Chen, Timothy M

Olivier Gruselle, Thierry Coche, Jamila Louahed

Reliable Transcript Quantification by Real-Time Reverse Transcriptase-Polymerase Chain Reaction in Primary Neuroblastoma Using Normalization to Averaged.

Detection of FLT3 Internal Tandem Duplication and D835 Mutations by a Multiplex Polymerase Chain Reaction and Capillary Electrophoresis Assay Kathleen.

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

Novel Fluorescent Ligase Detection Reaction and Flow Cytometric Analysis of SYT-SSX Fusions in Synovial Sarcoma Robyn Gaffney, Artemis Chakerian, John.

Molecular Monitoring of Chronic Myelogenous Leukemia

Consensus JH Gene Probes with Conjugated 3′-Minor Groove Binder for Monitoring Minimal Residual Disease in Acute Lymphoblastic Leukemia Michihiro Uchiyama,

Jianbo Song, Danielle Mercer, Xiaofeng Hu, Henry Liu, Marilyn M. Li

High-Throughput Identification and Quantification of Candida Species Using High Resolution Derivative Melt Analysis of Panfungal Amplicons Tasneem Mandviwala,

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

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

T Cell Receptor γ-Chain Gene Polymerase Chain Reaction to Diagnose Central Nervous System Involvement by Cutaneous T Cell Lymphoma Robert Taylor, Jo-Anne.

Mangalathu S. Rajeevan, Suzanne D

Rapid Polymerase Chain Reaction-Based Detection of Epidermal Growth Factor Receptor Gene Mutations in Lung Adenocarcinomas Qiulu Pan, William Pao, Marc.

Detection and Quantification of BCR-ABL1 Fusion Transcripts by Droplet Digital PCR Lawrence J. Jennings, David George, Juliann Czech, Min Yu, Loren Joseph

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

A Consensus on Fungal Polymerase Chain Reaction Diagnosis?

HPV Genotype Detection Using Hybrid Capture Sample Preparation Combined with Whole Genome Amplification and Multiplex Detection with Luminex XMAP Brian.

Quantification of bcl-2/JH Fusion Sequences and a Control Gene by Multiplex Real- Time PCR Coupled with Automated Amplicon Sizing by Capillary Electrophoresis

Mitochondrial DNA as a Cancer Biomarker

Presentation transcript:

Multiplex Detection of Ehrlichia and Anaplasma Species Pathogens in Peripheral Blood by Real-Time Reverse Transcriptase-Polymerase Chain Reaction Kamesh R. Sirigireddy, Roman R. Ganta The Journal of Molecular Diagnostics Volume 7, Issue 2, Pages 308-316 (May 2005) DOI: 10.1016/S1525-1578(10)60559-4 Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Sequence alignment of the 16S rRNA genes and the design of RT-PCR primers, capture primer, and TaqMan probes. The 16S rRNA gene sequences available in the GenBank database for Ehrlichia and Anaplasma species were analyzed using the Genetic Computer Group programs, PILEUP and PRETTY.25 GenBank numbers for the sequences are M73222, E. chaffeensis; M73221, E. canis; M73227, E. ewingii; X61659, E. ruminantium; M82801, A. platys; M73220, A. phagocytophilum; and M60313, A. marginale. The complete sequence of E. chaffeensis from nucleotides 1 to 151 and 426 to 482 are presented. The sequences for the other species are shown only when they differ from the E. chaffeensis sequence. A dot indicates identity with the E. chaffeensis sequence. Dashes refer to the gaps introduced by the PILEUP program to obtain the best alignment. The PCR primers and the capture primer were identified with their names. Lines with arrowheads refer to the orientation of the primers. The genera- and species-specific TaqMan probes are identified with shaded text. The Journal of Molecular Diagnostics 2005 7, 308-316DOI: (10.1016/S1525-1578(10)60559-4) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Schematic representation of the molecular strategy used to generate E. ewingii-positive control. Alignment shown in Figure 1 reveals that E. ewingii 16S rRNA sequence differs from E. chaffeensis mostly at the central variable region where the TaqMan probes were designed. The schematic representation of the sequences in this figure shows variable sequences as different shaped lines. To generate an E. ewingii 16S rRNA gene segment, a long forward primer was designed. It contains the Ehrlichia species common sequence at the 5′ and 3′ ends and E. ewingii-specific sequence in the middle. This primer and a genera-specific reverse primer, together with the E. chaffeensis 16S rRNA gene segment as the template were used in the PCR to generate amplicons containing E. ewingii sequence. The amplicons were cloned into a plasmid. A similar strategy was also used to clone A. platys-positive control plasmid using the A. phagocytophilum DNA segment as the template. The Journal of Molecular Diagnostics 2005 7, 308-316DOI: (10.1016/S1525-1578(10)60559-4) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Schematic representation of the 16S rRNA capture method. A sample containing Ehrlichia/Anaplasma species organisms is lysed in the GTC buffer and added to the conjugate of the avidin-coated magnetic particles coupled with the biotin-linked capture primer. A magnetic stand is used to capture and purify 16S rRNA from the lysed sample. The Journal of Molecular Diagnostics 2005 7, 308-316DOI: (10.1016/S1525-1578(10)60559-4) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Multiplex molecular test's detection sensitivity and linearity with RNA concentration. Serial 10-fold dilutions of the RNA transcripts, made from the positive control plasmids, were analyzed at equivalent molar concentrations of the three Ehrlichia species (A) and two Anaplasma species (B). The fluorescent emission from serial dilution templates for one species each (E. canis and A. platys) is shown in the insets. The average Ct values from three independent experiments for each template concentration of the three Ehrlichia species and two Anaplasma species were plotted against the log number of RNA molecules in A and B, respectively. The Journal of Molecular Diagnostics 2005 7, 308-316DOI: (10.1016/S1525-1578(10)60559-4) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions