Nucleic Acid Extraction Control in Real-Time PCR Assays Steve Hawkins Senior Global Product Manager Bioline.

Slides:



Advertisements
Similar presentations
Dr. Thaweesak Tirawatnapong Chula Medical Research Center (Chula MRC)
Advertisements

Pediatric Diagnosis of HIV-1 Infection Using Dried Blood Spots Chin-Yih Ou, PhD NCHSTP/DHAP Centers for Disease Control and Prevention.
REAL TIME PCR ………A step forward in medicine
National Center for Environmental Health Centers for Disease Control and Prevention DBS DNA Extraction, Validation & Quantitation NBS Molecular Training.
Tools for Molecular Biology Amplification. The PCR reaction is a way to quickly drive the exponential amplification of a small piece of DNA. PCR is a.
Assay I HLA-DQ Alpha Haplotype. Purpose n To determine which one of several known alleles is present at the HLA DQ-Alpha locus on each of an individual’s.
Assays Molecular Diagnostics CSULA. What’s a molecular diagnostic assay? n A laboratory test for the presence or absence of a particular type of molecule.
Real-Time PCR mRNA quantification. What do mRNA levels tell us? DNA  mRNA  protein Reflect level of gene expression Information about cell response.
Applied Biosystems 7900HT Fast Real-Time PCR System I. Real-time RT-PCR analysis of siRNA-induced knockdown in mammalian cells (Amit Berson, Mor Hanan.
Basic Procedures for DNA analysis I) DNA isolation & purification: –Sample: nucleated cells –Principle: A- PURIFICATION STEPS: 1.Cell lysis 2.Removal of.
What Can You Do With qPCR?
Genomic DNA purification
Plant virus infections induce tremendous economic losses for agricultural production each year in the U.S. Accurate and reliable diagnosis of the pathogens.
COBAS TaqMan 48 Sales Brochure
Real Time PCR = Quantitative PCR.
Real-Time PCR (Quantitative PCR)
Quantitative PCR Session 2: Overview of qPCR
Variants of PCR Lecture 4
Abstract Automated, High-Throughput Total and Viral RNA Purification Byung-in Lee 1, Seachol Oak 1, Sharon Khietala 2, Beate Schikora 2, and Karl H Hecker.
COBAS AmpliPrep/Cobas TaqMan HIV-1 Test
REAL-TIME RT-PCR BASED ASSAY ON BLOOD CLOT SPECIMENS FOR DIAGNOSIS OF HIV-1 INFECTION IN CHILDREN, MALAWI Hua Yang 1, Rita Helfand 2, Desiree Witte 3,
Real-Time Quantitative RT-PCR
The LightScanner ® System Achieve High Throughput Mutation Discovery and Genotyping.
DNA-based Methods for Quantifying Microbes in Atmospheric Samples Tom Hill, Helen Ahern and Bruce Moffett University of East London.
Quantitative Real-Time PCR Adrien Six Sophie Dulauroy Institut Pasteur & Université Pierre et Marie.
DNA Analysis Facility User Educational Series December 11, 2009.
Quantitative Real Time PCR USING SYBR GREEN. SYBR Green SYBR Green is a cyanine dye that binds to double stranded DNA. When it is bound to D.S. DNA it.
Real time RT-PCR Quantitating Gene Expression.
Results Alien Reference RNA QRT-PCR Detection Kit for Monitoring the Overall Performance of QRT-PCR Assays Bahram Arezi, Melissa McCarthy & Holly Hogrefe.
PCR reaction to be visualized “in real time” as the reaction progresses to quantify the amount of DNA in the sample at the start of the reaction REAL TIME.
Fecal Source Tracking Using Human and Animal DNA U.S. Department of the Interior U.S. Geological Survey Bane Schill- USGS Leetown Science Center.
Overview of the presentation 1.The LiMA technology 2.Sensitivity for ligase detection 3.Sensitivity for bacterial detection.
Chapter 13: DNA Quantitation.  Quantitation determines the amount of human DNA present in an extract  A narrow concentration range is required to “seed”
Journal Club Capture and Ligation Probe-PCR (CLIP-PCR) for Molecular Screening, with Application to Active Malaria Surveillance for Elimination Z. Cheng,
Impedance Sensor Arrays for Real Time and Label Free Bio-Affinity Assay Vena Haynes Mariya Smit & Andrei Ghindilis Holly M. Simon Laboratories of America.
Strand Displacement Amplification Presented by Lisa Smith & Apollo Kacsinta.
Real-Time Quantitative PCR Basis
DNA contamination can often occur in quantitative reverse transcription – polymerase chain reactions (QRT-PCR), and should be removed in order to avoid.
Julia Robbins August 11, Objectives Clinical Significance of MRSA in Healthcare Setting Principle of assay Assay Procedure Assay Perfomance.
All Rights Reserved AEIC Response Concentration 5% CV 20% CV Error bars for concentrations determined by the.
HCV PCR By Henrietta Orji July 31 st, 2010 Hepatitis C Virus by Polymerase Chain Reaction.
Invitrogen Corporation 1600 Faraday Ave. Carlsbad, CA USA Tel: FAX: Toll Free Tel:
Controls for Blood Septicemia Nucleic Acid Tests Mark Manak BBI Diagnostics, Inc. A Division of SeraCare Life Sciences, Inc. SoGAT XIX Meeting Berne, Switzerland.
All Rights Reserved AEIC 2004 Flanking Primer Transgene.
Molecular Testing and Clinical Diagnosis
PCR With PCR it is possible to amplify a single piece of DNA, or a very small number of pieces of DNA, over many cycles, generating millions of copies.
R EAL TIME P CR 1. L IMITATIONS OF E ND -P OINT PCR Poor Precision Low sensitivity Low resolution Non - Automated Size-based discrimination only Results.
The Factor II (Prothrombin) G20210A Detection and Genotyping
Kevin Chen.  A method of amplifying or copying DNA fragments.
Viro-chip Microarray Using a Pan-Viral Microarray Assay (Viro-chip) to Screen Clinical Samples for Viral Pathogens.
RT-PCR analysis 생화학 실험 2 조교 : 이 선 민 내선 7699, 첨단과학기술관 201-B 호 신과학원 S438 호.
Date of download: 6/24/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Interleukin 6 and Interleukin 8 as Potential Biomarkers.
Aim: To develop a new one-step RT-PCR assay to detect H1N1 by designing new primer to target NP gene Experimental approach: -nasopharyngeal swabs from.
Ligation In-situ Hybrdization Christopher Itoh 1, Joel Credle 1, Rajni Sharma 2, H. Benjamin Larman 1 1 Department of Immunopathology, Johns Hopkins University.
Immunological and DNA-methods
Result Introduction Methods
Gel electrophoresis analysis Automated DNA analyzer.
Diagnostic applications of the polymerase chain reaction (PCR). A
Tom Øystein Jonassen, Mona Holberg-Petersen, Einar S. Berg
Direct DNA Amplification from Crude Clinical Samples Using a PCR Enhancer Cocktail and Novel Mutants of Taq  Zhian Zhang, Milko B. Kermekchiev, Wayne.
Plasma Components Affect Accuracy of Circulating Cancer-Related MicroRNA Quantitation  Dong-Ja Kim, Sarah Linnstaedt, Jaime Palma, Joon Cheol Park, Evangelos.
Reverse Complement PCR: fast, low cost amplicon based NGS
Detection of bacteria in blood products
Direct DNA Amplification from Crude Clinical Samples Using a PCR Enhancer Cocktail and Novel Mutants of Taq  Zhian Zhang, Milko B. Kermekchiev, Wayne.
Plasma Components Affect Accuracy of Circulating Cancer-Related MicroRNA Quantitation  Dong-Ja Kim, Sarah Linnstaedt, Jaime Palma, Joon Cheol Park, Evangelos.
a b c Fold Change DeltaCt Cell Line ADAR1 ADAR2 Ratio H ,776
DNA Extraction from Blood
RealTime-PCR.
xTAG liquid-phase microarray.
Recovery template. Recovery template. The recovery template (internal control) has the same sequence as the PCR product except the probe region has been.
Presentation transcript:

Nucleic Acid Extraction Control in Real-Time PCR Assays Steve Hawkins Senior Global Product Manager Bioline

Real-Time PCR in Diagnostics Advantages Speed (hours vs. days) Sensitivity Specificity Throughput Multiplexing Challenges False Positive Results –Contamination False Negative Results – PCR Inhibition – Nucleic Acid Extraction Failure

DNA Extraction Control (DEC) Features Utilises bacteria as a vehicle for DNA extraction process monitoring Contains internal control sequence that has minimal homology to any known disease causing gene Suitable for common clinical samples (e.g. blood, sputum, swab) Monitors: DNA extraction efficiency PCR efficiency

Process of incorporating DEC into real-time PCR Assay Resuspend cells “Spike” into target sample “Spike” DEC Extract Target and Control DNA Extraction Add Target & Control primers and probes to real-time PCR Amplification Acquire Control sequence on cy-5 channel Detection and Analysis

Inefficient DNA extraction was simulated by substitution of either the lysis buffer or binding buffer with PBS. Inefficient DNA extraction Complete lysis step (red), no lysis (green) and no binding buffer (orange) showed significant “loss of sample” This demonstrate that the DEC can be used to monitor DNA extraction. Reference gene (green channel) / Internal control (red channel) SensiFAST SYBR

DEC vs. pure DNA DEC and DNA were spiked into cell resuspension. Extraction was carried out with or without lysis buffer in parallel for simulation of inefficient extraction. Sample with (red) or without a lysis step (violet), a difference, but there was no change of Ct in the naked DNA, with (green) or without a lysis step (blue) Spike pure DNA into sample as internal control may not function as extraction control due to the lack of lysis required. Gene of interest (green channel) / Internal control (red channel) SensiFAST SYBR

EDTA was added into elution buffer, as an inhibitory agent to show the effects on the internal control. PCR reaction inhibition DEC shows consistent inhibition level as with the sample target. Inhibition of PCR reaction can be identified using DEC. Gene of InterestInternal DEC control SensiFAST SYBR

A triplex reaction was compared with singleplex reactions to look for interference by the DEC. Minimal interference and consistency The results illustrate the consistency of the DEC SensiFAST SYBR

RNA Extraction Control (REC) Features Utilises an artificial cell as a vehicle to deliver stable ss-RNA Contains internal control sequence that has minimal homology to any known disease causing gene Suitable for common clinical samples (e.g. blood, sputum) Monitors: RNA extraction efficiency Reverse-transcription inhibition PCR inhibition

REC, spiked DNA and extraction control Spiked DNA control and REC were co-extracted out +/- lysis buffer or +/- EtOH (critical step in RNA Kit prep.). No DNase digestion was performed Spiked DNA REC Spiked DNA control will always produce a positive signal, so therefore no value as an extraction control Only REC demonstrates the effect of extraction quality on the Ct and is an indicator of RT inhibition SensiFAST SYBR One-Step

REC monitors PCR inhibition Different concentrations of EDTA were added prior to elution, as an inhibitory agent to test the monitoring capability of the internal control. The presence of PCR inhibitors result in a shift in Ct SensiFAST SYBR One-Step

REC provides reproducible results Three REC samples were extracted in parallel. The multiple extractions were performed and analyzed by real-time The results illustrate the reproducibility over several fold dilutions of template and over a number of extractions SensiFAST SYBR One-Step

Summary Nucleic acid extraction process monitoring is important in reducing false negative results Pure DNA controls not effective in monitoring nucleic acid extraction process DEC and REC effective in monitoring nucleic acid extraction and PCR inhibition

Monitors extraction efficiency Monitors PCR inhibition Consistent signal in a validated assay Easy incorporation steps Minimize exogenous contaminant introduction Summary

Acknowledgements Dr. Lopeti Lavulo Tom Lin Lisa Yang Dr. Sally Dubedat (Royal Prince Alfred Hospital, Sydney)