Loop-mediated Isothermal Amplification (LAMP) as a diagnostic tool in detection of infectious diseases

Size: px
Start display at page:

Download "Loop-mediated Isothermal Amplification (LAMP) as a diagnostic tool in detection of infectious diseases"

Transcription

1 Loop-mediated Isothermal Amplification (LAMP) as a diagnostic tool in detection of infectious diseases Dorota DRAPAŁA, Milena KORDALEWSKA Keywords: LAMP; DNA amplification; isothermal reaction Abstract: Loop-mediated isothermal amplification (LAMP) is a gene amplification method which amplifies DNA with high specificity and efficiency under isothermal conditions. Because of its rapidity and simplicity, it is a valuable diagnostic tool in the early detection and identification of infectious diseases. LAMP method is based on the use of a set of four to six specially designed primers spanning six to eight distinct sequences on the target DNA. The amplification is performed by DNA polymerase, which has strand-displacing activity. The whole reaction occurs in a single tube and is divided into two steps: non-cyclic and cyclic. The final products are artificial stem-loop DNA structures flanking the target sequences. 1. Introduction At present, techniques based on the detection of nucleic acids are used more and more often in the routine diagnostics of infectious diseases. PCR methods such as nested PCR, multiplex PCR, reverse transcription PCR or real-time PCR are commonly applied due to their sensitivity, specificity and rapidity (Van Belkum and Niesters, 1995). However, in spite of their many advantages, all the above-mentioned techniques have several significant limitations such as expensive amplification instruments, complicated methods of detection or complex analysis of the results. Loop-mediated isothermal amplification (LAMP) appeared to be an innovative technique which may be an alternative method for the diagnosis of some infectious diseases. It is sensitive, specific, fast (without time-consuming analysis) and, what is really significant, does not require expensive instruments and complicated methods of detection. 2. LAMP description LAMP was developed in 1998 by Eiken Chemical Company (Japan). It is a quite complex method which requires four to six different primers that are designed specifically Microbiology Department, Faculty of Chemistry, Gdańsk University of Technology, Narutowicza St. 11/12, Gdańsk

2 20 PhD Interdisciplinary Journal to recognize six to eight specific gene sequences. DNA amplification is accomplished with the use of DNA polymerase with strand-displacing activity (Notomi et al., 2000; Ushikubo, 2004). Thanks to this, there is no need for a thermal denaturation step in order to obtain single-stranded DNA. Thus, strand-displacing activity allows amplification under isothermal conditions in contrast to PCR where a thermal denaturation step is essential. LAMP technique is based on a generation of artificial stem-loop DNA structures flanking the target sequences. Cyclic strand displacement is performed at constant temperature, ca 65 C, at which double-stranded DNA remains in the dynamic equilibrium. This allows primers to anneal to the complementary sequence of DNA, so DNA polymerase (with strand-displacing activity) can start DNA synthesis. The whole LAMP procedure consists of two steps: non-cyclic and cyclic (Fig.1). The first step leads to the generation of artificial stem-loops used in the further steps (Fig. 1a). Firstly, the forward inner primer (FIP Primer) binds to the target sequence and initiates the polymerization. After that, the forward outer primer (F3 Primer) binds to the product and displaces it with a single artificial stem-loop appending to the target sequence. The single strand DNA serves as a temple for BIPinitiated (BIP Primer) DNA synthesis. Then, the reverse outer primer (B3 Primer) binds to the product and displaces the product with two artificial stem loops flanking the target reagent. This is the starting structure for the next, cyclic amplification step. The second cyclic step starts with the FIP primer hybridization to the loop on the product (Fig. 1b). Displacement DNA synthesis is initiated and leads to the generation of DNA with a new stem-loop structure at one end and an additional target sequence. These structures are used as templates in further DNA synthesis with the use of internal primers. Various-sized structures possessing the alternately inverted repeats of the target sequence on the same strand are formed. 3. LAMP advantages and disadvantages LAMP method, due to its sensitivity, specificity, high efficiency, rapidity and simplicity is a good molecular technique for identifying some infectious diseases. The main advantage of LAMP is the fact that amplification is performed under isothermal conditions. Therefore, it is possible to obtain high amplification efficiency, which is attributed to reduced time loss of thermal change. Moreover, there is no need for expensive and complicated equipment such as a thermal cycler. The process may be performed simply in a heating block or water bath. High specificity is obtained because of the use of 4-6 primers spanning 6-8 distinct sequences and all target genes must be present in order to initiate amplification. Additionally, the method is rapid and simple; amplification and detection may be carried out in one single tube. Gene amplification products can be detected not only by agarose gel electrophoresis and real-time monitoring in an inexpensive turbidimeter but also visually by the naked eye, either as turbidity or in the form of a colour change. In spite of its many advantages, LAMP also has some limitations. The primer designing is quite complicated, because it is essential to design 4-6 specific primers. Moreover, it must be remembered that LAMP is inadequate for the detection of unknown or unsequenced targets.

3 Special Issue: Biotech Conference 21 Tab. 1. LAMP applications in diagnostics Pathogens Disease Sequence for specific primers Method of detection References coronavirus SARS 1bRep gene spectrophotometric analysis by recording the optical density with a real-time turbidimeter (Hong et al., 2004) Mycobacterium tuberculosis,m. avium, M. intracellulare mycobacteriosis gyrb gene visualization by addition of SYBR Green I to the reaction tube (Iwamoto et al., 2003) Plasmodium falciparum malaria 18S ribosomal RNA gene analysis by turbidimeter in real-time or visually at the end of the assay (Poon, 2006) Staphylococcus aureus sepsis 16S ribosomal RNA gene analysis by agarose gel electrophoresis and visually by distinguished precipitation or turbidity (Zhang, 2013) Human immunodeficiency virus-1 HIV highly conserved sequences located within the protease and p24 gene regions identification by agarose gel electrophoresis and visually following the addition of the fluorescent nucleic acid stain (Curtis et al., 2008, 2009, 2012) Streptococcus pneumoniae pneumonia lyta gene analysis by agarose gel electrophoresis and visually by distinguished precipitation or turbidity (Seki et al., 2005)

4 22 PhD Interdisciplinary Journal Fig. 1. Non-cyclic (A) and cyclic (B) steps of LAMP Parida et al. (2008).

5 Special Issue: Biotech Conference 23 References Curtis, S. M., K. A. Rudolph and D. L. Owen (2008), Rapid detection of hiv-1 by reverse-transcription, loop-mediated isothermal amplification (rt-lamp), J Virol Methods 2, Curtis, S. M., K. A. Rudolph and D. L. Owen (2009), Sequence-specific detection method for reverse transcription, loop-mediated isothermal amplification of hiv-1, J Med Virol 81, Curtis, S. M., K. A. Rudoplh, I. Nejad, J. Sinleton, A. Beddoe, B. Weigl, P. La Barre and S. M. Owen (2012), Isothermal amplification using a chemical heating device for point-of-care detection of hiv-1, PLoS One 7, e Hong, T. C., Q. L. Mai, D. V. Cuong, M. Parida, H. Minekawa and T. Notomi (2004), Development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus, J Clin Microbiol 42, Iwamoto, T., T. Sonobe and K. Hayashi (2003), Loop-mediated isothermal amplification for direct detection of mycobacterium tuberculosis complex, m. avium, and m. intracellulare in sputum samples, J Clin Microbiol 41, Notomi, T., H. Okayama, H. Masubuchi, T. Yonekawa, K. Watanabe and N. Amino (2000), Loop-mediated isothermal amplification of dna, Nucleic Acids Res 28, e63. Parida, M., S. Sannarangaiah, P. K. Dash, P. V. Rao and K. Morita (2008), Loop mediated isothermal amplification (lamp): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases..18, Red Med Virol 18, Poon, L. L. (2006), Sensitive and inexpensive molecular test for falciparum malaria: detecting plasmodium falciparum dna directly from heat-treated blood by loopmediated isothermal amplification, Clin Chem 52, Seki, M., Y. Yamashita, H. Torigoe, H. Tsuda, S. Sato and M. Maeno (2005), Loopmediated isothermal amplification method targeting the lyta gene for detection of streptococcus pneumoniae, J Clin Microbiol 43, Ushikubo, H. (2004), Principle of lamp method a simple and rapid gene amplification method, Uirusu 54, Van Belkum, A. and H. G. Niesters (1995), Nucleic acid amplification and related techniques in microbiological diagnostics and epidemiology, Cell Mol Biol 41, Zhang, Y. C. (2013), Pathogen diagnosis of children sepsis by lamp technology., Asian Pac J 6,

Methods in virus diagnosis PCR techniques

Methods in virus diagnosis PCR techniques Methods in virus diagnosis PCR techniques 450 MBIO PRACTICAL LESSON 5 Molecular Methods Methods based on the detection of viral genome are also commonly known as molecular methods. It is often said that

More information

Current molecular diagnostic system

Current molecular diagnostic system LAMP-BART Name: Chris Wong Supervisor: Prof. Margaret Ip Joint Graduate Seminar Department of Microbiology The Chinese University of Hong Kong 18 th December 2012 Current molecular diagnostic system Detection

More information

INTRODUCTION TO REVERSE TRANSCRIPTION PCR (RT-PCR) ABCF 2016 BecA-ILRI Hub, Nairobi 21 st September 2016 Roger Pelle Principal Scientist

INTRODUCTION TO REVERSE TRANSCRIPTION PCR (RT-PCR) ABCF 2016 BecA-ILRI Hub, Nairobi 21 st September 2016 Roger Pelle Principal Scientist INTRODUCTION TO REVERSE TRANSCRIPTION PCR (RT-PCR) ABCF 2016 BecA-ILRI Hub, Nairobi 21 st September 2016 Roger Pelle Principal Scientist Objective of PCR To provide a solution to one of the most pressing

More information

The Polymerase Chain Reaction. Chapter 6: Background

The Polymerase Chain Reaction. Chapter 6: Background The Polymerase Chain Reaction Chapter 6: Background PCR Amplify= Polymerase Chain Reaction (PCR) Invented in 1984 Applications Invention of PCR Kary Mullis Mile marker 46.58 in April of 1983 Pulled off

More information

LAMP FOR FOOD Prof. Dr. Panagiotis Karanis Medical and Molecular Parasitology Medical School, University of Cologne

LAMP FOR FOOD Prof. Dr. Panagiotis Karanis Medical and Molecular Parasitology Medical School, University of Cologne LAMP FOR FOOD Prof. Dr. Panagiotis Karanis Medical and Molecular Parasitology Medical School, University of Cologne LAMP for Food Prof. Dr. Panagiotis Karanis 1 Loop mediated isothermal amplification (LAMP)

More information

Polymerase Chain Reaction

Polymerase Chain Reaction Polymerase Chain Reaction Variations of PCR in the Diagnostic Lab The most common variations of standard PCR used in the diagnostic laboratory are: Reverse Transcriptase PCR (RT-PCR) Nested PCR (n-pcr)

More information

Rapid and real-time detection technologies for emerging viruses of biomedical importance

Rapid and real-time detection technologies for emerging viruses of biomedical importance Rapid and real-time detection technologies for emerging viruses of biomedical importance 617 Rapid and real-time detection technologies for emerging viruses of biomedical importance M M PARIDA Department

More information

Technical Review. Real time PCR

Technical Review. Real time PCR Technical Review Real time PCR Normal PCR: Analyze with agarose gel Normal PCR vs Real time PCR Real-time PCR, also known as quantitative PCR (qpcr) or kinetic PCR Key feature: Used to amplify and simultaneously

More information

American Society of Cytopathology Core Curriculum in Molecular Biology

American Society of Cytopathology Core Curriculum in Molecular Biology American Society of Cytopathology Core Curriculum in Molecular Biology American Society of Cytopathology Core Curriculum in Molecular Biology Chapter 3 Molecular Techniques Alternatives to PCR, Part I

More information

Polymerase chain reaction: advantages and drawbacks

Polymerase chain reaction: advantages and drawbacks Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2015 Polymerase chain reaction: advantages and drawbacks Favrot, C Posted at

More information

Isothermal DNA Amplification

Isothermal DNA Amplification Isothermal DNA Amplification Update 2015/16 ROBUST TECHNOLOGIES FOR RAPID NUCLEIC ACID DETECTION What is isothermal DNA amplification? The Polymerase Chain Reaction (PCR) is a well-known approach for amplifying

More information

BIOLOGY Dr.Locke Lecture# 27 An Introduction to Polymerase Chain Reaction (PCR)

BIOLOGY Dr.Locke Lecture# 27 An Introduction to Polymerase Chain Reaction (PCR) BIOLOGY 207 - Dr.Locke Lecture# 27 An Introduction to Polymerase Chain Reaction (PCR) Required readings and problems: Reading: Open Genetics, Chapter 8.1 Problems: Chapter 8 Optional Griffiths (2008) 9

More information

Polymerase Chain Reaction PCR

Polymerase Chain Reaction PCR Polymerase Chain Reaction PCR What is PCR? An in vitro process that detects, identifies, and copies (amplifies) a specific piece of DNA in a biological sample. Discovered by Dr. Kary Mullis in 1983. A

More information

DNA Methods in Clinical Microbiology

DNA Methods in Clinical Microbiology DNA Methods in Clinical Microbiology DNA Methods Ill Clinical Microbiology by Paul Singleton SPRINGER-SCIENCE+BUSINESS MEDIA, B.V. A C.I.P. Catalogue record for this book is available from the Library

More information

HELINI Hepatitis B virus [HBV] Real-time PCR Kit (Genotype A to H)

HELINI Hepatitis B virus [HBV] Real-time PCR Kit (Genotype A to H) HELINI Hepatitis B virus [HBV] Real-time PCR Kit (Genotype A to H) Quantitative In vitro diagnostics Instruction manual Cat. No: 8001-25/50/100 tests Compatible with: Agilent, Bio-Rad, Applied Bio systems

More information

Reverse transcription-pcr (rt-pcr) Dr. Hani Alhadrami

Reverse transcription-pcr (rt-pcr) Dr. Hani Alhadrami Reverse transcription-pcr (rt-pcr) Dr. Hani Alhadrami hanialhadrami@kau.edu.sa www.hanialhadrami.kau.edu.sa Overview Several techniques are available to detect and analyse RNA. Examples of these techniques

More information

UltraFast Molecular Diagnostic System

UltraFast Molecular Diagnostic System UltraFast Molecular Diagnostic System CONTENTS 01 PCR vs Real-time PCR 02 NANOBIOSYS Sample Prep G2-16TU 03 NANOBIOSYS Real-time PCR G2-4 01 PCR vs Real-time PCR What is DNA & What is PCR? NANOBIOSYS 4

More information

Loop-Mediated Isothermal Amplification (LAMP) Primer Design and Assay Optimization

Loop-Mediated Isothermal Amplification (LAMP) Primer Design and Assay Optimization Loop-Mediated Isothermal Amplification (LAMP) Primer Design and Assay Optimization Tony Rockweiler, Diagnostic Research Scientist, Lucigen March, 2018 www.lucigen.com Agenda LAMP Overview Review the mechanics

More information

WarmStart LAMP Kit (DNA & RNA)

WarmStart LAMP Kit (DNA & RNA) POLYMERASES & AMPLIFICATION WarmStart LAMP Kit (DNA & RNA) Instruction Manual NEB #E1700S/L 100/500 reactions Version 1.0 9/16 This product is intended for research purposes only. This product is not intended

More information

Chapter 17. PCR the polymerase chain reaction and its many uses. Prepared by Woojoo Choi

Chapter 17. PCR the polymerase chain reaction and its many uses. Prepared by Woojoo Choi Chapter 17. PCR the polymerase chain reaction and its many uses Prepared by Woojoo Choi Polymerase chain reaction 1) Polymerase chain reaction (PCR): artificial amplification of a DNA sequence by repeated

More information

2 march 06 Seminar on RT-PCR. About Real-time PCR. Aurélie OLIVIER Université Catholique de Louvain Unité de pharmacologie cellulaire et moléculaire

2 march 06 Seminar on RT-PCR. About Real-time PCR. Aurélie OLIVIER Université Catholique de Louvain Unité de pharmacologie cellulaire et moléculaire 2 march 06 Seminar on RT-PCR About Real-time PCR Aurélie OLIVIER Université Catholique de Louvain Unité de pharmacologie cellulaire et moléculaire Target DNA PCR Applications: Gene Plasmide, phage Diagnostic

More information

Optimizing a Conventional Polymerase Chain Reaction (PCR) and Primer Design

Optimizing a Conventional Polymerase Chain Reaction (PCR) and Primer Design Optimizing a Conventional Polymerase Chain Reaction (PCR) and Primer Design The Polymerase Chain Reaction (PCR) is a powerful technique used for the amplification of a specific segment of a nucleic acid

More information

SuperScript IV Reverse Transcriptase as a better alternative to AMV-based enzymes

SuperScript IV Reverse Transcriptase as a better alternative to AMV-based enzymes WHITE PAPER SuperScript IV Reverse Transcriptase SuperScript IV Reverse Transcriptase as a better alternative to AMV-based enzymes Abstract Reverse transcriptases (RTs) from avian myeloblastosis virus

More information

Mobile nucleic acid amplification and detection systems. Animal Production and Health Sub-programme

Mobile nucleic acid amplification and detection systems. Animal Production and Health Sub-programme Mobile nucleic acid amplification and detection systems Animal Production and Health Sub-programme Hermann Unger OIE, Berlin 2013 Our mandate To improve livestock production through the support of problem-orientated

More information

LAMP User Guide Assay Design & Primers

LAMP User Guide Assay Design & Primers Contents Page Page Content 2 Assay Design overview 3 Assay Design Custom Design Service 4 Assay Design - Software 5 Assay Design Target Sequence 7 Primers Primer Concentrations 9 Primers Preparing Primer

More information

Principals of Real-Time PCR. Amira A. T. AL-Hosary Lecturer of Infectious Diseases, Faculty of Veterinary Medicine, Assiut University, Egypt

Principals of Real-Time PCR. Amira A. T. AL-Hosary Lecturer of Infectious Diseases, Faculty of Veterinary Medicine, Assiut University, Egypt Principals of Real-Time PCR Amira A. T. AL-Hosary Lecturer of Infectious Diseases, Faculty of Veterinary Medicine, Assiut University, Egypt What Is Real-Time PCR? Nucleic acid (DNA) amplification and detection

More information

APPLICATION OF MOLECULAR TECHNICS FOR DIAGNOSIS OF VIRAL INFECTIONS

APPLICATION OF MOLECULAR TECHNICS FOR DIAGNOSIS OF VIRAL INFECTIONS APPLICATION OF MOLECULAR TECHNICS FOR DIAGNOSIS OF VIRAL INFECTIONS Hossein Keyvani Basic Diagnostic Methods in Virology Immunology and serology techniques (Antigen-Antibody Reactions) 1 ELISA ( Enzyme

More information

Index 273 Index A Acrylamide gel electrophoresis Trichomonas vaginalis, 231, 234 Agarose gel electrophoresis amplification detection, 3 HCV detection,

Index 273 Index A Acrylamide gel electrophoresis Trichomonas vaginalis, 231, 234 Agarose gel electrophoresis amplification detection, 3 HCV detection, Index 273 Index A Acrylamide gel electrophoresis Trichomonas vaginalis, 231, 234 Agarose gel electrophoresis amplification detection, 3 HCV detection, 167, 169, 170 Legionella species, 175, 177, 179, 180

More information

POLYMERASE CHAIN REACTION (PCR) TECHNOLOGIES AND GLOBAL MARKETS

POLYMERASE CHAIN REACTION (PCR) TECHNOLOGIES AND GLOBAL MARKETS POLYMERASE CHAIN REACTION (PCR) TECHNOLOGIES AND GLOBAL MARKETS BIO087B March 2014 Jackson Highsmith Project Analyst ISBN: 1-56965-742-4 BCC Research 49 Walnut Park, Building 2 Wellesley, MA 02481 USA

More information

Texas A&M University-Corpus Christi CHEM4402 Biochemistry II Laboratory Laboratory 4 - Polymerase Chain Reaction (PCR)

Texas A&M University-Corpus Christi CHEM4402 Biochemistry II Laboratory Laboratory 4 - Polymerase Chain Reaction (PCR) Texas A&M University-Corpus Christi CHEM4402 Biochemistry II Laboratory Laboratory 4 - Polymerase Chain Reaction (PCR) Progressing with the sequence of experiments, we are now ready to amplify the green

More information

Introduction to Real-Time PCR: Basic Principles and Chemistries

Introduction to Real-Time PCR: Basic Principles and Chemistries Introduction to Real-Time PCR: Basic Principles and Chemistries Leta Steffen, PhD Applications Scientist Promega Corporation Outline I. Real-Time PCR overview Basics of Real-Time PCR Understanding the

More information

DiAGSure Mycobacterium tuberculosis (MTB) Detection Kitit

DiAGSure Mycobacterium tuberculosis (MTB) Detection Kitit DiAGSure Mycobacterium tuberculosis (MTB) Detection Kitit Description: Tuberculosis (TB) is caused by the acid-fast bacterium Mycobacterium tuberculosis. Although Mycobacterium tuberculosis most commonly

More information

AMPLIFICATION BY PCR. Target. 1. Denature. 2. Anneal primers. 3. Extend primers. Two copies of target. 1. Denature

AMPLIFICATION BY PCR. Target. 1. Denature. 2. Anneal primers. 3. Extend primers. Two copies of target. 1. Denature AMPLIFICATION BY PCR Target 5 3 2. Anneal primers 3 5 1. Denature Two copies of target 3. Extend primers 1. Denature 2. Anneal primers 3. Extend primers Four copies of target PCR: First 4 Cycles PCR: Completed

More information

Genetics and Genomics in Medicine Chapter 3. Questions & Answers

Genetics and Genomics in Medicine Chapter 3. Questions & Answers Genetics and Genomics in Medicine Chapter 3 Multiple Choice Questions Questions & Answers Question 3.1 Which of the following statements, if any, is false? a) Amplifying DNA means making many identical

More information

Applied Biosystems Real-Time PCR Rapid Assay Development Guidelines

Applied Biosystems Real-Time PCR Rapid Assay Development Guidelines Applied Biosystems Real-Time PCR Rapid Assay Development Guidelines Description This tutorial will discuss recommended guidelines for designing and running real-time PCR quantification and SNP Genotyping

More information

Sensitivity vs Specificity

Sensitivity vs Specificity Viral Detection Animal Inoculation Culturing the Virus Definitive Length of time Serology Detecting antibodies to the infectious agent Detecting Viral Proteins Western Blot ELISA Detecting the Viral Genome

More information

Polymerase Chain Reaction PCR

Polymerase Chain Reaction PCR 1 Description of Module Subject Name Paper Name Module Name/Title Dr. Vijaya Khader Dr. MC Varadaraj 2 1. Objectives 1. To understand principle of 2. Types 3. Applications 2. Lay Out 3 Types of Qualitative

More information

Supporting Information. Tandem Blocking of PCR Extension to Form Single-stranded Overhang for Facile, Visual, and Ultrasensitive Gene Detection

Supporting Information. Tandem Blocking of PCR Extension to Form Single-stranded Overhang for Facile, Visual, and Ultrasensitive Gene Detection Electronic Supplementary Material (ESI) for RSC Advances. This journal is The Royal Society of Chemistry 2018 Supporting Information Tandem Blocking of PCR Extension to Form Single-stranded Overhang for

More information

DETERMINATION OF THE Rh FACTOR BY PCR

DETERMINATION OF THE Rh FACTOR BY PCR DETERMINATION OF THE Rh FACTOR BY PCR Ref.: PCR2 1. EXPERIMENT OBJECTIVE The aim of this experiment is to introduce students to the principles and practice of the Polymerase Chain Reaction (PCR) by studying

More information

Real-Time PCR Principles and Applications

Real-Time PCR Principles and Applications Real-Time PCR Principles and Applications Dr Esam Ibraheem Azhar (BSc, MSc, Ph.D Molecular Medical Virology) Asst. Prof. Medical Laboratory Technology Department Objectives Real-Time PCR Principles and

More information

Contents... vii. List of Figures... xii. List of Tables... xiv. Abbreviatons... xv. Summary... xvii. 1. Introduction In vitro evolution...

Contents... vii. List of Figures... xii. List of Tables... xiv. Abbreviatons... xv. Summary... xvii. 1. Introduction In vitro evolution... vii Contents Contents... vii List of Figures... xii List of Tables... xiv Abbreviatons... xv Summary... xvii 1. Introduction...1 1.1 In vitro evolution... 1 1.2 Phage Display Technology... 3 1.3 Cell surface

More information

SunScript One Step RT-PCR Kit

SunScript One Step RT-PCR Kit SunScript ONE STEP R T-PCR KIT HANDBOOK SunScript One Step RT-PCR Kit INDEX Legal... 4 Intended use... 4 Kit contents... 5 Shipping and storage... 5 Handling... 6 Quality control... 6 Reagents and equipment...

More information

Quantitative Real time PCR. Only for teaching purposes - not for reproduction or sale

Quantitative Real time PCR. Only for teaching purposes - not for reproduction or sale Quantitative Real time PCR PCR reaction conventional versus real time PCR real time PCR principles threshold cycle C T efficiency relative quantification reference genes primers detection chemistry GLP

More information

Real-Time PCR: An Essential Guide

Real-Time PCR: An Essential Guide Real-Time PCR: An Essential Guide Publisher: Horizon Bioscience Editor: Kirstin Edwards, Julie Logan and Nick Saunders Genomics Proteomics and Bioinformatics Unit, Health Protection Agency, London Publication

More information

Kayhan Azadmanesh MD, Ph.D. Virology Department

Kayhan Azadmanesh MD, Ph.D. Virology Department Principles of molecular tests Kayhan Azadmanesh MD, Ph.D. Virology Department Pasteur Institute t of Iran 1 DNA molecule (1954) 2 Southern Blot (1975) 3 We needed to know the sequence of the genes and

More information

Taura Syndrome Virus (TSV) RT-PCR Kit

Taura Syndrome Virus (TSV) RT-PCR Kit Revision No.: ZJ0001 Issue Date: Aug 28th, 2007 Taura Syndrome Virus (TSV) RT-PCR Kit Cat. No.: AR-0200-03 For use with Conventional PCR Instrument or Real time PCR Instrument User Manual For in vitro

More information

Loop-Mediated Isothermal Amplification for Rapid and Reliable Diagnosis of Tuberculous Meningitis

Loop-Mediated Isothermal Amplification for Rapid and Reliable Diagnosis of Tuberculous Meningitis JOURNAL OF CLINICAL MICROBIOLOGY, May 2011, p. 1861 1865 Vol. 49, No. 5 0095-1137/11/$12.00 doi:10.1128/jcm.00824-10 Copyright 2011, American Society for Microbiology. All Rights Reserved. Loop-Mediated

More information

Sensitive and Rapid Detection of Herpes Simplex Virus and Varicella-Zoster Virus DNA by Loop-Mediated Isothermal Amplification

Sensitive and Rapid Detection of Herpes Simplex Virus and Varicella-Zoster Virus DNA by Loop-Mediated Isothermal Amplification JOURNAL OF CLINICAL MICROBIOLOGY, July 2005, p. 3290 3296 Vol. 43, No. 7 0095-1137/05/$08.00 0 doi:10.1128/jcm.43.7.3290 3296.2005 Copyright 2005, American Society for Microbiology. All Rights Reserved.

More information

The Biotechnology Toolbox

The Biotechnology Toolbox Chapter 15 The Biotechnology Toolbox Cutting and Pasting DNA Cutting DNA Restriction endonuclease or restriction enzymes Cellular protection mechanism for infected foreign DNA Recognition and cutting specific

More information

Factors affecting PCR

Factors affecting PCR Lec. 11 Dr. Ahmed K. Ali Factors affecting PCR The sequences of the primers are critical to the success of the experiment, as are the precise temperatures used in the heating and cooling stages of the

More information

Novel Diagnostic Methods of Tuberculosis

Novel Diagnostic Methods of Tuberculosis Novel Diagnostic Methods of Tuberculosis Qimin YOU USTAR Biotechnologies I. The TB Diagnosis Challenge My presentation will focus on the new diagnostic methods of tuberculosis, developed by USTAR Biotechnologies.

More information

Site directed mutagenesis, Insertional and Deletion Mutagenesis. Mitesh Shrestha

Site directed mutagenesis, Insertional and Deletion Mutagenesis. Mitesh Shrestha Site directed mutagenesis, Insertional and Deletion Mutagenesis Mitesh Shrestha Mutagenesis Mutagenesis (the creation or formation of a mutation) can be used as a powerful genetic tool. By inducing mutations

More information

Contents. 1 Basic Molecular Microbiology of Bacteria... 1 Exp. 1.1 Isolation of Genomic DNA Introduction Principle...

Contents. 1 Basic Molecular Microbiology of Bacteria... 1 Exp. 1.1 Isolation of Genomic DNA Introduction Principle... Contents 1 Basic Molecular Microbiology of Bacteria... 1 Exp. 1.1 Isolation of Genomic DNA... 1 Introduction... 1 Principle... 1 Reagents Required and Their Role... 2 Procedure... 3 Observation... 4 Result

More information

Quantitative Real time PCR. Only for teaching purposes - not for reproduction or sale

Quantitative Real time PCR. Only for teaching purposes - not for reproduction or sale Quantitative Real time PCR PCR reaction conventional versus real time PCR real time PCR principles threshold cycle C T efficiency relative quantification reference genes primers detection chemistry GLP

More information

Only for teaching purposes - not for reproduction or sale

Only for teaching purposes - not for reproduction or sale PCR reaction conventional versus real time PCR real time PCR principles threshold cycle C T efficiency relative quantification reference genes primers detection chemistry GLP in real time PCR Relative

More information

Polymerase Chain Reaction-361 BCH

Polymerase Chain Reaction-361 BCH Polymerase Chain Reaction-361 BCH 1-Polymerase Chain Reaction Nucleic acid amplification is an important process in biotechnology and molecular biology and has been widely used in research, medicine, agriculture

More information

Q-PCR QUANTITATIVE-PCR 세포생물학및실험 2 박태식교수님

Q-PCR QUANTITATIVE-PCR 세포생물학및실험 2 박태식교수님 Q-PCR QUANTITATIVE-PCR 세포생물학및실험 2 박태식교수님 Title : Study that the Drug A inhibits the accumulation of fat. Schedules 1. Mouse necropsy : take up the blood and major tissues(organs) ex) heart, liver, fat,

More information

Appendix A. Introduction to PCR

Appendix A. Introduction to PCR Appendix A Introduction to PR In 1983, Kary Mullis at etus orporation developed the molecular biology technique that has since revolutionized genetic research, earning him the Nobel Prize in 1993. This

More information

On-site detection of Potato spindle tuber viroid in a disease outbreak using loop-mediated isothermal amplification (LAMP)

On-site detection of Potato spindle tuber viroid in a disease outbreak using loop-mediated isothermal amplification (LAMP) On-site detection of Potato spindle tuber viroid in a disease outbreak using loop-mediated isothermal amplification (LAMP) Linda Zheng, Fiona Constable, Angela Freeman, Brendna Rodoni Department of Economic

More information

Stool GeneXpert MTB/Rif Assay

Stool GeneXpert MTB/Rif Assay Stool GeneXpert MTB/Rif Assay Standard Operating Procedure 1.0. Purpose The purpose of this standard operating procedure (SOP) is to detail the steps for correctly performing, interpreting, and documenting

More information

Polymerase chain reaction

Polymerase chain reaction Core course BMS361N Genetic Engineering Polymerase chain reaction Prof. Narkunaraja Shanmugam Dept. Of Biomedical Science School of Basic Medical Sciences Bharathidasan University The polymerase chain

More information

mirna QPCR Master Mix

mirna QPCR Master Mix mirna QPCR Master Mix Instruction Manual Catalog #600583 Revision B Research Use Only. Not for Use in Diagnostic Procedures. 600583-12 LIMITED PRODUCT WARRANTY This warranty limits our liability to replacement

More information

REVISED VERSION: January 7, Detection of Mycoplasma pneumoniae. Amy E. Ratliff, Lynn B. Duffy, and Ken B. Waites * Department of Pathology

REVISED VERSION: January 7, Detection of Mycoplasma pneumoniae. Amy E. Ratliff, Lynn B. Duffy, and Ken B. Waites * Department of Pathology JCM Accepts, published online ahead of print on 15 January 2014 J. Clin. Microbiol. doi:10.1128/jcm.02913-13 Copyright 2014, American Society for Microbiology. All Rights Reserved. REVISED VERSION: January

More information

HCV Genotype Primer Kit

HCV Genotype Primer Kit Instruction Manual for HCV Genotype Primer Kit HCV Genotype Determination Kit for Research Purpose Thoroughly read this instruction manual before use of this kit Background Study of nucleotide sequence

More information

mirna QPCR Master Mix

mirna QPCR Master Mix mirna QPCR Master Mix Instruction Manual Catalog #600583 Revision D.0 For Research Use Only. Not for use in diagnostic procedures. 600583-12 LIMITED PRODUCT WARRANTY This warranty limits our liability

More information

Real Time PCR (qpcr) Dott. Finetti Luca

Real Time PCR (qpcr) Dott. Finetti Luca Real Time PCR (qpcr) Dott. Finetti Luca fntlcu1@unife.it PCR (Polymerase Chain Reaction) PCR (Polymerase Chain Reaction) Theoretically: 2 n PCR (Polymerase Chain Reaction) It often used as a qualitative

More information

PCR for Fungal Infections

PCR for Fungal Infections PCR for Fungal Infections DR SUJATA DHANUKA Associate Vice President- Medico-Marketing METROPOLIS HEALTHCARE LTD Superficial & Invasive Fungal Infections While superficial and subcutaneous fungal infections

More information

Real Time PCR. Advanced Biotechnology Lab I Florida Atlantic University April 2, 2008

Real Time PCR. Advanced Biotechnology Lab I Florida Atlantic University April 2, 2008 Real Time PCR Advanced Biotechnology Lab I Florida Atlantic University April 2, 2008 Introduction We wish to compare the expression levels of our gene under study (Drosophila MsrA) for two different treatment

More information

Hepatitis C Virus Genemer Mix

Hepatitis C Virus Genemer Mix Product Manual Hepatitis C Virus Genemer Mix Primer Pair for amplification of HCV Specific DNA Fragment Includes Internal Negative Control Primers and Template Catalog No.: 60-2003-12 Store at 20 o C For

More information

Quantitation of mrna Using Real-Time Reverse Transcription PCR (RT-PCR)

Quantitation of mrna Using Real-Time Reverse Transcription PCR (RT-PCR) Quantitation of mrna Using Real-Time Reverse Transcription PCR (RT-PCR) Quantitative Real-Time RT-PCR Versus RT-PCR In Real-Time RT- PCR, DNA amplification monitored at each cycle but RT-PCR measures the

More information

Beginner s guide to Real-time PCR

Beginner s guide to Real-time PCR Beginner s guide to Real-time PCR Beginner s guide to Real-time PCR PCR or the Polymerase Chain Reaction has become the cornerstone of modern molecular biology the world over. Real-time PCR is a bespoke

More information

HiPer Real-Time PCR Teaching Kit

HiPer Real-Time PCR Teaching Kit HiPer Real-Time PCR Teaching Kit Product Code: HTBM032 Number of experiments that can be performed: 10 Duration of Experiment Protocol: 1.5 hours Storage Instructions: The kit is stable for 12 months from

More information

Genetic Testing Tool STH-PAS

Genetic Testing Tool STH-PAS Technical Information (20150930) Genetic Testing Tool STH-PAS ~ Easily used by anyone, anywhere ~ What is STH-PAS? STH(Single-stranded Tag Hybridization) Method: 1Conduct PCR amplification of target DNA

More information

RT-LAMP Test Kit: A New Generation of Molecular Quick Test Kit for Porcine Epidemic Diarrhea Virus (PEDV)

RT-LAMP Test Kit: A New Generation of Molecular Quick Test Kit for Porcine Epidemic Diarrhea Virus (PEDV) Journal of Agricultural Technology 2015 Vol. 11(8): 1987-2008 Available online http://www.ijat-aatsea.com ISSN 1686-9141 RT-LAMP Test Kit: A New Generation of Molecular Quick Test Kit for Porcine Epidemic

More information

Recitation CHAPTER 9 DNA Technologies

Recitation CHAPTER 9 DNA Technologies Recitation CHAPTER 9 DNA Technologies DNA Cloning: General Scheme A cloning vector and eukaryotic chromosomes are separately cleaved with the same restriction endonuclease. (A single chromosome is shown

More information

Molecular-level dengue fever diagnostic devices made out of paper

Molecular-level dengue fever diagnostic devices made out of paper Supplementary Information Molecular-level dengue fever diagnostic devices made out of paper Shih-Jie Lo a, Shih-Chun Yang b, Da-Jeng Yao a, Jiann-Hwa Chen b, Wu-Chun Tu c, Chao-Min Cheng a * a Institute

More information

DETECTION OF HEPATITIS C VIRUS RNA USING REVERSE TRANSCRIPTION PCR

DETECTION OF HEPATITIS C VIRUS RNA USING REVERSE TRANSCRIPTION PCR Chapter 10 XA9846743 10.1. INTRODUCTION DETECTION OF HEPATITIS C VIRUS RNA USING REVERSE TRANSCRIPTION PCR S.F. Yap Department of Allied Health Sciences, Faculty of Medicine, University of Malaya, Kuala

More information

By Dr. Zainab khalid

By Dr. Zainab khalid By Dr. Zainab khalid Introduction to PCR PCR was invented in 1984 by ( Kary mullis ) & he received the Nobel Prize in chemistry in 1993, for his invention What is PCR? PCR is an exponentially progressing

More information

DNA Amplification: A Comparison of Different Methods

DNA Amplification: A Comparison of Different Methods DNA Amplification: A Comparison of Different Methods Lisa Barton Abstract: The development of the Polymerase Chain Reaction by Kary Mullis has led to the establishment and refinement of many new DNA amplification

More information

21st International Conference on Virus and other Graft Transmissible Diseases of Fruit Crops

21st International Conference on Virus and other Graft Transmissible Diseases of Fruit Crops Improvement of the reverse transcription loop mediated isothermal amplification (RT- LAMP) method for the detection of Peach latent mosaic viroid (PLMVd) Boubourakas, I.N. 1 ; Fucuta, S. 2 ; Luigi, M.

More information

Principles of Real Time PCR Ameer Effat M. Elfarash

Principles of Real Time PCR Ameer Effat M. Elfarash Principles of Real Time PCR Ameer Effat M. Elfarash Dept. of Genetics Fac. of Agriculture, Assiut Univ. aelfarash@aun.edu.eg Types of PCR Standard PCR (conventional ) RT-PCR (Reverse Transcriptase PCR)

More information

Functional Genomics Research Stream. Research Meeting: June 19, 2012 SYBR Green qpcr, Research Update

Functional Genomics Research Stream. Research Meeting: June 19, 2012 SYBR Green qpcr, Research Update Functional Genomics Research Stream Research Meeting: June 19, 2012 SYBR Green qpcr, Research Update Updates Alternate Lab Meeting Fridays 11:30-1:00 WEL 4.224 Welcome to attend either one Lab Log thanks

More information

PrimeScript 1st strand cdna Synthesis Kit

PrimeScript 1st strand cdna Synthesis Kit Cat. # 6110A For Research Use PrimeScript 1st strand cdna Synthesis Kit Product Manual Table of Contents I. Description... 3 II. Components... 3 III. Materials Required but not Provided... 3 IV. Storage...

More information

B. Incorrect! Ligation is also a necessary step for cloning.

B. Incorrect! Ligation is also a necessary step for cloning. Genetics - Problem Drill 15: The Techniques in Molecular Genetics No. 1 of 10 1. Which of the following is not part of the normal process of cloning recombinant DNA in bacteria? (A) Restriction endonuclease

More information

LavaLAMP DNA Component Kit

LavaLAMP DNA Component Kit LavaLAMP DNA Component Kit Please read carefully and thoroughly before beginning FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE. Lucigen Corporation 295 Parmenter St, Middleton, WI 53562 USA Toll

More information

HiPer RT-PCR Teaching Kit

HiPer RT-PCR Teaching Kit HiPer RT-PCR Teaching Kit Product Code: HTBM024 Number of experiments that can be performed: 5 Duration of Experiment: Protocol: 4 hours Agarose Gel Electrophoresis: 45 minutes Storage Instructions: The

More information

Molecular LDT in Newborn Screening Laboratories

Molecular LDT in Newborn Screening Laboratories Molecular LDT in Newborn Screening Laboratories APHL/CDC Newborn Screening Molecular Workshop Atlanta, GA June 28-30, 2011 Mei Baker, M.D., FACMG Assistant Professor, Department of Pediatrics University

More information

Experiment (5): Polymerase Chain Reaction (PCR)

Experiment (5): Polymerase Chain Reaction (PCR) BCH361 [Practical] Experiment (5): Polymerase Chain Reaction (PCR) Aim: Amplification of a specific region on DNA. Primer design. Determine the parameters that may affect he specificity, fidelity and efficiency

More information

Roche Molecular Biochemicals Technical Note No. 4/99

Roche Molecular Biochemicals Technical Note No. 4/99 Roche Molecular Biochemicals Technical Note No. 4/99 LightCycler LightCycler-RNA Amplification Kit SYBR Green I (96 rxn) Cat. No. 2 05 37 Adaptation Protocol for Sequence-Independent Detection of RNA with

More information

Abstract... i. Committee Membership... iii. Foreword... vii. 1 Scope Introduction Standard Precautions References...

Abstract... i. Committee Membership... iii. Foreword... vii. 1 Scope Introduction Standard Precautions References... Vol. 28 No. 12 Replaces MM18-P Vol. 27 No. 22 Interpretive Criteria for Identification of Bacteria and Fungi by DNA Target Sequencing; Approved Guideline Sequencing DNA targets of cultured isolates provides

More information

In the name of God. Application of Molecular Methods for Detection of Viral Pathogens & Viral Resistance

In the name of God. Application of Molecular Methods for Detection of Viral Pathogens & Viral Resistance In the name of God Application of Molecular Methods for Detection of Viral Pathogens & Viral Resistance By: Kiana Shahzamani From :Digestive Disease Research Center (DDRC) Why detect microorganisms? Determine

More information

Te c htips. Simple Approaches for Optimization of RT-PCR TECH TIP 206

Te c htips. Simple Approaches for Optimization of RT-PCR TECH TIP 206 Te c htips TECH TIP 206 Fueling Innovation USB Corporation 26111 Miles Road Cleveland, Ohio 44128 800.321.9322 www.usbweb.com Simple Approaches for Optimization of RT-PCR Reverse transcription-polymerase

More information

Molecular Cell Biology - Problem Drill 11: Recombinant DNA

Molecular Cell Biology - Problem Drill 11: Recombinant DNA Molecular Cell Biology - Problem Drill 11: Recombinant DNA Question No. 1 of 10 1. Which of the following statements about the sources of DNA used for molecular cloning is correct? Question #1 (A) cdna

More information

Polymerase Chain Reaction (PCR) and Its Applications

Polymerase Chain Reaction (PCR) and Its Applications Polymerase Chain Reaction (PCR) and Its Applications What is PCR? PCR is an exponentially progressing synthesis of the defined target DNA sequences in vitro. It was invented in 1983 by Dr. Kary Mullis,

More information

FUTURE PROSPECTS IN MOLECULAR INFECTIOUS DISEASES DIAGNOSIS

FUTURE PROSPECTS IN MOLECULAR INFECTIOUS DISEASES DIAGNOSIS FUTURE PROSPECTS IN MOLECULAR INFECTIOUS DISEASES DIAGNOSIS Richard L. Hodinka, Ph.D. University of South Carolina School of Medicine Greenville Greenville Health System, Greenville, SC hodinka@greenvillemed.sc.edu

More information

Polymerase Chain Reaction (PCR) May 23, 2017

Polymerase Chain Reaction (PCR) May 23, 2017 Polymerase Chain Reaction (PCR) May 23, 2017 Outline History of PCR Uses of PCR How PCR works How to set up and run PCR The structure of DNA PCR Polymerase chain reaction Selective amplification of target

More information

Development of a multiplex RT-LAMP for the discrimination of FMD from other vesicular diseases

Development of a multiplex RT-LAMP for the discrimination of FMD from other vesicular diseases Development of a multiplex RT-LAMP for the discrimination of FMD from other vesicular diseases Dr. Veronica Fowler Applied Diagnostics Research Coordinator, The Pirbright Institute 1 The Problem FMD or

More information

Bootcamp: Molecular Biology Techniques and Interpretation

Bootcamp: Molecular Biology Techniques and Interpretation Bootcamp: Molecular Biology Techniques and Interpretation Bi8 Winter 2016 Today s outline Detecting and quantifying nucleic acids and proteins: Basic nucleic acid properties Hybridization PCR and Designing

More information

Ah, Lou! There really are differences between us!

Ah, Lou! There really are differences between us! Name Per Ah, Lou! There really are differences between us! Introduction The human genome (the total sum of our genetic makeup) is made up of approximately 6 billion base pairs distributed on 46 chromosomes.

More information