DOCSLIB.ORG

ABSTRACT Title of Document: NUCLEIC ACID

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
ABSTRACT Title of Document: NUCLEIC ACID ABSTRACT Title of Document: NUCLEIC ACID EXTRACTION AND DETECTION ACROSS TWO-DIMENSIONAL TISSUE SAMPLES Michael Daniel Armani, Doctor of Philosophy, 2010 Directed By (co-advisors): Professor Benjamin Shapiro, Fischell Department of Bioengineering, & Professor Elisabeth Smela, Department of Mechanical Engineering. Visualizing genetic changes throughout tissues can explain basic biological functions and molecular pathways in disease. However, over 90% of mammalian messenger RNA (mRNA) is in low abundance (<15 copies per cell) making them hard to see with existing techniques, such as in-situ hybridization (ISH). In the example of diagnosing cancer, a disease caused by genetic mutations, only a few cancer- associated mRNAs can be visualized in the clinic due to the poor sensitivity of ISH. To improve the detection of low-abundance mRNA, many researchers combine the cells across a tissue sample by taking a scrape. Mixing cells provides only one data point and masks the inherent heterogeneity of tissues. To address these challenges, we invented a sensitive method for mapping nucleic acids across tissues called 2D- PCR. 2D-PCR transfers a tissue section into an array of wells, confining and separating the tissue into subregions. Chemical steps are then used to free nucleic acids from the tissues subregions. If the freed genetic material is mRNA, a purification step is also performed. One or more nucleic acids are then amplified using PCR and detected across the tissue to produce a map. As an initial proof of concept, a DNA map was made from a frozen tissue section using 2D-PCR at the resolution of 1.6 mm per well. The technique was improved to perform the more challenging task of mapping three mRNA molecules from a frozen tissue section. Because the majority of clinical tissues are stored using formalin fixation and not freezing, 2D-PCR was improved once more to detect up to 24 mRNAs from formalin- fixed tissue microarrays. This last approach was used to validate genetic profiles in human normal and tumor prostate samples faster than with existing techniques. In conclusion, 2D-PCR is a robust method for detecting genetic changes across tissues or from many tissue samples. 2D-PCR can be used today for studying differences in nucleic acids between tumor and normal specimens or differences in subregions of the brain. NUCLEIC ACID EXTRACTION AND DETECTION ACROSS TWO- DIMENSIONAL TISSUE SAMPLES By Michael Daniel Armani Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2010 Advisory Committee: Professor Benjamin Shapiro, Chair Professor Elisabeth Smela Professor Yu Chen Professor Joonil Seog Professor Jason D. Kahn, Dean’s Representative Dr. Michael W. Phelan, M.D. © Copyright by Michael Daniel Armani 2010 Dedication To my loving parents, for all their hard work and motivation. ii Acknowledgements I am especially thankful for my wonderful advisors, Dr. Benjamin Shapiro, Dr. Elisabeth Smela, and Dr. Michael Emmert-Buck for guiding me for so many years. They taught me how question the status quo, formulate my own objectives logically, and keep from being biased by my own assumptions. Although I had three advisors at the same time, I am certain this created a unique mindset and proved that too many cooks do not spoil the broth. The skills they taught me branched out into all aspects of my life. I would like to thank Dr. Steven Hewitt and the TARP lab members Kris Ylaya and Jennifer Martinez for helpful discussions, sharing their time, and providing research support. A very special thanks also goes to Michael Tangrea, who shared countless hallway conversations and brainstorming sessions with me, resulting in many ideas that I pursued. I would also like to acknowledge the past and present members of Dr. Emmert-Buck’s lab (John Gillespie, Skye Kim, Brian Yang, Jaime Rodriguez- Canales, Jeff Hansen, Rodrigo Chuaqui, Heidi Erickson, Liang Zhu, Wusheng Yan, Sumana Mukherjee, Anneley Richardson, Alex Rosenberg), for discussing ideas with me, teaching me how to use the current molecular biology tools, doing experimental work with me, and for kindly sharing reagents or space. iii A very special thanks also goes to Belhu Metaferia, who essentially taught me a crash-course in organic chemistry, synthetic chemistry, and hydrogel chemistry even though he was not associated with our project or lab officially. I also must acknowledge many other colleagues, who helped me develop my ideas and supported my project in many ways including Disha Pant, Jeff Burke, Marc Dandin, Mark Kujawski, Mario Urdaneta, and Bavani Balakrisnan, from Dr. Smela’s lab; also Roland Probst, Pramod Mathai, Alek Nacev, Nir Kalush, and Derek Metzer from Dr. Shapiro’s lab. A special thanks to Eddie Chen and Maura Manion who were actually the first ones to work on 2D-PCR and who passed their project to me. I would also like to thank many more people who helped facilitate this project. This includes Tom Loughran and Jonathan Hummel at the UMD FabLab, Howie Grossenbacher at the UMD aerospace machine shop, Connie Newcome at Adhesives Research, who supplied us with specialty adhesive samples that were essential to this project, and Kelley Banfield at SAIC-Frederick who ran our Fluidigm experiments. I would like to thank people outside of my work who helped me and supported me, especially Sarah, and many others. Lastly, I have to thank the U.S. tax-payers, as their financial support made it to this project one way or another. iv Table of Contents DEDICATION..............................................................................................................II ACKNOWLEDGEMENTS........................................................................................ III TABLE OF CONTENTS............................................................................................. V LIST OF FIGURES .................................................................................................... IX LIST OF TABLES....................................................................................................XIII CHAPTER 1 : MOTIVATION AND BACKGROUND.............................................. 1 1.1 Introduction................................................................................................... 1 1.2 Background................................................................................................... 4 1.2.1 Nucleic acids......................................................................................... 4 1.2.2 Changes in nucleic acids....................................................................... 6 1.2.3 Detecting genetic changes by the polymerase chain reaction (PCR) ... 7 1.2.4 Typical methods of tissue sample preservation and storage............... 17 1.2.5 Typical methods for recovering nucleic acids .................................... 22 1.2.6 A common lab workflow to study changes in nucleic acids............... 28 1.2.7 Summary............................................................................................. 29 1.3 Current methods for localizing nucleic acids in tissue ............................... 29 1.3.1 In-situ hybridization (ISH).................................................................. 29 1.3.2 In-situ PCR (ISP)................................................................................ 33 1.3.3 Laser microdissection & PCR............................................................. 35 1.3.4 Macrodissection or voxelation & PCR............................................... 39 1.3.5 Immunohistochemistry (IHC)............................................................. 42 1.3.6 Summary............................................................................................. 43 1.4 Aims............................................................................................................ 44 1.4.1 A robust nucleic acid mapping method .............................................. 44 1.4.2 2D-PCR addresses all of these needs.................................................. 49 1.4.3 Aim 1 – Map DNA using 2D-PCR..................................................... 51 1.4.4 Aim 2 – Map RNA using 2D-PCR..................................................... 52 1.4.5 Aim 3 – Mapping total RNA in FFPE tissue using 2D-PCR.............. 52 CHAPTER 2 : DNA MAPS FROM FROZEN TISSUE ............................................ 55 2.1 Abstract....................................................................................................... 56 2.2 Introduction................................................................................................. 56 2.3 DNA Mapping Methodology...................................................................... 59 2.4 Materials and Methods................................................................................ 63 2.4.1 Device fabrication............................................................................... 63 2.4.2 Tissue sectioning and transfer............................................................. 64 2.4.3 Device heating and sealing ................................................................. 65 2.4.4 DNA amplification protocol............................................................... 67 2.4.5 Visualization....................................................................................... 67 2.5 Results........................................................................................................
Load more

Recommended publications
  • Evaluation of Six Methods for Extraction and Purification of Viral DNA from Urine and Serum Samples
    NEW MICROBIOLOGICA, 29, 111-119, 2006 Evaluation of six methods for extraction and purification of viral DNA from urine and serum samples Massimiliano Bergallo, Cristina Costa, Giorgio Gribaudo, Sonia Tarallo, Sara Baro, Alessandro Negro Ponzi, Rossana Cavallo Department of Public Health and Microbiology, Virology Unit, University of Turin, Italy SUMMARY The sensitivity and reliability of PCR for diagnostic and research purposes require efficient unbiased procedures of extraction and purification of nucleic acids. One of the major limitations of PCR-based tests is the inhibition of the amplification process by substances present in clinical samples. This study used specimens spiked with a known amount of plasmid pBKV (ATCC 33-1) to compare six methods for extraction and purification of viral DNA from urine and serum samples based on recovery efficiency in terms of yield of DNA and percentage of plasmid pBKV recovered, purity of extracted DNA, and percentage of inhibition. The most effective extraction methods were the phenol/chloroform technique and the silica gel extraction procedure for urine and serum samples, respectively. Considering DNA purity, the silica gel extraction procedure and the phenol/chloroform method produced the most satisfactory results in urine and serum samples, respectively. The presence of inhibitors was overcome by all DNA extraction techniques in urine samples, as evidenced by semiquantitative PCR amplification. In serum samples, the lysis method and the proteinase K procedure did not completely overcome the presence of inhibitors. KEY WORDS: PCR, DNA extraction, BKV, PCR inhibitors Received February 8, 2006 Accepted March 14, 2006 INTRODUCTION sequences has allowed the determination of pres- ence and quantification of specific viral genes and Molecular biology techniques represent a pow- sequences in biological matrices.
    [Show full text]
  • Advancing Gene Fusion Detection Towards Personalized Cancer Nanodiagnostics Kevin Maisheng Koo Bachelor of Science (Hons I)
    Advancing Gene Fusion Detection towards Personalized Cancer Nanodiagnostics Kevin Maisheng Koo Bachelor of Science (Hons I) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2017 Australian Institute for Bioengineering and Nanotechnology Abstract Prostate cancer (PCa) is one of the most prevalent non-cutaneous cancers in men, and is also one of the most lethal oncogenic diseases that accounts for a vast majority of male cancer-related deaths. Currently, widespread PCa screening is reliant on the prevailing usage of the FDA-approved blood- based prostate specific antigen (PSA) biomarker. Yet, landmark clinical trials in recent years have indicated that serum PSA screening holds a substantial risk of over-diagnosing low grade indolent PCa which are unlikely to result in mortality. Consequently, this paucity of accurate disease risk stratification during PCa screening has led to a variety of health burden associated with unnecessary biopsies, and over-treatment in a considerable fraction of patient population. Given that the screening shortcoming of the PSA test is outweighing its benefit, there is a clear need for better strategies to improve PCa risk stratification and accurately detect high-grade aggressive PCa molecular subtypes at an early stage for timely personalized treatment. To address this PCa screening conundrum, the research work described in this thesis primarily embodies a bipartite strategy which pairs together the use of next-generation PCa-specific molecular biomarkers, and the development of innovative nanodiagnostic technologies to target these superior biomarkers. In recent years, massive advances in next-generation sequencing techniques have led to the discoveries of novel PCa molecular targets which possess excellent PCa- specificity (i.e.
    [Show full text]
  • Virus Purification, Rna Extraction, and Targeted
    VIRUS PURIFICATION, RNA EXTRACTION, AND TARGETED GENOME CAPTURE IN ONE CHIP Miyako Niimi1*, Taisuke Masuda1, Kunihiro Kaihatsu2, Nobuo Kato2, and Fumihito Arai1 1Nagoya University, JAPAN and 2Osaka University, JAPAN ABSTRACT In this research, we demonstrated a microfluidic chip to pretreat the samples for viral genome assay. The microfluidic chip has the following three functions; (1) Virus purification and enrichment, (2) Viral RNA extraction, and (3) Capture of the targeted virus genome. (1) Hydroxyapatite chromatography, Boom method, and PNA (2) (Peptide Nucleic Acid) were used for the above three (3) functions, respectively. These three functions were integrated in one chip. Furthermore PNA immobilized on the glass can detect the targeted virus genome so that in situ virus detection would be possible by anybody, anywhere, anytime. KEYWORDS: Virus purification, RNA extraction and detection, Infectious disease diagnosis INTRODUCTION For the purpose of diagnosing the infectious diseases Figure 1. Concept of the microfluidic chip. The microflu- quickly and accurately, DNA sequencers for gene analysis idic chip consists of the three parts: (1) hydroxyapatite- of infectious viruses have been developed rapidly. The packed microcolumn for virus purification, (2) silica- latest DNA sequencers can treat the massive numbers of packed microcolumn for viral RNA extraction, and (3) samples such as saliva and nasal at one time. However, it PNA immobilized glass for capture of the targeted virus is necessary to purify and enrich the virus and extract the genome. viral RNA in the sample as the pretreatments before gene (1) (2) analysis. Hydroxyapatite chromatography[1] have been Sample Elution Buffer used extensively for purification and fractionation of Hydroxy- various biochemical substances such as protein and virus.
    [Show full text]
  • Amplication of 0.7Kb Fragment Katg Gene from Clinical Multi Drug Resistant Tuberculosis Isolate in Bali
    Indonesian Journal of Biomedical Sciences Volume 7, Number 2, July-December 2013: 69-72 Print-ISSN: 2085-4773, E-ISSN: 2302-2906. AMPLICATION OF 0.7KB FRAGMENT KATG GENE FROM CLINICAL MULTI DRUG RESISTANT TUBERCULOSIS ISOLATE IN BALI 1Dwiputri, A. W., 2Ratnayani, K., 1Yowani, S. C. 1Department of Pharmacy, Faculty of Mathematics and Natural Sciences, Udayana University, Bali-Indonesia 2Department of Chemistry, Faculty of Mathematics and Natural Sciences, Udayana University, Bali-Indonesia ABSTRACT During last decade has seen a particular increase in the occurrence of drug-resistant of tuberculosis (DR-TB) and multi-DR strains, such as Isoniazid (INH) resistant strains of M. tuberculosis. INH resistance is more frequently associated with mutations in the katG gene. Detection of katG gene mutations can be performed by PCR technique, followed by sequences. The aim of this study is to amplify katG gene region (0,7 Kb) from clinical isolate of MDR-TB in Bali. DNA isolation for PCR was done by Boom method and katG gene amplification was performed under the following conditions: predenaturation at 950C for 15 min; fourty cycles of denaturation at 940C for 1 min, annealing at 560C for 1 min, extension at 720C for 2 min; final extension at 720C for 10 min. The amplicons were detected by 1.5% agarose gel electrophoresis and showed a specific band size at 0.7 kb. This suggests that the fragment of katG gene has been successfully amplified in these areas. Keywords: amplification, katG gene, MDR-TB, 0.7 Kb INTRODUCTION we designed new primers with a longer area of Tuberculosis, caused by M.
    [Show full text]
  • Nederlandse Vereniging Voor Medische Microbiologie
    NEDERLANDS TIJDSCHRIFT VOOR Medische Microbiologie SUPPLEMENT BIJ TWAALFDE JAARGANG, APRIL 2004 Voorjaarsvergadering van de Nederlandse Vereniging voor Medische Microbiologie (NVMM) en de Nederlandse Vereniging voor Microbiologie (NVvM) in samenwerking met: Secties Algemene en Moleculaire Microbiologie, Microbiële Ecologie, Technische Microbiologie en Mycologie; Sectie Algemene Virologie; Sectie Levensmiddelenmicrobiologie; Nederlandse Vereniging voor Medische Mycologie; Werkgemeenschap Microbiële Pathogenese; Werkgroep Epidemiologische Typeringen; Werkgroepen Oost en West Medische Microbiologie; Nederlandse Werkgroep Klinische Virologie; Stichting Kwaliteitsbewaking Medische Microbiologie Papendal, 6 en 7 april 2004 Programma-overzicht Abstracts Auteursindex TWAALFDE J A A R G A N G . APRIL 2004 . SUPPLEMENT 041041_NtvMM_special_2004#20.indd 1 16-03-2004 14:58:04 adv Clindia 041041_NtvMM_special_2004#20.indd 2 16-03-2004 14:58:05 INLEIDING Inleiding Inmiddels is het traditie aan het worden: de Voorjaarsvergadering van de Nederlandse Vereniging voor Microbiologie (NVvM) en de Nederlandse Vereniging voor Medische Microbiologie (NVMM), en wordt op 6 en 7 april 2004 te Papendal gehouden. Ook de formule wordt een traditie: een plenair symposium op dinsdagochtend, dit jaar met als thema ‘Communication’, gevolgd door overwegend thematisch in- gedeelde parallelsessies. De multidisciplinaire, interactieve sessie van vorig jaar was dermate succesvol, dat deze sessievorm dit voorjaar wordt verdubbeld: één over mycobacteriële infecties bij kinderen en de ander over Chlamydia trachomatis en infertiliteit. AIO’s blijven zeer welkom op de Voorjaarsvergadering: zij worden vrijgesteld van inschrijfkosten, mid- dels de ‘Young Investigators Grant’, op voorwaarde dat zij een presentatie houden. Uiteraard is alleen de presenterende auteur van een voordracht of poster vrijgesteld. ‘Communication’ is het onderwerp van het plenaire symposium op dinsdagochtend: communicatie tus- sen micro-organismen onderling en tussen micro-organismen en gastheer.
    [Show full text]
  • Detection of Mycoplasma Hyopneumoniae by Polymerase Chain Reaction in Swine Presenting Respiratory Problems
    Brazilian Journal of Microbiology (2008) 39:471-476 ISSN 1517-8382 DETECTION OF MYCOPLASMA HYOPNEUMONIAE BY POLYMERASE CHAIN REACTION IN SWINE PRESENTING RESPIRATORY PROBLEMS Yamaguti, M.1*; Muller, E.E.1; Piffer, A.I.2; Kich, J.D.2; Klein, C.S.2; Kuchiishi, S.S.3 1Universidade Estadual de Londrina, Campus Universitário, Londrina, PR, Brasil; 2Embrapa Suínos e Aves, Concórdia, SC, Brasil; 3Universidade Estadual de São Paulo, Jaboticabal, SP, Brasil. Submitted: April 09, 2007; Returned to authors for corrections: January 23, 2008; Approved: July 04, 2008. ABSTRACT Since Mycoplasma hyopneumoniae isolation in appropriate media is a difficult task and impractical for daily routine diagnostics, Nested-PCR (N-PCR) techniques are currently used to improve the direct diagnostic sensitivity of Swine Enzootic Pneumonia. In a first experiment, this paper describes a N-PCR technique optimization based on three variables: different sampling sites, sample transport media, and DNA extraction methods, using eight pigs. Based on the optimization results, a second experiment was conducted for testing validity using 40 animals. In conclusion, the obtained results of the N-PCR optimization and validation allow us to recommend this test as a routine monitoring diagnostic method for Mycoplasma hyopneumoniae infection in swine herds. Key-words: Swine Enzootic Pneumonia, Mycoplasma hyopneumoniae, diagnosis, Nested- PCR. INTRODUÇÃO Nowadays, fastidious microorganisms have been detected by molecular biology techniques, such as DNA or RNA Mycoplasma hyopneumoniae (M. hyopneumoniae) is the hybridization and Polymerase Chain Reaction (PCR). These primary etiological agent of the Swine Enzootic Pneumonia (SEP), techniques are faster and more specific for microorganism that leads to a non-productive cough, mild fever, weight gain detection (6); and since mycoplasmas isolation is impractical in loss, high morbidity, and low mortality (12,17).
    [Show full text]
  • Design & Fabrication of Microfluidic DNA Extraction Device for Water
    DESIGN & FABRICATION OF MICROFLUIDIC DNA EXTRACTION DEVICE FOR WATER QUALITY MONITORING DESIGN & FABRICATION OF MICROFLUIDIC DNA EXTRACTION DEVICE FOR WATER QUALITY MONITORING By Bo Dang, B.ENG. B. Eng. (McMaster University, Ontario, Canada) A Thesis Submitted to the School of Graduate Studies In Partial Fulfillment of the Requirements For the Degree Master of Applied Science McMaster University © Copyright by Bo Dang, September 2012 MASTER OF APPLIED SCIENCE (2012) McMaster University (Mechanical Engineering) Hamilton, Ontario, Canada TITLE DESIGN & FABRICATION OF A MICROFLUIDIC DNA EXTRACTION DEVICE FOR WATER QUALITY MONITORING AUTHOR Bo Dang, B.Eng. SUPERVISOR Professor P. R. Selvaganapathy Department of Mechanical Engineering NUMBER OF PAGES XIX, 119 i ABSTRACT Continuous monitoring of pathogens that may be present in water is one of the key preventive measures that can be used in rural areas of developed countries and developing countries to reduce chances of the water borne diseases outbreak. Off-site testing of microbiological contamination of water is conventionally done for monitoring water quality. However, such a process is time consuming and involves using a variety of hazardous reagents. To address these issues, a portable device for rapid detection of unsafe water is needed. One of the key components in this system is to extract DNA from the pathogens. The primary consideration for DNA extraction is to separate DNA from proteins and other cell debris in the lysate solution. The pure population of DNA molecules are then sent downstream for subsequent processing such as real-time PCR (Polymerase Chain Reaction) and BioFET sensors for further identification and analysis. The focus of the thesis will be on the fabrication of a microfluidic DNA extraction system that can achieve high DNA extraction efficiency and a good repeatability.
    [Show full text]
  • Tularemia – Epidemiology
    This first edition of theWHO guidelines on tularaemia is the WHO GUIDELINES ON TULARAEMIA result of an international collaboration, initiated at a WHO meeting WHO GUIDELINES ON in Bath, UK in 2003. The target audience includes clinicians, laboratory personnel, public health workers, veterinarians, and any other person with an interest in zoonoses. Tularaemia Tularaemia is a bacterial zoonotic disease of the northern hemisphere. The bacterium (Francisella tularensis) is highly virulent for humans and a range of animals such as rodents, hares and rabbits. Humans can infect themselves by direct contact with infected animals, by arthropod bites, by ingestion of contaminated water or food, or by inhalation of infective aerosols. There is no human-to-human transmission. In addition to its natural occurrence, F. tularensis evokes great concern as a potential bioterrorism agent. F. tularensis subspecies tularensis is one of the most infectious pathogens known in human medicine. In order to avoid laboratory-associated infection, safety measures are needed and consequently, clinical laboratories do not generally accept specimens for culture. However, since clinical management of cases depends on early recognition, there is an urgent need for diagnostic services. The book provides background information on the disease, describes the current best practices for its diagnosis and treatment in humans, suggests measures to be taken in case of epidemics and provides guidance on how to handle F. tularensis in the laboratory. ISBN 978 92 4 154737 6 WHO EPIDEMIC AND PANDEMIC ALERT AND RESPONSE WHO Guidelines on Tularaemia EPIDEMIC AND PANDEMIC ALERT AND RESPONSE WHO Library Cataloguing-in-Publication Data WHO Guidelines on Tularaemia.
    [Show full text]
  • Branched Dna Nanostructures for Molecular Diagnostics
    BRANCHED DNA NANOSTRUCTURES FOR MOLECULAR DIAGNOSTICS A Dissertation Presented to the Faculty of the Graduate School of Cornell University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Mark Richard Hartman August 2013 © 2013 Mark Richard Hartman ALL RIGHTS RESERVED ii BRANCHED DNA NANOSTRUCTURES FOR MOLECULAR DIAGNOSTICS Mark Richard Hartman, Ph.D. Cornell University 2013 DNA nanotechnology has been enormously successful in using DNA as a nanoscale construction material, and consistent progress in using increasingly complex DNA- mediated assemblies and designs has been reported in the literature. However, the field of DNA nanostructures has sometimes been lacking for concrete applications. Meanwhile, DNA has also been widely used as probes and primers for nucleic acid diagnostics applications, and this provides an extremely critical application with potential for great medical benefits, and these DNA probes and primers are readily interfaced with DNA nanotechnology. Thus, in this Dissertation, I discuss the combination of (1) DNA in its role as a recognition element for nucleic acid detection, and (2) DNA as a structural material for nanoscale self-assembly. This combination represents a fusion of “structure” and “function”, and will ultimately lead to great advancements in the field of nucleic acid diagnostics. iii BIOGRAPHICAL SKETCH I received my B.S. degree in Chemical and Biomolecular Engineering from Cornell University in 2007. As an undergraduate with a minor in Biological Engineering, I worked with Prof. Dan Luo in the Biological and Environmental Engineering on the design and synthesis of DNA nanostructures and portable detection using DNA nanobarcodes. Upon graduation, I remained at Cornell and entered the Masters of Engineering program (2007 – 2008), where I worked on DNA-mediated self-assembly of gold nanoparticles.
    [Show full text]
  • Manual of Rotavirus Detection and Characterization Methods
    WHO/IVB/08.17 ORIGINAL: ENGLISH Manual of rotavirus detection and characterization methods Immunization, Vaccines and Biologicals WHO/IVB/08.17 ORIGINAL: ENGLISH Manual of rotavirus detection and characterization methods Immunization, Vaccines and Biologicals The Department of Immunization, Vaccines and Biologicals thanks the donors whose unspecified financial support has made the production of this document possible. This document was produced by the Expanded Programme on Immunization of the Department of Immunization, Vaccines and Biologicals Ordering code: WHO/IVB/08.17 Printed: October 2009 This publication is available on the Internet at: www.who.int/vaccines-documents/ Copies of this document as well as additional materials on immunization, vaccines and biologicals may be requested from: World Health Organization Department of Immunization, Vaccines and Biologicals CH-1211 Geneva 27, Switzerland • Fax: + 41 22 791 4227 • Email: [email protected] • © World Health Organization 2009 All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel: +41 22 791 3264; fax: +41 22 791 4857; email: [email protected]). Requests for permission to reproduce or translate WHO publications – whether for sale or for noncommercial distribution – should be addressed to WHO Press, at the above address (fax: +41 22 791 4806; email: [email protected]). The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries.
    [Show full text]
  • Highly Sensitive Sequence Specific Qpcr Detection of Mycobacterium
    Tuberculosis 101 (2016) 114e124 Contents lists available at ScienceDirect Tuberculosis journal homepage: http://intl.elsevierhealth.com/journals/tube Diagnostics Highly sensitive sequence specific qPCR detection of Mycobacterium tuberculosis complex in respiratory specimens Jennifer L. Reed a, Zachary J. Walker a, Debby Basu a, Veronica Allen b, Mark P. Nicol b, * David M. Kelso a, Sally M. McFall a, a Center for Innovation in Global Health Technologies (CIGHT), Department of Biomedical Engineering, Northwestern University, Evanston, IL 60208, USA b Division of Medical Microbiology, Department of Pathology, University of Cape Town and National Health Laboratory Service, Cape Town, South Africa article info summary Article history: Nucleic acid amplification tests for Mycobacterium tuberculosis (MTB) detection from sputum are highly Received 24 July 2016 sensitive and specific with smear microscopy positive specimens, but their sensitivity with smear- Received in revised form negative/culture-positive specimens is much lower; therefore, these tests cannot rule out a tubercu- 25 August 2016 losis diagnosis. Co-extraction of PCR inhibitors may be a cause of decreased test sensitivity. Here the Accepted 3 September 2016 design and early validation of a MTB screening assay with sample preparation and qPCR methods designed to specifically address this diagnostic gap is reported. First, human genomic DNA is identified as Keywords: a significant qPCR inhibitor. To circumvent this problem, a novel, streamlined sample preparation Mycobacterium tuberculosis fi Quantitative PCR method utilizing detergent and proteolysis to thin the sputum and DNA sequence speci c MTB DNA fi Sequence specific capture isolation was developed. Additionally, a multiplexed qPCR assay targeting two MTB complex-speci c Diagnosis loci: the potentially multi-copy IS6110 and the single-copy senX3-regX3, combined with the cotJC gene Sputum from Bacillus atrophaeus spores amplified as a process control was developed.
    [Show full text]
  • CHAPTER 10: PCR for Detection and Characterization of Bacterial
    CHAPTER 10 PCR for Detection and Characterization of Bacterial Meningitis Pathogens: Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae I. Overview of PCR technologies In developing countries, the most commonly used approaches for detection and characterization of bacterial meningitis pathogens include culture, Gram stain, and latex agglutination. Although culture is considered the gold standard for case confirmation in clinics, the positive rate is relatively low due to suboptimal storage and transportation conditions, culture practice, and/or antibiotic treatment administered before the specimen is collected. While Gram staining is important, inexpensive, and should be performed whenever possible, it merely gives a clue as to the genus and species of the etiological agent. The reading of latex agglutination results is subjective and can be difficult to interpret, especially when a specimen’s bacterial load is low. It is also not feasible to do quality control on latex agglutination. Culture should be kept as the gold standard as cultured bacteria are sources of data for antibiotic susceptibility, complete subtyping, the expression of antigens that are to be included in future vaccines, and pathophysiology of isolates. Specimens that do not yield any culture can still be analyzed by molecular methods (see below) that can be applied on DNA extracted from clinical specimens (typically, blood and CSF). A. Polymerase chain reaction (PCR) PCR was developed in the mid- to late 1980s (36, 42) and is considered one of the most important methodological inventions in molecular biology. It is designed to permit selective amplification of a specific target DNA sequence(s) within a heterogeneous collection of DNA sequences (e.g., total genomic DNA).
    [Show full text]