Wangh Laboratory

Introduction:  There is a growing need in veterinary and human medicine for diagnostic assays that can rapidly detect and analyse numerous species and strains of infectious organisms and viruses.  RNA viruses are particularly challenging because they evolve rapidly.  In the case of veterinary medicine, tests have to be doen at pen-side or in the field and the findings can have great economic impact, including slaughter of many animals.  Currently, the use of RT-PCR is limited in scope and few, if any, assays provide results from multiplex data in the field.  We have overcome these limitations by constructing assays based on LATE-PCR and (RT)-LATE-PCR.  We are collaborating with Smiths Detection to implement these assays on an automated portable sample preparation and PCR system.

June 2008
1^st Workshop on LATE-PCR and its Diagnostic Applications
National Veterinary Institute
Uppsala, Sweeden 

LATE-PCR and RT-LATE-PCR workshop

Speakers: Lawrence Wangh, Ken Pierce, Cristina Hartshorn, Bonnie Ronish

June 18-20, 2008
Cambridge Healthtech Institute
Baltimore, MD

LATE-PCR and the BioSeeq: Maximum Information from a Field Based Instrument

Lawrence J. Wangh, Ph.D., Laboratory Director, Brandeis University Department of Biology, in collaboration with Smiths Detection, Inc., Watford, UK

See Abstract

June 4-6, 2008
European Union (EU) Network of Excellence for Epizootic Disease Diagnose and Control
EPIZONE
Brescia, Italy

Rapid Detection and Identification of Avian Influenza Subtypes in a Single Tube Using RT-LATE-PCR.

Cristina Hartshorn, A.H. Reis, Jr., K.E. Pierce, L.J. Wangh

Introduction: Pathogenic strains of avian influenza, particularly subtypes H5 and H7 continue to spread and evolve through out Eurasia and Africa, causing death and requiring culling of millions of birds.  Several hundred people have died of avian influenza primarily following direct contact with infected poultry, but there is reason to fear that relatively few genetic changes could lead to efficient human-to-human transmission raising the specter of a devastating global pandemic.  Rapid, accurate detection of the presence and type of influenza in people, birds, and other species is urgently needed, particularly in remote rural areas where people leave very close to their livestock.  In collaboration with Smiths Detection, Inc. (Watford, U.K.) we are using two novel technologies developed in our laboratory at Brandeis University, LATE-PCR (U.S. Patent 7,198,897) and PrimeSafe^TM (a mis-priming preventing compound), to construct a highly multiplexed assay that can detect and distinguish between several subtypes of avian influenza and their strains in a single tube.  This assay is designed to work on a battery-powered point-of-care instrument, the BioSeeq, which is being designed and engineered by Smiths Detection, Inc.

April 8-13, 2008
Molecular Evolution as a Driving Force in Infectious Diseases
Beaver Run Resort - Breckenridge, Colorado
Keystone Symposia

Infectious diseases have been a key driving force in evolution, both of the hosts and the pathogens. The era of genomics has made it possible to evaluate both infectious agents and their hosts at the genomic level. The evolutionary dynamics of viruses, bacteria and parasites, in their adaptive evolution to changes in host response or adaptation to new hosts is enormously complex. The burden of infectious diseases on people and domestic animals is staggering. We are only now beginning to achieve an understanding of how infectious organisms cause disease and how the complex patterns of immune response generated by their infections mitigate it. By bringing together experts in the evolution of viruses, bacteria and parasites, plus those studying host adaptation and evolution of the immune response, we hope that this meeting will stimulate further interaction between widely disparate lines of investigation.

LATE-PCR for Detection and Analysis of Infectious Diseases - Presentation

Lawrence Wangh, Cristina Hartshorn, Arthur Reis, John Rice, Kenneth Pierce.  Department of Biology, Brandeis University, Waltham, MA, 02454.

LATE-PCR, invented in our laboratory, is an advanced form of asymmetric PCR that uses two primers of unequal concentration and differing melting temperatures to generate double-stranded DNA amplicons exponentially, followed by linear amplification of one strand of amplicon.  One of the primers can also be used to synthesize cDNA from RNA prior to the start of amplification, thus allowing for RT-PCR.

Cristina Hartshorn, Odelya Hartung, Yanwei Jia, and Lawrence J. Wangh, Brandeis University

Abstract: We are using three novel technologies invented in our laboratory, LATE-PCR, PrimeSafe, and PurAmp to construct multiplexed assays for quantitative analysis of specific mRNAs in single cells and small groups of cells. LATE-PCR is an advanced form of asymmetric PCR in which efficient exponential amplification of double-stranded DNA is followed by cycles of linear amplification of single-stranded DNA.  The resulting single strands are detected using fluorescent probes.

Using these technologies we have already constructed reactions for measurement of Xist RNA, Oct 4 mRNA, Cdx2 mRNA, and heatshock protein (hsp) 70i mRNA.  Duplex assays in various combinations have been used to quantify the levels of these gene transcripts in single blastomeres recovered from mouse embryos at the 4 cell and 8 cell stages of early development, as wells as from intact embryos.   These assays can also measure copy numbers of the respective genes when LATE-PCR is performed without RT.  These experiments serve as controls with which to validate that our methods are sensitive down to single molecules of genomic DNA in single cells.  They also allow us to compare several methods of reverse transcription in order to conveniently and reliably detect very low numbers of RNA molecules in single cells.

In collaboration with Brian Reid’s laboratory at the Fred Hutchinson Cancer Research Center we will use our optimized protocols to construction of duplex and multiplexed LATE-PCR assays for measurement of mRNAs levels in single normal and neoplastic esophageal cells.  The following transcripts, COX-2, hsp27, p53, are of particular interest because they are known to change in response to stress stimuli like heat, low pH, and bile salts.  In parallel with these studies we are also carrying out single-cell LATE-PCR assays to measure loss of heterozygosity (LOH) in the region of CDKN2A(p16) or TP53(p53) primers.  These genetic changes are hallmarks of Barretts esophagus, the precursor to esophageal adenocarcinoma.