Wangh Laboratory

May 21-25, 2007
107th General Meeting of the American Society of Microbiology
Toronto, ON, Canada

LATE-PCR Detection of Foot and Mouth Disease Virus (FMDV)

Kenneth E. Pierce, Juliet P. Dukes, Scott M. Reid, Donald P. King, Rohit Mistry, and Lawrence J. Wangh.  A collaboration between Brandeis University, Waltham, Massachusetts USA; the Institute for Animal Health, Pirbright, UK and Smiths Detection, Watford, UK.

Background: Foot and Mouth Disease is caused by a positive-strand RNA virus.  FMDV outbreaks are highly contagious and have serious financial consequences.  Detection of FMDV is complicated by teh presence of 7 serotypes and additional sequence variation within serotypes.  Here we describe a novel pan-FMDV assay which ultimately will be used in the field in combination with a handheld PCR Device.

Background:  A smallpox outbreak due to terrorist action would pose a serious health threat, but a false positive or false negative Variola warning would also have severe consequences.  This report describes a novel PCR-based orhopox assay that is sensitive, accurate, and able to distinguish among orthopox viruses. 

Background:  Diagnosis in critical care situations, such as sepsis-causing Gram-negative bacteria, could be greatly improved by use of multiplexed PCR assays that are rapid and able to distinguish among multiple organisms in a single reaction.  However, practical application of such assays is hampered by mis-priming, a problem that is inherent to all types of PCR and is exacerbated in reactions containing several pairs of primers.  We tested PrimeSafe^TM, a recently commercialized PCR additive, for its capacity to suppress mis-priming in multiplex assays for the identification of pathogenic bacteria.

Background: Highly pathogenic H5N1 avian flu has already directly and indirectly caused the deaths of millions of wild and domestic birds, as well as several hundred people, and threatens to evolve into a very lethal human pandemic influenza virus.  Identification of H5N1 and other subtypes of influenza is critical as a first line of defense and containment.  The challenge is to generate the maximum amount of reliable information in the minimum amount of time using a protable instrument.

Duplex RT-LATE-PCR Reveals Transcript Gradients in Sets of Single Cells Recovered from 8-Cell Mouse Embryos

Cristina Hartshorn, Odelya Hartung, and Lawrence J. Wangh

The formation of two distinctive cell lineages in preimplantation mouse embryos is characterized by differential gene expression. The cells of the inner cell mass (ICM) are pluripotent and express transcripts such as Oct4 RNA, which are down-regulated in the surrounding, differentiated trophectoderm (TE). Conversely, other genes are active in the TE and silenced in the ICM. These include Xist (expressed only in females) and Cdx2 (in both sexes). Prior to blastocyst formation, all these RNAs are ubiquitously found in blastomeres of embryos at the 8-cell stage. It is plausible, however, that transcript levels differ among blastomeres of the same embryo, and that these quantitative differences may presage the fate of their daughter cells. Testing this hypothesis presents numerous technical challenges because it requires simultaneous quantification of different RNAs in sets of single cells isolated from the same embryo. We have overcome these difficulties by combining PurAmp, a single-tube method for RNA preparation and quantification, with LATE-PCR, an advanced form of asymmetric PCR.

We initially constructed a duplex RT-LATE-PCR assay for real-time measurement of Oct4 and Xist templates and confirmed its specificity and quantitative accuracy using both biological samples and analysis of the LATE-PCR fluorescent signals. The linear slope of these signals is a sensitive tool to establish that amplification has been achieved with comparable efficiency for all templates analyzed. The Oct4/Xist duplex was an ideal test system, because comparison of data from males and females allowed us to determine that, due to the properties of LATE-PCR, Oct4 amplification was unaffected by sex-related differences in Xist expression (females: Oct4 +, Xist ++; males: Oct4 +, Xist -).

Our results show that both Oct4 and Xist RNA levels vary in individual blastomeres comprising the same embryo, with some cells having particularly elevated levels of either transcript. This is significant because all cells in the 8-cell embryo are believed to be developmentally equivalent. Our data also indicate that Xist and Oct4 expression levels are not correlated at this stage, although transcription of both genes is up-regulated at this time in development. We have now developed an additional assay for simultaneous measurements of Oct4 and Cdx2 RNA, in light of recent findings that these two genes are reciprocally regulated. This work describes the first example of RT-LATE-PCR and its utility for single-tube, multiplex quantitative analysis of transcripts in single cells. Levels of different RNAs can be accurately measured independently of their relative abundance; this is not possible with symmetric PCR. The techniques illustrated here are widely applicable, for instance to gene expression analysis in stem cells and cancer cells and to preimplantation genetic diagnostics. We are also employing these strategies for multiplex quantitative end-point detection of RNA viruses.

As reported previously LATE-PCR is an efficient form of asymmetric PCR which uses pairs of primers of unequal concentration designed such that the functional melting temperature of the Limiting Primer is equal to or greater than the functional melting temperature of the Excess Primer (Tm^L-Tm^X≥0). 

We are now extending this logic to construction of multiplex reactions for simultaneous amplification of sets of cDNA or genomic DNA target sequences. These multiplexed reactions generate copious levels of several single-stranded targets that can be detected in real-time or at end-point using combinations of sequence-specific probes, or mis-match tolerant probes that hybridize to variant sequences in a temperature-dependent manner. End-point ratios of such probe-target signals can then be computed as “Fluorescent Signatures”, one for each sequence variant.  In addition, multiple single-stranded amplicons in a multi-plex reactions can also be sequenced one-by-one, each with a different primer, using either Dilute-'N'-Go Pyrosequencing or Dilute-‘N’-Go Dideoxy-sequencing. For instance, heteroplasmic mixtures containing mixtures of SNP’s can be identified and quantified. All of these methods are sensitive down to the level of single starting molecules. In sum, these novel methods greatly enhance the information generated from a single closed-tube reactions and promise to revolutionize PCR based in vitro diagnostics. We are currently employing these technologies for construction of diagnostic assays in the fields of infectious diseases, cancer, forensics, and gene expression.