Browse Prior Art Database

AGGLUTINATION PCR AND ITS APPLICATION IN SEED HEALTH TESTING

IP.com Disclosure Number: IPCOM000228072D
Publication Date: 2013-Jun-05
Document File: 4 page(s) / 96K

Publishing Venue

The IP.com Prior Art Database

Abstract

The application of PCR detection for seed health testing is often hampered by the quality of the DNA sample. Typically, DNA samples from seed, or from seed extracts, contain many different chemical substances and microbial cells that interfere with the final PCR. In general, if seed-based DNA samples are retrieved and processed in a straight forward manner, as can done for example with many medical diagnostic samples such as blood or sputum, the PCR detection test will not meet the sensitivity that is required.

This text was extracted from a PDF file.
This is the abbreviated version, containing approximately 38% of the total text.

Page 01 of 4

AGGLUTINATION PCR AND ITS APPLICATION IN SEED HEALTH TESTING

Introduction

The application of PCR detection for seed health testing is often hampered by the quality of the DNA sample. Typically, DNA samples from seed, or from seed extracts, contain many different chemical substances and microbial cells that interfere with the final PCR. In general, if seed-based DNA samples are retrieved and processed in a straight forward manner, as can done for example with many medical diagnostic samples such as blood or sputum, the PCR detection test will not meet the sensitivity that is required.

    In order to use a seed-based sample for PCR detection testing, the sample will have to be pre-processed to get rid of substances that can inhibit the subsequent PCR reaction and/or to concentrate the target pathogen to increase test sensitivity. Various pre-processing technologies have been published already. For example, so called Bio-PCR or liquid enrichment PCR tests rely on a pre-growing step on/in growth medium before the sample is processed. This results in higher numbers of target cells and at the same time reduces the concentration of potential PCR inhibitors. Another technique, called Magnetic Capture Hybridization PCR (MCH-PCR), hybridizes the sequences of interest to magnetic beads after DNA isolation. Subsequently, the beads are fixed to the tube using a magnetic device and all competing DNA and PCR inhibitors are removed before PCR. Similarly, Immuno Magnetic Separation PCR (IMS-PCR) also uses magnetic beads to fix the target to the tube and clean the sample. However, in the latter case intact target cells are fixed to the magnetic beads using specific antibodies followed by sample cleaning and DNA isolation.

    The disadvantage of enrichment technologies such as Bio-PCR and liquid enrichment is the dependency on a growing step which takes time and often results in increased numbers of other microorganisms besides the target organism. Bead-based capturing techniques, such as MCH- and IMS-PCR, are not dependent on a target-multiplication step, but have as disadvantage that binding of the target to the beads is sometimes relatively weak. Experiments have shown that a lot of bacteria are lost as a result of the subsequent washing steps of the beads. This worsens the detection level and makes the method less robust. Moreover, the quality of the beads cannot be guaranteed. Whereas bead quality is crucial to the method, it is very difficult to prepare good, reproducible-quality beads.

    In this document the concept of a pre-processing step of difficult diagnostic samples is described, such as, but not limited to, seed-based samples, that are intended to be used for PCR detection. The method does neither depend on a pre-growth step, nor does it depend on magnetic beads. In short, this methodology is based on selective precipitation of the target organism using specific antibodies (agglutination).

Technical concept of agglutination PCR

The concept co...