Introduction

Immunocapture-polymerase chain reaction (IC-PCR) is a synthesis of two commonly used diagnostic tools. This method exploits the high-affinity binding of antibodies to provide a facile method of purification, usually from a complex matrix, supplying the substrate for PCR detection. PCR exponentially amplifies a deoxyribonucleic acid (DNA) template in a temperature-dependent fashion by the annealing of oligonucleotide primers, enzymatic extension of bound primers by a heat-stable polymerase, followed by denaturation of

From: Methods in Molecular Biology, vol. 295: Immunochemical Protocols, Third Edition. Edited by: R. Burns © Humana Press Inc., Totowa, NJ

the product. This three-stage process, performed in a thermocycler, is repeated sufficient times to allow amplification to proceed to levels, which will allow detection of the PCR product.

One advantage of this hybrid method is that the antibodies used in the immunocapture step do not need to provide the ultimate level of specificity required for the assay. For example, a genus-specific capture antibody may be used to provide the template for a species-specific PCR assay. In fact, each step of the technique can be tailored to provide the required level of discrimination.

IC-PCR has been used extensively in plant pathogen diagnostics, particularly in relation to viruses (1-11). The methodology outlined below was developed for use in the diagnostics of viral infection of potatoes (Solanum tuberosum). As the potato virus genomes involved were single-stranded ribonucleic acid (RNA), an additional step is needed between the immunocapture and PCR phases (RNA is not a suitable substrate for direct use in PCR). The action of reverse transcriptase (RT) on oligonucleotide-primed RNA allows the synthesis of complementary DNA (cDNA), required to permit detection using PCR. This technique is referred to as IC-RT-PCR.

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