Using Plasmid to Construct a Differentiable Positive Control for Polymerase Chain Reactions

LJ Ting*, YP Liu, LH Chen, WC Li, YP Chen, YJ Lin

Animal Health Research Institute, Council of Agriculture, Executive Yuan

Abstract

Polymerase chain reaction (PCR) has been a routine laboratory diagnostic method for the
detection of the etiological agents of infectious diseases. When employing this method, one of the
considerations is the potential of false-positive PCR results. To minimize the risk of producing
false-positive results, amplified nucleic acid positive controls are a high risk factor for
contamination, in addition to reagents, equipment, and working areas. The aim of this study was to
construct plasmid as differentiable positive controls when using PCR to detect neospora and bovine
parainfluenza virus 3, respectively. The positive control is a plasmid inserted with a fragment
composed of an inner sequence of avian influenza virus and the primer annealing regions of
neospora sequences at both ends. When the construct was employed in the neospora -AI PCR assay,
the reaction generates a 211-base-pair-long product, longer than that of the normal neospora
positive PCR product. In addition, the product can be identified by nested PCR which is used for
the routine identification of avian influenza viruses. The same strategy was used to construct a
parainfluenza 3-goatpox differentiable positive control, followed by transcribing plasmid DNA into
RNA with the MEGAscript™ SP6 Kit.

Keywords：differentiable positive control, polymerase chain reaction (PCR), contamination