A Comparison of Non-Radioactive Labeling and Detection Systems with Synthetic Oligonucleotide Probes for the Species Identification of Mosquitoes in the Anopheles Gambiae Complex

Abstract

Different non-radioactive probe labeling and detection systems were used with pAnaI, a species-specific oligonuleotide probe that distinguishes male Anopheles gambiae and An. arabiensis mosquitoes. Comparisons have been made between the performance of each technique with respect to sensitivity and specificity against DNA dot-blots and mosquito squashes. Their relative costs, economy, and ease of use were analyzed in an attempt to develop an appropriate non-radioactive system for use in the field. Enzyme-labeled probes that were detected directly by label activity proved more suitable than probes requiring reporter molecules for detection. Binding of reporter molecules to mosquito squashes caused the appearance of false positives and, in addition, their binding to nylon filters caused high background coloration. Chemiluminescent detection provided an attractive alternative to colorimetric detection. Both systems analyzed were rapid, simple, and economic. However, less severe treatment of filters was required for reprobing with chemiluminescence. The greatest sensitivity achieved was with chemiluminescent detection in which the limit of detection was 0.15 ng of target DNA. This study suggests that a synthetic DNA probe coupled to a chemiluminescent detection system should provide a sufficiently simple, sensitive, and reliable technique for insect vector identification in the field.