An immuno-PCR method for detecting Bacillus thuringiensis Cry1Ac toxin.
The Cry1Ac toxin is an insecticidal protein produced by Bacillus thuringiensis var. kurstaki. Recently, the gene encoding the toxin was genetically transformed into crop plants. A specific and sensitive method for detecting the Cry1Ac toxin would facilitate monitoring for this protein in crop and non-crop plants and also in foods. The purpose of this study was to develop an immuno-PCR technique for detecting this toxin. Immuno-PCR combines the specificity of an ELISA reaction with the sensitivity of assays that use a PCR-amplification step. In our assay, anti-Cry1Ac antibodies were covalently bound to reporter DNA via a linker molecule, succinimidyl-4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC). Antigen was coated onto the surfaces of polyvinyl chloride microtiter plates or onto streptavidin-coated beads. Each of these solid-surface platforms was tested in immuno-PCR reactions. Both the microtiter plate- and bead-based assays showed a high degree of specificity and sensitivity, with minimum detection limits of 21.6 and 432 ng of toxin, respectively. This sensitive immuno-PCR method could be modified for detecting a variety of other protein toxins.
Allen
Rebekah C
RC
Department of Biology, New Mexico Institute of Mining and Technology, Socorro, NM 87801, USA.
Rogelj
Snezna
S
Cordova
Susan E
SE
Kieft
Thomas L
TL
D000818:Animals; D000906:Antibodies; D001413:Bacillus thuringiensis / Q000235:genetics / Q000276:immunology; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology*; D001427:Bacterial Toxins / Q000032:analysis* / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D004274:DNA, Recombinant / Q000235:genetics; D004731:Endotoxins / Q000032:analysis* / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D017930:Genes, Reporter; D006460:Hemolysin Proteins; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D011137:Polystyrenes; D011817:Rabbits; D012680:Sensitivity and Specificity; D019809:Streptavidin
D000906:Antibodies; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004274:DNA, Recombinant; D004731:Endotoxins; D006460:Hemolysin Proteins; D011137:Polystyrenes; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D019809:Streptavidin