Extended screening by PCR for seven cry-group genes from field-collected strains of Bacillus thuringiensis.
An extended multiplex PCR method was established to rapidly identify and classify Bacillus thuringiensis strains containing cry (crystal protein) genes toxic to species of Lepidoptera, Coleoptera, and Diptera. The technique enriches current strategies and simplifies the initial stages of large-scale screening of cry genes by pinpointing isolates that contain specific genes or unique combinations of interest with potential insecticidal activities, thus facilitating subsequent toxicity assays. Five pairs of universal primers were designed to probe the highly conserved sequences and classify most (34 of about 60) genes known in the following groups: 20 cry1, 3 cry2, 4 cry3, 2 cry4, 2 cry7, and 3 cry8 genes. The DNA of each positive strain was probed with a set of specific primers designed for 20 of these genes and for cry11A. Twenty-two distinct cry-type profiles were identified from 126 field-collected B. thuringiensis strains. Several of them were found to be different from all published profiles. Some of the field-collected strains, but none of the 16 standard strains, were positive for cry2Ac. Three standard and 38 field-collected strains were positive by universal primers but negative by specific primers for all five known genes of cry7 and cry8. These field-collected strains seem to contain a new gene or genes that seem promising for biological control of insects and management of resistance.
Ben-Dov
E
E
Department of Life Sciences, Ben-Gurion University of the Negev, Be'er-Sheva, Israel.
Zaritsky
A
A
Dahan
E
E
Barak
Z
Z
Sinai
R
R
Manasherob
R
R
Khamraev
A
A
Troitskaya
E
E
Dubitsky
A
A
Berezina
N
N
Margalith
Y
Y
D000818:Animals; D001413:Bacillus thuringiensis / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004731:Endotoxins / Q000235:genetics*; D005798:Genes, Bacterial; D006460:Hemolysin Proteins; D007306:Insecticides; D010572:Pest Control, Biological; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017931:DNA Primers; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; C030956:insecticidal crystal protein, Bacillus Thuringiensis