Specific PCR primers directed to identify cryI and cryIII genes within a Bacillus thuringiensis strain collection

Abstract

In this paper we describe a PCR strategy that can be used to rapidly identify Bacillus thuringiensis strains that harbor any of the known cryI or cryIII genes. Four general PCR primers which amplify DNA fragments from the known cryI or cryIII genes were selected from conserved regions. Once a strain was identified as an organism that contains a particular type of cry gene, it could be easily characterized by performing additional PCR with specific cryI and cryIII primers selected from variable regions. The method described in this paper can be used to identify the 10 different cryI genes and the five different cryIII genes. One feature of this screening method is that each cry gene is expected to produce a PCR product having a precise molecular weight. The genes which produce PCR products having different sizes probably represent strains that harbor a potentially novel cry gene. Finally, we present evidence that novel crystal genes can be identified by the method described in this paper.

Identification of vip3A-type genes from Bacillus thuringiensis strains and characterization of a novel vip3A-type gene

“…To date, several PCR-based methods for cry gene identification have been developed (Ceron et al 1995;Juarez-Perez et al 1997;Ben-Dov et al 1999;Song et al 2003), including specific primer PCR, multiplex PCR, exclusive PCR and PCR-RFLP. These methods directly detect known cry genes from B. thuringiensis (Ceron et al 1995;Ben-Dov et al 1999) as well as identify novel cry genes (Juarez-Perez et al 1997). In the current study, we reported the large-scale use of PCR-RFLP with a pair of novel universal primers to identify vip3A-type genes from B. thuringiensis strains.…”

“…A variety of polymerase chain reaction (PCR)-based methods have been developed to detect different cry genes from B. thuringiensis strains (Ceron et al 1995;Ben-Dov et al 1999;Song et al 2003). Ce¢cile et al (2005) had described the identification of novel vip3 genes by PCR amplification using primers specific to conserved regions followed by restriction mapping of the PCR product.…”

Diversity of Colombian strains of Bacillus thuringiensis with insecticidal activity against dipteran and lepidopteran insects

“…The 445 isolates of B. thuringiensis were grown for 12 h at 30°C in Petri dishes with GEN nutrient medium (5 g yeast extract, 8AE8 g glucose, 10 g NaCl, 20 g agar in 1000 ml distilled water). A loop of each isolate was transferred to 100 ll sterile distilled water, the mixture was homogenized and stored at )20°C for 3 h, then immediately placed in a water bath at 100°C for 10 min to lyse the cells and finally centrifuged for 10 s at 9280 g. This supernatant was used for PCR as DNA template (Carozzi et al 1991;Ceró n et al 1994Ceró n et al , 1995Bravo et al 1998). General primers were used to characterize cry gene content (cry1, cry2, cry4 and cry11).…”

Characterization of novel Brazilian Bacillus thuringiensis strains active against Spodoptera frugiperda and other insect pests

“…). The conditions used for the PCR analyses were those described by Ceron et al (1994Ceron et al ( , 1995 and Bravo et al (1998). After amplification, a 15-lL aliquot of the product from each PCR reaction was electrophoresed on a 2% (w/v) agarose gel in Tris-borate buffer at 100 V for 1 h and stained with ethidium bromide.…”

“…Because of their high specificity and environmental safety (Krieg and Langenbruch, 1981), Cry proteins have been successfully used as bioinsecticides against lepidopteran, dipteran, and coleopteran pests (Feitelson et al, 1992;Aranda et al, 1996;Bohorova et al, 1996). Strains with toxic activity towards nematodes (Edwards et al, 1990) and other organisms, such as platyhelminths, protozoans, prokaryotes, and acarida have been identified (Feitelson, 1994;Payne et al, 1994;Ceron et al, 1995;Yudina and Burtseva, 1997), and the activity spectrum of the toxins of B. thuringiensis is increasing as the result of the continual isolation and characterization of new strains.…”