The real-time PCR technique has been applied to identify several species of Bacillus, such as B. anthracis (Wielinga et al., 2011)d cereus (Cattani et al., 2016). One possibility for basic differentiation of B. thuringiensis from other Bacillus is by identifying cry genes, because these are specific to B. thuringiensis.
The literature contains some reports that detect cry genes using the real-time PCR technique with hydrolysis probes, but none in triplex assays. The analyzed studies have detected cry genes under different experimental conditions. For example, Guidi et al. (2010) used hydrolysis probes to directly detect Bt in soil samples using the cry4Aa and cry4Ba genes as markers. Crighton et al. (2012) used a probe to detect the cry1 gene in studies about dispersion of Bt spores in a closed environment. Cry genes have also been used to detect transgenic events, such as the cry2Ae gene in cotton (Li et al., 2014) and cry1Ac in Oryza sativa (Sajjad et al., 2017).
In this work, the qPCR technique was used to identify multiple cry genes in a single reaction, reducing the time and cost of analyzes. The probes and primers were designed for the conserved regions of the N-terminal domain encoding part of the N endotoxin (pfam03945) of the selected cry genes. In the previously mentioned works, probes were designed primarily for the C-terminal regions of these genes. The in silico analysis demonstrated that the strategy adopted was able to detect the conserved regions of the genes under study with high specificity, as the values of identity (100%) and e-value (0.0) were within the expected range.
Both singleplex and triplex probe sets were sensitive in detecting cry genes (an important condition for this analysis). In the available literature, detection of cry genes by real-time PCR is linked to the quantity of spores per gram of soil, number of gene copies per square meter, or number of copies of the gene per microliter. Thus, the three cry genes could be detected simultaneously with LOD values in the range of 0.012 ng/µL to 0.065 ng/µl of total DNA. These results were obtained with efficiency tests that ranged from 100.987–102.027% in the singleplex. While in the triplex tests, the efficiency ranges from 100.940–102.875%, demonstrating the possibility of using TaqMan probes® to detection simultaneously three cry genes with high specificity and sensitivity from reduced amounts of DNA samples. In other analyzed studies (Li et al., 2014), the efficiency values ranged from 90–95% for probes in singleplex assays.
From the conditions established for the triplex, studies were carried out to verify the abundance, distribution, and diversity of the cry genes among the B. thuringiensis isolates. The search for cry genes is mostly based on the conventional PCR technique, which can detect the presence of new cry genes and direct bioassay work (Ceron et al., 1995; Bravo et al., 1998; Ibarra et al., 2003; Porcar; Juárez-Perez, 2003).
However, the use of real-time PCR to identify B. thuringiensis plus detect and analyze the distribution and diversity of cry genes is still poorly applied. In the assay, all cry1A (Cry1Aa1 to Cry1Aj1), cry1C (Cry1Ca1 to Cry1Cb3) and cry1F (Cry1Fa1 to Cry1Fb7) families were used in the cry gene bank to obtain the consensus sequence and design sets of primers and probes for the gene region that encodes the N-terminal portion of the Cry protein. This strategy, when applied to the Bt samples, identified cry1A family genes as the most abundant (55.37%) followed by cry1F (30.58%) and cry1C (14.05%) genes. These findings were similar to those found in the literature regarding the abundance of the cry1A gene, commonly reported in several articles. However, interestingly, the cry1F gene was more abundant than the cry1C gene, because the literature consulted indicated the opposite in terms of the abundance of these two genes. These results in the literature may be due to the use of PCR primers for the cry1Fa2 and cry1Fb genes only.
Several studies have screened for cry genes using conventional PCR primer oligonucleotides. For example, Porcar; Juárez-Perez (2003) used PCR primers to detect the genes of the cry1 family (cry1Aa, cry1Ab, cry1Ac, cry1Ad, and cry1Ae), cry1C (highly variable region) and cry1F (highly variable region). These researchers found that the cry genes most common in nature are from the cry1 family. Genes from the cry1A family were present in more than half of the analyzed strains (results were obtained from studies that analyzed from 58 to 500 strains of Bt). The cry1C genes also have well-reported abundance while the cry1F genes occur less. The findings of this work are corroborated with the H' = 0.96 and D = 0.58 indices found, indicating important diversity in the cry gene distribution.
Shishir et al. (2014) used PCR primers to detect several families of cry gene (cry1 to cry11) and observed that the cry1 genes were the most abundant (30.8%). Salama et al. (2015) also used PCR primers for diversity studies and found that cry1 family of genes were the most abundant (83.33%). In these two works, the universal primers used detected the genes of the families cry1Aa5, cry1Ac5, cry1Ad, cry1Ae, cry1Ba, cry1Bb, cry1Ca1, cry1Da, cry1Da, cry1Ea3, cry1Eb, cry1Fa2, cry1Fb, cry1G, cry1H, cry1Hb, cry1Ja, and cry1K, for example.
Our strategy for detecting cry genes by real-time PCR proved to be a robust strategy to detect several strains of Bt with several potential genes to be screened and studied.
The evaluation for the presence of the combination of cry genes in the analyzed Bt strain found that the cry1A/cry1F combination was the most common (39.34%), followed by cry1A/cry1C (27.89%) and cry1C/cry1F (16.39%). The presence of the three genes occurred in only 16.39% of the samples. These findings were corroborated by the Shannon (H') diversity indexes of 1.32 and Simpson (D) of 0.93, which suggests that these three genes are broadly distributed in the Bt strains.
These findings are corroborated by literature data. Porcar and Juarez-Perez (2003) reported a high frequency in the combination of certain cry genes such as cry1C-cry1D. The cry1C can be encountered independent of the cry1D (Kim et al., 1998). The cry1D gene has been frequently found alone, but the cry1C gene is always associated with cry1D. Cry1C-cry1D are located in the same replicon where the cry1C gene is downstream of an IS (Insertion Sequence). This may be responsible for the mobility of that gene in Bt strains. From this information and the qPCR results obtained in this study, the NGS results can be associated with two Bt strains that were already sequenced (data not shown). The results allowed identification of changes in the cry1C gene of the two strains. These two genes had alterations in the part of the gene that encodes the C-terminal portion of the Cry1C protein. One such change was the insertion of an IS of 150 bp and the other resulted from duplication of a portion of the cry1Ac gene in the final region of the cry1C gene. In addition, the cry1D gene in both strains could be identified. This gene was located upstream of the cry1C gene, as in the literature data.
Fiedoruk et al. (2017) described the origin of cry1 genes. These genes occur both individually and as part of an insect pathogenicity island (PAI). Cry1 genes occur as gene cassettes located in megaplasmids. Thus, the identification of a cry1 gene by the probe developed in this work may serve as a basis for exploration activities of other cry family genes.
Although many studies based on PCR are reported for cry gene screening, several factors limit their comparison, such as the use of different primers, variation in PCR conditions, and the number of strains and genes analyzed. Another important limitation is the lack of a precise preliminary characterization of the isolates. Some collections do not follow this preliminary step, and this may strongly influence the apparent genetic diversity of the collection (Porcar, Juárez-Perez, 2003).
Therefore, due to the results provided by the assays developed in this work, the use of Taqman probes could be an example of an approach to standard the assays to identify cry genes and their diversity and distribution. This diversity in cry gene content in a B. thuringiensis collection is influenced by the preselection process of the samples to be analyzed in a study and this procedure may only partially reflect the actual genetic diversity of the naturally occurring strains in a location. Another factor to be taken into consideration is the environmental diversity of the geographic area analyzed, which may also contribute to the high number of cry gene patterns in relation to the number of samples analyzed. Thus, due to the few studies that combined all these aspects, further studies are needed to obtain a more concise conclusion about cry gene diversity. An ideal study should combine the analysis of many analyzed samples with a high diversity of natural environments (Porcar, Juárez-Perez, 2003).
In addition, there is a need to establish an index that can better define diversity, not only by the positive reaction in a PCR reaction but following the analysis of some parameter of environmental analysis. In this work, we analyzed 240 Bt strains that resulted in 80 strains with positive amplification for one of the sampled cry genes. The design of the assays for the region of the cry gene to be detected can be a key factor in the amplification and definition of the identified gene score in a collection of sampled strains. This event is particularly impacted by the natural diversity of a particular geographic region, such as Brazil, which contains several vegetation regions that correspond to diverse biomes, from hot humid forest environments, through savanna-like vegetation, and temperate vegetation formations. The strategies of prospecting for the cry genes sought to contemplate all these biomes to cover the greatest possible diversity, observing a great diversity and variability in the distribution of these three cry genes from the performed AMOVA analyzes.
This diversity can also be influenced by the relationships of the Bt strains with the entomofauna in the environment, and populations may be present with naturally resistant or sensitivity to a certain Cry toxin (Monnerat et al., 2015).