Introduction

Vegetative insecticidal protein (Vip) is a novel class of insecticidal protein produced by a few strains of Bacillus thuringiensis (Bt) during the vegetative stage of their growth [1]. Bt is a gram-positive bacterium with a genome size of 2.4 million to 5.7 million base pairs. Various strains of Bt are known to produce several different classes of insecticidal proteins. The largest class consists of the δ-endotoxins, which often are produced as a 125−140-kDa protein precursor that is solubilized and proteolytically processed in the insect gut to yield a 55−75-kDa biologically active core protein. δ-Endotoxins have been expressed in many important crop plants, including cotton, potato, tomato, rice, and corn [2]. Bt organisms also produce two known classes of vegetatively expressed insecticidal proteins. These include the binary toxins Vip1 and Vip2 with coleopteran specificity and Vip3 with lepidopteran specificity [3]. A classification of these proteins into three classes, seven subclasses, and further subdivisions was recently proposed by the Bacillus thuringiensis Nomenclature Committee [4].

The 88.5-kDa (791 amino acids) Vip3 does not show any homology with the known Cry proteins and the structural dissimilarity of Vip3 is indicative of a possible divergent insecticidal mechanism from that of the other known Bt toxins. The structural divergence of Vip3 makes it an ideal candidate for deployment in an insect management program together with the other category of Bt toxins. Its different mode of action could play an effective role in resistance management and eventually offer growers an alternate refuge management opportunity. The Vip3 proteins act by binding to specific sites located in the midgut epithelium of susceptible insect species of the order Lepidoptera. Following binding, cation-specific pores are formed that disrupt ion flow in the midgut, thereby causing paralysis and death [5]. These characteristic features of Vip3 signify their importance and suitability for being part of insect resistance transgenic development. Vip3 has been grouped into Vip3A and Vip3B. Vip3A has several subgroups and Vip-S and Vip3Aa1 belong to such subgroups. As many as two synthetic vip3A-type genes with a plant-optimized codon have been used for stable expression in different transgenic crops. Field trials are currently at advance stages and the commercial release is expected very shortly. These are:

  1. 1.

    Transgenic cotton and tobacco bearing a synthetic vip-s gene, developed by the International Center for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India [6].

  2. 2.

    Transgenic maize and cotton lines (Cot102) carrying a synthetic vip3Aa1 gene (NCBI accession nos. DQ539887 and DQ539888) developed by Syngenta International, Switzerland [2].

The rapid increase in genetically modified (GM) crop cultivation worldwide, over 200 million acres in 2005 [7], has led regulatory authorities to implement a set of very strict procedures for the approval to grow, import and/or utilize GM organisms (GMOs) as food or food ingredients. The cultivation of vip3A-based GM crops is likely to increase exponentially and the produce is likely to reach the market globally. Hence, for regulatory compliance on GM labeling and identification of genetic trait, the need for an easy and reliable detection method for these transgenics is natural and necessary [8]. Many countries have implemented labeling thresholds for unintentional mixing of GM crops defined as 0.9% in the EU, 3% in Korea and 5% in Japan. Recently, Syngenta has submitted validated methods for extraction and direct ELISA analysis of Vip3A in cotton seed to the US Environmental Protection Agency and these were found to be acceptable by the US Environmental Protection Agency [9]. However, there is no PCR assay available for detection and characterization of vip3A-based transgenics.

PCR has proven to be a rapid and reliable method and has largely substituted bioassays in preliminary classification of Bt collections. Several PCR assays have been developed for the screening and identification of novel cry genes of Bt strains [10]. For vip3A-like gene screening in natural isolates of Bt, a PCR-based assay has been reported by Rice [11]. For characterization and species differentiation among the vip3A-bearing microbes, several PCR primers have been employed by many researchers [3]. However, the published Bt-centric primers would not be suitable for detection of GM plants, since during transgenic creation, synthetic genes are used which carry modified plant-preferred codons for the proper expression of the gene of interest. To overcome this hurdle, the main objective of this study was the design of primers and assay methods for detection of all existing vip3A-type synthetic genes in transgenic crops, food and food products.

In the present work, we propose a method based on standard, multiplex and construct-specific PCR followed by nested PCR assay for the complete characterization of vip3A-bearing transgenics. Our proposed standard PCR assay can be used as a universal method for vip3A screening in GMOs and native isolates, while multiplex and construct-specific PCR followed by nested PCR assays would be specific for characterization of vip3A-based GM plants.

Materials and methods

Reference material

The target DNA was sourced from transgenic cotton and tobacco leaves (VipCot14, VipCot29, VipTobacco1 and VipTobacco2) and recombinant Escherichia coli DH5α clones (pET29a and pQE30) (courtesy of ICGEB, New Delhi) and from Bacillus thuringiensis subsp. tolworthi HDB-8 (HD125) wild-type isolate (courtesy of Daniel R. Zeigler, Bacillus Genetic Stock Centre, Columbus, USA). For spiking assays, MECH-12Bt transgenic cotton seed, MON810 transgenic maize seed and Roundup ready Soya (a gift from S. Vasanthi, National Institute of Nutrition, Hyderabad, India) were used as a source for nontarget DNA. Owing to the lack of commercially available standard certified reference material for vip3A transgenic cotton and tobacco, the reference material was prepared in the laboratory as per the process outlined by Germini et. al. [12]. DNA samples extracted from GM VipCot and VipTobacco leaves (100% GM) were serially diluted with DNA (0% GM) isolated from their respective counterpart non-GM leaves, to obtain solutions with different percentages of GM trait (100,50,10,1.0,0.1 and 0.01%, respectively) and these solutions were used to establish the limit of detection (Table 1).

Table 1 The procedure for reference standard and calculation of “genetically modified organism” (“GMO”) genome copies in genetically modified (GM) cotton and tobacco
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DNA extraction

Isolation of DNA from the test material (i.e., GM and non-GM cotton and tobacco leaves) was carried out using the QIAGEN DNeasy plant mini kit (QIAGEN, Germany). To simulate processed food conditions, GM cotton and tobacco leaves were autoclaved at 121 °C and 15 psi for 20 min. The autoclaved leaf samples were then used for DNA isolation at room temperature. The plasmid DNA from E. coli recombinant clones and from Bacillus thuringiensis subsp. tolworthi HDB-8 (HD125) were isolated by the miniprep alkali lysis method [13].

The DNA concentration of the extracted DNA was measured by UV absorption at 260 nm and the purity was evaluated by the A 260 nm/A 280 nm ratio using a UV spectrophotometer (Spectronic Unicam, Genesys, USA). The isolated DNA was diluted to a final concentration of 50 ng/μl and used as stock solution for the PCR analysis.

Oligonucleotide primers

The oligonucleotide primer sets for vip3A used in this study were designed with help of the ABI Prism software Primer Express 2.0 (Applied Biosystems, USA). Prior to designing the primer, a highly conserved sequence of approximately 300 bp was selected by multiple sequence alignment using the ClustalW method of the MegAlign program of DNA STAR software (DNASTAR, USA). This conserved sequence was used for designing the primer for the vip-s gene and vip3A-like genes. The primer pairs for the Cauliflower mosaic virus (CaMV) 35S promoter, nopaline synthetase (nos) terminator and neomycin phosphotransferase (npt-II) marker gene were selected directly from published literature [14–16]. The proposed primers for synthetic vip-s and vip3Aa1 were designed on same backbone of the native vip-s primer set with minor modification and were verified by BLAST analysis. The characteristic features of the primer sets designed are given in Table 2.

Table 2 List of primer pairs employed for PCR assays
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Desalted oligonucleotide primers were purchased from Bangalore Genei, India, diluted to a final concentration of 20 μM with Milli-Q water and stored at −20 °C until use.

PCR conditions

The entire PCR assay was programmed in an epigradient Mastercycler (Eppendorf, Germany) and reagents for standard single PCR assays were procured from Bangalore Genei, India. The PCR comprised of 25 μl of 1× PCR buffer containing 10 mM tris(hydroxymethyl)aminomethane (Tris)–HCl pH 9.0, 50 mM KCl and 1.5 mM MgCl2. A typical optimal assay consisted of the following: 0.25 μM each primer, 200 μM dNTP, 100 ng template DNA and 1.0 U Taq DNA polymerase. The standard PCR cycle conditions were as follows: initial denaturation at 94 °C for 4 min, followed by 35 cycles, which involved a denaturation step at 94 °C for 30 s, annealing at 62 °C for 30 s and extension at 72 °C for 1 min, followed by a final extension at 72 °C for 7 min. The optimal annealing temperature of 62 °C was ascertained by a gradient PCR assay. To confirm the plant origin of extracted DNA, chloroplast DNA (trnl intron) amplification, with a universal primer for three noncoding regions of chloroplast t-RNA [15, 17] under the assay conditions stated above, was routinely done.

Multiplex PCR assay was performed using a primer concentration of 0.25 μM for vip-s, 0.20 μM for CaMV 35S promoter with duplicated enhanced region (P-e35S), 0.16 μM for npt-II and 0.14 μM for the 3′ nontranslated polyadenylation signal of Agrobacterium tumefaciens nos gene (T-nos) and the annealing temperature was maintained at 62 °C. The multiplex reaction sample was prepared using QIAGEN multiplex PCR master mix solution, which contains-HotStarTaq DNA polymerase, dNTP mix and multiplex PCR buffer with a final concentration of 3.0 mM MgCl2. The multiplex PCR cycling program was adjusted to have an initial denaturation step at 95 °C for 14 min followed by 35 cycles, which involved a denaturation step at 95 °C for 30 s, annealing at 62 °C for 1 min and extension at 72 °C for 1 min, followed by a final extension at 72 °C for 7 min.

Construct-specific PCR amplification was carried out by a long-run PCR assay. The reagents for long-run PCR assay were procured from Fermentas, USA. A typical reaction assay contains long PCR enzyme mix, 1× buffer, 1.5 mM MgCl2, 200 μM dNTP and 0.20 μM each primer. The PCR cycling program was modified to have an initial denaturation step at 95 °C for 2 min followed by 30 cycles, which involved a denaturation step at 95 °C for 30 s, annealing at 55 °C for 30 s and extension at 68 °C for 2.5 min, with autoextension of 2 s per cycle, followed by a final extension at 68 °C for 10 min. The extension time was ascertained by calculating the length of the construct-specific amplicon (Fig. 8a). For nested PCR assay, the long-run PCR amplicon was purified using the QIAGEN QIAquick gel extraction kit. The nested PCR program and assay conditions were same as those of the standard PCR assay.

Restriction endonuclease analysis

The restriction endonuclease analysis of the 284-bp PCR amplified product of vip-s was carried out to ascertain the specificity and confirmation of the result. The restriction map of the 284-bp PCR amplified product of vip-s was developed with the help of DNA STAR software (Fig. 6a)). The reagents used for restriction analysis were from New England Biolabs, USA. An aliquot of 25 μl (200 ng) 284-bp PCR-amplified vip-s DNA product was put into a microfuge tube and 10 U of restriction enzyme RsaI in 1× NEB buffer 1 (50 mM potassium acetate, 20 mM Tris acetate, 10 mM magnesium acetate and 1 mM dithiothreitol, pH 7.9) was added, and digestion was allowed to proceed for 3 h at 37 °C. In a parallel experiment, an aliquot of 25 μl (200 ng) 284-bp PCR-amplified vip-s DNA product was mixed with restriction enzyme DdeI (10 U) and digested similarly using 1× NEB buffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 and 1 mM dithiothreitol, pH 7.9).

Agarose gel electrophoresis

The amplified products were analyzed by electrophoresis on 2.0% (w/v) agarose gel in buffer containing 89 mM Tris, 89 mM boric acid, 2.0 mM EDTA (0.5× TBE buffer, pH 8.0) and visualized after ethidium bromide staining, with an Alphalmager 2200 gel documentation system (San Leandro, CA, USA). For multiplex PCR products, 2.5% (w/v) agarose gel was used, while for DNA quality checks and long-run PCR amplicons, 1.0% (w/v) agarose gel was used. The running conditions were constant voltage at 80 V for 2 h. The gene rulers of a 100-bp DNA ladder (1,000–100 bp) and a 1.0-kb DNA ladder (10–1.0 kb) were used as a marker. For restriction fragments analysis, a 10% (w/v) polyacrylamide gel was used in 0.5× TBE buffer for proper resolution of bands.

Results

The isolated cotton and tobacco leaf DNA samples were of good quality, displayed an A 260 nm/A 280 nm ratio of 1.7–1.9 and had an absolute concentration of 100–200 ng/μl. The consistent chloroplast t-RNA gene (the intron disrupting the trnl gene) amplification confirmed the plant origin of DNA and the proper DNA isolation [17]. The PCR assay generating the 500–600-bp amplicon of this gene served as a positive control.

A premise control (blank, no DNA) run with each assay confirmed the absence of laboratory contamination or any cross-contamination during sample processing and the assay run. The DNA isolated from nontransgenic cotton or tobacco leaf served as a negative control.

Optimal annealing temperature

To ascertain the optimal annealing temperature, template DNA from GM cotton (VipCot14) was employed and a gradient PCR assay performed. The vip-s gene fragment could be amplified with nearly equal intensity in the temperature range of 50–62 °C (Fig. 1). However, an annealing temperature of 62 °C was chosen for optimal concerted annealing, better amplification and better yield in multiplex PCR assays.

Fig. 1
figure 1

Effect of various annealing temperature on vip-s amplification. Lane M, 100-bp DNA ladder; 1, premise control; 2–12, genetically modified (GM) cotton (VipCot14) template at different annealing temperatures of 50.2, 51.1, 52.5, 54.3, 56.2, 58.3, 60.2, 62.0, 63.5, 64.6, 65.1 °C, respectively; 13, negative control (non-GM cotton); 14, positive control (chloroplast t-RNA gene)

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Specificity of the vip-s primer set

The PCR assay for vip-s detection with the target DNA material showed only one specific amplicon of the expected size, i.e., 284 bp. Four GM leaf samples (two cotton samples—VipCot14 and VipCot29—and two tobacco samples—VipTobacco1 and VipTobacco2) were tested under optimal PCR assay conditions and all of them specifically produced a 284-bp amplicon, attesting the presence of the vip-s gene (Fig. 2). Our assay could also effectively demonstrate the presence of vip3A in a wild-type natural isolate of Bacillus thuringiensis subsp. tolworthi HDB-8 (HD125) and in two E. coli recombinant clones (pQE30 and pET29a). The corresponding nontransformed cotton and tobacco leaves and E. coli DH5α served as respective negative controls.

Fig. 2
figure 2

Detection of vip-s in GM cotton, GM tobacco, Bacillus thuringiensis wild-type isolate and Escherichia coli recombinant clones Lane M, 100-bp DNA ladder. The numbers at the top indicate the template DNA used in each lane: 1, no template—premise control; 2, GM cotton VipCot14; 3, GM cotton VipCot29; 4, non-GM cotton; 5, GM tobacco VipTobacco1; 6, GM tobacco VipTobacco2; 7, non-GM tobacco; 8, B. thuringiensis subsp. tolworthi HDB-8 (HD125); 9, Escherichia coli recombinant clone pQE30; 10, E. coli recombinant clone pET29a; 11, negative control—E. coli DH5α; 12, positive control—chloroplast t-RNA gene)

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In addition to these experimental data, the multiple sequence alignment data (Fig. S1) are strongly suggestive that as many as 20 natural wild-type isolates of vip3A-like genes bearing Bt (NCBI accession nos. Y17158, AF373030, AF399667, AF500478, AF548629, AJ971413, AY044227, AY074706, AY074707, AY074708, AY187679, AY295778, AY489126, AY739647, AY743436, AY945939, DQ016968, DQ016969, L48811, L48812) could also be detected using the vip-s primer set under specified assay conditions.

To confirm specific amplification efficacy, the primer pair was analyzed with the NCBI BLASTN 2.2.12 program and the GenBank database was searched. The search indicated that this vip-s primer set has no sequence homology with existing DNA sequences of other agricultural plants and microorganisms. In order to further validate the specificity and efficiency of the vip-s primer set, 100 ng plant target DNA for the vip-s gene was spiked with an equal amount of nontarget DNA sourced from diverse GM plant seeds and amplification was done. The nontarget DNA samples used in this study were cotton (MECH12Bt), soybean (Round up ready) and maize (MON810). No amplification was observed with nontarget GM DNA samples. The gel run data clearly indicated that there was virtually no interference by spiked contaminants on PCR amplification and no nonspecific bands were present (Fig. 3). Thus, the ability of the primer pair to specifically amplify the vip-s gene was confirmed.

Fig. 3
figure 3

Effect of spiking with non-target DNA on vip-s detection. Lane M, 100-bp DNA ladder. The numbers at the top indicate the template DNA used in each lane: 1, no template—premise control; 2, GM cotton VipCot14; 3, non-GM cotton VipCot; 4, VipCot14 cotton spiked with MECH-12Bt cotton; 5, MECH-12Bt cotton; 6, VipCot14 spiked with RR Soya; 7, RR Soya; 8, VipCot14 spiked with MON810 maize; 9, MON810-maize; 10, positive control—chloroplast t RNA gene

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Limit of detection

The detection limit for any transgene is governed by four factors—transgene copy number, PCR amplicon size, primer pair efficiency and optimal assay conditions. To establish the limit of detection, reference material was prepared by mixing GM DNA with non-GM DNA in different proportions as detailed in Table 1. As is known, in any transgenic plant the presence of only a single copy of a transgene is expected in the nuclear DNA. The “GMO” genome copy number for each serially diluted reference DNA sample was calculated, on basis of the nuclear DNA content of cotton (Gossypium hirsutum) and tobacco (Nicotiana tabacum) [18]. The cotton nuclear DNA content is reported to be 6.1–6.5 pg/2C; thus, the 1C value would be 3.2 pg and the number of “GMO” genome copies in 100 ng VipCot DNA will be 3.1 × 104 (100 ng/3.2 pg). In the case of tobacco, the nuclear DNA content is 7.8–9.7 pg/2C; hence, the 1C value will be 4.4 pg and number of “GMO” genome copies in 100 ng VipTobacco DNA will be 2.3 × 104 (100 ng/4.4 pg) [19, 20]. Our data showed that 0.1% vip-s GM material, with 100 ng template DNA per assay, can easily be detected both in cotton and in tobacco (Fig. 4). This concentration represents 31 and 23 “GMO” copies for cotton and tobacco, respectively. However, theoretically, the presence of merely 1.0 copy of “GMO” genome for detection is adequate.

Fig. 4
figure 4

Limit of detection for vip-s transgene. Lane M, 100-bp DNA ladder; 1, premise control; 2–7, vip-s amplification of GM cotton containing 100, 50, 10, 1.0, 0.1 and 0.01% reference standard material, respectively; 8, negative control—non-GM cotton; 9, positive control—chloroplast t-RNA gene

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Detection in semiprocessed/processed GM food

In order to verify the detection efficacy of the vip-s primer set for processed food samples, VipCot and VipTobacco leaves were subjected to autoclaving at 121 °C and 15 psi for 20 min to simulate processed-food conditions. The DNA from thermally treated leaf samples was extracted at room temperature and the quality was checked by agarose gel electrophoresis. The DNA isolated from autoclaved samples appeared to be sheared and degraded (data not shown). This sheared DNA was employed as a template and marked amplification of the vip-s gene could still be seen. Though autoclaving diminished the amplification efficiency, the effect was very feeble and insignificant (Fig. 5). The limit of detection for DNA isolated from autoclaved GM samples remained at 0.1% despite decreased amplification (Fig. S2). The successful amplification of template DNA from thermally treated leaf samples, simulating processed food, validates the choice of a short amplicon for vip3A-like gene detection.

Fig. 5
figure 5

Effect of thermal treatment (simulated processed food condition) on vip-s detection. Lane M, 100-bp DNA ladder. The numbers at the top indicate the template DNA used in each lane: 1, no template—premise control; 2, VipCot cotton; 3, VipCot cotton autoclaved; 4, VipTobacco; 5, VipTobacco autoclaved; 6, negative control—non-GM cotton; 7, positive control—chloroplast t-RNA gene

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Confirmation of the vip-s amplicon

To confirm the identity of the vip-s amplicon, restriction endonuclease assay was done. The restriction map for the 284-bp amplicon of vip-s was developed using the MapDraw program of DNA STAR software. There are 25 unique sites in the 284-bp region of the vip-s amplicon which could be cut only at one place. However, only 19 unique sites were selected on the basis of restriction fragment size and their subsequent resolution by gel electrophoresis (Fig. 6a). This restriction unique map serves a very important and necessary function. During development of GM plants, synthetic genes with plant-preferred codons are employed which might cause alterations in the restriction sites. The reported map will assist in selection of the appropriate restriction enzymes for vip3A amplicon analysis. We chose RsaI and DdeI restriction enzymes, which were expected to yield two distinct fragments of 147 and 137 bp and 213 and 71 bp, respectively. The fragments generated, following restriction digestions, were visualized after a 10% polyacrylamide gel electrophoresis run. The data validated the vip-s amplicon identity (Fig. 6b). The synthetic vip-s, on RsaI digestion, is likely to generate two restriction fragments of 200 and 84 bp, whereas the DdeI digest would generate three restriction fragments of 178, 71 and 35 bp.

Fig. 6
figure 6

a Map illustrating unique restriction sites for vip-s amplicon analysis. b Polyacrylamide gel electrophoresis analysis of restriction fragments of the vip-s PCR amplicon. Lane M, 100-bp DNA ladder (MBI Fermentas); 1, 4, undigested vip-s PCR product (284 bp); 2, DdeI digested vip-s PCR product; 3, RsaI digested vip-s PCR product; 5, environmental control (no DNA); 7 100-bp DNA ladder (Bangalore Genei)

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Multiplex PCR assay for the vip-s transgene construct

In order to develop a qualitative PCR method for detecting the presence of more than one gene in GM samples in a single tube run, multiplex PCR assays were developed. The duplex, triplex and quadruplex PCR assays (Fig. 7) showed concurrent detection of the vip-s insert along with other GMO-specific sequences, i.e., npt-II marker gene, P-e35S and T-nos. The duplex and triplex assays were run using the vip-s primer set along with multiple combinations of primer pairs of npt-II marker, P-e35S and T-nos. The standard single PCR assays for all individual sequences, i.e., vip-s, P-e35S, T-nos and npt-II, showed a very high and clear amplification. In multiplex assays too the amplicons were well amplified and clearly visible. We observed that with an increase in the number of amplicons in each assay (duplex, triplex, and quadruplex) the band intensity showed a gradual decrease.

Fig. 7
figure 7

Standard single PCR and multiplex PCR analysis of cotton VipCot for amplification of vip-s, marker gene and regulatory sequences of the transgene construct. Lane M, 100-bp DNA ladder; 1, premise control, no template; 2–5, standard single PCR assay (2, vip-s; 3, P-e35S; 4, T-nos; 5, npt-II); 6–8, duplex PCR assay (6, vip-s + P-e35S; 7, vip-s + T-nos; 8, vip-s + npt-II); 9–11, triplex PCR assay (9, vip-s + npt-II + P-e35S; 10, vip-s + T-nos + P-e35S; 11, vip-s + npt-II + T-nos); 12 quadruplex PCR assay (vip-s + npt-II + P-e35S + T-nos); 13, negative control—non-GM cotton; 14, positive control—chloroplast t-RNA gene.

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Construct-specific amplification by long-run PCR assay

The whole vip-s transgene construct was amplified by employing a combination of the forward primer of P-e35S and the reverse primer of T-nos. The assay yielded an amplicon of approximately 3.1 kb, both for cotton and for tobacco (VipCot14, VipCot29, VipTobacco1, VipTobacco2) samples. The amplicon size matched the mathematically calculated value. The size of the vip-s gene is 2.63 kb (NCBI accession no. Y17158) and that of synthetic vip3Aa is 2.37 kb (NCBI accession no. DQ539888). The calculations included the 195-bp region of P-e35S, 180 bp of T-nos, 2.63 kb of the vip-s gene and some region of adjoining sequences, the sum being approximately 3.1 kb. The pBI121 plasmid DNA (CLONTECH, USA) having the gus gene flanked by CaMV 35S promotor (P-35S) and T-nos was used as a positive control in this experiment. The covering DNA sequence between the promoter and the terminator was calculated to be 2.281 kb (NCBI accession no. AF485783) and was confirmed by long-run assay (Fig. 8).

Fig. 8
figure 8

a Transgene construct for VipCot and VipTobacco, Binary vector (pBI121) and SYN-IR1ø2-7 (COT102) transgenic line. The arrowheads indicate the location and direction of the forward primer (to anneal with the promoter) and the reverse primer (to anneal with terminator) for entire transgene construct amplification. b Long-run PCR assay for whole transgene construct amplification. Lane M, 1.0-kb DNA ladder. The numbers at the top indicate the template DNA used in each lane: 1, no template—premise control; standard long-run PCR assay 2, GM cotton (VipCot14); 3, VipCot29; 4, GM tobacco 1; 5, GM tobacco 2 ; 6, negative control—non-GM cotton; 7, positive control—pBI121 plasmid DNA

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Confirmation of the construct-specific amplicon by nested PCR

For further confirmation, the approximately 3.1 kb long-run PCR amplicon was analyzed using a nested PCR assay. The same primer pairs of vip-s, P-e35S and T-nos, as used in standard PCR, were employed for nested PCR assay. The nested PCR yielded amplicons of 284, 195 and 180 bp corresponding to vip-s, P-e35S and T-nos respectively (Fig. 9). Thus, the long-run PCR data were fully confirmed and appropriately validated.

Fig. 9
figure 9

Nested PCR assay to confirm the long-run PCR product. Lane M, 100-bp DNA ladder; 1, premise environmental control, no template. The long-run PCR-generated amplicon served as template DNA; using specific primers, nested PCR assay showed amplification of 2, vip-s; 3, P-e35S; and 4, T-nos

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All the experiments detailed in this study were done more than 16 times, with at least four replicates of each sample. The results are exceptionally consistent and reproducible. The data depicted in the figures are of typical representative experiments.

Discussion

Despite their wide use, Cry protein based industrial formulations are not very effective against some of the agronomically important insects. The lepidopteron black cutworm (BCW; Agrotis ipsilon), a worldwide pest that attacks over 50 crops, including cereal grains, is one such example. Vip3 shows acute bioactivity against BCW, 260 times higher than other Cry proteins [21] and has broad-spectrum insecticidal activity against lepidopteron insects. Currently, field trials of crops carrying the vip3A gene are being done at several locations globally. In view of the commercial importance of cotton, intense research work on the introduction of the vip3A gene individually or in conjugation of a herbicide-resistance gene is being focused on in India, Australia and the USA [2, 6, 22]. One multinational company has developed cotton with the vip3Aa1 gene and is conducting field trials in India [23].

Thus, in the very near future, there will be a pressing need for detection and identification of vip3A-carrying products to comply with the regulatory needs for labeling of such GM produce and products thereof. Hence, the need for an easy and reliable detection method for vip3A-based transgenic plants is very urgent and crucial.

PCR-based assays are the preferred choice for such detection studies owing to their high sensitivity and specificity [24]. A PCR-based assay for vip3A-like gene screening in natural isolates of Bt has been proposed by Rice [11]. However, no PCR-based procedure to detect vip3A-bearing GM crops and produce is available in the public domain. This prompted us to develop a PCR assay to detect the presence of the vip3A gene in plants or food products. The primer sets designed are likely to detect as many as 20 natural wild-type vip3A-like-gene-bearing Bt isolates and two synthetic vip3A-carrying transgenics as is evident by our experimental data and the multiple sequence alignment data (Fig. S1).

Our study demonstrated the detection of the vip3A-type gene in transgenic cotton and tobacco leaves, as well as that in a wild-type natural isolate (Bacillus thuringiensis subsp. tolworthi HDB-8) and in two E. coli recombinant clones (pQE30 and pET29a). It is very likely that the vip-s primer set reported will be able to effectively detect newer naturally occurring vip3A isolates as well.

The amplicon length seems to be a crucial factor for detection in processed foods [12]. The short amplicon length and high annealing temperature of the assay are conducive for enhanced detection efficiency to detect vip3A in semiprocessed/processed foods.

We speculate that these vip3A primer sets could also be used to determine the stability of transgenic DNA in duodenal fluid as a part of human safety assessment [25].

The limit of detection, as established by standard single PCR assay, was found to be 0.1% for vip-s-bearing GM plants; thus, as little as 100 pg vip-s DNA originating from 100% GM cotton/tobacco samples could be detected sufficiently by this assay. Though the detection method is a qualitative one, it provides for identification of GMOs with a detection limit far below the requirement of the stringent EU regulations of 0.9%.

This unique primer set for vip-s, employed in single PCR, was equally effective in the multiplex PCR format. The concurrent amplification of vip-s along with other well-established GMO-specific sequences, i.e., npt-II, P-e35S and T-nos in GM cotton and tobacco, suggested that multiplex PCR (duplex, triplex and quadruplex) assay can assist in construct-specific vip-s sequence identification as well (Fig. 7). These GMO-specific sequences are important for the selection and expression of the transgene and are present in more than 80% of GM plants. These sequences do not occur in non-GM plants since P-e35S is of viral origin (CaMV) and T-nos is of bacterial origin (Agrobacterium tumefaciens).

The amplification of the whole transgene construct by long-run PCR assay was mediated by the predominant GM regulatory sequences, P-e35S and T-nos (Fig. 8), and such a construct-specific amplification followed by a nested PCR assay would be specific for vip3A-based transgenics only, being otherwise not present in nature. The amplification of the whole transgene, by long run, in different vip3A-bearing transgenic plants, would, however, yield transgene-specific amplicons of various sizes. During transgene inheritance different adverse effects such as deletion, duplication, rearrangement and repeated sequence recombination for transgenic loci have been reported [26]. Hence, such a construct-specific PCR assay could be employed to confirm the transgene structural stability over succeeding generations.

Future directions

Assay modifications for detection of synthetic vip3Aa1-gene-bearing transgenics

The PCR assays used in identifying all four GM leaf samples (two cotton and two tobacco, developed by ICGEB, but not yet commercially released) was based on a vip-s primer set. Recently, Syngenta has released GM cotton COT102 (SYN-IR1ø2-7), bearing a modified version of the native gene of vip3A, in the USA and Australia but it is not accessible to us and no PCR test for this vip3Aa1-carrying transgenic could be done. Nevertheless, owing to the commonality of crucial factors governing detection, we strongly believe that the proposed primers for vip3Aa1 (COT102) would allow amplification both in single and in multiplex assay formats. The nucleotide sequence of this gene is now available from the NCBI and on this basis we designed a primer set for amplifications of this gene as well. As stated earlier, the design of the proposed primer has the backbone of the vip-s primer set. The detection of this gene would require the use of the vip3Aa1 primer set instead of the vip-s primer set (Table 2). Our hypothesis regarding successful amplification of the vip3Aa1 gene is based on the facts that the major backbone of the primers is common, the annealing position is the same, i.e., at 333–356-bp regions for the forward primer and 594–616 bp for the reverse primer in the coding sequences part of the vip3A gene and even the amplicon size (284 bp) would be same. The statistical analysis of primer sequences, by DNA STAR software, showed the melting temperature of vip3Aa1 primer sets to be 73 °C, which is higher than that of vip-s (64 °C), and would be conducive for amplification.

To distinguish among all the known vip3A-bearing transgenics, apart from the use of distinct primer sets, restriction analysis can also be carried out. In the context of this study, a common set of the restriction enzymes could be used, but the fragments generated would be of different sizes. The enzyme RsaI would generate four restriction fragments, 17, 72, 111 and 84 bp, for the vip3Aa1 amplicon after complete digestion. The digestion of vip-s yielded fragments of 147 and 137 bp (Fig. 6b). The DdeI-mediated digestion of the vip-s amplicon yielded fragments of 213 and 71 bp. Similar action of DdeI would, however, generate four restriction fragments (41, 30, 21 and 192 bp) of vip3Aa1. Hence, with this approach, detection/distinction of either of the two vip3A genes in GMOs (seed, foods, powder, etc.) in a mixed lot would be feasible.

For identification and validation of construct-specific characterization of transgenic COT102, the long-run PCR and subsequent nested PCR amplification would need a minor modification. Since, the promoter sequence used in COT102 is the promoter and first intron of the A. thaliana ubiquitin-3 gene instead of the CaMV 35S promoter upstream of the vip-s gene, the long-run assay would require the forward primer from the A. thaliana ubiquitin-3 gene promoter and appropriate annealing time. The importance of construct-specific characterization for all GM crops is paramount to avoid the mistake made by Syngenta in marketing Bt-10 instead of Bt-11 maize in 2004 [27].

Conclusions

The proposed single PCR assay(s) can serve as a general screening method for all vip3A-like-gene-bearing GMOs. The multiplex PCR assays (duplex, triplex and quadruplex) can be employed for specific detection of vip3A-bearing GM plants, feed and foodstuff. The long-run PCR coupled with nested PCR would facilitate construct-specific identification. The assays will have immense application in vip3A gene labeling and identification studies and will facilitate faster regulatory compliance.