NAIMA as a solution for future GMO diagnostics challenges

Anal Bioanal Chem (2010) 396:2229–2233 DOI 10.1007/s00216-009-3197-7

TECHNICAL NOTE

NAIMA as a solution for future GMO diagnostics challenges David Dobnik & Dany Morisset & Kristina Gruden

Received: 29 July 2009 / Revised: 23 September 2009 / Accepted: 26 September 2009 / Published online: 12 October 2009 # Springer-Verlag 2009

Abstract In the field of genetically modified organism (GMO) diagnostics, real-time PCR has been the method of choice for target detection and quantification in most laboratories. Despite its numerous advantages, however, the lack of a true multiplexing option may render real-time PCR less practical in the face of future GMO detection challenges such as the multiplicity and increasing complexity of new transgenic events, as well as the repeated occurrence of unauthorized GMOs on the market. In this context, we recently reported the development of a novel multiplex quantitative DNA-based target amplification method, named NASBA implemented microarray analysis (NAIMA), which is suitable for sensitive, specific and quantitative detection of GMOs on a microarray. In this article, the performance of NAIMA is compared with that of real-time PCR, the focus being their performances in view of the upcoming challenge to detect/quantify an increasing number of possible GMOs at a sustainable cost and affordable staff effort. Finally, we present our conclusions concerning the applicability of NAIMA for future use in GMO diagnostics. Keywords Biochips . High-throughput screening . Multiplex . Genetically modified organisms . Detection . Quantification . Identification

D. Dobnik and D. Morisset contributed equally to this work D. Dobnik : D. Morisset (*) : K. Gruden Department of Biotechnology and Systems Biology, National Institute of Biology, Vecna pot 111, Ljubljana 1000, Slovenia e-mail: [email protected]

Introduction Regulations requiring traceability of GM products on the market have been adopted by many countries. This is sometimes accompanied by the compulsory labelling of products containing genetically modified organisms (GMOs) above a certain threshold. In the latter case, quantitative methods are required by GMO detection laboratories in order to accurately determine GMO content. Currently, PCR-based technologies are applied in the detection of GMOs, and real-time PCR is the method of choice for quantitative analysis in most GMO detection laboratories [1]. Real-time PCR allows sensitive and specific identification, as well as quantification of single GMOs [2]. To face the challenge of the increasing presence of GMOs on the market and their growing taxonomic and biotechnological diversity, it is necessary to introduce analytical technologies allowing highthroughput GMO diagnostics. Microarrays are already routinely used in several fields of research where a multiplex detection approach is required [3–5], and this technology can be adapted for the purpose of GMO detection and quantification. However, one limitation to microarray technology is that samples cannot be directly hybridized onto the microarray due to the high required sensitivity in terms of GMO target DNA copies within the background of non-transgenic genomic DNA [6]. This problem can be overcome by adding a PCR amplification step prior to hybridization onto the microarray. Nevertheless, the use of microarray hybridization may suffer from the lack of true multiplexing due to the limitations of amplification by PCR [7]. Consequently, a real need for development of novel alternatives to PCR-based technology, that would allow multiplexing with equivalent or improved performances, was identified [8, 9].

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Recently, several multiplexing methods have been developed or optimised for the purpose of GMO detection and/or quantification within the European Integrated project Co-Extra (http://www.coextra.eu). One of these is the NASBA implemented microarray analysis (NAIMA), a novel multiplex quantitative DNA-based target amplification method based on the NASBA principle, which is suitable for sensitive, specific and quantitative GMO detection on microarray technology [10]. In this article, a brief summary of the NAIMA technology and its principle is presented. NAIMA performances are then compared with those of the benchmarking technology (real-time PCR) the focus being on the performances in view of the upcoming challenge of efficiently detecting and quantifying the constantly increasing number of GMOs placed on the market.

NASBA implemented microarray analysis (NAIMA) Principle Nucleic acid sequence-based amplification (NASBA) is an isothermal technique that uses a combination of three enzymes: the avian myeloblastosis virus reverse transcriptase/DNA polymerase, T7 RNA polymerase and RNase H. A target-specific primer must be labelled with a T7 promoter to enable amplification [11]. Originally developed as an RNA amplification method, NASBA has been shown to be appropriate for DNA-target amplification [12] as well. Morisset and co-workers recently reported the development of a new NASBA-derived method which is suitable for use in combination with microarray detection, and was thus named NASBA implemented microarray analysis (NAIMA) [10]. NAIMA amplification occurs in two steps. The first is a multiplex template synthesis reaction during which several pairs of tailed primers are extended to produce templates bound to universal regions (Fig. 1). The use of tailed primers is required to obtain effective multiplex DNA target amplification using NASBA. The forward primers harbour the promoter sequence of the SP6 RNA polymerase (SP6 sequence) at their 5′-end. This sequence plays the role of a universal region in the second step of the method. At their 3′region, the forward primers consist of a target-specific sequence. The reverse primers, however, are tripartite. Their 3′-region is target specific, the central region is composed of an abiotic sequence and their 5′-end consists of the T7- RNA polymerase promoter sequence (T7 sequence) upon which NASBA amplification relies. The central abiotic sequence plays a role in the second step because it enables the restoration of the functional T7 promoter. The T7- and abiotic sequences represent the second universal region in the second step of NAIMA.

D. Dobnik et al.

The DNA templates synthesized during the first step are directly transferred in the second, universal amplification step using the SP6- and T7-universal primers (Fig. 2). These allow for a more uniform amplification of the different target sequences while conserving their initial ratio. After NASBA amplification, NAIMA products are directly ligated to 3DNA dendrimers harbouring 15 fluorescent dyes per molecule of dendrimer, conferring signal amplification in addition to the target amplification. Ligated products are subsequently hybridized to a customized oligonucleotide microarray [10]. NAIMA performance NAIMA was tested on different seed, food, feed and plant samples, including certified reference materials as well as complex samples from our laboratory’s routine diagnostics. On a triplex platform, NAIMA amplification reaches 108fold in as little as 45 min, when the reaction reaches a plateau. Both amplification and detection of NAIMA products on a microarray are specific, with no observed false positive results. NAIMA amplification is linear over a broad range of target concentrations, allowing for the quantitative analysis of the samples with acceptable repeatability, as shown by detection on the microarray, and indicating the quantitative properties of the on-chip detection system. The sensitivity or the absolute limit of detection (absolute LOD, the smallest amount of GM target copies that can be detected) of NAIMA is a few target copies [10]. Regarding the relative limit of detection (relative LOD, the smallest amount of GM target copies detectable relative to the amount of reference target copies), NAIMA is able to amplify and detect GM targets with as low as 0.1% of GM content, with a quantification range spanning from 0.1 to 25% GM content. Additionally, as little as 60 pg of starting DNA material is sufficient to perform the reaction. The method can easily cope with analysis of multiple samples per day. Sixteen to 24 samples can be analysed in multiplex reactions in our laboratory within a working day. Comparison with real-time PCR: pros and cons Performance PCR and especially its quantitative derivative, real-time PCR, are the standard testing technologies in most GMO detection laboratories. Real-time PCR can cope with simple and complex sample matrices, is very sensitive, specific and allows a wide dynamic range of quantification [13]. Amplification is followed in real-time with the use of fluorescently labelled molecular probes, allowing the results to be obtained in approximately 2 h.

NAIMA as a solution for future GMO diagnostics challenges

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Fig. 1 NAIMA template synthesis step. After denaturation of the target DNA, tailed primers with a sequence complementary to the target DNA and 5′-end sequences necessary for the multiplex amplification bind to the target DNA (1). Taq polymerase extends the tailed primers to produce the DNA template used in the amplification step (2). The pair of 5′-end sequences is identical to all the different targets (3). The DNA templates synthesized during this first step are flanked by a recognition site for DNAdependent T7 RNA polymerase (3)

The main drawbacks of real-time PCR involve the extent of multiplexing which is limited by the relatively small number of reporter dyes that can be simultaneously followed in a single reaction, by the poor sensitivity, the preferential amplification of certain specific targets and the possibility of unspecific amplification observed with the oligoplex real-time PCR methods [7, 14]. This explains why singleplex is the most commonly used format even though only a limited number of oligoplex real-time PCR methods have been reported [14, 15]. On the other hand, NAIMA offers the possibility

of multiplex amplification without loss of sensitivity or specificity. In a triplex format, NAIMA’s qualitative and quantitative performances are comparable to those of singleplex real-time PCR with similar specificity, an absolute LOD of a few target copies, a relative LOD of at least 0.1% and a relatively wide dynamic range of quantification [10]. With NAIMA, a shorter amplification time (25–45 min) than for real-time PCR is needed to reach the maximum target amplification with good sensitivity in a triplex format (Table 1) [10] (and unpublished data).

Fig. 2 NAIMA multiplex amplification step. The double-stranded DNA (dsDNA) templates synthesized during the first step are transcribed by the T7 RNA polymerase into numerous copies of antisense RNA molecules (1), which are later reverse-transcribed into single-stranded sense DNA (ssDNA) (2 and 3) to form an RNA–DNA duplex. This RNA–DNA duplex is degraded by RNAse H activity (4).

The ssDNA is then used as a template by the reverse transcriptase after T7-cap-extension primer annealing (5) to synthesize a second DNA strand (6). This dsDNA can be used as a template for several cycles of amplification. The final product of NAIMA is antisense cRNA. Only one pair of universal primers is needed in this amplification step for all the targets to be amplified

Several real-time PCR methods for specific detection/quantification of GM events have been validated in collaborative trials (see: http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htm). After in-house validation, NAIMA was transferred to a second laboratory to assess its reproducibility and robustness. No data available for the moment

f

Given as the maximum number of simultaneous amplifications in one reaction

Preliminary results (unpublished data) e

d

Given in absolute GM target copies, and content relative to the amount of reference target copies (%, in brackets)

As suitable for GMO diagnostics c

b

a Real-time PCR is the reference. In all tested samples, only the expected targets were amplified by NAIMA and qualitative results were identical to real-time PCR analysis of the same samples. See original publication for more details

NAIMA NASBA implemented microarray analysis, P plant material, S seed or seed flour, F food, f feed, PCR polymerase chain reaction, NASBA nucleic acid sequence-based amplification

[10] 25–45 10 (0.1%) No crossreactivity

Yes

3 (6e)

P, S, F, f

NASBA

Microarray

Collaborative trials validated Transfer in course Real-time 100 1 (0.1%) –

Real-time PCR NAIMA

Yes

1

P, S, F, f

PCR

Reproducibilityf Detection approach Amplification approach Tested matrices Amplification time (min) Multiplexingd Quantificationc Sensitivityb Specificitya Method name

Table 1 Properties of NAIMA and real-time PCR in GMO diagnostics

[13]

D. Dobnik et al. Sources

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NAIMA was thoroughly evaluated for its quantification performance on samples containing targets at different concentrations with high DNA background. Quantification results of the same samples were compared between singleplex real-time PCR analysis (for a GM contents range of 0.1% to 25%) and those analysed by NAIMA. For all analysed samples, NAIMA analyses results were in agreement with real-time PCR quantification [10]. These results show that NAIMA behaves similarly to real-time PCR in terms of quantitative analyses, even though slightly lower accuracy and higher variability are observed with NAIMA. For better comprehension, performances of real-time PCR and NAIMA methods are summarized in Table 1. Applicability and cost In comparison with real-time PCR, one limitation of NAIMA, as is the case for all other methods using microarray technology, is the time needed for hybridization (several hours) which significantly prolongs the analysis time. However, further optimization should allow for reasonable hybridization times. Indeed, the DualChip® GMO method only requires 1 h of hybridization [16] showing the possibility of reducing analysis time when using a microarray-based detection method. Microarrays and their theoretically unlimited number of spotted target-specific probes can provide high-throughput detection which compensates for the longer hybridization time. On a hexaplex platform (currently under development) NAIMA costs become competitive with the singleplex realtime PCR analysis, in terms of consumables and staff effort. Theoretically the costs would decrease even more with higher multiplexing of the technology. Therefore, owing to its microarray-based detection, NAIMA can become an advantageous alternative method to real-time PCR if the hybridization time were to be significantly reduced. One must keep in mind a drawback of microarray approaches as they are reported to lack reproducibility [17]. However, no such problem was observed in the case of NAIMA products detection on a microarray. This observation can be related to the good reproducibility obtained during the DualChip® GMO collaborative trial [16]: the microarray used in NAIMA experiments is based on the same technology as the DualChip® GMO microarray. Another possible pitfall of microarray application is that the data generated are prone to subjective analysis and interpretation. Several publications have already reviewed the various tools and concepts that help to properly interpret microarray results [17, 18]. Moreover, with smaller data sets as for GMO diagnostics, the use of ad hoc interpretation software could allow a less subjective data analysis, as is the case with the DualChip® GMO method [16].

NAIMA as a solution for future GMO diagnostics challenges

Finally, PCR needs multiple and precise thermal cycles, increasing the cost of the required equipment. On the other hand, in the case of the isothermal NAIMA amplification method, a simple thermoblock suffices. One of the drawbacks of NAIMA is the need for a laser scanner that is required for the detection of the hybridized products. A less costly solution for microarray equipment could be introduced with the use of non-fluorescent dyes (such as direct silver staining of NAIMA products or direct silver staining of biotin-labelled dendrimers). This solution would allow the use of simpler and cheaper equipment for microarray analysis.

Conclusion on the use of NAIMA in the future New and future GMO detection methods are foreseen to speed up and decrease the cost of qualitative detection and identification of GMOs. In this regard, NAIMA presents practical solutions for future GMO detection. NAIMA, owing to its potential for extended multiplex amplification and its theoretically unlimited microarray-based detection platform, shows good potential for high-throughput GMO analysis with the possibility of employing the matrix approach for UGM detection [9]. Owing to its high sensitivity and specificity, NAIMA equals real-time PCR’s performance. Finally, it is the only method so far that provides full quantitative results in a multiplex format. All these properties make NAIMA one of the most promising methods to confront the challenges of GMO detection in the future. However, higher amplification multiplexing must be proven in order to make the method cost-efficient in comparison with the current real-time PCR technology. Further improvements should also be made to this technology which would allow for shorter hybridization times to decrease the time needed for analysis. Better portability, in addition to simpler and cheaper equipment than the laser scanner needed for microarray analysis would also be beneficial. Acknowledgments This study was financially supported by the European Commission through the Integrated Project Co-Extra, Contract No. 007158, under the 6th Framework Programme, priority 5, food quality and safety and by Slovenian research agency (contract no. P4-0165). The authors thank Tina Likar for her critical review of the manuscript.

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