Detection of genetically modified plants using LAMP (loop-mediated amplification) technologies

Genetically modified (GM) plants, created using the recombinant DNA technologies, have been widely used all over the world since 1996. In 2019, 29 countries grew biotech crops on an area of 190.4 million hectares (Mha) [1]. The top producers of genetically modified plants are the United States (the total area occupied 71.5 Mha), Brazil (52.8 Mha), Argentina (24 Mha), Canada (12.5 Mha) and India (11, 9 Mha). In the global world market in 2019, the share of GM plants accounted for 79% from the area under all rapeseed crops in the world, 74% from the area under soybeans, 31% from the area under corn and 27% from the area under cotton [1]. The leader among biotech plants in terms of the total planted area was soybeans,


Introduction
Genetically modified (GM) plants, created using the recombinant DNA technologies, have been widely used all over the world since 1996. In 2019, 29 countries grew biotech crops on an area of 190.4 million hectares (Mha) [1]. The top producers of genetically modified plants are the United States (the total area occupied 71.5 Mha), Brazil (52.8 Mha), Argentina (24 Mha), Canada (12.5 Mha) and India (11,9 Mha). In the global world market in 2019, the share of GM plants accounted for 79% from the area under all rapeseed crops in the world, 74% from the area under soybeans, 31% from the area under corn and 27% from the area under cotton [1]. The leader among biotech plants in terms of the total planted area was soybeans, Borys Sorochynskyi https://orcid.org/0000-0002-6167-4071 which were cultivated on 91.9 Mha (while the area under crops in 2019 decreased by 4% compared to 2018), followed by corn (60.9 Mha), cotton (25.7 Mha), and rapeseed (10.1 Mha). The International Service for the Acquisition of Agri-biotech Applications (ISAAA) assumes that the level of use of the main GM crops has already reached its saturation [1]. It is likely that global producers of biotech plants will continue their further expansion into the seed market of the aforementioned crops using technologies other than recombinant DNA, like genome editing technologies. In addition to the above four main biotech crops (soybeans, corn, cotton and rapeseed), it is worth mentioning other genetically modified plants cultivated on an industrial scale -alfalfa (the area under crops in 2019 was 1.3 Mha), sugar beet (473 thousand hectares), sugar cane (20 thousand hectares), papaya (12 thousand hectares), sunflower (3.5  thousand hectares), as well as zucchini, apples and pineapples, which were grown on areas less than 1 thousand hectares. In general, the ISAAA database contains information on more than 300 different plants with genetically modified events "that have been approved for commercialization/planting and importation (food and feed)" (https://www.isaaa.org/gmapprovaldatabase/default.asp). When analyzing the list of authorized GM plants, it should be remembered that private and public research centers are actively working on the creation of new genetically engineered varieties for many plant species -rice, bananas, potatoes, wheat, mustard, chickpeas, peas, melons, flax, plums, etc., which will have various new beneficial properties and improved nutritional value. In addition to countries that grew GM plants, 42 countries around the world (26 EU member states, plus 16 other countries) also imported biotech plants for use as food, animal feed and for processing in 2019. Thus, in 2019, genetically modified plants were approved and used for various purposes in 71 countries of the world in total [1].
Despite the wide distribution of genetically modified plants, the attitude towards them in society continues to remain somewhat wary, which manifests in constant discussions regarding the possible risks to humans and animals from the use of GM plants and the products obtained with their use. About 50 countries have also introduced mandatory labeling of GM feed and products if their content exceeds a certain threshold value [2]. For example, the threshold for labeling GM products is 0.9% in the EU, 3% in South Korea, 5% in Japan [2][3][4]. It should be noted that GMO labeling in no way concerns the issue of their safety, but only informs the consumer about their presence.

Results
In order to meet labeling requirements, efficient and sensitive methods for detecting many known genetic modifications in a wide variety of plant materials, food products and animal feed must be developed and standardized. The most common methods for the determination of GMOs (genetically modified organisms) are based on the detection of specific proteins synthesized in transgenic plants due to the expression of new genes in their genome, and on the detection of the introduced genes themselves by analyzing certain nucleotide sequences of DNA isolated from a genetically modified organism. The developed enzyme immunoassay methods allow the determination of proteins encoded in transgenic plants by cp4-epsps, cry1Ab, cry1Ac, cry2A, cry2Ab, cry3A, cry9C, nptII, pat, gox, cpti genes [5]. Unfortunately, this inventory does not completely cover the entire list of GM plants currently presented on the market. Methods for the determination of GMOs based on the analysis of nucleic acids are the most common, since such methods are more sensitive and specific than the analysis of protein composition, and allow the detection of a significantly larger number of genetically modified plants. For the detection and monitoring of transgenic DNA, four main regions in the genome of a new organism are selected. These are specific nucleotide sequences characteristic of universal elements and marker genes that were inserted into the genetic cassette used for transformation; in fact, the newest introduced genes; genome section on the border between universal elements and introduced new genes; the boundary between the nucleotide sequence in the host genome and integrated new genes [6]. The use of methods for detection of these four regions in the genome makes it possible, respectively, to screen a particular sample for the presence of genetic modifications in it; determine the specific introduced gene; reveal the structure used for transformation; or identify a GM event. With the rapid emergence of new GM events, the most common methods of laboratory analysis are routine screening and determining the presence of a new gene or elements of the used construction. Since the 90s of the last century, most detecting laboratories began to actively use DNA-based polymerase chain reaction PCR) techniques. Since then, the polymerase chain reaction, through which a large number of copies of certain regions of the genome are possible to obtain, has become a routine method of molecular biology, biotechnology and genomics. PCR is widely used both in fundamental research and for solving various practical issues, including the detection of GMOs. Among the various PCR options used to detect genetically modified organisms, it is worth recalling the quantitative Polymerase Chain Reaction (qPCR), multiplex PCR, and digital drop PCR (ddPCR). Multiplex PCR attracts special attention, since it allows detecting several targets simultaneously. The publication [7] provides information on the use of multiplex PCR for the simultaneous detection from 4 to 9 different targets: • 4-Plex PCR (taxon specific soybean lectin (lec) gene, maize zein gene, element-specific sequence of 35s promoter of cauliflower mosaic virus and nos terminator (nopaline synthase gene terminator) are detected); • 5-Plex PCR (taxon specific gene of the maize alcohol dehydrogenases (adh) and GM events GA21, MON810, NK603, Bt11 are detected); • 6-Plex PCR (taxon specific acp1 gene -cotton gene that encodes an acyl carrier protein) and GM events Bollgard, Bollgard II, RR, 3006-210-23, 281-24-231 are detected); • 8-Plex PCR for the detection of 8 different GM events (Bt176, Bt11, HN1, RRS, T25, MON88913, MIR604, and MON1445) • 9-Plex PCR, where taxon specific hmg gene (highly mobile maize protein gene) and GMevents T25, GA21, TC1507, MON863, MON810, NK603, Bt176, Bt11 are determined.
Additional information on some other developed variants of multiplex PCR for GM plants detecting can be found in the review [5]. Further improvement of the methods for the simultaneous analysis of several different DNA targets was obtained thanks to the development of the so-called MPIC technologies (mul-tiplex Microdroplet PCR Implemented Capillary gel electrophoresis). This approach made it possible to detect simultaneously from 8 to 24 different GM events [8].
The main advantage of molecular detection methods based on the analysis of nucleic acids is their high specificity and sensitivity. Among the shortcomings of the methods, it is worth mentioning the need for sophisticated equipment that is not always available for laboratories with limited financial resources. The advantages and disadvantages of some methods for the determination of GMOs based on the use of the PCR are shown in Table 1.
The need for complex and expensive PCR equipment prompted the development of amplification methods that can be implemented at a constant temperature and, therefore, are an alternative to PCR. First of all, we are talking about isothermal amplification methods implemented at a constant temperature. Their main advantage is the absence of need for sophisticated equipment -you only need an ordinary laboratory thermostat, or a water bath. Among the methods of isothermal amplification of nucleic acids, it is worth mentioning stranddisplacement amplification (SDA) [10], helicasedependent amplification system (HAD) [11], rolling circle amplification (RCA) [12], loopmediated amplification (LAMP), Self-sustained sequence replication (3SR) [13] and nucleic acid sequence-bases amplification (NASBA) [14] and which is a further improvement of the 3SR method, Q beta replicase amplification, etc.
Isothermal amplification methods can be classified into two groups depending on how the nucleic acid is denatured -using enzymes or spontaneously, thanks to specific primers or probes. The methods are simple in terms of their implementation, and some of them can be used even in the field, since the main difference between isothermal amplification methods and PCR is that the reaction can occur at a constant temperature. The main purpose of this article is to analyze the experience of using the LAMP reaction for detecting genetically modified plants.
Loop-mediated isothermal amplification method was first developed by Japanese scientists in 2000 [15] and is based on the unique feature of DNA polymerase from the bacterium Bacillus stearothermophillus (Geobacillus), which, in addition to DNA polymerase, also has a high revertase activity. Usually, the reaction itself is carried out at a temperature in the range of 55-65 °C. LAMP reaction was first described using 4 primers, however, later it was found that the use of an additional pair of primers for loop formation significantly increases the sensitivity of the method [16].
The LAMP study can be implemented rather quickly, since in this amplification reaction, unlike PCR, there are no separate stages of denaturation, hybridization and synthesis. At the same time, the loop-mediated amplification Á³îòåõíîëîã³ÿ òà á³îáåçïåêà can be divided into the following conditional stages -initiation, cyclic amplification and elongation. For loop-mediated amplification DNA polymerase that can replace the strand during synthesis (B. stearothermophillus Bst DNA polymerase), Forward Inner Primer (FIP) and Backward Inner Primer, (BIP) and external primers (F3, B3) that recognize 6 different regions on the target are needed (Figure 1). Two looping primers are needed to form a loop, and two pairs of stripping primers are needed to synthesize linear nucleic acid strands.

Fig. 1. Description of primers used in (LAMP)
Internal primers FIP and BIP correspond to regions F2 (B2) and F1c (B1c At the beginning of the reaction, the primers forming the loop hybridize with F2 or B2 regions in order to initiate the synthesis of complementary DNA strands (Fig. 2, stage 2). After that, primers necessary for the synthesis of linear strands of nucleic acids hybridize with loci F3 or B3, and amplification of complementary DNA strands begins, which further leads to the release of the synthesized chains of the molecule (stage 3).
At this stage, the single-stranded chain of the nucleic acid molecule already has a nucleotide sequence that allows the formation of a loop-like structure (stage 4). The F1 and B1 regions at the 5'-end act as primers for generating a double-stranded loop (step 5). The regions containing the loop (F1 and B1) are single stranded, so new primers that generate the loop can hybridize with these regions.
As a result, a new complementary DNA strand is formed (stage 6, stage 7 and stage 8). At the 5'-end, a loop is formed (step 9), similar to how it happened in step 4. The synthesis of the molecule from regions F2 and B2, and the synthesis, which is caused by primers that generate a structure in the form of a loop, occurs alternately (steps 9, 10 and 11), which leads to the formation of a largesize reaction product containing nucleotide sequences that correspond to the target (step 11).
Further improvement of LAMP method was aimed at the development of its variants, allowing the detection of RNA. As a result, conditions for performing LAMP along with reverse transcription, (RT-LAMP) were developed. Reverse transcriptase is added to the reaction mixture in order to provide reverse transcription, which is carried out with the participation of primers that form a loop and primers necessary for the synthesis of linear strands of nucleic acids. RT-LAMP is used primarily for the detection of RNA-containing viruses, while the «traditional» LAMP is successfully used to determine various DNA-containing pathogenic microorganisms (viruses, bacteria, fungi) and parasites, to determine the sex of embryos, in research on the study of cancer [18].
Using the LAMP method, a very small amount of target DNA can be detected; it is also important that the amplification reaction can also occur in the presence of foreign nucleic acids. These circumstances make the method quite suitable for detecting GMOs. As in the case of PCR, the use of LAMP for detecting GMOs can be implemented in several ways: routine screening to study the presence of GMOs due to the detection of universal and most common regulatory elements, such as the 35s promoter and nos terminator, and the identification of a GM event, what requires specific primers.
The results of the detection of many transgenic plants by the method of loop-mediated isothermal amplification have been published in the scientific literature ( Table 2).
The minimum sensitivity of the LAMP met hod in the experiments of various authors, which are referenced in Table 2, was extremely high and made it possible to determine from 10 to 4 copies of the target gene.
The study [32] described the structure of primers and reaction conditions for the LAMP detection of universal elements (using the example of a reference material of transgenic soybean with resistance to Roundup (Roundup ready soybean), which is often used to create genetic constructs, such as the cauliflower mosaic vi-  GM event -specific sequence [34,35] rus (CaMV) 35S promoter, as well as the promoter and terminator of the nopaline synthase gene from Agrobacterium spp. The authors also showed the possibility of detecting transgenic MS8 and RF3 lines of oilseed rape (Brassica napus L.) using primers with affinity to a GM event of a specific locus. It was found that the sensitivity limit of the LAMP method for determining both the nos terminator and the  F1c

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35s promoter was 0.01%, what is well below the certain limit for labeling (0.9%), declared in the European Union regulations.
In publication [36], commercialized GM plants were analyzed and it was shown that the sensitivity of the LAMP method is 10 haploid genome equivalents (HGE) for the fmv35s promoter, cry1Ac gene, and pat gene. At the same time, when detecting the 35s promoter of the cauliflower mosaic virus, bar gene, nos terminator, cp4-epsps and nptII genes, the sensitivity limit of the method corresponded to 5 HGE. The authors successfully confirmed the potential of the LAMP method for screening studies using samples of commercialized varieties of GM rapeseed, soybeans, and corn.
In the paper [34], the possibility of visual detection of amplicons synthesized in loop-mediated isothermal amplification reaction was shown. For this, intercalating dyes were added to the reaction mixture, such as the asymmetric cyanine dye SYBR green, or HNB (hydroxynaphthol blue), which, by binding to the fragments of the double DNA strand synthesized in the reaction, can change their color. This appro-ach makes electrophoretic analysis of amplification products unnecessary and significantly reduces the time for research. In the cited work, the entire experiment lasted only about one hour, and in some publications cited above in Table 2, the LAMP reaction time was even less than one hour. The authors applied the developed technique to detect GM events in rice KMD1, TT51-1, and KF6. The obtained results allowed us to conclude that the LAMP method was more sensitive and specific in comparison with traditional PCR and can be used even in the field [34].
The authors of the article [28] published the results of studies where the bar gene in transgenic sugar cane was determined by LAMP and traditional PCR methods. It was shown that the use of LAMP made it possible to detect the transgene in 100 cases out of 100 studied samples (100%) and in 97 cases out of 100 studied samples (97%) in the case of PCR.
Taking into account the high sensitivity of the loop-mediated isothermal amplification met hod, it is logical to compare its advantages and disadvantages with the polymerase chain reaction method (Table 3). Attempts to improve the conditions for carrying out loop-mediated isothermal amplification continue constantly. Thus, an attempt was described to use, instead of the rather valuable Bst DNA polymerase from B. stearothermophillus, another Bsm DNA polymerase, which has similar properties, but is cheaper and more accessible to the user [38]. An example of creating a consumable device, which is able to maintain a constant temperature for a certain time, has been published, which allows further use of LAMP as an express method of analysis in the field [39]. Attempts to create biosensors based on the use of LAMP [40] and the search for new stable dyes for detecting nucleic acid fragments synthesized in the reaction are also continuing [24].
It should be noted that the improvement of existing and development of new methods for GMO detection concerns not only loop-mediated isothermal amplification. One can recall the introduction of capillary electrophoresis for amplicon analysis; the use of "peptide nucleic acids" (PNAs are synthetic homologues of nuc-leic acids that contain standard DNA nucleotides, but in this case the polyamide chain is replaced by repetitive units of N- (2-aminoethyl) glycine to which the nucleotide pairs are attached via a methyl carbonyl linker. The neutral chain in this form does not have the ability to repel when hybridized. This explains that PNA can bind to DNA or RNA with a high degree of specificity); development of various biosensors; application of microarray technology and the development of DNA chips; use of new genome sequencing (NGS) technologies for detecting GMOs, etc. [5,8,41].

Conclusion
Summarizing, we can say that the main criterion for the application of a particular method of GMO detection analysis is, first of all, its sensitivity, time of reaction, availability and ease of implementation, the cost of reagents and equipment, as well as the possibility for simultaneous detection of many samples. LAMP technologies fully meet many of these requirements.