As more and more genetically modified (GM) crops are approved for commercialization and planting, safety issues of GM crops have become hot topics worldwide. For both regulatory and academic purposes, development of rapid, economic and effective on-site detection methods for GM components is indispensible. Up to now, the most effective and sensitive techniques used for GMO detection are based on polymerase chain reaction (PCR). PCR method needs expensive, heavy instruments and gel electrophoresis, therefore, it is commonly used in laboratory test, and unsuitable for on-site detection. Loop-mediated isothermal amplification (LAMP), an isothermal nucleic acid amplification technique, has been extensively used in many areas such as food safety and clinic diagnosis. Advantageous characteristics of LAMP, such as high specificity and sensitivity, simple operation, low cost, eye visualization, particularly free of special equipment, renders it with high potential to be used for GMO on-site detection. In this review, we summarized current status of the application of LAMP in GMO detection, and discussed possible improvements needed for its adaptability regarding to on-site GMO detection. Hopefully, the information present here would facilitate the practical risk assessment of GMO.
Commercialization of genetically modified (GM) crops started in 1996 in USA, and now GM crops are increasingly gained acceptance worldwide, which makes GM crops the fastest adopted crop technology in the history of modern agriculture and biotechnology [
According to a recent report released from International Service for The Acquisition of Agri-Biotech Applications (ISAAA), ISAAA Brief 46-2013 [
Along with the rapid adoption of GM crops in modern agriculture, the debate over the biosafety of GMOs has never ceased. Public fears about possible food and environment risk of GM crops, which is a matter of concern. It is well-known that there is no biotechnology that is absolutely safe; therefore, GM crop could bring their risks together with their benefits to the environment and human being. In an attempt to relieve public concerns about GM food safety, a series of regulations and legislations have been established in more than 50 countries and regions to strengthen the management of GMOs and label the products containing GM contents [
Generally, the detection methods for GM crops and their products were developed to detect the presence of either protein or nucleic acid sequence of the exogenous insertion. Protein-based detection methods include enzyme-linked immunosorbent assay (ELISA) and lateral flow strips (LFS) [
The intrinsic drawbacks of protein-based detection methods have been well documented in many excellent reviews [
Among these analytical methods for GMO detection, LAMP, one PCR-free method for isothermal nucleic acid amplification, developed by Notomi et al. in 2000 [
As an alternative to normal PCR, the amplification of target DNAs with LAMP is obviously faster than conventional PCR-based technologies. It is less sensitive to inhibitors [
Because of its advantages, LAMP has been tested for its applicability in GMO detection. Generally, it is used mainly in following three sections (for detailed information, please refer to
In this case, the LAMP targets mainly the general exogenous elements; therefore, the developed method can be used to screen for the presence of GMO components. A GMO screening method using LAMP targeting GM- element Cauliflower Mosaic Virus 35S (P-35S) was first reported by Fukuta et al. (2004) [
Some LAMP assay also targets exogenous genes. For example, LAMP for cry1Ab gene in GM rice has been recently developed and proved to be better than qPCR detection [
Just like PCR, most of LAMP techniques are just suitable for single gene detection. Recently, Randhawa et al. (2013) [
Purpose | Detected targets/events | Detection limit | Reference | |
---|---|---|---|---|
Materials | LOD | |||
Screening common elements | P-CaMV35S | 5%, 2%, 1%, 0.5%, 0% roundup ready soybeans | 0.5% | [ |
Soya Roundup Ready™ GMO Reference Material | 10 copies | [ | ||
MON15985 | 4 copies | [ | ||
T-nos | Soya Roundup Ready™ GMO Reference Material | 10 copies | [ | |
P-nos | The plasmid pGreenII | 6 copies | [ | |
P-FMV | MON88913 | 4 copies | [ | |
Screening marker genes | nptII | MON15985 | 4 copies | [ |
cry1Ab | Transgenic rice line KMD1 | 0.5% GM contents | [ | |
cry2Ab | GM maize MON89034 | 5 copies | [ | |
cry3A | GM maize MIR604 | 5 copies | [ | |
pat | Plasmid pGreen0229 DNA | 0.65 fg/tube | [ | |
Phytase gene | Transgenic maize with phytase (BVLA 430101) | 30 copies | [ | |
Event specific detection | Rice | TT51-1 | 0.01% | [ |
15 copies | [ | |||
KMD1 | 0.01% | [ | ||
KF6 | 0.005% | [ | ||
10 copies | [ | |||
T1C-19 | 10 copies | [ | ||
Maize | DAS-59122-7 | 4 copies | [ | |
T25 | 4 copies | [ | ||
BT176 | 4 copies | [ | ||
TC1507 | 4 copies | [ | ||
BT11 | 4 copies | [ | ||
MON863 | 4 copies | [ | ||
MON810 | 40 copies | [ | ||
Soybean | GTS 40-3-2 | 4 copies | [ | |
MON89788 | 4 copies | [ | ||
Cotton | cry1A(b) gene and chi gene in T2 transgenic cotton | 10−8 dilutions of chi gene | [ | |
Wheat | B73-6-1 | 6 copies | [ | |
Oilseed rape (OSR) | MS8 | 8 copies | [ | |
RF3 | 8 copies | [ |
These result implied a high potential for the application of LAMP in GMO detection, in a single or multiplex manner.
LAMP is increasingly used for the detection of GM events. It was successfully used for the detection of two GM soybean events (GTS 40-3-2 and MON89788) in 2010 [
The identification of seven GM maize events (DAS-59122-7, T25, Bt176, TC1507, MON810, Bt11, and MON863) with LAMP was reported in 2011, and the LOD was as low as four copies of maize haploid genomic DNA, with the exception of MON 810 [
In 2012, a visual and rapid LAMP method has been developed for three GM rice events (KMD1, TT51-1, and KF6) [
In 2013, wheat B73-6-1 has also been detected by LAMP [
Recently in 2014, phytase gene in GM maize was detected within less than 20 min using LAMP, and its sensitivity was about 30 copies of phytase genomic DNA, which is 30 times more than that of conventional PCR method. Its limit of quantification (LOQ) was 60 copies [
No GMO detection method is perfect, neither is LAMP. LAMP is undoubtedly much sensitive than other GMO detection methods; however, obvious shortages of it also emerge along the analysis process. Firstly, LAMP needs at least 4 primers target 6 distinct regions of target gene. Therefore, the LAMP primer design is time- consuming and difficult, particularly when the available target sequence is too short. Usually, the specific LAMP primer design website can generate hundreds of primers, and it take a long time to screen for suitable primer pairs. Secondly, the products are easily contaminated by aerosol when the lid is taken off because of the high sensitivity of LAMP. Thirdly, LAMP positive reaction displays ladder-like bands on the gel, and it is difficult to distinguish the non-specificity amplification from specificity amplification and false positive results are easy to appear; and fourthly, the LAMP products can’t be sequenced for the confirmation of the amplification of target sequences. In addition, when the quantification of the amplification products are needed, heavy instrument such as fluorescent quantitation PCR equipment or turbidity meter has to be invested. Furthermore, in the case of the result visualization by inflorescent dying, current SYBR is generally an inhibitor of the template DNA, therefore, it is not possible to add the dying before the amplification. But, when addition of dying after the amplification, the lid of reaction tube has to be open, and which generates cross-contamination. All those disadvantages impeded the application of LAMP in GMO detection, specifically for on-spot detection.
LAMP detection procedure consists mainly of three parts, namely nucleic acid extraction, LAMP amplification and result judgments. Any improvements in each of these three parts will contribute to the applicability and of LAMP to on-site GMO detection.
The development of a rapid and efficient DNA extraction method is essential for LAMP based on-site GMO detection. DNA extraction and purification methods widely used in the laboratory, such as, CTAB method, SDS method, and commercial kits including DNeasy Plant Mini Kit from Qiagen, do not fit for this purpose. Zhang et al. [
A highly efficient, low cost and less time-consuming LAMP amplification facility is one of the key points for its application on GMO on-site testing. For LAMP amplification of target DNA on-field, an isothermal apparatus that can reach to and maintain 60˚C - 65˚C is indispensable. In the laboratory, LAMP amplification of target DNA can be achieved through isothermal equipment such as a water bath pot or a thermal cycler. However, when testing on-site without electricity, novel portable isothermal apparatus independent on direct electricity resource will be essential. In order to apply LAMP to on-site GMO detection, chargeable isothermal equipment dependent on battery or vehicle-mounted heater would be also useful alternatives.
For an effective on-site GMO detection, the identification and quantification of LAMP products should also be rapid and sensitive. In the laboratory, amplified products can be observed directly by naked eye in the reaction tube by adding SYBR Green I stain or picogreen dye, or by gel electrophoresis under UV trans-illuminator to see a ladder-like structure, or by turbidity assay based on chemical reactions [
With the increasing number and complexity of GM events globally, judging for the presence or absence of GM contents in crops with high specificity and sensitivity, cost-effective methods, has becoming a pressing need. LAMP proves to be more sensitive than conventional PCR methods, and its result is visible; therefore, it is of highly potential to be an alternative to conventional PCR for GMO detection, especially as a field detection alternative in scenes where a fast screen is needed while it lacks complex laboratory equipment. The practicability of LAMP will facilitate GMO detection in the laboratory and also on spot, if it is combined with other techniques, such as a simple and rapid DNA extraction device, a portable isothermal apparatus or a simple heating block. And from a long-term perspective, strategies and methods to avoid the opening of the cap of the reaction tubes will make the LAMP method more convenient for GMO detection in both lab and outdoors. Further emphasis should also go to the development of high throughput LAMP methods such as multiple LAMP methods. The last but not least is its capacity for automatic quantification. With the current characteristics, together with the future improvement in the assay, we strongly believe that LAMP will contribute significantly to GMO detection both on laboratory and on-site.
This work was partially supported by the National Transgenic Plant Special Fund (2013-2014ZX08012-002) and EU FP7 project DECATHLON (613908).
The authors declare that there are no conflicts of interest.
Rong Li,Cong Wang,Lilian Ji,Xiangxiang Zhao,Man Liu,Dabing Zhang,Jianxin Shi, (2015) Loop-Mediated Isothermal Amplification (LAMP) Assay for GMO on: Recent Progresses and Future Perspectives. Open Access Library Journal,02,1-8. doi: 10.4236/oalib.1101264