Abstract
Genetically modified (GM) rice KMD1, TT51-1, and KF6 are three of the most well known transgenic Bt rice lines in China. A rapid and sensitive molecular assay for risk assessment of GM rice is needed. Polymerase chain reaction (PCR), currently the most common method for detecting genetically modified organisms, requires temperature cycling and relatively complex procedures. Here we developed a visual and rapid loop-mediated isothermal amplification (LAMP) method to amplify three GM rice event-specific junction sequences. Target DNA was amplified and visualized by two indicators (SYBR green or hydroxy naphthol blue [HNB]) within 60 min at an isothermal temperature of 63 °C. Different kinds of plants were selected to ensure the specificity of detection and the results of the non-target samples were negative, indicating that the primer sets for the three GM rice varieties had good levels of specificity. The sensitivity of LAMP, with detection limits at low concentration levels (0.01%–0.005% GM), was 10- to 100-fold greater than that of conventional PCR. Additionally, the LAMP assay coupled with an indicator (SYBR green or HNB) facilitated analysis. These findings revealed that the rapid detection method was suitable as a simple field-based test to determine the status of GM crops.
Highlights
The production of genetically modified (GM) crops is increasing annually worldwide
The results indicated that the developed Loop-mediated isothermal amplification (LAMP) assays were more specific and sensitive than conventional Polymerase chain reaction (PCR) assays and could be used for genetically modified organism (GMO) detection in the field
The specificities of the LAMP assays for detecting phospholipase D (PLD), KMD1, TT51-1, and KF6 were confirmed by checking the reaction with various plant DNA samples
Summary
The production of genetically modified (GM) crops is increasing annually worldwide. By 2011, genetically modified (GM) crops were being grown by 16.7 million farmers distributed across. Polymerase chain reaction (PCR) or real-time PCR is currently the most sensitive and specific method of GM detection. Taq DNA polymerase in PCR assays can be inactivated by inhibitors present in crude biological samples [24] and may not be applicable for field trials of GM detection. Another rapid, simple, and effective assay is needed to supplement the current PCR methods. The results indicated that the developed LAMP assays were more specific and sensitive than conventional PCR assays and could be used for GMO detection in the field
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