The Agricultural Biosafety Evaluation Research Center of the Biotechnology Institute of SAAS has developed and constructed a variety of new, accurate, rapid, and visual detection technology systems based on nucleic acid molecular characteristics of key target traits of biological breeding materials, combined with nanomaterials, CRISPR and lateral-flow chromatography technology. The relevant research results were published in the international academic journals 《Food Science and Human Wellness》 ((JCR Q1, IF:8.022), 《Food Control》 (TOP 1, IF:6.652), and《Current Research in Food Science》 (JCR Q1, IF:6.269).

As of 2021, the cumulative planting area of global genetically modified (GM) crops has reached 1.955 billion hectares worldwide, which is about 112 times more than that in 1996. The large-scale application of GM crops has aroused widespread social concern, and the market regulation of GM crops is crucial to ensuring food security and biosafety. Different GM organism regulatory policies have been developed by different countries and regions. The implementation of management policies requires technical support. Transgenic assays tend to be rapid, visual, and can be used in field-portable on-site testing settings.

1. A dual-RPA based lateral flow strip for sensitive, on-site detection of CP4-EPSPS and Cry1Ab/Ac genes in genetically modified crops

In response to the current situation that the proportion of GM crops planted with insect resistant/herbicide-tolerant traits is increasing, a dual RPA-based strip detection system (named "Dual-RPA-LFD") was established targeting the major insect-resistant/herbicide-tolerant genes Cry1Ab/Ac and CP4 EPSPS. In this study, the dual RPA-based strip strategy was realized by primer labeling and primer optimization, and the detection results were characterized by control line (C line), test line 1 (T1 line), and test line 2 (T2 line). The whole analysis process could be completed within 20 min at 37℃. The results showed that the strip method can specifically identify Cry1Ab/Ac and CP4 EPSPS genes, not affected by other genes, and was suitable for random samples in the market and GM crops of different components, with good specificity and practicality. Meanwhile, plasmids containing Cry1Ab/Ac gene and CP4 EPSPS gene as low as 100 copies/µL (223 fg/mL) could be detected, indicating high sensitivity of the Dual-RPA, LFD strategy.

2. Rapid detection of genetically modified products based on CRISPR-Cas12a combined with recombinase polymerase amplification

A visualization system (named "RPA-Cas12a-LFB") was established based on RPA technology, Cas12a "trans-cleavage" reaction and lateral flow strips targeting transgenic screening elements P-CaMV 35S and T-NOS. The assay could be completed within 40 min at 37℃. The results showed that the "RPA-Cas12A-LFB" method was highly sensitive and could detect 10 copies of standard plasmids and 0.01% of GM crops with ultra-low content. The method was successfully applied to GM crops with different transgenic components and contents as well as random samples in the market and showed good specificity, accuracy, and practicality. The "RPA-Cas12a-LFB" system provided a new and highly sensitive visualization method for the detection of GM crops, while making up for the shortcomings of the strip method in terms of detection sensitivity.

3. An ultra-sensitive test strip combining with RPA and CRISPR/Cas12a system for the rapid detection of GM crops

To further improve the detection performance of the "RPA-Cas12a-LFB" method, a colloidal gold-based lateral flow strip based on CRISPR/Cas12a "trans-cleavage" was developed and evaluated. In this study, the preparation parameters and using conditions of the strip were optimized, and the optimized strip was applied to sample analysis. The whole procedure included RPA reaction, Cas12a cleavage and lateral flow immunochromatography, all of which could be performed at 37℃, and the total time was less than 40 min. The results showed that the prepared strips were successfully applied to GM crops with excellent detection performance. The sensitivity of the strips for P-CaMV 35S and T-NOS screening elements reached 1 and 10 copies, respectively, with good specificity, applicability, and stability for actual samples. The successful preparation of the Cas12a-based strips provided a powerful tool for highly sensitive and visualized field detection of GM crops.