Full
Outline
To develop a dual recombinase polymerase amplification (RPA)-mediated colloidal gold strip assay for the rapid and visible simultaneous detection of 2 kinds of marker genes, the neomycin phosphotransferase II gene (NPT II) and the hygromycin phosphotransferase II gene (HPT II), in transgenic plants.
In this study, highly specific gene fragments of NPT II and HPT II were selected to design corresponding amplification primers. Using RPA technology, the 2 kinds of target nucleic acids were rapidly and stably amplified exponentially. The amplification products were subsequently detected by a dual test strip for rapid visual interpretation. On the test strip, the corresponding detection lines (test line N, test line, TN and TH lines) and the quality control line (C line) developed visible signals, allowing qualitative analysis based on the color intensity of the TN, TH, and C lines.
The assay was performed under isothermal conditions at 40 °C, involving a 15 min dual RPA reaction followed by a 5 min antigen‑antibody recognition step on the test strip. It achieved precise detection with limits of detection of 615 copies/µL for the NPT II gene and 314 copies/µL for the HPT II gene, and enabled rapid screening in simulated reald samples down to 0.10% for NPT II and 0.05% for HPT II.
The dual RPA-mediated colloidal gold strip method developed in this study enables visual detection of the marker genes NPT II and HPT II. Characterized by user-friendly operation, rapid detection and high specificity, this platform demonstrates significant potential for broad application in point-of-care testing.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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