Development of the sensor with dual-probe nucleic acid paper-based for detection of Foc4 of early banana fusarium wilt

Banana fusarium wilt has a significant adverse effect on the banana industry. It is worth noting that fusarium oxysporum f.sp. cubense 4(Foc4) is one of the most devastating and widespread pathogens of banana fusarium wilt in the world. It is a catastrophic threat to the global banana industry. At present, there is still no technology that can cure bananas infected with banana fusarium wilt. Therefore, it is extremely important for banana planting industry to detect banana fusarium wilt in time and do a good job in prevention and control. In order to realize the early accurate detection of banana fusarium wilt race 4, this study proposed a detection method of double probe paper-based sensor based on colloidal gold, which increased the use of signal enhancement probes to increase the binding amount of colloidal gold, thereby reducing the detection limit. Two kinds of colloidal gold particles with different particle sizes were combined with the corresponding detection probes and signal enhancement probes to form a double gold label probe system. The large particle size colloidal gold was used with the detection probe, while the small particle size colloidal gold was paired with the signal enhancement probe. The sample being tested was mixed with this double gold-labeled probe system. The Foc4 target sequence in the sample would be paired with the double gold-labeled probe to form a ' gold-labeled probe-target sequence-T-line probe ' complex, which continued to migrate to the water-absorbing pad. The complex was captured on the test area of the sensor. Within 10 minutes, the test paper would generate a visible target product that could be observed by the naked eye at both the test line and the quality control line areas, thus achieving qualitative detection. For quantitative detection of Foc4, the light intensity of the test strip would be further analyzed using software. The light intensity peak area was obtained and substituted into the standard curve to obtain the concentration of Foc4 detected and realize quantitative detection. Experimental results demonstrate that the detection limit of the dual-probe paper-based sensor is 0.001 nmol/L, which is 100 times of that of the traditional paper-based sensor, and the detection sensitivity is improved. The sensitivity test results demonstrated a strong linear relationship between the concentration of Foc4 and the peak area of the test line light intensity within the concentration range of 0.001-1000.000 nmol/L. This finding suggests that the sensor can be effectively employed for quantitative detection. To assess specificity, a high concentration of non-complementary probe was utilized as the target sequence for detection and compared with the detection results of samples containing Foc4. The experiment revealed the absence of a red band at the test line when the test paper was detected by the non-complementary sequence. Importantly, this observation indicated that the presence of the non-complementary sequence had minimal impact on the detection effect, thus affirming the sensor's excellent specificity. Finally, the paper-based sensor was used to detect Foc4 in banana leaves. The average recovery rate was 77.6%-102.3%, and the relative standard deviation was 7.4%-7.7%, indicating that it can be used for on-site detection of Foc4. The detection technology possesses the characteristics of being low-cost and easy to operate. In comparison to traditional detection methods, it enables timely, rapid and accurately determination of the presence of Foc4. Compared with the existing molecular detection technology, the detection can be completed without the need of expensive equipment or specialized personnel. This feature makes it highly suitable for widespread use. The dual-probe paper-based sensor realizes the combination of nucleic acid lateral chromatography detection technology and banana fusarium wilt detection. Its potential for widespread adoption in the early disease warning system of the banana industry is substantial. Furthermore, this technology can be combined with traditional polymerase chain reaction (PCR) and other techniques to facilitate on-site detection of Foc4.