La 320c realtime turbidimeter

Nanoparticle-based lateral flow biosensor, Isothermal amplification kit, Visual Detection Reagent (VDR) were purchased from BeiJing-HaiTaiZhengYuan Technology Co., Ltd (Beijing, China). QIAamp DNA Mini Kits were purchased from Qiagen Co., ltd (Beijing, China). Primers used in this report were synthesized by Tianyi Huiyuan Biotechnology Co., Ltd (Beijing, China). LA-320C Realtime Turbidimeter was purchased from Eiken Chemical Co., Ltd (Tokyo, Japan). Gel Doc XR + Imaging System was purchased from Bio-Rad Co., Ltd (USA). PCR instrument was purchased from Dongsheng Innovation Biotechnology Co., Ltd (Beijing, China).

The reagents were added to the 200-μL PCR tube according to the LAMP reaction system shown in Table 4. The entire reaction process was conducted on an ice bath. The PCR tube with the added reagents was placed in the L-A-320C real-time turbidimeter (Eiken Chemical Co., Ltd., Tokyo, Japan) for the reaction duration under the following reaction conditions. An L-A-320C real-time turbidity meter was used to incubate the tube at 65 °C for 60 min, followed by heat inactivation at 80 °C for 5 min. After the reaction was terminated, SYBR Green I (Shenggong Bioengineering (Shanghai) Co., Ltd., Shanghai, China) was added to the amplified product for direct fluorescent visual analysis, and the result was judged based on the colour change in the PCR tube. The fluorescent green colour of the reaction solution indicated that the sample had undergone LAMP amplification and was positive; that is, it contained the pathogenic bacteria of interest. The orange colour of the reaction solution indicated no amplification product and hence negative reaction; that is, the sample did not contain the target pathogen. Meanwhile, 5 μL of the amplified product was used for 1.2% agarose gel electrophoresis detection, and the positive amplification result showed a trapezoidal characteristic band, while the negative result showed no band amplification. Each experiment was repeated three times.