关键词: 纳米孔靶向测序, PCR, 一致性, 猪病原

Abstract: This study was conducted to develop a primer set for Nanopore targeting sequencing (NTS) to simultaneously detect ten porcine pathogens and explore the potential for detecting all porcine pathogens on this platform. Primers for ten pathogens were designed using Primer Premier 6 software. The specificity of the ten primers was verified by PCR and agarose gel electrophoresis. The optimal primer concentration in the multiplex PCR system was explored using nanopore sequencing technology by comparing the detection numbers of pathogens at different concentration gradients. The sensitivity of multiplex PCR was verified using the optimal primer concentration obtained in the previous step. The detection sensitivity was determined by amplifying pathogen templates at different concentration gradients and analyzing the corresponding results. Finally, a conformity test between fluorescence quantitative PCR and NTS was performed using clinical samples (n=201). The agarose gel electrophoresis images for verifying primer specificity all showed a single, bright, and clear band, and no bands were amplified for the other ten porcine pathogens not selected in this study. All ten pathogens could be detected in the reaction system when the final concentration of primers was 3 and 5 μmol·L^-1, while all other tested concentrations failed to detect all ten pathogens. All positive samples in the four replicates could be detected when 5 μL of the nucleic acid template with a concentration of 9×10² copies·mL^-1 was added. In the detection of clinical samples, more pathogen-positive samples were detected by NTS. After the chi-square test, there were statistically significant differences in the detection results of five pathogens (P<0.05), while no statistically significant differences were found in the detection results of the other five pathogens (P>0.05). After kappa value analysis, excellent consistency was observed in the detection of six pathogens, and good consistency was observed in the detection of four pathogens. The results indicate that the NTS detection method can simultaneously detect ten different porcine pathogens. For samples with inconsistent detection results compared to the control method, further verification by a third-party detection method is required.

Key words: nanopore targeted sequencing, PCR, consistency, porcine pathogens