Label-Free Ratiometric Fluorescence Detection of Norovirus Nucleic Acids Based on G-quadruplex Integrated PCR Strategy
摘要
In order to optimize the detection method of genogroup II (GII) norovirus in food, this study is to construct a G-quadruplex integrated polymerase chain reaction (PCR) strategy for GII norovirus genomic DNA. Based on this strategy, a ratiometric fluorescent assay for GII norovirus genomic DNA was innovatively developed using two labeling-free, dual-emitting nucleic acid dyes. The PCR procedure and system, and the ratiometric fluorescence detection system, were optimized to improve the amplification efficiency and enhance the signal response. The results showed that, under the optimal conditions, the ratiometric fluorescence signal value of the detection system showed a linear relationship with the logarithmic value of the concentration of GII norovirus genomic DNA fragments in the concentration range of 10 ~ 250 pmol·L−1 (R2 = 0.983), and the detection limit was 1.19 pmol·L−1. In the range of 10 pmol·L−1 ~ 1 nmol·L−1, the method can achieve quantitative detection of GII norovirus genomic DNA, and in the range of 10 ~ 250 pmol L−1, the method can achieve quantitative detection, and in the saturated concentration interval of 250 pmol·L−1 ~ 1 nmol·L−1 outside the linear range, the method shows good specificity. This method is based on the mature PCR technology system, which achieves the simultaneous output of dual fluorescence signals through clever primer design, and establishes a ratiometric fluorescence PCR detection method, which is expected to achieve the detection of GII norovirus in food.