PRV和CAstV-Ⅰ及CIAV纳米PCR检测方法与常规PCR检测方法敏感度的比较

Sensitivity comparison of nano-PCR with conventional PCR method for detection of PRV,CAstV-Ⅰand CIAV

为了比较PRV、CAstV-Ⅰ和CIAV的单重纳米PCR方法和单重PCR方法的敏感性,探讨靶基因GC含量和引物T_m值对纳米PCR和常规PCR的影响,本研究根据PRV的gE基因、CIAV的VP1基因和CAstV-Ⅰ的ORF1b基因设计了12对引物,运用这12对引物建立单重的纳米PCR方法和常规PCR方法,分别优化纳米PCR和常规PCR方法的退火温度和引物浓度,比较两者的敏感性。结果显示,纳米PCR和常规PCR方法在最佳的反应条件下的敏感度基本一致,纳米PCR方法在敏感度上没有明显的优势;纳米PCR反应液对高GC含量靶基因的扩增比常规的PCR反应液更容易;纳米PCR的可选退火温度范围宽松,而常规PCR则需要严格的退火温度才能增强扩增效果或减少非特异性扩增;常规PCR的退火温度只与靶基因的GC含量呈现正显著相关(P<0.01),纳米PCR的退火温度与靶基因的GC含量和引物T_m值无相关性。以上数据为相关的技术人员对于病原体检测技术的选择上提供了数据参考。

To compare the sensitivity of nano-PCR and conventional PCR method for detection of PRV,CAst V-Ⅰand CIAV,and investigate the effect of GC content of target genes and T_m value of primers on nano-PCR and conventional PCR.Twelve pairs of primers were designed based on g E gene of PRV,VP1 gene of CIAV and ORF1 b gene of CAst V-Ⅰ,and a single nano-PCR method and conventional PCR method were established using 12 pairs of primers.The annealing temperature and primer concentration of the nano-PCR methods and the conventional PCR methods were optimized respectively,and the sensitivity of the two methods was compared.The results showed that the sensitivity of nano-PCR and conventional PCR detection of PRV,CAst V-Ⅰand CIAV was basic consistent under the optimal reaction conditions,and nano-PCR had no obvious advantage in sensitivity;compared with the conventional PCR reaction solution,the nano-PCR is easier to amplify the target genes with high GC content;the annealing temperature range of nano-PCR is wide,while conventional PCR needs a strict annealing temperature to enhance amplification effect or reduce non-specific amplification;the annealing temperature of conventional PCR reaction was positively correlated with GC content of target gene(P<0.01),but the annealing temperature of nanoPCR was no correlation with GC content of target gene and T_m value of primers.The above data provide data reference for the choice of pathogen detection method of technical personnel.