核酸检测是医疗、食品、卫生等领域的关键技术，而实际样品检测中很难解决高灵敏度与高特异性的矛盾，易产生误判。特别是对于食品或病体组织中病菌病毒含量极低的情况，因大量背景核酸的干扰，检测灵敏度降低，假阳性频发。目前主要采用荧光定量PCR法解决检测精确度不足的问题，但对样品要求严格，结果仍不稳定。本项目将进行逆向思维，在预扩增中有效利用大量的背景核酸来提高扩增的灵敏度；然后进行特异性扩增，解决特异性和灵敏度不能共同提高的矛盾。同时，利用上述原理对滚环扩增进行根本上的改进，实现常温检测，达到简便、经济、准确等实用要求。本方法的特点是分两步进行，预扩增中利用酶切后部分背景核酸与靶DNA具有同样粘性末端的性质，使扩增灵敏度提高数百倍以上；特异性扩增中采用比较温和的条件达到高特异性。本项目将研究理论模型建立、序列设计、检测效果评价等内容，为高核酸背景下的核酸检测提供技术基础。

Nucleic acids amplification is the key technology widely used in medical treatment, food safety, and sanitation field. However, the existing techniques can hardly balance high sensitivity and specificity which may lead to misjudgement. Especially in actual food sample, the large amount of background nucleic acids usually lowers the sensitivity and easily to give a false-positive result, which limits its practical application. One strategy commonly used to overcome this problem at present is fluorescent quantitative PCR, also along with some disadvantages such as high cost, requiring high quality sample, and low reproducibility. By reverse thinking, take advantage of the high background, this study is aimed to solve the problem that high sensitivity and specificity cannot be easily achieved at the same time. Furthermore, by using the same principle, rolling circle amplification (RCA) will be greatly improved to achieve DNA target amplification under room temperature and satisfy the requirements of facile, low cost, and accuracy. The feature of this method is to separate the amplification to two steps. In the first step, some fragments of background nucleic acids having the same sticky ends with the target DNA fragment were ligated together with the universal adaptor. After ligation, these newly formed fragments can be served as template for pre-amplification along with the target fragment, thus the template number for pre-amplification is increased and high sensitivity is obtained. In the second step, a pair of target-specific primers is used to obtain the high specificity. For supplying basic technique, the main contents of this study include establishment of theoretical model, design of nucleotide sequences, and evaluation of detection effect.