Research On Novel Methods For MicroRNA Detection Based On Isothermal Nucleic Acid Amplification Technology

Colorectal cancer is the third most frequently diagnosed malignant tumor in the world,and is associated with significant mortality as many patients are diagnosed with advanced stage disease.Early accurate diagnosis and standardized treatment of colorectal cancer are the keys to improve the survival rate.Micro RNA is a kind of non-coding small RNA molecule with key biological regulatory functions,whose abnormal expression is closely related to the occurrence and development of tumor.The mi R-200 family has a central role in epithelial-mesenchymal transition(EMT),and the expression level of mi R-152 is related to lymph node metastasis and tumor-node-metastasis(TNM)stage,both of which have the potential for early diagnosis and prediction of survival time of colorectal cancer patients.Therefore,it is of great significance to develop mi RNA detection methods with high sensitivity,high selectivity and strong anti-interference ability.Isothermal nucleic acid amplification technology has become the mainstream technology for mi RNA detection in recent years due to its high efficiency and stability.Based on phosphorothioated-terminal hairpin formation and self-priming extension(PS-THSP),we have established two new mi RNA detection methods,with mi R-200 a and mi R-152 as the research objects respectively.The two methods are universal for different target mi RNAs.Firstly,taking mi R-200 a as the research object,we simplified the detection system by introducing phosphorothioates into the 5’ terminal of the probe which enabled the repetitive refolding of self-priming amplicons.The detection of mi RNA could be achieved under constant temperature without any other complicated primers,providing a simpler idea for the establishment of other new methods.The new method had a detection limit of 4 fmol.Besides,the method had strong resistance to biological matrix interference.By diluting the serum sample to 50% with reaction buffer,mi RNA in serum could be detected without extra pre-treatment.In order to improve the detection sensitivity,we combined the PS-THSP strategy with loop-mediated isothermal amplification(LAMP)technology to establish the reverse transcription-phosphorothioated LAMP(RT-PS-LAMP)method.Inspired by the “dumbbell” amplicon produced during LAMP,probe A and 5’ phosphorothioated probe B were well designed to converte the amplification of short-chain mi RNA to the amplification of long-chain stem-loop DNA.The combination of LAMP technology and PS-THSP strategy greatly improved the amplification efficiency.Moreover,we used sequence-specific one-step strand displacement(OSD)probes as signal molecules,which contributed to the reduced background interference caused by non-specific products.RTPS-LAMP showed a wide linear range from 50 fmol/L to 3 nmol/L with the lower detection limit 10 fmol/L.In this section,we successfully applied this method to the detection of mi R-152 in human colorectal cancer and adjacent tissues,which indicated the great prospect of RT-PS-LAMP assay in clinical application.