Construction Of Auto-cycling Primer Extension Amplification Platform And Its Application In MiRNA Detection

MicroRNA(miRNA)is a class of short non-coding RNA,whose abnormal expression is closely related to many diseases.As an important member of miR-17-92 family,miR-19 a shows a tendency of high expression in a variety of tumor cells.Due to the low concentration of miRNA,it is very important to develop highly sensitive miRNA detection methods.At present,a good deal of nucleic acid signal amplification methods have been developed.Among them,polymerase-based methods have the advantages of high amplification efficiency and selectivity,so they are widely used in miRNA detection.The amplification efficiency depends on the the continuous amplification of the polymerization reaction,however,none of these nucleic acid amplification techniques can maintain the continuous polymerization reaction unless by changing the temperature or adding additional reagents(ex.nickase or betaine),which undoubtedly increases the cost and operation steps.Therefore,a new nucleic acid amplification method based on auto-cycling primer extension(APE)was proposed,and the sensitive detection of miRNA in serum and other complex samples was achieved.The details are as follows:(1)A target-triggered auto-cycling primer extension amplification method was developed for miR-19 a detection.In this method,copy-and-release hairpin(CRH)is introduced,and palindromic sequences and amplification termination sites are designed in the stem of CRH.Before the reaction begins,CRH is blocked.In the presence of the target,a toehold mediated chain replacement reaction activates the reaction and releases CRH.At this point,the primers take CRH as the template for intermolecular polymerization extension,and the polymerization stops when it reaches the termination site.Because the extended sequences are palindromic sequences,the primers will bend to form intramolecular hairpins,and then take themselves as templates for intramolecular polymerization extension,which finally form hairpins with blunt ends and then release from CRH.CRH allows for the next cycle,thus enabling continuous amplification of the reaction.In this method,the probe was ingeniously designed,and the intermolecular to intramolecular polymerization can be realized only through the design of the probe.Also,the continuous amplification process can be realized,which simplifies the reaction system.Meanwhile,this method had high sensitivity and selectivity,and 5.0 p M miR-19 a can be detected.(2)A bio-barcode and auto-cycling primer extension cascade amplification platform was constructed for miR-19 a detection.Bio-barcode technology is introduced in order to realize target detection in complex samples.In this method,the target is enriched and separated by magnetic beads,and then co-incubate with the modified gold nanoparticles to form the sandwich composite structures of magnetic beads-target-gold nanoparticles.The target is transformed into barcode probes on gold nanoparticles,and the amplification of the target is realized.Then the barcode probes are used to perform auto-cycling primer extension reaction according to the previous work,thus realizing the cascade amplification detection of miR-19 a.Through the introduction of this cascade amplification strategy,the selectivity of miRNA detection was further improved.Also,single base mismatch can be distinguished,and 50.0 f M miR-19 a can be detected,which can be used for target detection in serum samples.Compared with the previous work,the detection range of this method was wider,the detection limit was reduced by two orders of magnitude and the sensitivity was significantly improved.