Development Of A Novel Rolling Circle Amplification Based On Target Circularization And Its Application

Nucleic acid amplification is a basic biotechnology that plays an irreplaceablerole in all aspects such as health care, agriculture development, environmentalprotection and forensics analysis. As a classical amplification technology, PCR withits evolved fashion named real-time quantitative PCR (RT-PCR), are widely used inthose fields. However, vulnerability to the contamination that generates false positives,inhibition caused by polymerase inhibitor, impaired sensitivity delivered by massivebackground nucleic acid in, discrediting PCR as a powerful technology for thedetection of exogenous microbes in food. Meanwhile, as a non-thermal amplificationtechnique, PCR relies on precise thermostable equipments, which does not meet therequirements of the on-spot-rapid detection. Therefore, the great demand for anoriginal amplification technology that is free from expensive equipment and immuneto the interference from background nucleic acids, which can be applied for foodsecurity administration, is enhancedAs an isothermal detection method for overcoming the shortcomings of PCR,RCA (rolling circle amplification) has gained a great attention over the past decade.For the feasibility and sensitivity, padlock-RCA has been investigated in many labors.The specificity of padlock-RCA is based on the specific hybridization of two ends of apre-circular DNA (probe) with the target sequence. Nonetheless, even in the case thatseveral mismatches are present at the position not close to the ligation site, the probecan also be circularized and end up with false positive results. Moreover, theamplification reaction of padlock-RCA only generates tandem copies of the addedprobe itself but not the DNA target.Here we present a novel DNA detection method, termed “Target-CircularizationRolling Circle Amplification”(TC-RCA) based on rolling circle amplification, Adigestion step is employed to generate a number of shorter fragments including theDNA target with9nt sticky ends. Then a ligase is utilized to ligate the adaptor and the target DNA form a double stranded circle with a gap. Phi29DNA polymerase is thenused to amplify the circle by RCA using the integral strand as the template and theopen strand as the primer. The hyper-branched RCA is achieved with the help of twoprimers complementary to the target sequence. Finally, the products consisting oftandem copies of dsDNA target were digested by restriction endonuclease (TspR I) atthe cutting site as5’-NNCASTGNN-3’. Hence, the target DNA meant to be replicatedor detected is obtained with the augmentation of several magnitudes. Based onTC-RCA, several strategies such as high fidelity DNA ligase, biotinylated adaptor,streptavidin magnetic bead (SMB) and magnetic separation have been taken toimprove the sensitivity and specificity of TC-RCA to be an isothermal amplificationtechnology for detection harmful bacteria in food product. The following gists havebeen studied in this paper.1) The feasibility of TC-RCA has been proved. TC-RCA is capable of amplifyingat least6×104copies of target molecule in the absence of background DNA. Besides,the adaptor can be immobilized on solid phase surface of magnetic beads, andimmobilization does not affect the ligation and afterward amplification. This providestechnical support and theoretical basis for TC-RCA based on chip isothermalamplification.2) Detection of the target sequence in the presence of background DNA isachieved by TC-RCA. The detection limit is6×106copies of target molecule alongwith nonspecific amplifications. Taq DNA ligase featuring high ligation fidelity failsto promote the detection specificity, since Taq DNA ligase tolerates mismatches underabnormal conditions. This indicates that TC-RCA can be used for primary detection infood security control and intrigues us to contrive for new solutions for the problems.3) By utilizing SMB that realizes the separation between circular amplicon andinhibitors such as Taq DNA ligase and background DNA, SMB-assisted TC-RCAupgrade to a level that can detect at least60copies of target sequence in the presenceof massive background DNA. The desirable specificity is accomplished for nounexpected amplification occurred. With effort towards efficiency improvement, thedetection result can be obtained with24h, which paves the way for SMB-assisted TC-RCA to a general-use, rapid and precise detection protocol.4) SMB-assisted TC-RCA is proved to be applicable to detect harmful bacteriafrom aquatic food under isothermal conditions with great sensitivity and specificity.SYBR Green I displays the same results with electrophoresis. Comparing to PCR andother protocols in food control, this strategy can be conducted away from enrichmentculture, so the original infection status is allowed to investigate. In addition, ourprotocol avoids complicated thermal cycling program and expensive equipment, andheating in a period of time using a simple water incubator is sufficient to amplifyDNA to detectable levels. SMB-assisted TC-RCA is considered to be promoted as afast, rapid, real-time detection strategy for detecting harmful microbes in food andrelated products.