Research On Loop-mediated Isothermal Amplification Based Novel Bioanalytical Method

Recently,rapid development of biotechnology has greatly promoted the development of life science and analytical chemistry.Nucleic acid amplification,an important technology in molecular biology,has the advantages of being high specific and rapid and is used in the field of biomedicine and clinical diagnosis.However,quantitative detection of analytes with high robustness,sensitivity and selectivity still presents a challenge.In this doctoral thesis,we have developed many novel LAMP(loop-mediated isothermal amplification)based methods for the detection of microRNA,UDG activity,SNP and bacterial pathogens.The developed methods are simple,rapid,highly sensitive and selective.The specific contents are as follows:MicroRNAs(miRNAs)play important roles as post-transcriptional regulators for gene expression and serve as promising biomarkers for diagnosis and prognosis of disease.In chapter 2,we develop a novel ligation-based loop-mediated isothermal amplifica-tion(Ligation-LAMP)method for highly selective miRNA detection based on reverse transcription of target miRNA into cDNA and cDNA templated ligation of two hairpin probes followed by a LAMP mediated detection of the ligated product.Thus,a large number of long stem-loop DNA replicates are generated,achieving exponential amplification of the target miRNA.Employing SYBR Green I,a double-stranded specific dye,as the readout signal of the amplification reaction,the real-time monitoring of miRNA is achieved.This method is demonstrated to allow quantitative detection of miR-21 in a dynamic range from 1 fM to 1 nM with a detection limit of 0.2 fM.Moreover,this assay has the ability of discriminating single-base mismatch with a discrimination ratio over 300,and shows good performance for miRNA detection in complex biological samples.Therefore,the proposed strategy may provide an isothermal and cost-effective approach for highly selective and sensitive detection of miRNA,affording useful platform for miRNA based diagnostics.Uracil-DNA glycosylase(UDG)plays essential roles in base excision repair pathway by eliminating uracil from DNA to sustain the genome integrity.Sensitive detection of UDG activity is of great significance in the study of many fundamental biochemical processes and clinical applications.In chaper 3,we develop a label-free method for UDG activity detection using stem-loop primer-mediated exponential amplification(SPEA).In the presence of active UDG,the uracil base in helper hairpin probe(HP)can be excised to generate an abasic site(AP site),which can be cleaved by endonuclease IV(Endo IV)with a blocked primer released.This primer then triggers the strand displacement reaction to produce a dumb-bell structure DNA,which can initiate the cycling amplificarion step of LAMP reaction.This reaction generates a large number of long stem-loop DNA replicates,which can be stained by SYBR Green I to deliver enhanced fluorescence for quantitative detection of UDG activity.A linear range from 1 mU/mL to 1 U/mL and a detection limit down to 0.68 mU/mL are achieved.This strategy has also been demonstrated for UDG assay in complex cell lysates,implying its great potential for UDG based clinical diagnostics and therapeutics.The relation of single-nucleotide polymorphisms(SNPs)to many diseases emphasizes the importance of fast and accurate SNP detection for biomedical research and diagnosis.However,sensitive and selective detection of SNP remains a chal-lenge due to the small amount of mutant sequences in a high background of wild-type genes.In chapter 4,we develop a simple,highly sensitive and specific strategy for SNP detection named PA-LAMP on the basis of LAMP incorporating RNase H2 enzyme that has SNP discrimination capability.BIP is modified with a single RNA residue complementary with the mutant target(MT)near the 3' end and a C3 spacer at the 3' terminal to prevent the polymerase extension.When the BIP forms a perfect match with the target DNA at the RNA base,the phosphodiester bond upstream to the RNA base is then cleaved by RNase H2,generating a hydroxyl group at the 3' end which makes the BIP to be able to function as a primer again to trigger the LAMP reaction.Via repeat allele discrimination by RNase H2 in the process of LAMP,only the mutant target sequence can be amplified with an enhanced fluorescence signal.The developed assay is demonstrated to give a discrimination ratio of close to 10000,and the lowest detectable concentration for the mutant target is down to 22 aM.Moreover,this method shows desirable performance for specific SNP detection in real biological samples.The results confirm that this simple and reliable technique holds great potential application in biomedical diagnosis and therapy.In chapter 5,we develop a dual-LAMP method based on the PA-LAMP reaction for the detection of C.difficile and L.pneumophila at the same time.Firstly,two sets of LAMP primers were designed for the TcdB gene of C.difficile and the mip gene of L.pneumophila,respectively.The FIPs is modified with a single RNA residue near the 3' end which flanked with a fluorophore and a quencher.The CD-FIP is modified with ROX and the LP-FIP is modified with FAM.In the presence of the target sequence,the RNA base in the FIP can be cleaved by RNase H2 enzyme,initiating the LAMP reaction and generating fluorescence signal.Via distinguishing the ROX or FAM signal to achieve the dual detection of the two bacterial pathogens.This method is demonstrated to show good performance for dual detection of bacterial pathogens in real samples.