Research On Nucleic Acid Detection Technologies Based On Loop-mediated Isothermal Amplification And Their Application In Biological Sample Analysis

Nucleic acids are biomarkers for biological research and medical diagnosis,and their analysis can assist in the screening,diagnosis,risk assessment,and treatment planning of diseases in clinical practice.Driven by pressing clinical needs,nucleic acid tests based on different detection principles have evolved to be a rapidly advancing field.Therefore,improving the detection specificity,sensitivity,and simplicity of nucleic acid tests are the key to their clinical application.In view of the high sensitivity and specificity of loopmediated isothermal amplification(LAMP)technology,this study established several LAMP-based analytical methods for detecting SARS-CoV-2 RNA and miRNA.Firstly,we established a reverse transcription loop-mediated isothermal amplification(RT-LAMP)assay for the simultaneous detection of ORF1ab and N gene fragments of SARS-CoV-2 RNA in one pot.In the presence of AMV reverse transcriptase which possesses activity in a large range of temperatures and Bst DNA polymerase with chain displacement activity,the reverse transcription and LAMP could be carried out simultaneously.Using two primer sets and two molecular beacon(MB)probes respectively labelled with different fluorophore,positive results were obtained in one pot with a limit of detection of 20 and 2 copies/μL for ORF1ab and N gene fragments,respectively.The proposed method is more sensitive than reverse transcription quantitative polymerase chain reaction(RT-qPCR)method,and has good selectivity and matrix interference ability.Subsequently,using universal LAMP amplification primer sets and placing the P681 site of the SARS-CoV-2 S gene at the recognition site of detection probe to design two OSD probes targeting wild type and Delta strain,the RTLAMP based assay was applied to differentiate the S gene of wild type,Delta and Alpha strains.The proposed method is based on the difference in the single base of the mutation site,which results in different efficiency of the amplification product in opening the OSD probe,and achieves the distinguishing of the mutant strains based on fluorescence intensity.At last,a ligation-initiated phosphorothioated primer-based LAMP(LI-PS-LAMP)strategy was established to quantify miRNAs.In this method,two linker probes designed from dumbbell-shaped amplicons of LAMP are ligated by SplintR Ligase to obtain an active amplicon only when they are perfectly hybridized with target miRNA,exhibiting high specificity.Then,under the catalysis of Bst DNA polymerase,the amplicon binds to FIP/BIP inner primers to complete LAMP amplification.The introduction of PS modification into inner primers facilitated extra efficient hairpin formation and extension for LAMP,which significantly improved sensitivity.Using two sets of linker probes,inner primers and MB probes,simultaneous quantification of two miRNAs in one pot with limit of detection of 100 amol/L was achieved.The proposed method was more sensitive than stem-loop RT-qPCR method and was successfully applied to the detection of miR-451a and miR-146b-5p in tissue samples.To further demonstrate the clinical application value for detecting miRNAs in biological samples,the LI-PS-LAMP method was applied to detect low abundance miRNAs in fine-needle aspiration(FNA)washout precipitation specimens of thyroid nodules.By screening candidate miRNAs through public database analysis and tissue samples validation,then detecting the expression levels of these candidate miRNAs in FNA washout precipitation specimens of thyroid nodules using the LI-PS-LAMP method,a diagnostic model for distinguishing papillary thyroid carcinoma(PTC)from benign nodule was established.Then,the diagnostic efficiency of the model was evaluated in the validation cohort samples.The results indicated that the combination of miR-146b-5p and miR-222-3p in the FNA washout precipitation specimens has good diagnostic efficiency in distinguishing PTC from benign nodules,and has the potential to become biomarkers for PTC liquid biopsy.This process also reflects the advantages of the LI-PS-LAMP method as a miRNA detection method in biological samples,demonstrating its good application prospects.