Establishment And Application Of Novel Isothermal Amplification And Portable Signal Output

In recent years,there have been frequent outbreaks of various viruses,such as the severe acute respiratory syndrome coronavirus(SARS-CoV)that broke out in 2002,the influenza A H1N1 virus that broke out in 2009,the Middle East respiratory syndrome coronavirus(MERS-CoV)that broke out in 2012,and the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)that broke out in 2020.Above viruses are highly contagious and pathogenic,and rapid,accurate,and sensitive detection can greatly reduce their exposure to damage to human society.Besides,cancer is also one of the major diseases affecting human health.Studies have found that early-stage patients may be cured with timely treatment,while middle-advanced patients are more difficult to cure,and have higher recurrence and metastasis rates.Therefore,early detection,early diagnosis and early treatment are extremely important for cancer patients.Conventional etiological detection methods include direct smear microscopy,separation culture and biochemical reaction,immunological detection,nucleic acid detection,etc.Among them,the nucleic acid detection directly detects nucleic acid.After the low-abundance nucleic acid undergoes an amplification reaction,a large number of amplification products can be produced,so that the detection has high accuracy,sensitivity and specificity.It is one of the most used detection techniques.Common nucleic acid amplification methods include polymerase chain reaction(PCR),loop-mediated isothermal amplification(LAMP),and nucleic acid sequence-based amplification(NASBA).These methods have good sensitivity,but all require timeconsuming and labor-intensive primer design and optimization process,which increases the detection cycle and cost.And the length of the targeted nucleic acid fragment should be no less than 100 nucleotides,which cannot be applied to the detection of microRNA(miRNA)containing 20-25 nucleotides,so these methods also lack universality.In addition,nucleic acid amplification products need to have a matching signal output method to assist in the realization of efficient,sensitive,and portable detection.Common signal output methods include fluorescence,electrochemistry,colorimetry,and portable nursing diagnosis platform(POCT)devices.Among them,POCT devices(such as blood glucose meters)have the advantages of simple operation,miniaturization of instruments,instant results,and low cost.It can play a huge role in responding to sudden outbreaks and controlling the spread of infectious viruses.Upon to now,detection of nucleic acid-based targets has been achieved using blood glucose meters.However,in these tests,invertase needs to be expressed and purified in advance,and the operation is cumbersome.Moreover,affected by factors such as the difference of magnetic ball batches,the reproducibility and stability of the results are not ideal.Therefore,there is an urgent need to establish a label-free and separation-free signal output method.This topic will be carried out from the following three aspects:1.A dual hairpin ligation-induced isothermal amplification(DHLA)is proposed for the first time.Introduced a SplintR ligase,which can use complementary DNA or RNA as a "bridge" to efficiently catalyze the connection of adjacent deoxyribonucleotides,so that the single hairpin probe(SHP)is connected to form a double hairpin probe(DHP)to start the exponential amplification reaction.And the detection of new targets can be realized without changing primers.Using miRNA and viral RNA fragments as model targets,it is found that DHLA is suitable for the detection of nucleic acids with a length of 20 nucleotides or more,with a detection limit as low as aM level,and meets the clinical requirements of miRNA.MiRNA-21 and miRNA-10b,which are both overexpressed in HER2+breast cancer,are used as two model targets to verify the effectiveness of multiplex detection based on DHLA.This multiplex detection can provide information on the expression levels of two or more miRNAs,thereby assisting the early diagnosis and treatment of cancer.Furthermore,SARS-CoV-2 is detected using a DHLA-based "multi-probe strategy".By simultaneously anchoring multiple regions of the long target,the risk of false negatives due to RNA degradation is greatly reduced.2.Upgrade DHLA to DHLApro.After sequence design(including signal probe,target length and base type of ligation position)and system optimization(including hybridization buffer and SplintR ligase concentration),DHLA’s sensitivity has been increased by about 2000 times,meeting the detection needs of pathogens.Replace the F:Q probe with the F*:Q*probe that hybridizes to the BL*fragment.BL*is only generated after the exponential amplification reaction is turned on,which greatly reduces the possibility of background signal,and the sensitivity is improved from aM to zM level.On this basis,DHLApro is used to detect the highly infectious S ARS-CoV2 Omicron variant and Delta variant with high sensitivity and universality.In addition,N pathogens in 1 screening,Two-tube two-channel quadruple detection and One-tube three-channel triple detection are successfully carried out based on DHLApro,which minimizes the development cycle,development cost and cross-interference between primers for primary screening and multiplex analysis.3.Introduce the Toehold Switch element in synthetic biology to realize portable signal output.The sensor plasmid containing the Toehold Switch element can controllably express the reporter protein,thereby realizing the detection of the target.Thermotoga maritima Invertase(TmINV)is used instead of the original β-galactosidase(LacZ)as the reporter protein,and a sensor plasmid with the reporter gene TmINV is successfully constructed.TmINV catalyzes the conversion of the substrate sucrose into glucose,and the signal can be read by a personal blood glucose meter(PGM),realizing the portable detection of the target.In addition,in order to avoid complicated Toehold Switch element design,a four-way transport probe(composed of TP1 and TP2)is introduced between the target and the Toehold Switch element,and the target hybridized with the four-way transport probe instead of directly with the Toehold Switch element sequence.This transduction method can input different targets into a high-performance sensor plasmid,which greatly reduces the design difficulty and cost of Toehold Switch elements,and greatly enhances the universality of detection.In summary,this paper establishes a new nucleic acid isothermal amplification method,which can not only detect nucleic acids with a length of 20 nucleotides or more,but also realize multiplex analysis,primary screening,genotyping,and mutation detection.It has excellent universality,sensitivity,and flexibility.At the same time,this paper successfully couples the four-way transport probe to the sensor plasmid whose reporter protein is TmINV,and constructs a general,portable,and efficient signal output method.This greatly reduces the design difficulty and cost of the Toehold Switch element,and the signal output can be performed by using a blood glucose meter,which provides a new idea for the development of the POCT.