Study Of Construction And Performance Of Highly Sensitive Detection System For E.coil O157:H7 Based On Aptamer And Rolling Cycle Amplification

E.coli O157:H7 is one of the most common foodborne pathogenic bacteria,which can cause serious diseases such as diarrhea,colic and even intestinal bleeding and has a low pathogenicity limit(10-100 CFU/m L),so it is important to monitor E.coli O157:H7 in food or the environment.Traditional detection methods take too long,are cumbersome,costly,and require professionals to operate large instruments to meet the demand for rapid,low-cost,and sensitive detection,so the development of sensitive,rapid,low-cost,and simple detection methods in complex samples is essential.Aptamer is a functional nucleic acid that is specifically identified from a DNA library by in vitro ligand index enrichment evolution(SELEX)and can bind to a wide range of targets such as bacterial toxins,proteins and even whole cells.Aptamers have been widely used as recognition elements in biosensors because of their programmability,non-volatility,temperature tolerance,long shelf life and low cost.The current types of aptamer-based biosensor signal outputs are fluorescent,colorimetric,electrochemical,chemiluminescent,and other aptamer sensors,but many methods cannot meet the need for sensitive and simple detection in complex samples.Rolling cycle amplification(RCA)is an efficient isothermal amplification strategy that eliminates the need for large instruments and complex operations for warming and cooling.Rolling cycle amplification requires primers that are strictly complementary to the cycle template and therefore highly sp ecific.And exponential signal amplification can be easily achieved by adding primers,which has been widely used for signal amplification in biosensing.In this paper,two simple,low-cost and highly sensitive fluorescent aptamer sensors for the detection of E.coli O157:H7 were constructed and their detection performance was investigated by using a variant aptamer probe with an aptamer sequence as the recognition element with a high-efficiency signal amplification strategy of rolling cycle amplification as follows.(1)Sensitive detection of E.coli O157:H7 based on variant aptamer probes and rolling cycle amplificationIn this chapter,a hairpin-shaped DNA recognition element was designed to specifically identify the target E.coli O157:H7 and to enable a rolling cycle amplification signal amplification strategy.The recognition element is designed into three regions:an aptamer region with specific recognition and a primer region complementary to the aptamer base(hairpin stem),and a region connecting th e two(hairpin cycle).The presence of the target E.coli O157:H7 will bind the aptamer region to change the double-stranded portion of the hairpin DNA to single-stranded,exposing the primer region.In this study,we designed DNA dumbbell probe templates that can be complementarily paired with primer bases at both ends of the cycle,and when the variant aptamer probe recognizes and opens to bind to it,a long linear DNA product with repeated sequences is extended at the3′end of the primer using the dumbbell probe as a template by the action of polymerase.By designing a long DNA strand modified with a FAM fluorescent motif at the 5′end and another short DNA strand modified with a Dabcyl burst motif at the 3′end complementary to it as the signal molecule,the fluorescent modified DNA strand binds to it when there is an amplification product due to the extremely low signal at the fluorescence resonance energy transfer(FRET)norm,and the FRET disappears releasing the FAM fluorescent signal.The signal intensity is proportional to the target E.coli O157:H7.Through feasibility verification,condition optimization,detection performance analysis and specificity verification,this fluorescence sensing system has a good linear response in the range of 10~2-10~5 CFU/m L of E.coli O157:H7 concentration,and the linear response equation is y=21912.40+12417.26lgc(R~2=0.990),with good selectivity and can be applied in the detection of E.coli O157:H7 in milk.The method is simple,sensitive and selective,and can be applied to the detection of other substances by replacing the aptamers and their primers and template binding sequences,which has good application prospects.(2)Construction and performance analysis of a fluorescent aptamer sensor for the detection of E.coli O157:H7 based on enzymatic feedback double rolling cycle amplification cascade signal amplification techniqueTo further improve the detection sensitivity,this chapter improves the signal amplification strategy and signal molecules based on the prev ious chapter.In this study,a variant recognition element with complementary aptamer and primer chains was designed to convert the detection of bacterial single cells into a strategy of nucleic acid amplification.The aptamer is captured when the target E.coli O157:H7 is present,releasing the primer DNA strand.The dumbbell template for the first amplification is designed with a double primer complementary pairing region and an enzymatic antisense sequence for the endonuclease at each end of the cycle.When the primers are released,they bind to the cycle region of the dumbbell template,and polymerase causes the 3′end of both primer strands to extend continuously along the template,and the product has the recognition site of endonuclease,which will turn the product into a large amount of repetitive single-stranded DNA under the action of endonuclease,and the single-stranded DNA can be combined with the dumbbell template for feedback amplification to generate more single-stranded DNA for the first amplification.The single-stranded DNA acts as a primer in the next cycle amplification step,producing a long linear DNA product in the presence of the cycle template and polymerase,resulting in an overall exponential signal amplification.The product is des igned with a region that binds specifically to the molecular beacon through the cycle template,which is modified with a FAM fluorescent group and a Dabcyl burst group at the 5′and 3′ends,respectively,so that the hairpin is closed in the absence of the second amplified product because there is no signal from FRET,and the molecular beacon is turned on to generate a fluorescent signal in the presence of the product.The signal intensity was proportional to the detection target E.coli O157:H7.Through feasibility verification,condition optimization,detection performance analysis and specificity verification,the fluorescence sensing system had good linear response in the range of 10-10~6 CFU/m L of E.coli O157:H7 concentration,and the linear response equation was y=18045.71+10539.01lgc(R~2=0.996),and the limit of detection(LOD)reached 1.6 CFU/m L,with good sensitivity and selectivity.The recoveries were in the range of 96.25%-105.62%with RSD less than 5%in the actual milk samples with good practical application,and it can be applied to the detection of other substances by changing the aptamer and subsequent corresponding primers and template sequences,which has good application prospects.