Sensitivity Study Of Denatured Vesicle-mediated Strand Exchange Amplification (SEA) Nucleic Acid Detection Method

In this article,we will conduct preliminary research on the design rules of denatured bubble-mediated strand exchange amplification?Strand Exchange Amplifi-cation,SEA?primers,preliminary application of rapid SEA?accelerated SEA,ASEA?,and specificity of nucleic acid hybridization.The main research contents and conclusions are as follows:1.Denatured bubble-mediated strand exchange amplification primer?SEA?design strategy:using a series of M.pneumoniae primers with different Tm values to study the optimal combination of primer Tm value and reaction temperature.The effect of the Tm difference of primers on the amplification efficiency was studied via C.trachoma and S.domestica specific primers'amplification efficiency.M.pneumoniae and C.trachoma specific primers were designed to study the effect of GC content on amplification efficiency.Based on the amplification efficiency of C.trachomatis and B.cereus specific primers,the effects of primer complementarity and 3?-end complementarity on amplification efficiency were studied.The priority of Tm value and 3'-end G/C content was determined according to the amplification efficiency of S.aureus.Results:SEA had the highest amplification efficiency when the primer Tm value and reaction temperature were both 61?.The primer pair with the smallest difference in Tm value has the lowest threshold time?Tt?value,and the primer pair with the greatest difference in Tm value has the highest Tt value.Primers with higher G/C content at the 3'-end showed lower Tt values.The number of complementary sites is positively correlated with the Tt value.The primers have the lowest total potential complementary sites and the lowest Tt value.The difference between the Tm value and the Tm value should take precedence over the 3'-end G/C content.Conclusion:The design of SEA primers should obey the following principles:?1?The Tm value of the primer should be close to 61?;?2?The difference in primer Tm value should not be greater than 1?;?3?The last five nucleotides should have at least two G/C,and the terminal nucleotides should be G/C;?4?Primer self-complementarity and 3'-end complementarity should be avoided;?5?The factors mentioned above should be considered in order of priority for self-complementarity and 3'complementarity,differences in Tm and Tm values,and 3'-end CG content.2.Study of rapid SEA?ASEA?detection method:Design ASEA primers with M.pneumoniae as the target,and use short targets and PCR products to evaluate the sensitivity,respectively.The ability of ASEA to detect single base mutations was evaluated by SNP detective experiments.Design Mycoplasma haemofelis,feline herpes virus and canine herpes virus ASEA primers to assess sensitivity of ASEA.Results:The sensitivity of M.pneumoniae primers to short targets and PCR products was 1.0×10^-15 M and 1.0×10^-14 M,respectively.The ASEA detection method can detect 1%of the correct targets in the 1.0×10^-12 M mutation background.The sensitivity of H.haemophilus primers was 1.0×10^-15 M,the sensitivity of feline herpes virus primers was 1.0×10^-16 M,and the sensitivity of canine herpes virus primers was 1.0×10^-15 M.Conclusion:ASEA can finish the detection within 15 minutes,which takes only 50%of SEA,while 100times more sensitive than SEA.3.Nucleic acid hybridization specificity research:Based on the prediction of Gibbs free energy,calculate the discrimination factors of toehold probes and molecular beacons,design the molecular beacons of toehold and calculate the discrimination factors.Using Let-7a as a target,the effects of mutation positions and the number of complementary bases on??G were studied.Results:The maximum value of toehold probe discrimination factors was 2486,while the median was 621.The maximum molecular beacon discrimination factors is 21.25,while the median is 5.71.The maximum toehold molecular beacon discrimination factors is 693,while the median is 29.92.Regarding to Y-shaped probes,when the mutation sites are at positions 5 and 18,the??G has the greatest effect on the reaction,and the number of probe bases has no effect on??G.Conclusion:Compared to molecular beacons,the toehold molecular beacon's discrimination factors can increase by up to 72.85 folds;compared to the toehold probe,the toehold molecular beacon hybridization yield is increased by 1.40 times.When designing a Y-shaped probe,the mutation site should be designed in the center of the hybridization region.