Research Of A New Method Of Staggered Strand Exchange Amplification And Its Application In The Detection Of Staphylococcus Aureus

Staphylococcus aureus is one of the bacterial microorganisms that causes foodborne diseases nationwide,and its high pathogenicity and high mortality rate of patients pose a great threat to human life and health.Rapid and sensitive detection of S.aureus is important to prevent and control the spread of this pathogen.In this study,a new isothermal nucleic acid amplification technique termed staggered strand exchange amplification（SSEA）was established.This technique uses Bst 2.0 Warm Start DNA polymerase and two sets of forward and reverse primers arranged in tandem to invade denaturation bubbles of double-stranded DNA to achieve exponential amplification of the target fragment.The optimal reaction temperature of60°C was determined by experimental optimization,as well as 0.75μM primer F1/R1,1.5μM primer F2/R2,3.2 U Bst 2.0 Warm Start DNA polymerase,2.0 m M Mg SO₄ and60 m M guanidine hydrochloride as the optimal reaction component concentrations.The reaction time was less than 1 h.In order to get rid of the dependence on precision instruments,a visualization assay was established in this paper,with the system color as rose red for positive and orange for negative,and the whole process can be completed with only a small metal bath.The technique could detect a minimum of 20 pg/μL genomic DNA,and the sensitivity of SSEA technique was increased by 20-fold compared with denaturation bubble-mediated strand exchange amplification（SEA）.Based on the reverse transcription activity of Bst 2.0 Warm Start DNA polymerase,SSEA could be used for one-step detection of RNA,solving the current chalenge of multi-step amplification detection of RNA and providing a new means for rapid nucleic acid detection of alive S.aureus.In this study,al-in-one S.aureus detection protocol was established by combining the silica-hydroxy magnetic bead extraction method with the SSEA technique.100μg of magnetic beads and p H 5.0 adsorption buffer were identified as the optimal extraction conditions.The adsorption process only required 5 min,and all the enriched DNA could be used for subsequent amplification,thus improving the sensitivity of the protocol.The al-in-one protocol had good specificity and could detect pure cultures of S.aureus at10² CFU/m L.The detection limits of the protocol were 10²,10³ and 10³ CFU/m L in artificially spiked S.aureus pork,duck and scallop samples,respectively,with sensitivity comparable to that of Loop-mediated isothermal amplification technique.In summary,the novel isothermal nucleic acid amplification assay established in this thesis has the advantages of easy operation,short time,high sensitivity and specificity,and can achieve nucleic acid enrichment detection in low concentration samples,which is expected to meet the demand for immediate detection of pathogenic bacteria in the field under low-resource conditions,and is of great significance for solving the worldwide outbreak of foodborne diseases.