Preparation Of Hot-start DNA Polymerase And Its Preliminary Application In Food Detection

Direct PCR technology has a wide application prospects in the field of rapid food detection with the advantages of time and labor saving.However,the direct PCR technology still has some problems,such as the insufficient detection sensitivity and the low amplification rate.DNA polymerase is the key component for the direct PCR reaction system.It is of great significance to promote the progress of direct PCR technology by modifying the DNA polymerase to improve its resistance to inhibitors,amplification rate and sensitivity.In this paper,a Taq DNA polymerase mutant?STaq?was prepared by genetically engineering,and the enzymatic properties of STaq were studied.STaq was further modified to hot-start DNA polymerase?HS-STaq?by the incubation with its monoclonal antibody,the PCR amplification characteristics of HS-STaq was investigated,and then HS-STaq was applied to the detection of adulteration of dairy products.The main research contents and conclusions are as follows:?1?The recombinant expression of STaq.The staq gene was successfully designed and synthesized,p ET-28a recombinant expression vector for STaq expression was constructed,and STaq was expressed in a recombinant E.coli.The conditions for inducing STaq expression were optimized:induction temperature of 25?,IPTG concentration of 0.1 mmol/L,induction time of 6 h.Electrophoretic pure STaq stock solution with the enzyme activity concentration of9.4×104 U/m L,was prepared by nickel ion affinity chromatography predominantly.?2?The enzymatic charactersiteics of STaq.The constitute of the optimal PCR buffer for STaq:p H9.0,20 mmol/L Tris-HCl,2 mmol/L Mg SO₄,20 mmol/L KCl,10 mmol/L?NH₄?₂SO₄,0.05%Triton X-100.After heat treatment at 95?for 2 h,STaq could still maintain more than75%of the initial enzyme activity.The PCR extension rate with STaq reached 1 s/kb at the optimal PCR extension temperature of 72?,and the target gene with a GC content of 73.1%could be amplified from the genomic template.In addition,STaq could tolerate the PCR system with the inhibiters of 0.04%SDS or 100 mmol/L Na Cl,and the direct PCR amplification of rat tail lysate and 50%?v/v?blood was achieved.?3?Preparation and properties of HS-STaq.The monoclonal antibody 6C1 was screened to block STaq enzymatic activity reversibly with the highest efficiency.The incubating conditions for preparing HS-STaq from STaq and antibody 6C1 were optimized:molar ratio of1:2,temperature of 0?,incubation time of 60 min.The hot start temperature of the prepared HS-STaq was greater than 70?,and the DNA polymerase activity could be released completely by heat-treatment at 95?for 80 s.The enzyme activity lost only 5.2%rate after STaq was modified to HS-STaq.Compared with STaq,the sensitivity of HS-STaq for PCR amplification was increased by more than 10 times,and the specificity was significantly improved.?4?Application of HS-STaq in the detection of adulteration of dairy products.The direct PCR detection methods with HS-STaq were established preliminarily for the detection of cow-derived genes in goat milk products or soybean-derived genes in cow milk products.In the direct PCR detection system,the highest addition amount of goat milk samples was 15%?v/v?,and the highest addition amount of cow milk samples was 20%?v/v?;the detection sensitivity of both detections was both 0.1 ng genomic DNA/?L.