Establishment Of Helicase-dependent Isothermal PCR For The Detection Of Several Pathogens

In the past ten years,high-risk influenza infectious diseases have gradually increased,showing a trend of repeated attacks,and it is difficult to carry out medical rescue tasks in some remote mountainous areas and unstable power supply areas in China.In this context,high requirements are placed on the rapid detection of pathogens.Helicase-dependent amplification(HDA)is a novel molecular amplification technology,which can use helicase to efficiently unravel the DNA double strand under constant temperature conditions,and cooperate with DNA polymerase to achieve exponential amplification of target genes in a relatively short time.This technique differs significantly from traditional thermal cycling PCR,reducing the test environment and equipment requirements while ensuring the accuracy of test results.This project aims to develop an efficient isothermal PCR reaction system that relies on helicase and detect several pathogens.In this paper,recombinant DNA polymerase,DNA helicase derived from the hyperthermophilic archaeon Thermococcus kodakarensis were heterologously expressed using Escherichia.coli,And the recombinant enzyme purified by anion exchange chromatography column was used to construct the HDA multi enzyme reaction system.The following pathogens were used: a high GC content ropb fragment(B.tuberculosis),a medium GC content ttp47 fragment(syphilis),and a low GC content tac1fragment(Candida albicans).In addition,an inorganic pyrophosphatase TK1700 recombinant expression plasmid p ET28a-TK1700 derived from T.kodakarensis was constructed and inducibly expressed in E.col I,purified,characterized,and applied to the HDA system to enhance its amplification efficiency.The helicase encoded by KOD polymerase with TK0178 was successfully prepared and the purity was above 95%,The concentration of KOD pol was 0.54 mg/m L,Helicase TK0178 at a concentration of 318 μg/m L.The constructed HDA multi-enzyme reaction system can effectively amplify DNA fragments with high,medium and low GC content at 77 °C,and realize the amplification of pathogen genes with a full range of GC content.Inorganic pyrophosphatase TK1700 was successfully expressed and prepared,and the purity of TK1700 gene was 90% by expressing and purifying it in E.coli in large quantities.With a concentration of 380 μg/m L and a specific activity of 180 U/mg,the optimal p H of the enzyme was 9.5 and the optimal temperature for the reaction was 65 °C.In the HDA reaction system,the HDA reaction system with inorganic pyrophosphatase TK1700 added increased the amplification efficiency by 1.47 times compared with the system without pyrophosphatase TK1700,and the detection was shortened to 30 min.The HDA multi-enzyme reaction system constructed by the experimental results that can detect a full range of GC content pathogens solves the problem that the current commercially available HDA kits cannot detect high GC content pathogens,and provides a new tool for rapid in vitro detection of DNA.TK1700 inorganic pyrophosphatase added to the isothermal PCR system can be used as a novel PCR enhancer for molecular biology research and applications.