Rapid Extraction Of Nucleic Acid From Pathogenic Microorganisms Based On Nanomagnetic Bead

Diseases caused by pathogenic microorganisms are collectively referred to as infectious diseases,among which the highly contagious ones that can cause transmission are called infectious diseases,and their large-scale transmission usually causes public health security incidents or even serious crises,such as the global outbreak of 2019.The novel coronavirus pneumonia(COVID-19)epidemic has infected hundreds of millions of people and killed millions,causing severe damage to the global economy.Rapid and accurate detection of pathogenic microorganisms is an important measure to prevent and diagnose various infectious diseases,and rapid acquisition of high-quality pathogen nucleic acid is the premise to ensure the efficiency and accuracy of nucleic acid detection.The current nucleic acid extraction methods have problems such as cumbersome steps,long time-consuming and organic reagents used in the extraction process have toxic effects on the human body.Therefore,it is essential to establish a simple and rapid nucleic acid extraction method for pathogenic microorganisms with high nucleic acid yield.The first part is the establishment of Pasteur Pipette-Nanomagnetic Beads(PPNB)nucleic acid extraction system.In this study,nano-magnetic beads were combined with Pasteur pipettes,and Salmonella typhimurium was used as the research object.Using the new rapid nucleic acid detection technology-Accelerated Strand Exchange Amplification(ASEA)developed by the research group,The key factors of the extraction process,including the specifications of Pasteur pipettes,the type and amount of magnetic beads,lysis adsorption conditions,washing solution components and elution conditions,were optimized.Finally,a PPNB system with short extraction time(15 min),high recovery efficiency(93%-98%),simple operation and no electrical equipment was established.The second part evaluated the applicability of PPNB to different types of samples and pathogenic microorganisms and the sensitivity of ASEA to PPNB to extract nucleic acid products.The PPNB system extracted four artificial simulated samples,including SARS-Co V-2 false virus-positive throat swabs,Staphylococcus aureus-positive serum,Salmonella typhimurium-positive milk,and Vibrio parahaemolyticus-positive pork,respectively.Using ASEA to amplify the above-mentioned sample nucleic acid products extracted by PPNB,the results show that the PPNB system is suitable for various types of samples.At the same time,the sensitivity of ASEA to PPNB to extract the nucleic acid products of the above samples was analyzed.The results showed that the detection limit of SARS-Co V-2 pseudovirus in throat swabs was as low as 1.0×10~3 copies/m L.The detection limit of Staphylococcus aureus in serum,Salmonella typhimurium in milk and Vibrio parahaemolyticus in pork were as low as 10 CFU/m L,These data show that the PPNB system has strong concentration and enrichment ability when extracting the above-mentioned sample pathogen nucleic acid.In addition,30 clinical samples collected from hospitals were extracted using PPNB,The samples included Mycoplasma pneumoniae-positive throat swabs,human papillomavirus 16-positive cervical swabs,and gastric mucosa from patients with Helicobacter pylori infection,Using ASEA to amplify the nucleic acid products of the samples extracted from PPNB,the amplification results are consistent with the hospital diagnosis results.It is further proved that the extraction method is suitable for different types of samples and pathogenic microorganisms,and is expected to be used for clinical diagnosis.The third part compares and analyzes the PPNB system and the magnetic bead commercial kit,the spin column commercial kit and the boiling method kit to extract nucleic acid,the time and the equipment used in the extraction process.Three bacteria(Staphylococcus aureus,Salmonella typhimurium,Vibrio parahaemolyticus)were used as targets,and the ability of each method to extract nucleic acid was evaluated by ultra-trace spectrophotometer and ASEA.From the analysis of DNA concentration and purity,the nucleic acid concentration extracted by PPNB is 10-40 ng/μL(10%-37%)higher than the above three kits,and the nucleic acid purity is higher;On the other hand,the average Ct value of the nucleic acid products extracted by ASEA for the PPNB system was 2-5 earlier than that of the three kits,which further verified that PPNB has a strong ability to extract nucleic acids.Using serial dilutions of Staphylococcus aureus,the sensitivity of ASEA to the extraction of nucleic acid products by four methods was determined,The results showed that ASEA had the highest sensitivity for PPNB extraction of nucleic acid products,and the detection limit was 10 CFU/m L,indicating that the ability of PPNB system to concentrate and enrich target nucleic acid from samples was higher than that of the three kits.In addition,compared with the three kits,the extraction time of PPNB is only 15 minutes,and the whole process is carried out in the tube.It does not require steps such as liquid transfer,and also does not require electrical equipment such as metal baths,vortex mixers,centrifuges,etc.,which achieves the goal of simple and rapid extraction of pathogenic microorganisms.The results of this paper show that the PPNB extraction system can be applied to the extraction of nucleic acid from different types of samples and pathogenic microorganisms,with high recovery efficiency and good extraction effect.The nucleic acid extraction can be completed within 15 minutes without electrical equipment.This fast,easy and efficient extraction method is more conducive to the application of pathogen nucleic acid detection on a global scale.