Detection Of Bioaerosol Nucleic Acid Based On Recombinant Polymerase Amplification And Electrochemiluminescence

Aerosol pathogens mainly include viruses and bacteria(biological aerosols).Because aerosol size is small and often suspended in the air,biological aerosol pathogens are liable to cause large-scale outbreaks of respiratory infectious diseases.Biological aerosol sampling is particularly important for the prevention and control of infectious diseases.At present,the traditional biological aerosol sampling methods mainly include natural sedimentation method,impact method,sampling membrane method and so on.Because of the high sampling efficiency,the sampling membrane method is often used in air sampling.FTA card(flinders technology associates)is suitable for the preservation of blood,oral swabs,epithelial cells,bacteria,viruses and culture cells,which can bind to nucleic acids and maintain the integrity of DNA in the samples.FTA card has not been reported for the collection of biological aerosol pathogens.The common methods for detection of biological aerosol pathogens include cell culture colony count,immunoassay and nucleic acid detection.The method of cell culture colony count is dependent of manual operation dependent,time-consuming and laborious and needs cell culture more than 24 hours,which can not realize automatic and rapid detection.The method of immune detection is relatively simple to deal with the sample,but there are some problems in the detection of complex samples,such as cross contamination and so on.In recent years,nucleic acid molecular detection technology plays a more and more important role in clinical treatment,disease prevention and control,such as polymerase chain reaction(PCR),fluorescence in situ hybridization(FISH),gene sequencing technology and so on.But the traditional PCR method depends on the precision instrument to control the temperature change accurately,and it is operated and analyzed by the professional technicians,which limits its application in the field of detection with limited conditions to a great extent.In recent years,the outbreak of various airborne infectious diseases(such as SARS,H1N1,H7N9)in our country has brought unprecedented challenges to the prevention of infectious diseases,in order to effectively prevent the outbreak of infectious diseases.The research on rapid detection of biological aerosol pathogens is of great significance.A FTA card sampling method for biological aerosol pathogens was constructed in this study,which realized the integration of aerosol pathogen sampling and nucleic acid extraction.Combined recombinant polymerase amplification with paper based electrochemiluminescence(RPA-ECL),a sensitive,specific and simple method for the detection of biological aerosol pathogens was established.The serum samples of clinical HBV were detected and compared with real-time fluorescence quantitative PCR.The main results are as follows:1.By FTA card,sampling can be continued for 8 minutes under the initial negative pressure of 40 kpa,98%RH of air humidity and tempreture at 26?.The maximum unit interception of the sample is 0.96 mg/cm~2.FTA card can be used to collect aerosol HBV plasmid,and the collection effect can be semi-quantitatively analyzed by RPA+gel electrophoresis ultraviolet imaging method.2.The labeled rate of tripyridine ruthenium labeled primers was 74.62%,which provided the technical basis for the preparation of tripyridine ruthenium labeled primers.3.The optimal reaction conditions for RPA of HBV plasmids were 0.8?mol/L primer,280 mmol/l magnesium acetate,and 20 min at 30?.4.The experimental results of sensitivity and linear detection range about RPA-ECL method showed that the ECL response intensity show a good linear relationship between concentration of 0.12-1200 ng/ml HBV plasmid samples.Detection limit was 1.2pg/ml(S/N>3)and the relative standard deviations were 5.18%and 6.40%,respectively.The results suggest that RPA-ECL method has good reproducibility,sensitivity and wide linear range for detection of HBV gene.5.FTA-RPA-ECL method was used to detect the clinical HBV serum samples.The ECL response intensity showed a good linear relationship in the concentration of 20-2×10~5 IU/mL and the detection limit was 0.2 IU/mL(S/N>3).The relative standard deviations were 1.94%,2.03%and 2.32%for three groups of samples with different concentrations,respectively.The relative deviations between the results of detection and real-time quantitative PCR were-8%—12%.The experimental results show that the FTA-RPA-ECL method has high accuracy and repeatability for the detection of clinical HBV serum samples.In a word,the integrated operation of sampling and nucleic acid extraction can be realized by using FTA card to sample air aerosol pathogens,and the whole detection process can be simplified.Nucleic acid detection based on RPA and paper-based ECL has the characteristics of specificity,simplicity,sensitivity and rapidity.Therefore,the biological aerosol sampling method based on FTA card,combined with RPA and paper based electroluminescent(ECL)technology,is expected to provide a new way for the rapid and real-time detection of virus in biological aerosol.