The Development Of An Infectious Disease Detection System Based On The Fully-Enclosed Cartridge

In recent years,due to environmental changes,natural disasters,changes in species diversity and other factors,a variety of infectious diseases caused by various virus break out continuously and appear repetitively,such as SARS virus,H1N1,H7N9 Avian influenza virus,MERS-CoV(Middle East Respiratory Syndrome Coronavirus),as well as the once-feared Ebola virus and Zika virus in these two years.With the acceleration of the globalization process and the changefulness of the route of transmission of infectious diseases,the outbreak of these infectious diseases no longer appears in a local area.It is bursting out all over the world,which brings great harm to human health and society.Therefore,the prevention and control,diagnosis and treatment of infectious diseases have become a global public health's hotspot and focus.However,most of the traditional detection methods for infectious agents are suitable for the laboratory deployment environment,which has the disadvantages of dispersed and complex detection process,complex experimental operation and environmental sensitivity etc.Besides,it has high requirements for the experimental skills of laboratory staff.All these disadvantages limit the application of the traditional detection technology in the field of on-site testing of infectious diseases.The rapid detection of the pathogen of infectious diseases has the characteristics of simple operation,low-cost,portability,automation and so on.Therefore,it is urgent to find a new technology platform to meet the needs of the field of infectious diseases.In this paper,an enclosed testing cartridge was designed and manufactured using 3D printing technology.A fully automatic and portable nucleic acid detection system was developed on this basis.The performance test of the instrument and the detection of the infectious disease form clinical samples were carried out,which demonstrated the automatic system has the ability to detect the infectious disease from sample to answer.The specific contents include:1.The design and manufacture of the enclosed testing cartridge based on 3D printing technologyFirstly,according to the experimental procedure of nucleic acid testing,the design requirements of the encloded cartridge are defined.The cartridge structure based on the structure of a flexible slide and its internal function modules are presented.A high precision 3D printing technology and medical materials(PC-ABS)are used throughout the design and manufacturing process,experiencing a total of 5 batches of rapid iterative development processes.The structure of 7 special parts of cartridge is designed and optimized and its size is 140 mm×90 mm × 30 mm.Liquid handling module,areas of sample preparation,amplification and detection are integrated internally.Sample can range from 20 ?L to 1000 ?L,and multiplex realtime PCR detection of at most six kinds pathogens could be carried out.The liquid handling range is 2?200 ?L.The relative errors measured at 10 ?L and 100 ?L are 1.71%and 0.76%respectively.The repeatability CV values are 2.3%and 0.77%respectively,which are better than the national standard.At the same time,the method of nucleic acid extraction based on magnetic separation and PCR preparation process in the cartridge have been optimized.Finally,the experimental results show that the upstream nucleic acid extraction process has no inhibitory effect on the downstream real-time PCR,which suggests that it is feasible to carry out integrated nucleic acid testing in the enclosed cartridge.2.The research and development of the on-site pathogen detection system based on the enclosed cartridgeThe automatic driving system was developed based on the enclosed cartridge.The internal system is composed of functional modules such as liquid handling control module,magnetic separation and heating module,thermal cycle module,fluorescence detection module and auxiliary circuit etc.Among them,the maximum operating volume of the liquid handling control module is 260 ?L,with the accuracy of 0.118 ?L,which contains the filter element to prevent cross contamination.Magnetic separation part of the magnetic separation and heating module adopts the inclined sliding structure and it could control the position of the magnetic enrichment programmatically.The temperature control precision of heating part is±0.5? and it could rise to the target temperature within 2 min.The thermal cycle module could provide 3 channels for PCR reaction,and the heating and cooling speeds are 4.6?/s and 4?/s respectively,with a control precision of±0.3?.Fluorescence acquisition module provides dual color fluorescence channels(FAM/SYBR Green and Texas Red)using monochromatic LED as the excitation light source,the volatility of which is less than 1?W.The collection of the fluorescence signal can be completed within 2.5 s.The overall size of the instrument is 270 mm ×275 mm × 250 mm,and the weight is 6.5 kg.The cartridge with the sample just put into the instrument with the experimental parameters configured by software,and then the whole process could be completed automatically.3.The performance testing of the on-site pathogen detection systemFirstly,the contrast experiment of the extraction efficiency of nucleic acid was carried out by using the plasmid DNA of Pertussis.The efficiencies of the systematic and manual extraction were 95.49%and 84.33%respectively.The extraction limit of 104 copies/mL was obtained using the HBV plasmid,suggesting the efficiency and sensitivity of the system could meet the needs of the downstream PCR detection.At the same time,clinical samples of E.coli and HBV were used to extract the nucleic acid.The results show that the systematic operation is 6.6 ng/L and 0.93 ng/L higher compared with the manual operation respectively.The detecting difference between holes of the realtime PCR is 0.023(CV value)with the detection limit of 104 copies/mL.Finally,the testing results are consistent with the expected results of the interval experiment of 20 consecutive runs of negative and positive samples,which suggests that there is no cross contamination between batches.4.The practical application of the on-site infectious disease detection systemThe detection of 30 cases of HBV virus was carried out in the GuLou hospital of Nanjing city.The order of magnitude of HBV-DNA used in the sample was greater than 104 copies/mL,meanings that the detection of HBV virus was positive in all patients.And all results were consistent with the testing result of the hospitals.The testing experiment of adenovirus was carried out in Jiangsu provincial center for disease control and prevention,and the Roche LightCycler 2.0 fluorescence quantitative PCR instrument was used for the experimental control.The Ct values of the unknown samples were 31.15 and 31.68 respectively.The Ct values of positive control were 27.52 and 28.01 respectively.The comparison of the shape of the amplification curve and the Ct value showed that the detection results of the two systems were consistent.Finally,the cartridge system was used for the multiple-detection of Salmonella and Shigella.The suspension sampling method was used for the preparation of the PCR reagents in the cartridge.At the same time,the ABI SteponePlus PCR system was used for the contrast experiment.The shapes of the two amplification curves were consistent.The mean difference of Ct values between the two fluorescence detection groups(FAM and Texas Red)was 0.71 and 0.98 respectively,just has a difference of 0.29.The result shows that the cartridge system is consistent with the test results of the commercial system in multiple-detection.