| The novel coronavirus disease(COVID-19)caused by the novel coronavirus(SARS-CoV-2)poses a great threat to global public health security.Rapid detection of the virus is crucial in the prevention and control of such emerging infectious diseases.The traditional virus detection methods have the disadvantages of complex operation,high requirement of experimental environment and long detection time.The current development trend is rapid detection,simple operation,short detection period,and no need for complex instruments and equipment,which is conducive to the use in local unit and resource-poor areas.The most commonly used methods for SARS-CoV-2 detection are viral antigen detection and nucleic acid detection.The immunochromatographic assay using viral antigen as the detection target has the advantages of simple operation and rapid reaction.It can read the results immediately without equipment and is very suitable for field application.However,due to the low detection sensitivity,it can only be used as an auxiliary diagnostic method for pathogenic infection.Real-time fluorescence quantitative PCR(RT-q PCR)based on nucleic acid detection has the advantages of high sensitivity and accurate results,and has become the gold standard for the diagnosis of pathogenic infections.However,it requires professional laboratory conditions and equipment,as well as time-consuming nucleic acid extraction and amplification process.Therefore,RT-q PCR is not suitable for on-site and grassroots detection applications.At present,it is urgent to develop highly sensitive virus antigen detection technology and more simple and rapid nucleic acid detection technology.In this study,SARS-CoV-2 was taken as the research object.In view of the low sensitivity of virus antigen detection and the long time-consuming problem of nucleic acid detection,the high-sensitivity automatic magnetic particle immunoassay technology based on luciferase-labeled antibody and the integrated rapid nucleic acid detection technology based on recombinase polymerase isothermal amplification were studied respectively.In the research process of highly sensitive automatic magnetic particle immunoassay(Nluc-AMCA)based on Nanoluc luciferase-labeled antibody,in order to improve the sensitivity of virus antigen detection,we innovatively used Nanoluc luciferase as the labeling molecules,which is small molecular weight,high sensitivity and bio-markable,and adopted the following technical optimization strategies:(1)The eukaryotic system recombinant expression strategy was used to realize the efficient directional labeling of Nanoluc luciferase on the detection antibody,so as to avoid the uneven antibody labeling and uncontrollable labeling sites caused by traditional chemical labeling methods,which affected the binding activity of Fab fragments.(2)Explore different plasmid types,plasmid construction methods and transfection methods.The genes of antibody light chain,heavy chain and Nanoluc luciferase were connected in series to form a single plasmid stably transfected cells to sustainably obtain labeled antibodies.(3)An automatic,sensitive magnetic particles immunoassay for the novel coronavirus antigen was established.By screening a broad-spectrum capture antibody,combining with the automatic magnetic particle chemiluminescence instrument,optimizing the detection process,the Nluc-AMCA was established in double-antibody sandwich mode.To evaluate the role of intracellular expression of luciferase-labeled antibody(Nluc-ch2C5)in improving the sensitivity of viral antigen detection,we compared it with Nluc-antibody using the heterobifunctional crosslinker reagent m-maleimidobenzoyl-N-hydroxysuccinimide(MBS)(Nluc-MBS-ch2C5),and HRP-antibody labeled by the heterobifunctional crosslinker reagent MBS(HRP-ch2C5).The results showed that Nluc-ch2C5 had the highest sensitivity,the LOD was 79 pfu/reaction.The sensitivity of Nluc-ch2C5 was 16 times higher than that of Nluc-MBS-ch2C5(1315.3pfu/reaction)and 42 times higher than that of HRP-ch2C5(3339 pfu/reaction).To ensure the sensitivity and specificity of Nluc-AMCA detection results,we evaluated its LOD,sensitivity and specificity.The system is used for the detection of novel coronavirus,and the LOD can reach 68 pfu/reaction.It does not cross-react with other respiratory-associated viruses.For clinical samples,the specificity was 100% and the sensitivity was 75%,which was higher than that of colloidal gold method(68.75%)and ELISA(62.5%).In the research process of integrated rapid nucleic acid detection technology(I-RPA)based on recombinase polymerase isothermal amplification,in order to shorten the nucleic acid detection time and simplify the experimental operation,we have carried out technical exploration from the following aspects:(1)Preparation of sample treatment solution,direct release of viral nucleic acid to the reaction system,simplify the sample processing process,and realize the sample processing steps without nucleic acid purification;(2)Design and screen sensitive and specific primers and probes for recombinase polymerase isothermal amplification(RPA)to achieve exponential amplification of detection targets;(3)The closed detection cassette and detection process were optimized.The RPA isothermal amplification process was combined with the sample processing process without nucleic acid extraction to establish an integrated RPA detection technology for novel coronavirus.The whole sample processing and amplification process can be completed in 30 minutes.In order to ensure the sensitivity and specificity of the test results,we evaluated the limit of detection(LOD),sensitivity and specificity of I-RPA detection.The LOD of the I-RPA technique was determined to be 35copies/reaction by using multiple cultures of novel coronavirus strains.The specificity experiment revealed that I-RPA had no cross-reaction with other 20 respiratory viruses,including the common coronaviruses.The detection results for clinical samples of I-RPA were consistent with those of conventional q PCR,with a specificity of 100% and a sensitivity of 92.8%.Nluc-AMCA and I-RPA methods established in this study can not only be used for antigens detection and rapid nucleic acid detection of novel coronavirus,but also can be used as a technical platform for the detection of other infectious pathogens,thus providing new detection methods and ideas for infectious disease detection. |
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