Application Of Nanoplasmonic-Enhanced Isothermal Amplification Technology In SARS-CoV-2 Nucleic Acid Detection

The continuing pandemic of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)and its variants has a huge impact on the global economy and public health security,thus timely and effective diagnostic strategies are essential for epidemic prevention.Reverse transcription and quantitative polymerase chain reaction(RTqPCR)technology is most common method for diagnosing SARS-CoV-2 infection,which shows excellent sensitivity and specificity.However,long time cost,complicated operation,expensive equipment essential,and professional technician required making it limited for widely use,especially applications for home self-testing or rapid on-site virus screening in remote areas.Therefore,it is urgent to develop an accurate,rapid,sensitive and portable nucleic acid detection platform.To overcome the shortcomings of traditional nucleic-acid detection methods in sensitivity and detection speed,this study proposes a nanoplasmonic-enhanced isothermal amplification(NanoPEIA)strategy for accurate,sensitive,specific and rapid SARS-CoV-2 nucleic acid real time detection and immediate diagnosis(Point-Of-Care Testing,POCT).The method modifies the nanoplasmonic array chip surface(NanoACS)by thiolated forward(F-SH)primers to build an asymmetric enzymatic recombinase amplification(ERA)isothermal amplification technique,and uses surface fluorescence enhancement mechanism to build a NanoPEIA platform.This NanoPEIA platform enables high-throughput,real-time detection,visualization,or real-time POCT diagnostics.The research content includes the following three aspects:(1)Integration of nanoplasmonic sensing chip and ERA to build the NanoPEIA platform.The nanoplasmonic sensing chip was prepared using nanoimprint technology and electron beam evaporation process and was integrated into a 3D-printed bottomless96-well microplate to build a high-throughput NanoPEIA platform which can detect 96 samples simultaneously.The NanoPEIA platform exhibits splendid sensitivity and specificity for SARS-CoV-2 nucleic acid-related standards(plasmid DNA,RNA standard,pseudoviruses,β-inactivated and γ-inactivated viruses)and cross-inactivated viruses(H3N2,H1N1,H5N1,H7N9,and SARS),with a minimum detection concentration of 76 copies/mL for the N gene and orf1 ab gene of β-inactivated and γ-inactivated viruses.(2)Detection performance of clinical samples were evaluated by the highthroughput NanoPEIA platform.To verify the adaptability of the NanoPEIA platform to SARS-CoV-2 clinical samples,clinical samples from 21 SARS-CoV-2 infected patients and 31 healthy patients were tested using the NanoPEIA platform.The sensitivity of the NanoPEIA platform were 100% for the N gene and the orf1 ab gene in52 clinical samples,while sensitivity of the RT-qPCR method was 88.9%(N gene)and90.0%(orf1ab gene),respectively.The specificities of the NanoPEIA platform were92.3%(N gene)and 91.7%(orf1ab gene)for 31 negative samples.Limits of detection based NanoPEIA platform were 28.3 copies/mL and 23.3 copies/mL for the N gene and orf1 ab genes,respectively.Therefore,the NanoPEIA platform exhibits good performance for clinical samples.(3)Real-time and visual NanoPEIA platform were constructed for POCT diagnosis.To explore application scenario of NanoPEIA platform for POCT diagnosis,a POCT diagnostic solution that can be used for visualization and real-time detection was built by using a small blue-light glue separator and a portable real-time fluorescence detector,which meets the needs of different users and regions.The nucleic acid was released by the heating the virus lysis,and the amplification reagents were prefrozen in a PCR tube.The whole NanoPEIA test can be conducted by simply adding the template.This method greatly facilitates the defects of methods by eliminating the requirement of expensive instruments,tedious operations or professional technicians for RT-qPCR.In summary,the NanoPEIA platform simultaneously enables high-throughput,real-time,laboratory detection or visual POCT diagnostics combined with the use of portable instruments.Its convenience makes it feasible for application in remote areas and home self-examinations.