Development And Evaluation Of Nucleic Acid Tests For Respiratory Viruses Based On Isothermal Amplification And Microfluidic Technology

Objective:The acute infections caused by respiratory viruses such as 2019-n Co V have become a public health problem affecting human health.However,in many cases various pathogens are often indistinguishable from one another by clinical diagnosis.Rapid and reliable methods are critically important for the recognition of the pathogens and blocking the dissemination of the pathogens.This study combined microfluidic technology and isothermal amplification technologies including thermophilic helicase?dependent amplification（t HDA）and multienzyme isothermal rapid amplification（MIRA）to develop sensitive and specific methods for respiratory viruses’rapid detection.Methods:（1）The conventional RT-MIRA-LFD assay:Primers were designed for each specific gene of 2019-n Co V,and the amplicons were analyzed to screen out the most efficient of RT-t HDA.The amplification conditions were optimized.Performance evaluation including precision,cross-specificity and limit of detection was carried out.（2）The conventional RT-MIRA-LFD assay:Primers and probes were designed for each specific gene of 2019-n Co V and screened out the most efficient of RT-MIRA.The amplification conditions were optimized.Performance evaluation including precision,cross-specificity and limit of detection were performed.（3）The isothermal amplification system suitable for microfluids:The effects of reaction volumes and virus preservation solutions on amplification efficacy were tested.The established system was used to develop the new extraction-free RT-MIRA-LFD assay.Performance evaluation were performed.The extraction-free RT-MIRA-LFD assay was used to detect 153 clinical specimens and 90 simulated clinical specimens.（4）The multiplex microfluidic paper-based chip assay:Primers were designed for other four viruses and screened out the most efficient of MIRA.The SYBR Green I concentration,amplification temperature and time were optimized.The paper-based platform for multiplex respiratory viruses’detection was established by combining the MIRA out of chip and microfluidic paper-based chip with five channels designed and fabricated before.Test the feasibility of the paper-based platform to detect 2019-n Co V,influenza A（H1 and H3）,influenza B and human adenovirus simultaneously.Results:（1）The established conventional RT-t HDA method combined with LFD was adopted,which could be conducted within 60 min at a constant temperature（65°C）.The C50-20%to C50+20%concentration range encompassed C5-C95 interval.The 95%limit of detection（Lo D）of the conventional RT-t HDA-LFD assay was 383.0（95%CI,362.7-411.2）copies/m L for N gene and 357.1（95%CI,348.9-392.6）copies/m L for E gene.The assay showed no cross reaction with other eight human respiratory pathogens.（2）The conventional RT-MIRA-LFD duplex assay was developed,which could be performed within 20 min at 38°C.The concentration of primers was 200 n M.And the concentration of probes was 100 n M.The precision evaluation revealed that the C50-20%to C50+20%range bounded the C5-C95 interval.The 95%limit of detection（Lo D）of the conventional RT-MIRA-LFD assay was 47.7（95%CI,45.4-51.1）copies/m L for ORF1ab gene and 47.9（95%CI,44.7-57.0）copies/m L for N gene.There is no cross-reaction with other human respiratory pathogens.（3）The RT-MIRA system was suitable for microfluidics.Solutions without guanidinium have no effect on its amplification efficiency.The extraction-free RT-MIRA-LFD assay which used the system suitable for microfluidics was developed.The turnaround time was only 25 min.The reaction volume was 10μL.The C50-20%to C50+20%concentration range encompassed C5-C95 interval.The 95%limit of detection（Lo D）of the extraction-free RT-MIRA-LFD assay was 49.5（95%CI,46.8-52.7）copies/m L for ORF1ab gene and 48.8（95%CI,46.5-52.6）copies/m L for N gene.No cross-reaction with other human respiratory pathogens was observed by this assay.The extraction-free RT-MIRA and q PCR detection results of 243 nucleic acid specimens from suspected patients or national references showed a 100.00%（95%CI,96.70%-100.00%）sensitivity and a 100.00%（95%CI,98.10%-100.00%）specificity.Compared with q PCR,the kappa value of the two assays was 1.00（P<0.05）.The positive predicted value was 100.00%（95%CI,96.70%-100.00%）and the negative predicted value was 100.00%（95%CI,98.10%-100.00%）.（4）The paper-based platform for multiplex respiratory viruses’visualized detection was conducted at 40℃for 20 min.The most optimal concentration of SYBR Green I was 6×10^-2 times its original concentration.The paper-based platform could be used to detect 2019-n Co V,influenza A（H1 and H3）,influenza B and human adenovirus simultaneously.Conclusion:（1）This study provided two novel visualized method for detection of 2019-n Co V including the conventional RT-t HDA-LFD assay and the conventional RT-MIRA-LFD assay.（2）This study developed the isothermal amplification system which suitable for microfluidics based on the two conventional assays.And the system was used to establish the extraction-free assay for 2019-n Co V.This system is expected to be employed for various microfluidic chips.（3）This study initially established a paper-based platform for multiplex respiratory viruses’visualized detection and it is promising for further application toward complex environments.