Research On Nucleic Acid Visualization And Digital Detection For MERS-CoV

Middle East Respiratory Syndrome(MERS)is a respiratory infectious disease caused by the Middle East Respiratory Syndrome Coronavirus(MERS-Co V).Since the first case was confirmed in September 2012,the number of confirmed cases has continued to increase.As of11 Match 2021,there have been 2574 confirmed cases of MERS,including 886 deaths,reported to WHO,and with a mortality rate of approximately 34%.Dromedary camels are the main intermediate host of MERS-Co V transmission and the source of human infection with MERS-Co V,which can repeatedly transmit the virus to humans.This condition resulted in a long-term risk of potential infection for people who worked in camel farms,slaughterhouses and markets.Because dromedary camel products are the main agricultural and livestock products in the Middle East countries,it is difficult to prevent virus shedding and human infection by prohibiting the trade or culling of camel products.Additionly,there is currently no approved vaccine against MERS-Co V.Given this,timely and effective early detection is helpful to prevent the MERS pandemic.RT-qPCR was the golden standard for MERS-Co V detection recommended by WHO.Among many RNA-based detection assays,RT-q PCR had outstanding advantages in terms of sensitivity and maturity.However,samples from the low-resource setting have to be transported to a laboratory with sufficient and precision equipment to test.The time for obtaining the results is prolonged and is not conducive to the early control of MERS and the prompt treatment of infected patients.This study utilize MS2 phage capsid protein to specifically encapsulate m RNA mediated by phage RNA operon RNA sequence(Pac),and phage-like particles(PLP)containing MERS-Co V RNA are preparedto be the quality control of the MERS-Co V nucleic acid detection.A nucleic acid visual assay(RT-RPA-VF)for MERS-Co V Up E and N genes suitable for basic laboratories in low-resource areas has been established,which combines reverse transcription recombinase polymerase amplification with a closed vertical flow visualization strip.In addition,we firstly used a portable glucose meter(PGM)to detect RNA virus,and established a PGM digital detection method for MERS-Co V Up E gene.The main results were described as follows:1.Preparation of quality control products for MERS-Co V nucleic acid detection.To contruct a single plasmid double expression system was constructed for packaging MERS-Co V PLP,the gene of capsid protein dimer,gene of mature enzyme protein,mutant Pac site and MERS-Co V Up E and N genes were cloned into p ACYCDuet-1 vector,respectively.Four different MERS-Co V PLPs containing Up E and N gene sequence were successfully constructed by using E.coli expression system.The results of enzyme resistance test and RT-q PCR suggested that the purified PLP can protect MERS-Co V RNA from degradation by RNase enzyme,and can be used as a quality control for the MERS-Co V RNA detection;2.Establishment of MERS-Co V RT-RPA-VF assay.Specific PRA primers and probes for human and dromedary sources were designed for the MERS-Co V Up E and N genes which recommended targets by the WHO.The RT-RPA-VF assay for Up E and N genes had been established.The RT-RPA-VF assays for Up E gene can detect 1.2×10~1 copies/?L of MERS-Co V RNA within 30 minutes at 33°C,and the RT-RPA-VF assay for N gene can detect 1.2 copies/?L of MERS-Co V RNA within 30 minutes at 35°C.210 human,camel,alpaca respiratory tract swab samples spiked with MERS-Co V PLP and 30 negative swab samples were used to evaluate RT-RPA-VF assays,compared with RT-q PCR,the sensitivity and specificity of the RT-RPA-VF assay for N gene were 100%,while the sensitivity and specificity of the RT-RPA-VF assay for Up E gene were 86%and 100%,respectively.Therefore,we recommend that the N assay to screen MERS-Co V,and the Up E assay for MERS-Co V diagnosis.3.Explored the digital detection of MERS-Co V Up E gene based on portable PGM.Based on the principle that sucrose invertase can catalyze sucrose to produce glucose,this study prepared a MERS-Co V detection probe labeled with sucrose invertase,and combined with RPA technology to realize the digital detection of MERS-Co VRNA.ND-PGM assay can detect 1.2×10~2 copies/?L MERS-Co V RNA within 2h at 37?,and it had no cross reaction with SARA-Co V-2,HKU4,TGEV,PEDV,FCo V.210 respiratory swab samples of human,camel and alpaca spiked with MERS-Co V PLP and 30 negative swab samples were evaluated for this method.Compared with RT-q PCR,the sensitivity of this method was 75.2%and the specificity was 100%.In summary,this study prepared a PLP quality control product containing MERS-Co V RNA,which provides important guidance and guarantee for the effective monitoring of the quality of MERS-Co V nucleic acid detection.A RT-RPA-VF method suitable for human and dromedary MERS-Co V has been established.It has the advantages of rapidness,no special equipment dependence,high sensitivity,and high specificity,and is suitable for the grassroots in resource-poor areas laboratory.The digital detection method of MERS-Co V Up E gene nucleic acid based on home PGM was explored,and its lower cost is expected to be applied to the screening of suspected infected animals.The RT-RPA-VF assay suitable for human and dromedary-derived MERS-Co V has been established.It has the advantages of rapidness,no special equipment dependence,high sensitivity and specificity.It is suitable for resource-poor areas laboratory.Explored the application of domestic PGM in RNA virus detection,and established a PGM-based digital detection assay of MERS-Co V Up E gene,its lower cost is expected to be used in screening of suspected infected animals.