Development Of MERS-CoV Rapid Detection Methods

Middle East respiratory syndrome coronavirus?MERS-CoV?,as a novel human coronavirus causes high mortality rate of respiratory diseases.MERS-CoV has been confirmed to infect people,bats,dromedary camels,and alpacas.However,from September 2012 to the end of April 2018,2206 laboratory-confirmed cases of MERS-CoV infection were reported,at least 787 related deaths?mortality rate of approximately 35.7%?.In 2015,an imported case was reported in Guangzhou,which sounded an alarm for the prevention and control of the MERS epidemic in China.As no available vaccines or specific treatments currently exist,rapid and accurate diagnosis is significant for the prevention of MERS outbreaks and prevalent.Current diagnostic tests for MERS-CoV are based on nucleic acid detection,real-time RT-PCR was recommend by WHO in Laboratory Testing for Middle East Respiratory Syndrome Coronavirus Interim guidance;To detect MERS-CoV antigens,double-antibody sandwich ELISA and colloidal gold immunochromatographic test strips have been developed.These methods have the disadvantages of require special equipment,complicated procedures and time-consuming,low sensitivity and poor stability.To overcome the deficiencies of the above detection methods,a simple,rapid,and specific detection method for MERS-CoV nucleic acids and antigens was established in this study.1?We combined RT-LAMP with a vertical flow visualization strip to detect MERS-CoV N gene.Six specific primers in conservative sequence of N gene were designed for LAMP.The amplified product was observed by a disposable nucleic acid visualization device.The RT-LAMP-VF assay were evaluated sensitivity by RNA transcripts and MERS-CoV RNA,and were evaluated specificity by SARS-related coronavirus?SARSr-CoV?,HKU4,HKU1,OC43 and 229E.The results showed that the optimal conditions for RT-LAMP were:when the final concentrations of internal primers in the amplification system were 0.4?M,the synthesized RNA transcripts were amplified in 61°C for 30 min.This method specifically detects 2×10¹ copies/?l copies/ul of RNA transcripts and 1×10¹ copies/?l MERS-CoV RNA within 35min.There was no cross-reactivity with other coronaviruses.2?Development of quantum dots based immunochromatographic strip for detection of Middle East respiratory syndrome coronavirus.We used traditional EDC coupling method to covalently couple carboxylated CdTe/ZnSe QDs with anti-MERS-CoV RBD monoclonal antibody?mAb/MERS-CoV?.The activity of the conjugates was tested by dot blot,and QDs-mAb/MERS-CoV was used as the capture antibody.The horse anti-MERS-CoV IgG was used as the detection antibody.The immunochromatographytechniquewasusedtopreparetheQD immunochromatographic strips on the basis of the double antibody sandwich method principle.The sensitivity of the QD immunochromatographic test paper was evaluated by MERS-CoV pseudovirus,and the specificity was evaluated by commercial SARS-CoV S1 subprotein,porcine epidemic diarrhea virus,and H5N1 influenza virus-like particles.The results showed that quantum dots were successfully coupled to mAb/MERS-CoV,and the conjugates had good biological activity.The optimal coupling condition was that 5?l QDs was efficiently coupled with 16?g mAb/MERS-CoV after activated by 46.8?g EDC in pH=6.4 system.This method can specifically detect MERS-CoV pseudovirus with a detection limit of 2×5³TCID₅₀/ml within 15min,and has good specificity.