Colorimetry And Fluorescence Dual-mode Nucleic Acid Diagnosis Method Construction For African Swine Fever Based On A Magnetic Beads-enzyme Reporting System

African swine fever(ASF)is a highly infectious and deadly disease caused by the African swine fever virus(ASFV).To date,there is no widely available vaccine or treatment for African swine fever,so rapid diagnosis and culling of infected pigs are often used to control the epidemic effectively.At present,the diagnosis of African swine fever mainly relies on PCR(Polymerase chain reaction)and other laboratory diagnostic technologies,which are highly sensitive,but strongly rely on laboratory instruments and equipment and professional operators,and cannot meet the needs of point-of-care(POC)diagnosis.RPA(Recombinase polymerase amplification)technique can achieve constant temperature nucleic acid amplification,but its sensitivity is lower than PCR.The CRISPR/Cas system discovered in recent years,whose trans-cleavage activity can be used as a signal amplifier for nucleic acid detection,combining CRISPR/Cas system with RPA technology,can greatly improve the sensitivity of nucleic acid detection.CRISPR/Cas detection technology often uses fluorescent probes or Au NPs(Gold nanoparticles)as signaling components to realize colorimetry or fluorescent detection of targets.Such a single-mode signaling system is prone to environmental interference,leading to inaccurate detection results.Dual-mode detection mode can solve this problem well.Based on a novel magnetic bead-enzyme reporting system,this study constructed colorimetry-fluorescence dual-mode signal reporting system.Using this reporting system,combining recombinase polymerase amplification(RPA)and CRISPR-Cas12 a technology to construct a colorimetry-fluorescence dual-mode molecular diagnostic method for African swine fever virus.The research content is divided into two parts:1)Construction of a novel magnetic bead-enzyme dual-mode reporting system.A MB-ss DNA-ALP probe based on Alkaline phosphatase(ALP)was synthesized,The ALP on the probe hydrolyzes p NPP(p-Nitrophenyl phosphate)to produce p NP(p-Nitrophenol)efficiently,resulting in substantial changes in absorbance.At the same time,four quantum dots(QDs)with different emission wavelengths were synthesized in this study.Due to the Inner filtering effect(IFE),p NP quenched the fluorescence of QDs and changed the fluorescence signal.Further experiments showed that the blue Cd Zn Se QDs had the best quenching efficiencies,so a novel magnetic bead-enzyme reporting system was constructed.The system can realized the output of colorimetry-fluorescence dual-mode signal output,and can be used as a signal element for in vitro molecular diagnosis to achieve high sensitivity and accuracy of colorimetry and fluorescence dual-mode detection.2)Based on magnetic bead-enzyme reporting system,RPA-CRISPR/Cas12 a system and magnetic separation technique,a dual-mode analysis for African swine fever virus gene was constructed,including colorimetry and fluorescence.The ASFV gene was selected as the initial RPA template to generate the amplicon.The RPA amplicon was then recognized by CRISPR-associated RNA(cr RNA),activating the trans-cleavage activity of Cas12 a and leading to the non-specific cleavage of ss DNA as well as a significant release of ALP in the ALP-ss DNA modified magnetic bead.After magnetic separation,the ALP released in the supernatant catalyzed p NPP hydrolysis to generate p NP,changing both the fluorescence and colorimetry through the additions of QDs as potential fluorescence reporters,and the change of colorimetric signal and fluorescence signal can be observed through the naked eye.This strategy allows the sensitive detection of ASFV DNA,with a 20 copies/m L detection limit;and no cross-reactivity with other viruses was observed.A good detection performance was obtained in serum.In addition,this sensor displayed 100% specificity and sensitivity for clinical samples analysis.In conclusion,this study combined with the construction of a novel magnetic beadsenzyme reporting system to develop a highly sensitive,and low-cost dual-mode detection of viral nucleic acid,thereby providing a broad prospect for the practical application in the diagnosis of virus infection.