Ntawm no, weestablish sCRAM (suboptimal protospacer adjacent motif (PAM)-mediated CRISPR RNA thiab Cas12a nuclease ua ke nrog multienzyme isothermal nrawm amplification), ceev thiab rho tawm - dawb kuaj platform uas kuaj pom ASFV li ntawm 20 min. sCRAM integrates ib tug ceev nucleic acid tso cov kauj ruam (40℃, 5 min) nrog ib tug - raj cov tshuaj tiv thaiv combining multienzyme isothermal amplification (MIRA) thiab ib tug suboptimal PAM -mediated CRISPR/
Cas12a system (37℃, 15 min), ua kom cov cuab yeej siv tau - nyeem ntawv pub dawb ntawm UV lub teeb los yog sab nraud ntws strips (LFS).sCRAM qhia ib zaug -copy nrhiav kom pom tseeb thiab tsis muaj qhov cuam tshuam -reactivity nrog tsib hom kab mob swine (PRRSV, PCV, PREV, J. Kev soj ntsuam ntawm 111 simulated thiab kuaj kuaj mob (xws li ntshav, ntshav, thiab swabs) qhia 100% concordance nrog qPCR rau UV nyeem ntawv thiab 98.20% rau LFS
nyeem ntawv, nrog rau ob txoj hauv kev nthuav tawm 100% tshwj xeeb. Qhov yooj yim, ceev, thiab kev hloov pauv ntawm thaj chaw ntawm sCRAM qhia txog nws lub peev xwm rau kev saib xyuas ASFV hauv cov peev txheej hauv cov peev txheej- txwv tsis pub, muab cov cuab yeej muaj zog rau kev tiv thaiv kev sib kis.
Nyob rau hauv txoj kev tshawb no, peb tsim sCRAM, ib tug ceev thiab teb -deployable ASFV nrhiav kom tau platform integrating suboptimal PAM-mediated CRISPR/Cas12a nrog multienzyme isothermal amplification. Qhov kev ntsuam xyuas ua tiav ib zaug -copy 1 daim / uL rhiab heev nyob rau hauv 20 min thiab qhia tias tsis muaj tus ntoo khaub lig-reactivity nrog swine kab mob loj. Kev soj ntsuam kev lees paub thoob plaws cov ntshav, ntshav, thiab cov qauv swab tau pom tias muaj kev sib haum xeeb nrog qPCR siv UV lub teeb nyeem ntawv thiab 98.20% qhov tseeb ntawm lateral flow strips. Nrog rau kev ua haujlwm yooj yim, muaj kev nkag siab zoo thiab qhov tshwj xeeb, thiab kev txhais cov txiaj ntsig pom, sCRAM muaj peev xwm muaj zog rau ntawm -site ASFV kev soj ntsuam thiab cov ntsiab lus dav dua - ntawm- daim ntawv thov kev kuaj mob.
