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Amasistimu e-Lab-on-a-chip anamakhono akusayithi anikeza amandla okuxilonga ngokushesha futhi okunembile futhi awusizo kuzilungiselelo ezicindezelwe yizinsiza lapho okokusebenza kwe-biomedical kanye nochwepheshe abaqeqeshiwe bengatholakali.Kodwa-ke, ukudala uhlelo lokuhlola indawo yokunakekelwa okuhambisana nazo zonke izici ezidingekayo zokukhishwa kwemisebenzi eminingi, ukukhululwa okudingekayo, ukusebenza okuthembekile, nokugcinwa kwesikhathi eside kwama-reagents kuseyinselele enkulu.Lapha sichaza ubuchwepheshe bokushintshwa kwe-lever-actuated micro travel obungakwazi ukulawula uketshezi kunoma iyiphi indlela, inikeze impendulo enembile nelinganayo kumfutho womoya osetshenzisiwe, futhi uhlale uzinzile ngokumelene nokunyakaza okungazelelwe nokudlidliza.Ngokusekelwe kubuchwepheshe, siphinde sichaze ukuthuthukiswa kwesistimu ye-polymerase chain reaction ehlanganisa isingeniso se-reagent, ukuxuba kanye nokusebenza kokusabela konke ngenqubo eyodwa, eyenza "isampula-in-answer-out" ukusebenza kwawo wonke amasampula omtholampilo avela ezigulini eziyi-18 Umkhuhlane kanye nezilawuli ngazinye eziyi-18, ngokuhambisana kahle kokuqina kwe-fluorescence nokusabela okujwayelekile kwe-polymerase chain (ama-Pearson coefficients> 0.9).Ngokusekelwe kubuchwepheshe, siphinde sichaze ukuthuthukiswa kwesistimu ye-polymerase chain reaction ehlanganisa isingeniso se-reagent, ukuxuba kanye nokusebenza kokusabela konke ngenqubo eyodwa, efeza ukusebenza "kwesampula-in-answer-out" kuwo wonke amasampula omtholampilo wamakhala ezigulini eziyi-18. ngoMfuluwenza kanye nezilawuli ngazinye eziyi-18, ngokuhambisana kahle kokuqina kwe-fluorescence nokusabela okujwayelekile kwe-polymerase chain (ama-Pearson coefficients> 0.9).Основываясь на этой технологии, мы также описываем разработку системы полимеразной цепной реакции, которая объединяет фуединяет фунгиняет фуединяет фуединяет фуединяет функции реакции в одном процессе, что обеспечивает выполнение «образец-в-ответ-выход» для всех клинических образцов образцов из 18 сположение futhi 18 отдельных контролей, хорошем соответствии интенсивности флуоресценции со стандартной полимеразной цепной реакцией (Пиэфенцией)Ngokusekelwe kulobu buchwepheshe, siphinde sichaze ukuthuthukiswa kwesistimu ye-polymerase chain reaction ehlanganisa imisebenzi yokujova, ukuxuba, nokusabela ngenqubo eyodwa, okuvumela isampula-in-response-out kuzo zonke izibonelo zamakhala ezigulini eziyi-18 zomkhuhlane.kanye nezilawuli ezingazodwana eziyi-18, ezivumelana kahle nesilinganiso se-polymerase chain reaction fluorescence intensity (ama-coefficient kaPearson> 0.9).Ngokusekelwe kulobu buchwepheshe, siphinde sichaze ukuthuthukiswa kwesistimu ye-polymerase chain reaction ehlanganisa umjovo we-reagent, ukuxuba, kanye nemisebenzi yokusabela ukuze kuhlaziywe zonke izibonelo zemitholampilo yamakhala kusukela kumasampuli angu-18 esampula esiguli sesiguli. kahle nge-polymerase chain reaction evamile (i-coefficient ka-Pearson> 0.9).Inkundla ehlongozwayo iqinisekisa ukuzenzekelayo okuthembekile kokuhlaziywa kwe-biomedical futhi ngaleyo ndlela ingasheshisa ukuthengiswa kwezinhlobonhlobo zamadivayisi okuhlola indawo yokunakekelwa.
Izifo zabantu ezisafufusa, njengobhadane lwango-2020 lwe-COVID-19 oselubulale izigidi zabantu, lubeka engcupheni enkulu impilo yomhlaba kanye nempucuko yabantu1.Ukutholwa kwezifo kusenesikhathi, ngokushesha nangokunembile kubalulekile ukuze kulawulwe ukusabalala kwegciwane kanye nokwenza ngcono imiphumela yokwelashwa.I-ecosystem eyinhloko yokuxilonga esekelwe kumalebhu aphakathi nendawo lapho amasampula okuhlola athunyelwa ezibhedlela noma emitholampilo yokuxilonga futhi eqhutshwa ochwepheshe okwamanje ikhawulela ukufinyelela cishe kubantu abayizigidi eziyizinkulungwane ezingu-5.8 emhlabeni wonke, ikakhulukazi labo abahlala ezindaweni ezivinjezelwe yizinsiza.lapho kunokuntuleka kwemishini ebizayo ye-biomedical kanye nochwepheshe abaqeqeshiwe.odokotela 2. Ngakho, kunesidingo esiphuthumayo sokuthuthukisa uhlelo lwe-lab-on-a-chip olungabizi futhi olusebenziseka kalula olunekhono lokuhlola indawo yokunakekelwa (POCT) olunganikeza odokotela ulwazi lokuxilonga ngesikhathi ukuze benze izinqumo zokuxilonga benolwazi. .kanye nokwelashwa 3.
Imihlahlandlela yeNhlangano Yezempilo Yomhlaba (i-WHO) ithi i-POCT ekahle kufanele ithengeke, isebenziseke kalula (kulula ukuyisebenzisa ngokuqeqeshwa okuncane), inembe (gwema ukuphika okungamanga noma imibono engamanga), isheshe futhi ithembeke (inikeze izici ezinhle zokuphindaphinda), futhi ukulethwa (okukwazi ukugcina isikhathi eside futhi kutholakala kalula kubasebenzisi bokugcina)4.Ukuze kuhlangatshezwane nalezi zidingo, amasistimu e-POCT kufanele ahlinzeke ngezici ezilandelayo: umthamo oguquguqukayo wokunciphisa ukungenelela okwenziwa ngesandla, ukukhululwa okudingekayo ukuze kukale ukuthutha kwe-reagent ukuze uthole imiphumela yokuhlolwa enembile, nokusebenza okuthembekile ukumelana nokudlidliza kwemvelo.Njengamanje, idivayisi ye-POCT esetshenziswa kakhulu i-lateral flow strip5,6 ehlanganisa izingqimba ezimbalwa ze-porous nitrocellulose membranes eziphusha inani elincane kakhulu lesampula phambili, lisabela ngama-reagents angaphambi kokuvinjelwa ngamandla e-capillary.Nakuba zinenzuzo yezindleko eziphansi, ukusetshenziswa kalula, kanye nemiphumela esheshayo, amadivaysi e-POCT asuselwa ku-flow strip angasetshenziswa kuphela ekuhloleni kwebhayoloji (isb., ukuhlolwa kwe-glucose7,8 kanye nokuhlolwa kokukhulelwa9,10) ngaphandle kokudinga ukuhlaziya okunezigaba eziningi.ukusabela (isb ukulayisha ama-reagents amaningi, ukuxuba, ukuphindaphinda).Ukwengeza, amandla okushayela alawula ukunyakaza koketshezi (okungukuthi, amandla e-capillary) awanikezi ukuvumelana okuhle, ikakhulukazi phakathi kwamaqoqo, okuholela ekukhiqizeni okungalungile11 nokwenza amabhande okugeleza asemaceleni abe usizo ngokuyinhloko ekutholeni okuhle12,13.
Amandla okukhiqiza anwetshiwe ku-micro- kanye ne-nanoscale adale amathuba okuthuthukiswa kwemishini ye-POCT ye-microfluidic yezilinganiso zobuningi14,15,16,17.Ngokulungisa izakhiwo ze-interface 18, 19 kanye nejometri yeziteshi 20, 21, 22, amandla e-capillary kanye nesilinganiso sokugeleza kwalawa madivayisi angalawulwa.Kodwa-ke, ukwethembeka kwazo, ikakhulukazi oketshezini olumanzi kakhulu, kuhlala kungamukelekile ngenxa yokungalungi kokukhiqiza, ukonakala kwezinto, kanye nokuzwela ukudlidliza kwemvelo.Ukwengeza, njengoba ukugeleza kwe-capillary kudalwe esibonakalayo se-liquid-gas, akukho ukugeleza okwengeziwe okungangeniswa, ikakhulukazi ngemva kokugcwalisa isiteshi se-microfluidic ngoketshezi.Ngakho-ke, ukuze kutholakale okuyinkimbinkimbi ngokwengeziwe, izinyathelo ezimbalwa zomjovo wesampula kufanele zenziwe24,25.
Phakathi kwamadivayisi we-microfluidic, amadivayisi we-centrifugal microfluidic okwamanje angenye yezixazululo ezingcono kakhulu ze-POCT26,27.Indlela yayo yokushayela inenzuzo ngoba amandla okushayela angalawulwa ngokulungisa isivinini sokujikeleza.Kodwa-ke, okubi ukuthi amandla aphakathi nendawo ahlala eqondiswe ngasemaphethelweni angaphandle edivayisi, okwenza kube nzima ukusebenzisa ukusabela okuyizinyathelo eziningi ezidingekayo ukuze kuhlaziywe okuyinkimbinkimbi.Nakuba amandla okushayela engeziwe (isb. ama-capillary 28, 29 namanye amaningi 30, 31, 32, 33, 34, 35) ngaphezu kwamandla aphakathi nendawo ethulwa ukuze kuthathwe umthamo wemisebenzi eminingi, ukudluliswa koketshezi okungalindelekile kusengenzeka ngoba la mandla engeziwe ngokuvamile ama-oda. yobukhulu obungaphansi kwamandla we-centrifugal, okuwenza asebenze kuphela kumabanga amancane okusebenza noma angatholakali lapho kudingeka khona nokukhululwa koketshezi.Ukufaka ama-manipulations womoya kuma-microfluidis e-centrifugal njengezindlela ze-centrifugal kinetic 36, 37, 38, izindlela ze-thermopneumatic 39 kanye nezindlela ezisebenzayo ze-pneumatic 40 kuye kwafakazela ukuthi kungenye indlela ekhangayo.Ngendlela ye-counterfugodynamic, i-cavity eyengeziwe kanye nama-microchannels axhumayo ahlanganiswe kudivayisi kokubili isenzo sangaphandle nangaphakathi, nakuba ukusebenza kahle kokupompa kwayo (kusukela ku-75% kuya ku-90%) kuncike kakhulu kwinani lokujikeleza kokupompa kanye ne-viscosity. koketshezi.Endleleni ye-thermopneumatic, ulwelwesi lwe-latex negumbi lokudlulisa uketshezi kuklanyelwe ngokukhethekile ukuvala noma ukuvula kabusha i-inlet lapho ivolumu yomoya evaleleke ishisa noma ipholiswa.Kodwa-ke, ukusethwa kokushisisa/ukupholisa kwethula izinkinga zokuphendula kancane futhi kukhawulele ukusetshenziswa kwakho ekuhlolweni kwe-thermosensitive (isb., ukukhulisa i-polymerase chain reaction (PCR)).Ngendlela esebenzayo ye-pneumatic, ukukhululwa okufunwayo kanye nokunyakaza kwangaphakathi kufinyelelwa ngokusetshenziswa kanyekanye kwengcindezi enhle kanye nesivinini sokujikeleza esifaniswe ngokunembile ngamamotho anesivinini esikhulu.Kunezinye izindlela eziphumelelayo ezisebenzisa ama-actuator womoya kuphela (ingcindezi eqondile engu-41, 42 noma ingcindezi engemihle 43) kanye nemiklamo evalekile yamavalvu.Ngokufaka ingcindezi ngokulandelana egumbini le-pneumatic, uketshezi luphonswa phambili nge-peristaltically, futhi i-valve evamise ukuvaleka ivimbela ukugeleza koketshezi ngenxa ye-peristalsis, ngaleyo ndlela ibone ukusebenza koketshezi oluyinkimbinkimbi.Kodwa-ke, okwamanje kunenani elilinganiselwe kuphela lobuchwepheshe be-microfluidic obungakwazi ukwenza imisebenzi yoketshezi eyinkimbinkimbi kudivayisi eyodwa ye-POCT, okuhlanganisa ukukhishwa kwemisebenzi eminingi, ukukhishwa okudingekayo, ukusebenza okuthembekile, ukugcinwa kwesikhathi eside, ukuphatha uketshezi olune-viscosity ephezulu, kanye nokukhiqiza okungabizi kakhulu.Konke ngesikhathi esisodwa.Ukuntuleka kokusebenza kwezinyathelo eziningi kungase futhi kube esinye sezizathu ezenza kube yimikhiqizo embalwa ye-POCT yokuhweba efana ne-Cepheid, Binx, Visby, Cobas Liat, kanye ne-Rhonda eye yethulwa ngempumelelo emakethe evulekile kuze kube manje.
Kuleli phepha, siphakamisa i-actuator ye-pneumatic microfluidic esekelwe kubuchwepheshe be-green ring micro switch (FAST).I-FAST ihlanganisa zonke izakhiwo ezidingekayo ngesikhathi esisodwa ezinhlobonhlobo zama-reagents kusuka kuma-microliters kuya kumamililitha.I-FAST iqukethe ulwelwesi olunwebekayo, izinsimbi namabhlogo.Ngaphandle kokusetshenziswa komfutho womoya, ama-membrane, ama-levers namabhlogo angavalwa ngokuqinile futhi uketshezi olungaphakathi lungagcinwa isikhathi eside.Lapho kusetshenziswa ingcindezi efanele futhi ilungiselelwa ubude be-lever, i-diaphragm iyakhula futhi iphushele i-lever endaweni evulekile, ivumela uketshezi ukuba ludlule.Lokhu kuvumela ukulinganiswa koketshezi okunemisebenzi eminingi ngendlela ye-cascade, kanyekanye, elandelanayo noma ngendlela ekhethiwe.
Senze uhlelo lwe-PCR sisebenzisa i-FAST ukuze sikhiqize imiphumela yokusabela ngesampula ukuze kutholwe amagciwane omkhuhlane A no-B (IAV kanye ne-IBV).Sithole umkhawulo ophansi wokutholwa (i-LOD) wamakhophi angu-102/mL, ukuhlola kwethu okuphindaphindiwe kwabonisa ukucaciswa kwe-IAV ne-IBV futhi kwavumela i-pathotyping yegciwane lomkhuhlane.Imiphumela yokuhlolwa komtholampilo kusetshenziswa isampula ye-swab yamakhala evela ezigulini eziyi-18 kanye nabantu abangu-18 abanempilo enhle ikhombisa ukuvumelana okuhle kumfutho we-fluorescence nge-RT-PCR ejwayelekile (ama-Pearson coefficients> 0.9).Imiphumela yokuhlolwa komtholampilo kusetshenziswa isampula ye-swab yamakhala evela ezigulini eziyi-18 kanye nabantu abangu-18 abanempilo enhle ikhombisa ukuvumelana okuhle kumfutho we-fluorescence nge-RT-PCR ejwayelekile (ama-Pearson coefficients> 0.9).I-Результаты клинических испытаний с использованием образца мазка из носа от 18 пациентов и 18 здоровых лиц показывает хостентовыт хосифентые хостовыт хоров сценции стандартной ОТ-ПЦР (коэффициенты Пирсона > 0,9).Imiphumela yokuhlolwa komtholampilo kusetshenziswa isampula ye-swab yamakhala evela ezigulini eziyi-18 kanye nabantu abangu-18 abanempilo ikhombisa ukuvumelana okuhle phakathi kokuqina kwe-fluorescence kwe-RT-PCR ejwayelekile (ama-coefficients kaPearson> 0.9).0.9................ I-Результаты клинических испытаний с использованием образцов назальных мазков от 18 пациентов и 18 здоровых лиц показать достовиный песни ю флуоресценции и стандартной ОТ-ПЦР (коэфффициент Пирсона > 0,9).Imiphumela yokuhlolwa komtholampilo kusetshenziswa izibonelo ze-swab yamakhala ezigulini eziyi-18 kanye nabantu abangu-18 abanempilo yabonisa ukuvumelana okuhle phakathi kokuqina kwe-fluorescence kanye ne-RT-PCR evamile (i-coefficient kaPearson> 0.9).Izindleko zempahla ezilinganiselwe zedivayisi ye-FAST-POCT cishe i-US$1 (Ithebula Lokwengeza 1) futhi ingabuye yehliswe ngokusebenzisa izindlela zokukhiqiza ezinkulu (isb. ukubumba umjovo).Eqinisweni, amadivaysi e-POCT asekelwe ku-FAST anazo zonke izici ezidingekayo ezigunyazwe yi-WHO futhi ahambisana nezindlela ezintsha zokuhlola amakhemikhali ezinto eziphilayo njenge-plasma thermal cycling44, i-amplification-free immunoassays45 kanye nokuhlolwa kokusebenza kwe-nanobody46 okuwumgogodla wezinhlelo ze-POCT.kungenzeka.
Emkhiwaneni.I-1a ibonisa ukwakheka kweplatifomu ye-FAST-POCT, equkethe amakamelo oketshezi amane: igumbi lesitoreji sangaphambili, igumbi lokuxuba, igumbi lokuphendula, kanye negumbi lokulahla.Isihluthulelo sokulawula ukugeleza koketshezi yiklamo ye-FAST (ehlanganisa ulwelwesi olunwebekayo, amaleveli namabhulokhi) atholakala ekamelweni langaphambi kwesitoreji kanye negumbi lokuxutshwa.Njengendlela ye-pneumatically actuated, umklamo we-FAST uhlinzeka ngokulawula okunembile kokugeleza koketshezi, okuhlanganisa ukushintsha okuvaliwe/okuvulekile, umthamo oguquguqukayo, ukukhishwa koketshezi olufunekayo, ukusebenza okuthembekile (isb, ukungezwani nokudlidliza kwemvelo), kanye nokugcinwa kwesikhathi eside.Inkundla ye-FAST-POCT iqukethe izendlalelo ezine: isendlalelo esisekelayo, isendlalelo sefilimu esinwebekayo, isendlalelo sefilimu yepulasitiki, kanye nesendlalelo sekhava, njengoba kuboniswe ekubukeni okunwetshiwe ku-Fig. 1b (futhi kuboniswe ngokuningiliziwe kokuthi Izibalo Ezingeziwe S1 kanye ne-S2 ).Zonke iziteshi namagumbi okuthuthwa okuwuketshezi (njengamakamelo angaphambi kwesitoreji nezokusabela) ashumekwe kuma-PLA (i-polylactic acid) engaphansi esuka ku-0.2 mm (ingxenye encane kakhulu) ukuya ku-5 mm ubukhulu.Izinto zefilimu ezinwebekayo ziyi-PDMS engu-300 µm ewugqinsi enwebeka kalula lapho ukucindezela komoya kusetshenziswa ngenxa “yokuqina okuzacile” kanye nemodulus ephansi yokunwebeka (cishe 2.25 MPa47).Ungqimba lwefilimu ye-polyethylene yenziwe nge-polyethylene terephthalate (PET) enogqinsi oluyi-100 µm ukuze kuvikelwe ifilimu enwebekayo ekuwohlokeni ngokwedlulele ngenxa yomfutho womoya.Ngokuhambisana namakamelo, ungqimba lwe-substrate lunamaleveli axhunywe kungqimba lwekhava (eyenziwe nge-PLA) ngamahinge ukuze kulawulwe ukugeleza koketshezi.Ifilimu elinwebekayo lalinanyathiselwe kungqimba olusekelayo kusetshenziswa itheyiphu yokunamathela enamacala amabili (i-ARseal 90880) futhi yambozwa ngefilimu yepulasitiki.Izendlalelo ezintathu zahlanganiswa ku-substrate kusetshenziswa i-T-clip design isendlalelo sekhava.I-T-clamp inegebe phakathi kwemilenze emibili.Lapho isiqeshana sishuthekwa emseleni, imilenze yomibili yagoba kancane, yabe isibuyela esimweni sayo sasekuqaleni futhi yasibopha ngokuqinile isivalo kanye nokusekela njengoba idlula emseleni (Supplementary Fig. S1).Izendlalelo ezine zibe sezihlanganiswa kusetshenziswa izixhumi.
Umdwebo wohlelo weplathifomu obonisa amakhompathimenti ahlukahlukene asebenzayo nezici ze-FAST.b Umdwebo okhulisiwe weplathifomu ye-FAST-POCT.c Isithombe seplatifomu eduze kohlamvu lwemali lwekota yase-US.
Indlela yokusebenza yeplathifomu ye-FAST-POCT iboniswa kuMfanekiso 2. Izingxenye ezibalulekile amabhulokhi asesendlalelo sesisekelo namahinge esendlaleni sekhava, okuholela ekwakhiweni kokuphazamiseka lapho izendlalelo ezine zihlanganiswa kusetshenziswa umumo we-T. .Uma kungekho mfutho womoya osetshenziswayo (umkhiwane 2a), ukulingana kokuphazamiseka kubangela ukuthi ihinge igobe futhi igxumeke, futhi amandla okuvala asetshenziswa nge-lever ukuze ucindezele ifilimu enwebekayo ngokumelene ne-block, futhi uketshezi olusemgodleni wophawu luyachazwa. njengesimo esivaliwe.Kumele kuqashelwe ukuthi kulesi simo, i-lever igobe ngaphandle, njengoba kuboniswe ekubukeni ohlangothini ku-Fig. 2a.Lapho kunikezwa umoya (Fig. 2b), ulwelwesi olunwebekayo lunwebeka lubheke ngaphandle bese luphushela i-lever phezulu, ngaleyo ndlela kuvule igebe phakathi kwe-lever kanye ne-block ukuze uketshezi lugelezele ekamelweni elilandelayo, elichazwa njengendawo evulekile. .Ngemva kokukhululwa komfutho womoya, i-lever ingabuyela endaweni yayo yokuqala futhi ihlale iqinile ngenxa yokunwebeka kwehinge.Amavidiyo okunyakaza kwe-lever ethulwa ku-movie eyengeziwe ethi S1.
A. Umdwebo wohlelo nezithombe uma ivaliwe.Uma kungabikho ukucindezela, i-lever icindezela i-membrane ngokumelene ne-block, futhi uketshezi luvalwe.b Isesimweni esihle.Lapho ukucindezela kusetshenziswa, ulwelwesi luyakhula futhi luphushele i-lever phezulu, ngakho isiteshi siyavuleka futhi uketshezi lungageleza.c Thola ubukhulu besici somfutho obalulekile.Ubukhulu besici buhlanganisa ubude be-lever (L), ibanga phakathi kwesilayidi nehinji (l) kanye nobukhulu be-lever protrusion (t).I-Fs amandla okuhlanganisa endaweni ye-throttle B. q umthwalo osabalaliswe ngokulinganayo ku-lever.I-Tx* imele i-torque ethuthukiswe i-lever ehinged.Ukucindezela okubalulekile yingcindezi edingekayo ukuze uphakamise i-lever futhi wenze ukugeleza kwamanzi.d Imiphumela yetiyetha neyokuhlola yobudlelwano phakathi kwengcindezi ebalulekile kanye nosayizi wesici.n = Ukuhlolwa okuzimele okungu-6 kwenziwa futhi idatha ikhonjiswa njengokuchezuka okujwayelekile ±.Idatha engahluziwe yethulwa njengamafayela edatha eluhlaza.
Imodeli yokuhlaziya esekelwe kumbono we-beam iye yasungulwa ukuze kuhlaziywe ukuncika kwengcindezi ebalulekile ye-Pc lapho igebe livuleka khona kumapharamitha wejometri (isibonelo, u-L ubude be-lever, l ibanga phakathi kwe-block kanye i-hinge, i-S yi-lever Indawo yokuxhumana ne-liquid t ubukhulu be-lever protrusion, njengoba kuboniswe ku-Fig. 2c).Njengoba kuchazwe ku-Supplementary Notes naku-Supplementary Figure S3, igebe livuleka uma \({P}_{c}\ge \frac{2{F}_{s}l}{SL}\), lapho u-Fs eyitorque \ ({T}_{x}^{\ast}(={F}_{s}l)\) ukuze kuqedwe amandla ahlobene nokulingana kokuphazamiseka futhi kubangele ukuthi ihinji igobe.Impendulo yokuhlola kanye nemodeli yokuhlaziya ibonisa ukuvumelana okuhle (Fig. 2d), okubonisa ukuthi ingcindezi ebalulekile ye-Pc iyanda ngokukhula kwe-t / l kanye nokwehla kwe-L, okuchazwa kalula ngemodeli ye-classical beam, okungukuthi i-torque iyanda nge-t / Lift. .Ngakho-ke, ukuhlaziywa kwethu kwethiyori kubonisa ngokucacile ukuthi ukucindezela okubucayi kungalawulwa ngokuphumelelayo ngokulungisa ubude be-lever L kanye nesilinganiso se-t / l, esihlinzeka ngesisekelo esibalulekile sokuklama kwesiteji se-FAST-POCT.
Inkundla ye-FAST-POCT ihlinzeka ngokusabalaliswa okusebenzayo (okuboniswe kuMfanekiso 3a ngokufakwa nokuhlola), okuyisici esibaluleke kakhulu se-POCT ephumelelayo, lapho uketshezi lungageleza kunoma iyiphi indlela nanoma iyiphi i-oda (i-cascade, ngesikhathi esisodwa, ngokulandelana) noma i-multichannel ekhethiwe. ukuhambisa .- umsebenzi womthamo.Emkhiwaneni.I-3a(i) ibonisa imodi yokudosa eyi-cascade lapho amagumbi amabili noma ngaphezulu achithwa kusetshenziswa amabhulokhi ukuhlukanisa ama-reactants ahlukahlukene kanye neleva ukuze kulawulwe izifunda ezivulekile nezivaliwe.Lapho kufakwa ingcindezi, uketshezi lugeleza lusuka phezulu luye egumbini elingaphansi ngendlela ehlayo.Kumele kuqashelwe ukuthi amakamelo e-cascade angagcwaliswa ngamakhemikhali amanzi noma amakhemikhali omile njenge-lyophilized powders.Esivivinyweni esikuFig. 3a(i), uyinki obomvu ovela ekamelweni eliphezulu ugeleza kanye nempushana yodayi oluhlaza okwesibhakabhaka (i-copper sulfate) ungene ekamelweni lesibili futhi uguqule ube luhlaza okwesibhakabhaka okumnyama uma ufika ekamelweni eliphansi.Iphinde ibonise ingcindezi yokulawula uketshezi olumpontshwayo.Ngokufanayo, lapho i-lever eyodwa ixhunywe emakamelweni amabili, iba imodi yomjovo ngesikhathi esisodwa, njengoba kuboniswe kumfanekiso.3a(ii), lapho uketshezi lungasatshalaliswa ngokulinganayo phezu kwamakamelo amabili noma ngaphezulu uma kusetshenziswa ingcindezi.Njengoba ukucindezela okubalulekile kuncike kubude be-lever, ubude be-lever bungalungiswa ukuze kuzuzwe iphethini yomjovo olandelanayo njengoba kuboniswe kumfanekiso.3a(iii).I-lever ende (ene-pressure ebucayi Pc_long) ixhunywe ku-chamber B kanye ne-lever emfushane (enengcindezi ebalulekile i-Pc_short > Pc_long) ixhunywe ekamelweni A. Njengoba i-pressure P1 (Pc_long < P1 < Pc_short) isetshenzisiwe, kuphela uketshezi olubomvu. ingagelezela ku-chamber B futhi lapho ukucindezela kwenyuswa ku-P2 (> Pc_short), uketshezi oluluhlaza okwesibhakabhaka lungagelezela ku-chamber A. Le modi yomjovo olandelanayo isebenza oketshezini oluhlukene oludlulisela emakamelweni azo ahlobene ngokulandelana, okubalulekile ku-POCT ephumelelayo. idivayisi.I-lever ende (ene-pressure ebucayi Pc_long) ixhunywe ku-chamber B kanye ne-lever emfushane (enengcindezi ebalulekile i-Pc_short > Pc_long) ixhunywe ekamelweni A. Njengoba i-pressure P1 (Pc_long < P1 < Pc_short) isetshenzisiwe, kuphela uketshezi olubomvu. ingagelezela ku-chamber B futhi lapho ukucindezela kwenyuswa ku-P2 (> Pc_short), uketshezi oluluhlaza okwesibhakabhaka lungagelezela ku-chamber A. Le modi yomjovo olandelanayo isebenza oketshezini oluhlukene oludlulisela emakamelweni azo ahlobene ngokulandelana, okubalulekile ku-POCT ephumelelayo. idivayisi.Длинный рычаг (с критическим давлением Pc_long) был соединен с камерой B, а короткий рычаг (с критическим давлением Pc_short > Pc_long) и давления P1 (Pc_long < P1 < Pc_short) только жидкость, выделенная красным может течь в камеру B, ne-когда давление было увеличено до P2 (> Pc_short), синяя жидкость может течь в камеру A. Этот режим последователькость зпруська костям, последовательно перемещаемым в соответствующие камеры, что имеет решающее значение для успешной POCT.I-lever ende (enengcindezi ebalulekile engu-Pc_long) ixhunywe ku-chamber B, futhi i-lever emfushane (enengcindezi ebalulekile Pc_short > Pc_long) ixhunywe ku-chamber A. Uma i-pressure P1 (Pc_long < P1 < Pc_short) isetshenziswa, kuphela uketshezi olugqanyisiwe. ngokubomvu kungagelezela kugumbi B, futhi lapho umfutho unyusiwe waba ngu-P2 (> Pc_short), uketshezi oluluhlaza okwesibhakabhaka lungagelezela kugumbi A. Le ndlela yokujova elandelanayo isetshenziswa oketshezini oluhlukene oludluliselwa ngokulandelana emakamelweni afanele, okubalulekile. nge-POCT ephumelelayo.idivayisi. Длинный рычаг (критическое давление Pc_long) соединен с камерой B, а короткий рычаг (критическое давление Pc_short > Pc_long) соедийн с камерой.Ingalo ende (ingcindezi ebalulekile Pc_long) ixhunywe ku-chamber B bese ingalo emfushane (ingcindezi ebalulekile Pc_short > Pc_long) ixhunywe ku-chamber A.При приложении давления P1 (Pc_long < P1 < Pc_short) в камеру B может поступать только красная жидкость, а при увеличении давления до пушен программы (>) синяя жидкость.Uma kufakwa i-pressure P1 (Pc_long < P1 < Pc_short ), uketshezi olubomvu kuphela olungangena kugum B, futhi uma ukucindezela kwenyuka kuya ku-P2 (> Pc_short), uketshezi oluluhlaza okwesibhakabhaka lungangena kugum A. Le modi yomjovo olandelanayo ifanele ukudluliswa okulandelanayo kwe- uketshezi oluhlukahlukene emakamelweni afanele, okubalulekile ekusebenzeni ngempumelelo kwedivayisi ye-POCT.Umfanekiso we-3a (iv) ubonisa imodi yomjovo ekhethiwe, lapho igumbi eliyinhloko linemfushane (ene-Pc_short yokucindezela okubucayi) kanye ne-lever ende (ene-pressure ebucayi i-Pc_long <Pc_short) exhunywe ku-chamber A ne-chamber B, ngokulandelana, ngaphezu kwalokho. kwesinye isiteshi somoya esixhunywe ku-chamber B. Ukuze udlulisele uketshezi ku-chamber A kuqala, ukucindezela okungu-P1 (Pc_long < P1 < Pc_short) no-P2 (P2 > P1) nge-P1 + P2 > Pc_short kwasetshenziswa ngesikhathi esifanayo.Umfanekiso we-3a (iv) ubonisa imodi yomjovo ekhethiwe, lapho igumbi eliyinhloko linemfushane (ene-Pc_short yokucindezela okubucayi) kanye ne-lever ende (ene-pressure ebucayi i-Pc_long
Umdwebo wemisebenzi eminingi eyabelwe, okungukuthi (i) i-cascading, (ii) kanyekanye, (iii) ngokulandelanayo, kanye (iv) nesabelo esikhethiwe.Amajika amele ukuhamba komsebenzi namapharamitha alezi zindlela ezine zokusabalalisa.b Imiphumela yokuhlolwa kwesitoreji sesikhathi eside emanzini e-deionized kanye ne-ethanol.n = 5 ukuhlola okuzimele kwenziwa futhi idatha ikhonjiswe njenge ± sd c.Imibukiso yokuhlola ukuzinza lapho idivayisi ye-FAST nedivayisi ye-capillary valve (CV) bekuku-(i) static kanye (ii) nezimo ezidlidlizayo.(iii) Ivolumu uma iqhathaniswa nesikhathi samadivayisi we-FAST ne-CV kumafrikhwensi ama-angular ahlukahlukene.d Ukushicilelwa kwemiphumela yokuhlolwa ngokufunwa (i) kwedivayisi ye-FAST kanye (ii) nedivayisi ye-CV.(iii) Ubudlelwano phakathi kwevolumu nesikhathi samadivayisi we-FAST ne-CV asebenzisa imodi yokucindezela okuphakathi.Yonke imigoqo yesikali, i-1 cm.Idatha eluhlaza inikezwa njengamafayela edatha eluhlaza.
Ukugcinwa isikhathi eside kwama-reagents kungenye into ebalulekile yedivayisi ye-POCT ephumelelayo ezovumela abasebenzi abangaqeqeshiwe ukuthi baphathe ama-reagents amaningi.Nakuba ubuchwepheshe obuningi bubonise amandla abo okugcina isikhathi eside (isb., ama-microdispensers angu-35, amaphakethe angama-blister angu-48, namaphakethe we-stick angama-49), igumbi lokwamukela elizinikele liyadingeka ukuze kufakwe iphakheji, okwandisa izindleko nokuba yinkimbinkimbi;ngaphezu kwalokho, lezi zindlela zokulondoloza azikuvumeli ukukhishwa kwesidingo futhi kubangele ukumosheka kwama-reagents ngenxa yezinto ezisele emaphaketheni.Amandla okugcina isikhathi eside aqinisekiswa ngokwenza ukuhlolwa kwempilo okusheshisiwe kusetshenziswa impahla ye-PMMA yomshini we-CNC ngenxa yokuma kwayo okuncane kanye nokumelana nokugeleza kwegesi (Supplementary Figure S5).Imishini yokuhlola yagcwaliswa ngamanzi angcolile (amanzi akhishwe yi-deionized) kanye ne-ethanol engu-70% (ekulingisa ama-reagents aguquguqukayo) ku-65 ° C izinsuku ezingu-9.Kokubili amanzi enziwe i-deionized kanye ne-ethanol agcinwe kusetshenziswa i-aluminium foil ukuvimba ukufinyelela kusuka phezulu.I-equation ye-Arrhenius namandla okwenza kusebenze ukungena okubikwe ezincwadini50,51 zisetshenziswe ukubala okulingana nesikhathi sangempela.Emkhiwaneni.I-3b ibonisa isilinganiso semiphumela yokulahlekelwa kwesisindo samasampuli angu-5 agcinwe ku-65°C izinsuku ezingu-9, okulingana no-0.30% wamanzi akhishwe yi-deionized kanye no-0.72% we-ethanol engu-70 eminyakeni engu-2 ku-23°C.
Emkhiwaneni.I-3c ibonisa ukuhlolwa kokudlidliza.Njengoba i-capillary valve (CV) iyindlela yokuphatha uketshezi edume kakhulu phakathi kwamadivayisi akhona e-POCT28,29, idivayisi ye-CV engu-300 µm ububanzi no-200 µm ukushona yasetshenziswa ukuze kuqhathaniswe.Kungabonakala ukuthi lapho womabili amadivaysi ehlala emile, uketshezi oluku-FAST-POCT platform luvala futhi uketshezi olusedivayisini ye-CV luyavaleka ngenxa yokwanda okungazelelwe kwesiteshi, okunciphisa amandla e-capillary.Nokho, njengoba imvamisa ye-angular ye-orbital vibrator ikhula, uketshezi oluku-FAST-POCT luhlala luvaliwe, kodwa uketshezi olukudivayisi ye-CV lugelezela ekamelweni eliphansi (bheka futhi I-Supplementary Movie S3).Lokhu kusikisela ukuthi amahinge aguga weplathifomu ye-FAST-POCT angasebenzisa amandla anamandla emishini kumojula ukuze avale ngokuqinile uketshezi ekamelweni.Kodwa-ke, kumadivayisi e-CV, uketshezi luyagcinwa ngenxa yebhalansi phakathi kwezigaba eziqinile, zomoya, neziwuketshezi, ukudala ukungazinzi, nokudlidliza kungaphazamisa ibhalansi futhi kubangele ukuziphatha kokugeleza okungalindelekile.Inzuzo yesikhulumi se-FAST-POCT ukuthi inikeza ukusebenza okuthembekile futhi igwema ukwehluleka phambi kokudlidliza okuvame ukwenzeka ngesikhathi sokulethwa nokusebenza.
Esinye isici esibalulekile seplathifomu ye-FAST-POCT ukukhululwa kwayo ngokufunwa, okuyisidingo esibalulekile sokuhlaziywa komthamo.Emkhiwaneni.I-3d iqhathanisa ukukhishwa okudingekayo kwengxenyekazi ye-FAST-POCT kanye nedivayisi ye-CV.Kusukela emkhiwaneni.I-3d (iii) sibona ukuthi idivayisi ye-FAST isabela ngokushesha esignali yokucindezela.Lapho ingcindezi isetshenziswa endaweni yesikhulumi se-FAST-POCT, uketshezi lwageleza, lapho ukucindezela kukhishwa, ukugeleza kuma ngokushesha (Fig. 3d (i)).Lesi senzo singachazwa ngokubuya okunwebekayo okusheshayo kwehinge, okucindezela i-lever emuva ngokumelene ne-block, ukuvala igumbi.Kodwa-ke, uketshezi lwaqhubeka lugeleza kudivayisi ye-CV, lwagcina lubangele umthamo woketshezi olungalindelekile olulinganiselwa ku-100 µl ngemva kokukhishwa kwengcindezi (Umfanekiso 3d(ii) kanye ne-Supplementary Movie S4).Lokhu kungachazwa ngokunyamalala komphumela wokuphina i-capillary ekumanziseni okuphelele kwe-CV ngemva komjovo wokuqala.
Ikhono lokuphatha uketshezi lokumanzisa okuhlukahlukene kanye ne-viscosity kudivayisi efanayo lihlala liyinselele ezinhlelweni ze-POCT.Ukumanzisa okungekuhle kungaholela ekuvuzeni noma ekuziphatheni kokugeleza okungalindelekile eziteshini, kanye nemishini ehambisanayo efana nama-vortex mixers, ama-centrifuges kanye nezihlungi ngokuvamile kuyadingeka ukuze kulungiswe uketshezi olune-viscous kakhulu 52.Sihlole ubudlelwano phakathi kwengcindezi ebalulekile kanye nezakhiwo eziwuketshezi (ezinobubanzi obubanzi bokumanzisa kanye ne-viscosity).Imiphumela ikhonjiswe kuThebula 1 naku-Video S5.Kungabonakala ukuthi uketshezi lwe-wettability ehlukene kanye ne-viscosity lungavalwa ekamelweni, futhi lapho kufakwa ingcindezi, ngisho noketshezi olune-viscosity efika ku-5500 cP lungadluliselwa ekamelweni eliseduze, okwenza kube lula ukuthola amasampula aphezulu. i-viscosity (okungukuthi, isikhwehlela, isampula ebonakalayo esetshenziselwa ukuhlonza izifo zokuphefumula).
Ngokuhlanganisa la madivayisi angenhla anemisebenzi eminingi eminingi, kungathuthukiswa anhlobonhlobo amadivaysi e-POCT asuselwa ku-FAST.Isibonelo sikhonjiswe kuMfanekiso 1. Lesi sitshalo siqukethe igumbi lokugcina, igumbi lokuxuba, igumbi lokuphendula kanye negumbi lokulahla.Ama-reagents angase agcinwe ekamelweni langaphambi kwesitoreji isikhathi eside futhi akhishwe ekamelweni lokuxutshwa.Ngokucindezela okulungile, ama-reactants axubile angadluliselwa ngokukhetha ekamelweni likadoti noma ekamelweni lokusabela.
Ngenxa yokuthi ukutholwa kwe-PCR kuyizinga eligolide lokuthola amagciwane afana ne-H1N1 ne-COVID-19 futhi kuhilela izinyathelo zokusabela eziningi, sisebenzise inkundla ye-FAST-POCT yokutholwa kwe-PCR njengohlelo lokusebenza.Emkhiwaneni.4 ikhombisa inqubo yokuhlola i-PCR isebenzisa inkundla ye-FAST-POCT.Okokuqala, i-reagent eluting, i-magnetic microbead reagent, isixazululo sokugeza A, nesisombululo sokugeza u-W safakwa ngamapayipi emakamelweni angaphambi kwesitoreji u-E, M, W1 kanye no-W2, ngokulandelana.Izigaba ze-RNA adsorption ziboniswa kumfanekiso.4a futhi zimi kanje: (1) lapho ingcindezi P1 (=0.26 bar) isetshenziswa, isampula ithuthela ekamelweni M futhi ikhishelwe ekamelweni lokuxutshwa.(2) Umfutho womoya P2 (= 0.12 bar) unikezwa ngembobo A exhunywe phansi kwegumbi lokuxutshwa.Nakuba izindlela eziningi zokuxuba zibonise amandla azo ekuhlanganiseni uketshezi ezisekelweni ze-POCT (isb. ukuxuba inyoka engu-53, ukuxuba okungahleliwe 54 kanye nokuxuba inqwaba 55), ukusebenza kahle kwazo kokuxuba kanye nempumelelo akugculisi namanje.Yamukela indlela yokuxuba ibhamuza, lapho umoya ungeniswa khona ngaphansi kwegumbi lokuxuba ukuze udale amabhamuza oketshezini, emva kwalokho i-vortex enamandla ingafinyelela ukuxuba okuphelele ngemizuzwana.Ukuhlolwa kokuxuba amabhamuza kwenziwa futhi imiphumela yethulwa ku-Supplementary Figure S6.Kungabonakala ukuthi uma ingcindezi ye-0.10 bar isetshenziswa, ukuxuba okuphelele kuthatha cishe imizuzwana engu-8.Ngokwandisa ukucindezela kubha ye-0.20, ukuxuba okuphelele kufinyelelwa cishe imizuzwana emi-2.Izindlela zokubala ukusebenza kahle kokuhlanganisa zivezwa esigabeni Sezindlela.(3) Sebenzisa uzibuthe we-rubidium ukuze ukhiphe ubuhlalu, bese ucindezela u-P3 (= ibha engu-0.17) ngembobo engu-P ukuze uhambise ama-reagents ekamelweni likadoti.Emkhiwaneni.I-4b,c ikhombisa izinyathelo zokugeza ukuze kukhishwe ukungcola kwisampula ngendlela elandelayo: (1) Isixazululo sokugeza A esivela ekamelweni u-W1 sikhishelwa ekamelweni lokuxuba ingcindezi P1.(2) Bese wenza inqubo yokuxuba ibhamuza.(3) Isixazululo sokugeza A sidluliselwa ekamelweni le-liquid engcolile, futhi ama-microbeads ekamelweni lokuxuba akhishwa uzibuthe.Ukugeza W (Fig. 4c) kwakufana nokugeza A (Fig. 4b).Kumele kuqashelwe ukuthi isinyathelo ngasinye sokugeza A no-W senziwe kabili.Umdwebo 4d ubonisa izinyathelo zokunciphisa i-RNA ebuhlalu;Izinyathelo ze-lution kanye nokuxuba isingeniso ziyefana ne-RNA adsorption kanye nezinyathelo zokugeza ezichazwe ngenhla.Njengoba ama-reagents e-lution edluliselwa ekamelweni lokusabela le-PCR ngaphansi kwengcindezi P3 kanye ne-P4 (=0.23 bar), ukucindezela okubalulekile kufinyelelwa ukuvala ingalo yegumbi lokuphendula le-PCR.Ngokufanayo, ingcindezi ye-P4 iphinde isize ukuvala indlela eya ekamelweni likadoti.Ngakho-ke, wonke ama-reagents e-elution asatshalaliswa ngokulinganayo phakathi kwamagumbi amane okusabela e-PCR ukuze aqalise ukusabela kwe-PCR ephindaphindwayo.Inqubo engenhla yethulwa ku-Supplementary Movie S6.
Esinyathelweni se-adsorption ye-RNA, isampula yethulwa endaweni yokungena engu-M bese ijovwa egunjini lokuxutshwa kanye nesixazululo sobuhlalu esigcinwe ngaphambilini.Ngemuva kokuxuba nokukhipha ama-granules, ama-reagents asakazwa ekamelweni lokulahla.b kanye c nezinyathelo zokugeza, ngenisa ama-reagents ahlukahlukene okugeza agcinwe ngaphambilini ekamelweni lokuxuba, futhi ngemva kokuxuba nokukhipha ubuhlalu, dlulisela ama-reagents ekamelweni le-liquid yemfucuza.d Isinyathelo se-Elution: Ngemva kokwethula ama-reagents e-lution, ukuxuba nokukhipha ubuhlalu, ama-reagents adluliselwa ekamelweni lokusabela le-PCR.Amajika abonisa ukuhamba komsebenzi kanye nemingcele ehlobene yezigaba ezihlukahlukene.Ingcindezi iwukucindezela okwenziwa egunjini ngalinye.Umthamo umthamo woketshezi egumbini lokuxutshwa.Yonke imigoqo yesikali ingu-1 cm.Idatha eluhlaza inikezwa njengamafayela edatha eluhlaza.
Inqubo yokuhlola ye-PCR yenziwe futhi Umfanekiso Owengeziwe we-S7 wethula amaphrofayli ashisayo afaka amaminithi angu-20 esikhathi sokuhlehla sokuloba kanye nemizuzu engu-60 yesikhathi sokuhamba ngebhayisikili elishisayo (95 kanye no-60 °C), kanti umjikelezo owodwa wokushisa ube ama-90 s (I-Supplementary Movie S7)..I-FAST-POCT idinga isikhathi esincane ukuqedela umjikelezo owodwa oshisayo (amasekhondi angu-90) kune-RT-PCR evamile (amasekhondi angu-180 omjikelezo owodwa oshisayo).Lokhu kungachazwa ngendawo ephezulu nesilinganiso sevolumu kanye ne-inertia ephansi eshisayo yegumbi lokusabela le-micro-PCR.Ingaphezulu legumbi lingu-96.6 mm2 kanti umthamo wegumbi ungu-25 mm3, okwenza isilinganiso esiphezulu sevolumu sibe ngu-3.86.Njengoba kubonakala ku-Supplementary Figure S10, indawo yokuhlola ye-PCR yesikhulumi sethu inomsele kuphaneli engemuva, okwenza indawo ephansi yegumbi le-PCR ibe ngu-200 µm ubukhulu.Iphedi yokunwebeka ye-thermally conductive inamathiselwe endaweni yokushisisa yesilawuli sezinga lokushisa, iqinisekisa ukuthintana okuqinile nengemuva lebhokisi lokuhlola.Lokhu kunciphisa inertia eshisayo yeplathifomu futhi kuthuthukisa ukusebenza kahle kokushisisa/ukupholisa.Ngesikhathi sokuhamba ngebhayisikili elishisayo, upharafini oshumekwe emsamo uyancibilika bese ugeleza ungene egumbini lokusabela le-PCR, usebenza njengesivalo ukuvimbela ukuhwamuka kwe-reagent kanye nokungcoliswa kwemvelo (bona i-Supplementary Movie S8).
Zonke izinqubo zokutholwa kwe-PCR ezichazwe ngenhla bezizenzekela ngokugcwele kusetshenziswa ithuluzi elenziwe ngokwezifiso le-FAST-POCT, elihlanganisa iyunithi yokulawula ingcindezi ehleliwe, iyunithi yokukhipha kazibuthe, iyunithi yokulawula izinga lokushisa, kanye neyunithi yokuthwebula isignali ye-fluorescent kanye nokucubungula.Okuqaphelekayo, sisebenzise inkundla ye-FAST-POCT yokuhlukaniswa kwe-RNA sabe sesisebenzisa amasampula e-RNA akhishiwe kokusabela kwe-PCR sisebenzisa uhlelo lwe-FAST-POCT kanye nesistimu ye-PCR yedeskithophu ukuze siqhathanise.Imiphumela ibicishe ifane naleyo ekhonjiswe ku-Supplementary Figure S8.Umsebenzisi wenza umsebenzi olula: wethula isampula ku-M-chamber futhi afake iplatifomu entweni.Imiphumela yokuhlolwa yobuningi iyatholakala cishe emizuzwini engama-82.Ulwazi oluningiliziwe mayelana namathuluzi we-FAST-POCT lungatholakala kusibalo esengeziwe.C9, C10 kanye ne-C11.
Umkhuhlane obangelwa amagciwane omkhuhlane A (IAV), B (IBV), C (ICV) kanye no-D (IDV) uyinto evamile emhlabeni wonke.Kulokhu, i-IAV kanye ne-IBV yibo ababhekene nezigameko ezimbi kakhulu kanye nezifo eziwumshayabhuqe zesizini, zithelela abantu abangu-5-15% emhlabeni, okubangela amacala amabi ayizigidi ezi-3-5 futhi kubangele ukufa kwabantu abangu-290,000-650,000 ngonyaka.Izifo zokuphefumula56,57.Ukuxilongwa kusenesikhathi kwe-IAV ne-IB kubalulekile ukuze kuncishiswe ukugula kanye nomthwalo wezomnotho ohlobene.Phakathi kwamasu okuxilonga atholakalayo, i-reverse transcriptase polymerase chain reaction (RT-PCR) ithathwa njengokuzwela kakhulu, okuqondile, nokunembe kakhulu (>99%)58,59.Phakathi kwamasu okuxilonga atholakalayo, i-reverse transcriptase polymerase chain reaction (RT-PCR) ithathwa njengokuzwela kakhulu, okuqondile, nokunembe kakhulu (>99%)58,59.Среди доступных диагностических методов полимеразная цепная реакция с обратной транскриптазой (ОТ-ПЦР) считается наийболее наиболее > 99%)58,59.Phakathi kwezindlela zokuxilonga ezitholakalayo, i-reverse transcriptase polymerase chain reaction (RT-PCR) ithathwa njengokuzwela kakhulu, okuqondile futhi okunembile (> 99%)58,59. Из доступных диагностических методов полимеразная цепная реакция с обратной транскриптазой (ОТ-ПЦР) считается наийболее чулее, сположение 99%)58,59.Ezindleleni ezitholakalayo zokuxilonga, i-reverse transcriptase polymerase chain reaction (RT-PCR) ithathwa njengokuzwela kakhulu, okuqondile futhi okunembile (>99%)58,59.Kodwa-ke, izindlela zendabuko ze-RT-PCR zidinga ukufakwa kwamapayipi okuphindaphindiwe, ukuxutshwa, ukukhipha nokudluliselwa koketshezi, kukhawulelwe ukusetshenziswa kwazo ngochwepheshe kuzilungiselelo ezinomkhawulo wezinsiza.Lapha, inkundla ye-FAST-POCT yasetshenziselwa ukutholwa kwe-PCR ye-IAV ne-IBV, ngokulandelana, ukuze kutholwe umkhawulo wabo ophansi wokutholwa (LOD).Ukwengeza, i-IAV ne-IBV ziye zaphindwaphindwa ukuze zihlukanise phakathi kwezinhlobo zezifo ezihlukahlukene kuzo zonke izinhlobo zezilwane, okuhlinzeka ngenkundla ethembisayo yokuhlaziywa kofuzo kanye nekhono lokwelapha ngokunembile isifo.
Emkhiwaneni.5a ibonisa imiphumela yokuhlolwa kwe-HAV PCR kusetshenziswa u-150 µl we-purified viral RNA njengesampula.Emkhiwaneni.I-5a(i) ibonisa ukuthi ekugxilisweni kwe-HAV kwamakhophi angu-106/ml, amandla e-fluorescence (ΔRn) angafinyelela ku-0.830, futhi uma ukugxiliswa kwehliselwa kumakhophi angu-102/ml, i-ΔRn isengafinyelela ku-0.365, ehambisana nokungaphezulu kwalokho yeqembu elingenalutho lokulawula elingalungile (0.002), cishe izikhathi eziyi-100 ngaphezulu.Ngokulinganisa okusekelwe ekuhloleni okuyisithupha okuzimele, ijika lokulinganisa elilinganayo lakhiqizwa phakathi kokugxilisa ilogu nomkhawulo womjikelezo (Ct) we-IAV (Fig. 5a(ii)), R2 = 0.993, kusukela ku-102-106 amakhophi/mL.imiphumela ivumelana kahle nezindlela ezivamile ze-RT-PCR.Emkhiwaneni.I-5a(iii) ibonisa izithombe ze-fluorescent zemiphumela yokuhlolwa ngemva kwemijikelezo engu-40 yeplathifomu ye-FAST-POCT.Sithole ukuthi inkundla ye-FAST-POCT ingathola i-HAV iphansi njengamakhophi angu-102/mL.Nokho, indlela yendabuko ayinalo inani le-Ct kumakhophi angu-102/mL, okuyenza i-LOD yamakhophi angaba ngu-103/mL.Sicabange ukuthi lokhu kungenzeka kungenxa yokusebenza kahle okuphezulu kokuxutshwa kwamabhamuza.Ukuhlolwa kokuhlolwa kwe-PCR kwenziwa ku-IAV RNA ehlanzekile ukuze kuhlolwe izindlela ezihlukahlukene zokuxuba, okuhlanganisa ukuxutshwa kwe-shake (indlela yokuxuba efanayo njengasekusebenzeni okuvamile kwe-RT-PCR), ukuxutshwa kwe-vial (le ndlela, ama-3 kubha engu-0.12) futhi akukho ukuxutshwa njengeqembu elilawulayo. ..Imiphumela ingatholakala ku-Supplementary Figure S12.Kungabonakala ukuthi ekugxiliseni okuphezulu kwe-RNA (amakhophi angu-106/mL), amanani e-Ct ezindlela ezihlukene zokuhlanganisa acishe afane nokuxutshwa kwamabhamuza.Lapho ukugxiliswa kwe-RNA kwehlela kumakhophi angu-102/mL, ingxube ye-shake nezilawuli zazingenazo amanani e-Ct, kuyilapho indlela yokuxuba ibhamuza isanikeza inani le-Ct elingu-36.9, elalingaphansi komkhawulo we-Ct ongu-38. Imiphumela ibonisa isici sokuhlanganisa esivelele. ama-vesicles, okuye kwaboniswa nakwezinye izincwadi, okungase futhi kuchaze ukuthi kungani ukuzwela kwesiteji se-FAST-POCT kuphakeme kancane kune-RT-PCR evamile.Emkhiwaneni.I-5b ibonisa imiphumela yokuhlaziywa kwe-PCR yamasampula e-IBV RNA ahlanzekile asukela ku-101 kuya ku-106 amakhophi/ml.Imiphumela yayifana nokuhlolwa kwe-IAV, yathola u-R2 = 0.994 kanye ne-LOD yamakhophi angu-102/mL.
ukuhlaziywa kwe-PCR kwegciwane lomkhuhlane A (IAV) ngokugxila kwe-IAV kusuka ku-106 kuya ku-101 amakhophi/mL kusetshenziswa i-TE buffer njengesilawuli esingesihle (NC).(i) Ijika le-fluorescence lesikhathi sangempela.(ii) Ijika lokulinganisa elingumugqa phakathi kokugxilisa kwe-logarithmic IAV RNA kanye nomkhawulo womjikelezo (Ct) we-FAST nezindlela zokuhlola ezivamile.(iii) Isithombe sefluorescent se-IAV FAST-POCT ngemva kwemijikelezo engama-40.b, ukutholwa kwe-PCR yegciwane le-influenza B (IBV) nge-(i) yesikhathi sangempela se-fluorescence spectrum.(ii) Ijika lokulinganiswa komugqa kanye (iii) nesithombe se-FAST-POCT IBV se-fluorescence ngemva kwemijikelezo engu-40.Umkhawulo ophansi wokutholwa (LOD) we-IAV ne-IBV usebenzisa inkundla ye-FAST-POCT ubungamakhophi angu-102/mL, ephansi kunezindlela ezivamile (amakhophi angu-103/mL).c Imiphumela yokuhlolwa eminingi ye-IAV ne-IBV.I-GAPDH yasetshenziswa njengesilawuli esihle futhi isigcinalwazi se-TE sasetshenziswa njengesilawuli esingalungile ukuvimbela ukungcola okungenzeka kanye nokukhulisa ingemuva.Izinhlobo zesampula ezine ezihlukene zingahlukaniswa: (1) amasampula angalungile e-GAPDH kuphela (“IAV-/IBV-”);(2) Ukutheleleka kwe-IAV (“IAV+/IBV-”) nge-IAV ne-GAPDH;(3) Ukutheleleka kwe-IBV (“IAV-/IBV+”) nge-IBV ne-GAPDH;(4) Ukutheleleka kwe-IAV/IBV (“IAV+/IBV+”) nge-IAV, IBV ne-GAPDH.Ulayini onamachashazi umele umngcele.n = 6 ukuhlola okuzimele ngokwebhayoloji kwenziwa, idatha ikhonjiswa njengokuchezuka okujwayelekile ±.Idatha engahluziwe yethulwa njengamafayela edatha eluhlaza.
Emkhiwaneni.I-5c ibonisa imiphumela yokuhlolwa kokuphindaphinda kwe-IAV/IBV.Lapha, i-virus lysate isetshenziswe njengesixazululo sesampula esikhundleni se-RNA ehlanzekile, futhi ama-primers amane e-IAV, IBV, GAPDH (ukulawula okuhle) kanye ne-TE buffer (ukulawula okungalungile) kwengezwe emakamelweni amane okusabela ahlukene esiteji se-FAST-POCT.Izilawuli ezinhle nezingezinhle zisetshenziswa lapha ukuvimbela ukungcola okungaba khona kanye nokuthuthukisa ingemuva.Ukuhlolwa kwahlukaniswa ngamaqembu amane: (1) amasampula e-GAPDH-negative (“IAV-/IBV-”);(2) I-IAV ethelelekile (“IAV+/IBV-”) iqhathaniswa ne-IAV ne-GAPDH;(3) IBV-.utheleleke (“IAV-”) -/IBV+”) IBV kanye ne-GAPDH;(4) IAV/IBV (“IAV+/IBV+”) ukutheleleka nge-IAV, IBV ne-GAPDH.Emkhiwaneni.I-5c ibonisa ukuthi uma amasampula angalungile esetshenziswa, ukushuba kwe-fluorescence ΔRn kwegumbi lokulawula elihle lalingu-0.860, futhi i-ΔRn ye-IAV ne-IBV yayifana nokulawula okungekuhle (0.002).Emaqenjini e-IAV+/IBV-, IAV-/IBV+ kanye ne-IAV+/IBV+, amakhamera e-IAV/GAPDH, IBV/GAPDH kanye ne-IAV/IBV/GAPDH abonise ukushuba okukhulu kwe-fluorescence, ngokulandelana, kuyilapho amanye amakhamera aze abonisa ukushuba kwe-fluorescence ngemuva. izinga lama-40 ngemva kokuhamba ngebhayisikili elishisayo.Kusukela ekuhlolweni okungenhla, inkundla ye-FAST-POCT yabonisa ukucaciswa okuvelele futhi yasivumela ukuba kanyekanye sihlanganise amagciwane omkhuhlane ahlukahlukene.
Ukuze kuqinisekiswe ukusebenza komtholampilo kwe-FAST-POCT, sihlole izibonelo zomtholampilo ezingama-36 (izibonelo ze-swab yekhala) kusuka kuziguli ze-IB (n=18) nezilawuli ezingezona ze-IB (n=18) (Umfanekiso 6a).Ulwazi lwesiguli lwethulwe kuThebula Lokwengeza 3. Isimo sokutheleleka nge-IB saqinisekiswa ngokuzimele futhi umthetho olandelwayo wocwaningo wagunyazwa i-Zhejiang University First Affiliated Hospital (Hangzhou, Zhejiang).Isampula ngayinye yeziguli yahlukaniswa izigaba ezimbili.Enye yacutshungulwa kusetshenziswa i-FAST-POCT kanti enye yacutshungulwa kusetshenziswa isistimu ye-PCR yedeskithophu (SLAN-96P, China).Zombili izivivinyo zisebenzisa izinsiza ezifanayo zokuhlanza nezokubona.Emkhiwaneni.I-6b ibonisa imiphumela ye-FAST-POCT kanye nokulotshwayo okujwayelekile okuhlanekezela kwe-PCR (RT-PCR).Siqhathanise amandla e-fluorescence (FAST-POCT) ne -log2(Ct), lapho i-Ct ingumkhawulo womjikelezo we-RT-PCR evamile.Kwakukhona ukuvumelana okuhle phakathi kwalezi zindlela ezimbili.I-FAST-POCT ne-RT-PCR ibonise ukuhlobana okuhle okuqinile nenani le-Pearson's ratio (r) elingu-0.90 (Umfanekiso 6b).Sibe sesihlola ukunemba kokuxilonga kwe-FAST-POCT.Ukusatshalaliswa kwe-Fluorescence intensity (FL) kwamasampuli amahle noma angalungile kunikezwe njengesilinganiso sokuhlaziya esizimele (Fig. 6c).Amanani e-FL ayephezulu kakhulu ezigulini ze-IB kunezilawuli (****P = 3.31 × 10-19; ukuhlolwa kwe-t-tailed two) (Fig. 6d).Okulandelayo, i-IBV receiver operating features (ROC) curves yahlelwa.Sithole ukuthi ukunemba kokuxilonga kwakukuhle kakhulu, kunendawo engaphansi kwejika le-1 (Fig. 6e).Sicela uqaphele ukuthi ngenxa yoku-oda imaski okuyisibopho e-China ngenxa ye-COVID-19 kusukela ngo-2020, asikazihlonzi iziguli ezine-IBD, ngakho zonke izibonelo zomtholampilo ezinhle (okungukuthi, izibonelo ze-swab zamakhala) bezingeze-IBV kuphela.
Idizayini yocwaningo lomtholampilo.Isamba samasampula angama-36, okuhlanganisa amasampula esiguli se-18 kanye nezilawuli ezingezona zomkhuhlane we-18, ahlaziywa kusetshenziswa inkundla ye-FAST-POCT kanye ne-RT-PCR evamile.b Hlola ukuvumelana kokuhlaziya phakathi kwe-FAST-POCT PCR kanye ne-RT-PCR evamile.Imiphumela yayihlotshaniswa kahle (Pearson r = 0.90).c Amazinga we-Fluorescence intensity ezigulini eziyi-18 ze-IB kanye nezilawuli eziyi-18.d Ezigulini ze-IB (+), amanani e-FL ayephezulu kakhulu kuneqembu lokulawula (-) (****P = 3.31 × 10-19; ukuhlolwa kwe-t-tailed; n = 36).Esigcawini ngasinye esiyisikwele, umaka omnyama ophakathi nendawo umele i-median, futhi imigqa engezansi nephezulu yebhokisi imele amaphesenti angama-25 kanye nama-75, ngokulandelana.Amadevu adlulela kumaphoyinti edatha amancane naphezulu, angathathwa njengamangaphandle.e ROC ijika.Umugqa wamachashazi ongu-d umele inani lomkhawulo elilinganiselwe kusukela ekuhlaziyweni kwe-ROC.I-AUC ye-IBV ithi 1. Idatha eluhlaza inikezwa njengamafayela edatha eluhlaza.
Kulesi sihloko, sethula i-FAST, enezici ezidingekayo ze-POCT ekahle.Izinzuzo zobuchwepheshe bethu zihlanganisa: (1) Umthamo oguquguqukayo (i-cascade, ngesikhathi esisodwa, ngokulandelana futhi ekhethiwe), ukukhululwa ngokufunwa (ukukhululwa okusheshayo nokulinganiselwe kwengcindezi esetshenzisiwe) kanye nokusebenza okuthembekile (ukudlidliza ngamadigri angu-150) (2) isitoreji sesikhathi eside (iminyaka engu-2 yokuhlolwa okusheshayo, ukulahlekelwa isisindo mayelana ne-0.3%);(3) ikhono lokusebenza ngoketshezi olunezinhlobonhlobo zokumanzisa kanye ne-viscosity (i-viscosity kufika ku-5500 cP);(4) Ezomnotho (Izindleko zezinto ezibonakalayo ezilinganiselwe zedivayisi ye-FAST-POCT PCR cishe zingu-$1).Ngokuhlanganisa ama-multifunctional dispensers, inkundla ehlanganisiwe ye-FAST-POCT yokutholwa kwe-PCR yamagciwane omkhuhlane A no-B yaboniswa futhi yasetshenziswa.I-FAST-POCT ithatha imizuzu engu-82 kuphela.Ukuhlolwa komtholampilo okunamasampula e-nasal swab angama-36 kubonise ukuvumelana okuhle kumfutho we-fluorescence nge-RT-PCR ejwayelekile (ama-coefficients ka-Pearson> 0.9).Ukuhlolwa komtholampilo okunamasampula e-nasal swab angama-36 kubonise ukuvumelana okuhle kumfutho we-fluorescence nge-RT-PCR ejwayelekile (ama-coefficients ka-Pearson> 0.9).Клинические тесты с 36 образцами мазков из носа показали хорошее соответствие интенсивности флуоресценции стандартной ОТ-ПЦенций ОТ-ПЦ (коэртсценции ОТ-ПЦРи,финций ОТ-ПЦР0)Ukuhlolwa komtholampilo okunamasampula angu-36 e-nasal swabs kubonise ukuvumelana okuhle nokuqina kwe-fluorescence ye-RT-PCR evamile (ama-coefficients ka-Pearson> 0.9).RT-PCR Клинические испытания 36 образцов мазков из носа показали хорошее совпадение интенсивности флуоресценции со стандартной ОТ-Пофинции стандартной ОТ-Пофинция ОТ-ПофинцийUkuhlolwa komtholampilo kwama-36 nasal swab specimens kubonise ukuvumelana okuhle kokuqina kwe-fluorescence ne-RT-PCR evamile (i-coefficient ka-Pearson> 0.9).Ngokuhambisana nalo msebenzi, izindlela ezahlukahlukene ezivelayo zamakhemikhali ezinto eziphilayo (isb., ukuhamba ngebhayisikili okushisayo kwe-plasma, ukuhlola ama-immunoassay angenawo ama-amplification, kanye nokuhlolwa kokusebenza kwe-nanobody) kubonise amandla azo ku-POCT.Kodwa-ke, ngenxa yokuntuleka kweplatifomu ye-POCT edidiyelwe ngokugcwele futhi eqinile, lezi zindlela nakanjani zidinga izinqubo ezihlukene zokucubungula (isb, i-RNA isolation44, incubation45 kanye ne-washing46), ehambisana nomsebenzi wamanje ngalezi zindlela zokuqalisa imisebenzi ye-POCT ethuthukisiwe nge. amapharamitha adingekayo.landa-in-impendulo-okukhiphayo ukusebenza.Kulo msebenzi, nakuba iphampu yomoya esetshenziselwa ukuvula i-valve ye-FAST incane ngokwanele ukuba ihlanganiswe nensimbi ye-benchtop (Fig. S9, S10), isasebenzisa amandla abalulekile futhi idala umsindo.Empeleni, amaphampu e-pneumatic amafomu amancane angashintshwa ngezinye izindlela, njengokusebenzisa amandla kazibuthe kagesi noma ukwenza umunwe kusebenze.Okunye ukuthuthukiswa kungase kuhlanganise, ngokwesibonelo, ukulungisa amakhithi okuhlola amakhemikhali e-biochemical ahlukene futhi athile, kusetshenziswa izindlela zokuthola ezintsha ezingadingi amasistimu okushisa/okupholisa, ngaleyo ndlela kunikeze inkundla ye-POCT engenamathuluzi yezinhlelo zokusebenza ze-PCR.Sikholelwa ukuthi uma kubhekwa inkundla ye-FAST inikeza indlela yokulawula uketshezi, sikholelwa ukuthi ubuchwepheshe be-FAST obuhlongozwayo bunikeza amandla okudala inkundla evamile hhayi kuphela yokuhlolwa kwe-biomedical, kodwa futhi nokuqapha imvelo, ukuhlolwa kwekhwalithi yokudla, ukuhlanganiswa kwezinto ezibonakalayo kanye nezidakamizwa. ..
Ukuqoqwa nokusetshenziswa kwezibonelo ze-swab yamakhala yomuntu kuvunywe Ikomidi Lezimiso Zokuziphatha le-Zhejiang University First Affiliated Hospital (IIT20220330B).Kwaqoqwa amasampula e-nasal swab angama-36, ahlanganisa abantu abadala abangu-16 abaneminyaka engu-30 ubudala, abadala abangu-7> abaneminyaka engu-40 ubudala, nabesilisa abangu-19, abesifazane abangu-17.Kwaqoqwa amasampula e-nasal swab angama-36, ahlanganisa abantu abadala abangu-16 abaneminyaka engu-30 ubudala, abadala abangu-7> abaneminyaka engu-40 ubudala, nabesilisa abangu-19, abesifazane abangu-17.Было собрано 36 образцов мазков из носа, в которых приняли участие 16 взрослых < 30 лет, 7 взрослых старше 40 лемун17 инге 19Kwaqoqwa ama-specimens angama-36 we-nasal swab kubantu abadala abangu-16 abaneminyaka engu-30 ubudala, abadala abangu-7 abangaphezu kweminyaka engama-40 ubudala, amadoda angu-19 nabesifazane abangu-17.Idatha yezibalo zabantu yethulwe kuThebula Lokwengeza 3. Imvume enolwazi itholwe kubo bonke ababambiqhaza.Bonke ababambiqhaza babesolwa ngokuba nomkhuhlane futhi bahlolwe ngokuzithandela ngaphandle kwesinxephezelo.
Isisekelo se-FAST nesivalo zenziwe nge-polylactic acid (PLA) futhi zaphrintwa iphrinta ye-Ender 3 Pro 3D (Shenzhen Transcend 3D Technology Co., Ltd.).Itheyiphu enamacala amabili yathengwa kwa-Adhesives Research, Inc. Model 90880. Ifilimu ye-PET engu-100 µm obukhulu yathengwa kwaMcMaster-Carr.Kokubili okunamathelayo nefilimu ye-PET kusikwe kusetshenziswa isisiki se-Silhouette Cameo 2 esivela ku-Silhouette America, Inc. Ifilimu elinwebekayo lenziwe ngezinto ze-PDMS ngokubumba umjovo.Okokuqala, uhlaka lwe-PET oluwugqinsi luka-200 µm lwasikwa kusetshenziswa isistimu ye-laser futhi lwanamathiselwa eshidini le-PMMA elingu-3 mm eliwugqinsi kusetshenziswa itheyipu yokunamathela enezinhlangothi ezimbili engu-100 µm.Isandulela se-PDMS (i-Sylgard 184; Ingxenye A: Ingxenye B = 10:1, i-Dow Corning) yabe isithelwa esikhunjeni futhi kwasetshenziswa induku yengilazi ukuze kukhishwe i-PDMS eyeqile.Ngemva kokwelapha ku-70° C. amahora angu-3, ifilimu ye-PDMS engu-300 μm ewugqinsi ingaxebuka esikhunjeni.
Izithombe zokusabalalisa okuhlukahlukene, ukushicilela okudingekayo nokusebenza okuthembekile zithathwa ngekhamera enesivinini esikhulu (Sony AX700 1000 fps).I-orbital shaker esetshenziswe ekuhlolweni kokwethembeka ithengwe kwa-SCILOGEX (SCI-O180).Umfutho womoya ukhiqizwa i-compressor yomoya, futhi izilawuli eziningana ze-digital precision pressure zisetshenziselwa ukulungisa inani lokucindezela.Inqubo yokuhlola ukuziphatha kokugeleza imi kanje.Inani elinqunywe kusengaphambili loketshezi lijovwe kudivayisi yokuhlola futhi ikhamera enesivinini esiphezulu yasetshenziswa ukurekhoda ukuziphatha kokugeleza.Izithombe ezimile zabe sezithathwa kumavidiyo okuziphatha kokugeleza ngezikhathi ezimisiwe, futhi indawo esele yabalwa kusetshenziswa isofthiwe ye-Image-Pro Plus, eyabe iphindaphindwa ngokujula kwekhamera ukuze kubalwe ivolumu.Imininingwane yesistimu yokuhlola ukuziphatha kokugeleza ingatholakala kokuthi Supplementary Figure S4.
Jova u-50 µl wama-microbeads kanye no-100 µl wamanzi enziwe i-deionized kudivayisi yokuxuba i-vial.Izithombe zokusebenza okuxubile zithathwe ngekhamera enesivinini esiphezulu njalo ngemizuzwana engu-0.1 ngezingcindezi zebha engu-0.1, ibha engu-0.15 nebha engu-0.2.Ulwazi lwephikseli phakathi nenqubo yokuhlanganisa lungatholwa kulezi zithombe kusetshenziswa isofthiwe yokucubungula izithombe (Photoshop CS6).Futhi ukuhlanganisa ukusebenza kahle kungazuzwa nge-Equation 53 elandelayo.
lapho u-M esebenza kahle ekuhlanganiseni, u-N uyisamba senani lamaphikseli esampula, futhi u-ci kanye \(\ibha{c}\) kuwukugxilisa okujwayelekile nokulindelwe okujwayelekile.Ukusebenza kahle kokuxuba kusuka ku-0 (0%, okungaxutshiwe) kuye ku-1 (100%, okuxutshwe ngokugcwele).Imiphumela ikhonjiswe ku-Supplementary Figure S6.
Ikhithi yesikhathi sangempela ye-RT-PCR ye-IAV ne-IBV, okuhlanganisa amasampuli e-IAV ne-IBV RNA (cat. no. RR-0051-02/RR-0052-02, Liferiver, China), Tris-EDTA buffer (TE buffer no. B541019 , Sangon Biotech, China), Positive Control RNA Purification Kit (Ingxenye No. Z-ME-0010, Liferiver, China) kanye ne-GAPDH Solution (Ingxenye No. M591101, Sangon Biotech, China) ziyatholakala ngokwezentengiso.Ikhithi yokuhlanza i-RNA ihlanganisa isigcinalwazi esibophayo, i-wash A, i-wash W, i-eluent, i-microbeads kazibuthe, kanye ne-acrylic carrier.I-IAV kanye ne-IBV yesikhathi sangempela se-RT-PCR kits ihlanganisa i-IFVA nucleic acid PCR ukuthola imiksi kanye ne-enzyme ye-RT-PCR.Engeza u-6 µl we-AcrylCarrier kanye no-20 µl wobuhlalu obuzibuthe ku-500 µl wesixazululo sebhafa esibophayo, nyakazisa kahle bese ulungisa isisombululo sobuhlalu.Faka u-21 ml we-ethanol ukuze ugeze u-A no-W, unyakazise kahle ukuze uthole izixazululo zokugeza A no-W, ngokulandelana.Bese, u-18 µl wengxube ye-PCR ye-fluorescent ne-IFVA nucleic acid ne-1 µl ye-enzyme ye-RT-PCR yengezwe ku-1 µl yesisombululo se-TE, yanyakaziswa futhi yafakwa phakathi imizuzwana embalwa, kwatholwa u-20 µl we-IAV kanye neziqalo ze-IBV.
Landela le nqubo elandelayo yokuhlanza i-RNA: (1) I-RNA adsorption.I-Pipette 526 µl yesisombululo se-pellet ku-1.5 ml centrifuge tube bese wengeza u-150 µl wesampula, bese unyakazisa ishubhu ngesandla phezulu naphansi izikhathi ezingu-10.Dlulisela u-676 µl wengxube kukholamu ye-affinity kanye ne-centrifuge ku-1.88 x 104 g amasekhondi angu-60.Amadreni alandelayo abe eselahlwa.(2) Isigaba sokuqala sokugeza.Engeza u-500 µl wesisombululo sokugeza A kukholamu ye-affinity, i-centrifuge ku-1.88 x 104 g amasekhondi angu-40, bese ulahla isixazululo esichithiwe.Le nqubo yokugeza iphindwe kabili.(3) isigaba sesibili sokugeza.Engeza u-500 µl wesisombululo sokugeza W kukholamu ye-affinity, i-centrifuge ku-1.88×104 g amasekhondi angu-15 bese ulahla isisombululo esichithiwe.Le nqubo yokugeza iphindwe kabili.(4) Isixazululo.Engeza u-200 µl we-eluate kukholomu ye-affinity kanye ne-centrifuge ku-1.88 x 104 g imizuzu engu-2.(5) I-RT-PCR: I-eluate yajovwa ku-20 μl wesixazululo se-primer kushubhu ye-PCR, bese ishubhu yafakwa kumshini wokuhlola we-PCR wesikhathi sangempela (SLAN-96P) ukuze kwenziwe inqubo ye-RT-PCR.Yonke inqubo yokuthola ithatha cishe imizuzu eyi-140 (imizuzu engama-20 yokuhlanzwa kwe-RNA kanye nemizuzu eyi-120 yokutholwa kwe-PCR).
U-526 µl wesixazululo sobuhlalu, u-1000 µl wekhambi lokugeza A, 1000 µl wesisombululo sokugeza u-W, 200 µl we-eluate kanye no-20 µl wesixazululo se-primer angeziwe futhi agcinwa egunjini lokutholwa elingu-M, W1, W2, E kanye ne-PCR.Umhlangano weplathifomu.Bese, u-150 µl wesampula wafakwa ngepayipi kugumbi M futhi inkundla ye-FAST-POCT yafakwa ethuluzini lokuhlola eliboniswe Kumfanekiso Owengeziwe S9.Ngemva kwemizuzu engaba ngu-82, imiphumela yokuhlolwa yayisitholakala.
Ngaphandle kwalapho kuphawulwe ngenye indlela, yonke imiphumela yokuhlolwa yethulwa njengesho ukuthi ± SD ngemva kokuphindaphinda okungenani okuyisithupha kusetshenziswa inkundla ye-FAST-POCT kuphela namasampuli azimele ngokwebhayoloji.Ayikho idatha ekhishiwe ekuhlaziyweni.Ukuhlolwa akukona okungahleliwe.Abacwaningi bebengaphuphutheki emisebenzini yeqembu ngesikhathi sokuhlolwa.
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