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Sisitemu ya laboratoire ifite ubushobozi bwo kurubuga itanga amahirwe yo kwisuzumisha byihuse kandi nyabyo kandi ni ingirakamaro mumikoreshereze yimikoreshereze yumutungo aho ibikoresho bya biomedical hamwe nababigize umwuga bahuguwe bitabonetse.Ariko, gushiraho ingingo-y-ibizamini bya sisitemu icyarimwe ifite ibintu byose bikenewe kugirango itangwe ryimikorere myinshi, kurekurwa kubisabwa, imikorere yizewe, hamwe no kubika igihe kirekire reagent biracyari ikibazo gikomeye.Hano turasobanura uburyo bukoreshwa na mikorobe ikora ingendo ya tekinoroji ishobora gukoresha amazi mu cyerekezo icyo aricyo cyose, igatanga igisubizo nyacyo kandi kigereranije nigitutu cyumuyaga gikoreshwa, kandi kigakomeza guhagarara neza mukigenda gitunguranye no kunyeganyega.Dushingiye ku ikoranabuhanga, turasobanura kandi iterambere rya sisitemu ya reaction ya polymerase ihuza ibikorwa bya reagent, kuvanga no gukora ibikorwa byose muburyo bumwe, bigasohoza imikorere ya "sample-in-Answer-out" kubikorwa byose by’amavuriro y’amavuriro yaturutse ku barwayi 18 bafite Ibicurane na 18 bigenzura buri muntu, muburyo bwiza bwa fluorescence hamwe nimbaraga zisanzwe za polymerase (Coefficients Pearson> 0.9).Dushingiye ku ikoranabuhanga, turasobanura kandi iterambere rya sisitemu ya polymerase yerekana uburyo bwo guhuza ibikorwa, kuvanga no gukora ibintu byose muburyo bumwe, bigasohoza imikorere ya "sample-in-Answer-out" kubikorwa byose by’amavuriro y’amavuriro yaturutse ku barwayi 18 hamwe na ibicurane hamwe na 18 bigenzura buri muntu, muburyo bwiza bwa fluorescence ubukana hamwe na polymerase isanzwe yerekana (coefficient Pearson> 0.9).Основываяь на этой технологии, мы также описываем разработку пы полимазной цепной зциции, которая одединяетункции введения реагентов, смешивания и одном всес в в в в в ѕ клинических обццов из носа от 18 аданов с Грипп и 18 отдельных зиналы, в хорошем соответствии интеншности флуоресценции со башной полимеразной цепной матцией (коэффа Пир раа> 0,9).Dushingiye kuri iri koranabuhanga, turasobanura kandi iterambere rya sisitemu ya polymerase yerekana uburyo bwo guhuza inshinge, kuvanga, no kubyitwaramo muburyo bumwe, bigafasha icyitegererezo-cyo-gusubiza ibisubizo byose by’amavuriro y’abarwayi ba grippe 18.na 18 kugenzura kugiti cye, mubyumvikanyweho neza na polymerase isanzwe ya reaction ya fluorescence ubukana (Coefficient ya Pearson> 0.9).Dushingiye kuri iryo koranabuhanga, turasobanura kandi iterambere rya sisitemu ya reaction ya polymerase ihuza inshinge za reagent, kuvanga, hamwe nigikorwa cyo gusesengura ingero zose zamavuriro zivuye muri 18 zicyitegererezo cy’abarwayi b’izuru.Influenza na 18 kugenzura buri muntu, ubukana bwa fluorescence burahuye neza hamwe na polymerase isanzwe ikora (coefficient ya Pearson> 0.9).Ihuriro ryateganijwe ryemeza ko ryizewe ryisesengura ryibinyabuzima bityo rikaba ryihutisha ubucuruzi bwibikoresho bitandukanye byo gupima.
Indwara z’abantu zivuka, nk’icyorezo cya COVID-19 cyahitanye ubuzima bwa miliyoni z’abantu, kibangamiye cyane ubuzima bw’isi ndetse n’umuco w’abantu1.Kumenya hakiri kare, byihuse kandi neza indwara ni ngombwa mu kurwanya ikwirakwizwa rya virusi no kunoza ibisubizo by’ubuvuzi.Urusobe rw’ibinyabuzima rusuzumwa rushingiye kuri laboratoire ikomatanyirijwe hamwe aho ibizamini byoherezwa mu bitaro cyangwa ku mavuriro yo kwisuzumisha kandi bigakorwa n’inzobere kuri ubu birabuza abantu kugera kuri miliyari 5.8 ku isi yose, cyane cyane abatuye ahantu hafite amikoro.aho habuze ibikoresho bya biomedical bihenze ninzobere zibishoboye.abaganga 2. Kubwibyo, harakenewe byihutirwa guteza imbere laboratoire ihendutse kandi yorohereza abakoresha-kuri-chip ifite ubushobozi bwo gupima (POCT) ubushobozi bushobora guha abaganga amakuru yo kwisuzumisha mugihe cyo gufata ibyemezo byo gusuzuma neza. .no kuvura 3.
Amabwiriza y’umuryango w’ubuzima ku isi (OMS) avuga ko POCT nziza igomba kuba ihendutse, yorohereza abakoresha (byoroshye gukoresha hamwe n’amahugurwa make), yuzuye (irinde ibibi cyangwa ibyiza bitari byo), byihuse kandi byizewe (bitanga ibintu byiza bisubirwamo), kandi gutangwa (bishoboye kubika igihe kirekire kandi byoroshye kuboneka kubakoresha amaherezo) 4.Kugira ngo ibyo bisabwa bishoboke, sisitemu ya POCT igomba gutanga ibintu bikurikira: gufata imiti myinshi kugirango igabanye intoki, kurekurwa kubisabwa kugirango habeho ubwikorezi bwa reagent kubisubizo nyabyo byikizamini, nibikorwa byizewe kugirango bihangane n’ibidukikije.Kugeza ubu, igikoresho gikoreshwa cyane muri POCT ni urujya n'uruza rw'ibice 5,6 bigizwe n'ibice byinshi bya poroteyine ya nitrocellulose isunika urugero ruto cyane rw'icyitegererezo imbere, ikagira reaction ya pre-immobilisation na capillary force.Nubwo bafite ibyiza byigiciro gito, koroshya imikoreshereze, nibisubizo byihuse, ibikoresho bya POCT byifashishwa mugupima ibinyabuzima gusa (urugero, ibizamini bya glucose 7,8 nibizamini byo gutwita9,10) bidasabye gusesengura ibyiciro byinshi.ibisubizo (urugero: gupakira ibintu byinshi, kuvanga, kugwiza).Byongeye kandi, imbaraga zitwara zigenzura umuvuduko wamazi (ni ukuvuga imbaraga za capillary) ntabwo zitanga ubudahwema bwiza, cyane cyane hagati yitsinda, bikavamo imyororokere mibi11 no gukora imirongo itembera cyane cyane mugutahura neza12,13.
Ubushobozi bwagutse bwo gukora muri micro- na nanoscale byatanze amahirwe yo guteza imbere ibikoresho bya microfluidic POCT yo gupima ingano 14,15,16,17.Muguhindura imiterere yimiterere 18, 19 hamwe na geometrie yimiyoboro 20, 21, 22, imbaraga za capillary nigipimo cyibi bikoresho birashobora kugenzurwa.Nyamara, kwizerwa kwabo, cyane cyane kumazi yatose cyane, ntagishobora kwemerwa kubera gukora ibintu bidahwitse, inenge yibintu, hamwe no kumva neza ibidukikije.Mubyongeyeho, kubera ko capillary itemba ikorerwa kumurongo wa gazi-gazi, ntayandi masoko ashobora gutangizwa, cyane cyane nyuma yo kuzuza umuyoboro wa microfluidic n'amazi.Kubwibyo, kugirango tumenye byinshi, intambwe nyinshi zo gutera inshinge zigomba gukorwa 24,25.
Mubikoresho bya microfluidic, ibikoresho bya microfluidic centrifugal kuri ubu ni kimwe mubisubizo byiza kuri POCT26,27.Uburyo bwayo bwo gutwara ni bwiza kuberako imbaraga zo gutwara zishobora kugenzurwa no guhindura umuvuduko wo kuzenguruka.Ariko, ibibi ni uko imbaraga za centrifugal zihora zerekeza kumpera yinyuma yigikoresho, bikagorana gushyira mubikorwa intambwe nyinshi zisabwa kugirango hasesengurwe byinshi.Nubwo imbaraga zinyongera zitwara (urugero: capillaries 28, 29 nizindi nyinshi 30, 31, 32, 33, 34, 35) usibye imbaraga za centrifugal zashyizweho kugirango zikoreshe inshuro nyinshi, ihererekanyabubasha ritunguranye rishobora kubaho kubera ko izo mbaraga zinyongera ari itegeko y'ubunini buri munsi ya centrifugal imbaraga, bigatuma ikora neza kurwego ruto ruto cyangwa ntiruboneka kubisabwa hamwe no kurekura amazi.Kwinjiza pneumatic manipulation muri microfluidics ya centrifugal nka centrifugal kinetic uburyo 36, 37, 38, uburyo bwa termopneumatike 39 hamwe nuburyo bukoreshwa bwa pneumatike 40 byagaragaye ko aribwo buryo bushimishije.Hamwe nuburyo bwa anti-fugodinamike, umwobo wongeyeho hamwe na microchannel ihuza byinjizwa mubikoresho haba mubikorwa byo hanze ndetse n’imbere, nubwo imikorere yayo yo kuvoma (iri hagati ya 75% na 90%) iterwa cyane numubare wikizunguruka hamwe nubwiza. y'amazi.Muburyo bwa thermopneumatike, latex membrane hamwe nicyumba cyohereza amazi byateguwe byumwihariko kugirango ushireho cyangwa wugurure inleti mugihe umuyaga wafashwe ushushe cyangwa ukonje.Nyamara, gushyushya / gukonjesha bizana ibibazo byo gusubiza buhoro kandi bigabanya imikoreshereze yabyo (urugero, polymerase ya reaction ya PCR).Hamwe nuburyo bukora pneumatike, kubisabwa kurekurwa no kugenda imbere bigerwaho hifashishijwe icyarimwe icyarimwe cyumuvuduko mwiza kandi bihuye neza numuvuduko wo kuzunguruka na moteri yihuta.Hariho ubundi buryo bwiza bwogukoresha ukoresheje pneumatike gusa (umuvuduko mwiza 41, 42 cyangwa umuvuduko mubi 43) kandi mubisanzwe bifunze valve.Mugukurikirana gukurikiraho igitutu mucyumba cya pneumatike, amazi asunikwa imbere perisitique, kandi ubusanzwe valve ifunze irinda gusubira inyuma kwamazi bitewe na peristalisiti, bityo bikamenya ibikorwa byamazi bigoye.Ariko, kuri ubu hariho umubare muto gusa wa tekinoroji ya microfluidic ishobora gukora ibikorwa byamazi bigoye mugikoresho kimwe cya POCT, harimo gutanga ibikorwa byinshi, kurekurwa kubisabwa, imikorere yizewe, kubika igihe kirekire, gutunganya amazi y’amazi menshi cyane, no gukora neza.Byose icyarimwe.Kubura imikorere yintambwe nyinshi birashobora kandi kuba imwe mumpamvu zituma ibicuruzwa bike byubucuruzi bya POCT nka Cepheid, Binx, Visby, Cobas Liat, na Rhonda byamenyekanye neza kumasoko afunguye kugeza ubu.
Muri iyi nyandiko, turasaba icyuma cyitwa microfluidic pneumatic actuator gishingiye ku ikoranabuhanga ryicyatsi kibisi (FAST).FAST ikomatanya ibintu byose nkenerwa icyarimwe kumurongo mugari wa reagent kuva microliter kugeza kuri mililitiro.FAST igizwe na elastique ya membrane, levers na blok.Hatabayeho gushyiramo ingufu zumwuka, membrane, levers na blok birashobora gufungwa cyane kandi amazi yimbere arashobora kubikwa igihe kirekire.Iyo igitutu gikwiye gishyizwe hamwe kandi kigahinduka muburebure bwa lever, diaphragm iraguka kandi igasunika leveri kumwanya ufunguye, bigatuma amazi anyuramo.Ibi bituma ibipimo byinshi byamazi byamazi muri kaskade, icyarimwe, bikurikiranye cyangwa byatoranijwe.
Twateje imbere sisitemu ya PCR dukoresheje FAST kugirango tubone ibisubizo-by-ibisubizo kugirango tumenye virusi ya grippe A na B (IAV na IBV).Twageze ku ntera yo hasi yo kumenya (LOD) ya kopi 102 / mL, ubushakashatsi bwakozwe na multiplex bwerekanye umwihariko wa IAV na IBV kandi twemerera virusi ya grippe.Ibisubizo byo kwipimisha kwa clinique ukoresheje icyitegererezo cyizuru cyatanzwe nabarwayi 18 nabantu 18 bafite ubuzima bwiza bagaragaza guhuza neza muburemere bwa fluorescence hamwe na RT-PCR isanzwe (coefficient Pearson> 0.9).Ibisubizo byo kwipimisha kwa clinique ukoresheje icyitegererezo cyizuru cyatanzwe nabarwayi 18 nabantu 18 bafite ubuzima bwiza bagaragaza guhuza neza muburemere bwa fluorescence hamwe na RT-PCR isanzwe (coefficient Pearson> 0.9).Результатя клинических испытаний с использованием оца мазка из носа от 18 отов и 18 здоровых лиц показывают хорошее соответствие интинсности флуоресценции внной ОТ-ПЦР (коэч.Ibisubizo by'ibizamini byo kwa muganga ukoresheje icyitegererezo cy'izuru ry’abarwayi 18 n'abantu 18 bafite ubuzima bwiza byerekana amasezerano meza hagati ya fluorescence ubukana bwa RT-PCR isanzwe (coefficient ya Pearson> 0.9).0.9） 。。。。。。。。。。。。。。。。。。。。。。。。。 Результаты клинических испытаний с использованием оцов назальных мазков от 18 матов и 18 здоровых лиц показали хорошее соответствие между интвенностве флуоресценции и кной ОТ-ПЦент ПIbisubizo by'ibizamini byo kwa muganga ukoresheje ingero zo mu mazuru biturutse ku barwayi 18 n'abantu 18 bafite ubuzima bwiza byagaragaje amasezerano meza hagati y’ingufu za fluorescence hamwe na RT-PCR isanzwe (coefficient ya Pearson> 0.9).Ikigereranyo cyibikoresho byigikoresho cya FAST-POCT ni hafi US $ 1 (Imbonerahamwe yinyongera 1) kandi birashobora kugabanuka ukoresheje uburyo bunini bwo gukora (urugero nko gutera inshinge).Mubyukuri, ibikoresho bya PAST bishingiye kuri FAST bifite ibintu byose nkenerwa byateganijwe na OMS kandi birahujwe nuburyo bushya bwo gupima ibinyabuzima nka plasma yumuriro cycling44, amplification-immunoassays45 hamwe na nanobody ikora ibizamini 46 aribyo nkingi ya sisitemu ya POCT.birashoboka.
Ku mutini.1a yerekana imiterere ya platform ya FAST-POCT, igizwe nibyumba bine byamazi: icyumba kibanziriza ububiko, icyumba kivanga, icyumba cyakira, nicyumba cy’imyanda.Urufunguzo rwo kugenzura ibicuruzwa bitemba ni igishushanyo cya FAST (kigizwe na elastike ya elastique, levers na bloks) giherereye mubyumba byabitswe mbere no kuvanga icyumba.Nuburyo bukoreshwa muburyo bwa pneumatike, igishushanyo cya FAST gitanga kugenzura neza gutembera neza kwamazi, harimo gufunga / gufungura ibintu, guhinduranya ibintu byinshi, kurekura ibintu bisabwa, gukora byizewe (urugero, kutumva neza ibidukikije), no kubika igihe kirekire.Ihuriro FAST-POCT rigizwe nibice bine: urwego rwinyuma, urwego rwa firime ya elastike, igipande cya firime ya plastike, hamwe nigipfukisho, nkuko bigaragara muburyo bwagutse ku gishushanyo 1b (nacyo cyerekanwe muburyo burambuye mumashusho yinyongera S1 na S2 ).Imiyoboro yose hamwe nibyumba bitwara amazi (nkibibanza byabitswe mbere nibyumba byerekana) byinjijwe muri substrate ya PLA (aside polylactique) kuva kuri mm 0.2 (igice cyoroshye) kugeza kuri mm 5 z'ubugari.Ibikoresho bya firime ya elastike ni 300 µm yuburebure bwa PDMS yaguka byoroshye mugihe umuvuduko wumwuka ushyizwe mubikorwa kubera "ubunini buke" hamwe na modulus nkeya ya elastique (hafi 2.25 MPa47).Filime ya polyethylene ikozwe muri polyethylene terephthalate (PET) ifite umubyimba wa 100 µm kugirango irinde firime ya elastique kutagira ihinduka ryinshi kubera umuvuduko wumwuka.Bihuye nibyumba, substrate layer ifite levers ihujwe nigipfukisho (gikozwe muri PLA) hinges kugirango igenzure imigendekere yamazi.Filime ya elastike yometse kumurongo winyuma ukoresheje kaseti zifata impande zombi (ARseal 90880) hanyuma zipfundikirwa na plastiki.Ibice bitatu byakusanyirijwe kuri substrate hifashishijwe igishushanyo cya T-clip murwego rwo hejuru.T-clamp ifite icyuho hagati yamaguru abiri.Iyo clip yinjijwe muri ruhago, amaguru yombi yunamye gato, hanyuma asubira uko yari ameze hanyuma ahambira cyane umupfundikizo no kumugongo igihe banyuraga muri ruhago (Ishusho yinyongera. S1).Ibice bine noneho biraterana hakoreshejwe umuhuza.
Igishushanyo mbonera cya platifomu yerekana ibice bitandukanye bikora nibiranga FAST.b Igishushanyo kinini cyurubuga rwa FAST-POCT.c Ifoto yikibuga iruhande rwigiceri cyamadorari yAmerika.
Uburyo bukora bwibikorwa bya FAST-POCT bwerekanwe ku gishushanyo cya 2. Ibice byingenzi ni ibibujijwe kurwego rwibanze hamwe na hinges ku gipfukisho, bivamo igishushanyo mbonera iyo ibice bine byateranijwe hakoreshejwe T-shusho. .Iyo nta muvuduko ukabije w’umwuka ushyizwe (igishushanyo cya 2a), guhuza kwiza gutera impeta kwunama no guhinduka, kandi hashyirwaho imbaraga zo gufunga binyuze mu cyuma kugira ngo ukande firime ya elastike ihagarike, kandi amazi yo mu cyuho cya kashe arasobanurwa. nk'igihugu gifunze.Twabibutsa ko muri iyi leta, leveri yunamye hanze, nkuko bigaragara kuruhande rwishusho ya 2a.Iyo umwuka utanzwe (Igicapo 2b), membrane ya elastike yaguka hanze yerekeza ku gipfukisho hanyuma igasunika leveri hejuru, bityo igafungura icyuho kiri hagati yigitereko nigitereko kugirango amazi atembera mucyumba gikurikira, bisobanurwa nkigihugu gifunguye. .Nyuma yo kurekura umuvuduko wumwuka, lever irashobora gusubira kumwanya wambere kandi igakomeza gukomera kubera ubworoherane bwa hinge.Amashusho yimikorere ya lever yerekanwe muri firime yinyongera S1.
A. Igishushanyo mbonera n'amafoto iyo bifunze.Mugihe habuze umuvuduko, lever ikanda membrane irwanya blok, hanyuma amazi arafunze.b Mumeze neza.Iyo igitutu gishyizwe, membrane iraguka kandi igasunika leveri hejuru, bityo umuyoboro urakinguka kandi amazi ashobora gutemba.c Menya ingano iranga igitutu gikomeye.Ibipimo biranga harimo uburebure bwa lever (L), intera iri hagati yigitambambuga na hinge (l) nubunini bwikigina (t).Fs nimbaraga zo gukwega kuri trottle point B. q numutwaro wagabanijwe kimwe kuri lever.Tx * yerekana itara ryakozwe na levering.Umuvuduko wingenzi nigitutu gisabwa kugirango uzamure leveri kandi utume amazi atemba.d Ibisubizo byubushakashatsi nubushakashatsi byerekana isano iri hagati yigitutu gikomeye nubunini bwibintu.n = Ubushakashatsi 6 bwigenga bwakozwe kandi amakuru yerekanwe nka ± gutandukana bisanzwe.Ibyibanze bitangwa nkamadosiye yamakuru yibanze.
Icyitegererezo cyisesengura gishingiye ku gitekerezo cya beam cyateguwe kugira ngo gisesengure biterwa n’umuvuduko ukabije Pc aho icyuho gifungura ku bipimo bya geometrike (urugero, L ni uburebure bwa lever, l ni intera iri hagati yikibanza na hinge, S ni leveri Ahantu ho guhurira hamwe na t t ni ubunini bwikigina, nkuko bigaragara ku gishushanyo 2c).Nkuko bisobanuwe mubisobanuro byinyongera hamwe ninyongera Igicapo S3, icyuho gifungura iyo \ ({P} _ {c} \ ge \ frac {2 {F} _ {s} l} {SL} \), aho Fs ari itara \ ({}}Igisubizo cyubushakashatsi hamwe nuburyo bwo gusesengura byerekana amasezerano meza (Igishusho 2d), byerekana ko igitutu gikomeye Pc yiyongera hamwe no kwiyongera kwa t / l no kugabanuka L, ibyo bikaba bisobanurwa byoroshye nicyitegererezo cyibiti bya kera, ni ukuvuga ko umuriro wiyongera hamwe na t / Lift .Rero, isesengura ryibintu byerekana neza ko igitutu gikomeye gishobora kugenzurwa neza muguhindura uburebure bwa L na igipimo cya t / l, gitanga urufatiro rukomeye rwo gushushanya urubuga FAST-POCT.
Ihuriro rya FAST-POCT ritanga uburyo bwinshi bwo gutanga (bwerekanwe ku gishushanyo cya 3a hamwe no gushiramo no kugerageza), kikaba aricyo kintu cyingenzi kiranga POCT igenda neza, aho amazi ashobora gutembera mu cyerekezo icyo ari cyo cyose no muburyo ubwo aribwo bwose (casade, icyarimwe, ikurikiranye) cyangwa uburyo bwinshi bwo guhitamo gutanga.- imikorere yo gukuramo.Ku mutini.3a (i) yerekana uburyo bwo kunywa bwa kasike aho ibyumba bibiri cyangwa byinshi byuzuzanya hifashishijwe ibibari kugirango bitandukane reaction zitandukanye hamwe na leveri yo kugenzura leta zifunguye kandi zifunze.Iyo igitutu gishyizwe mubikorwa, amazi atemba ava hejuru yerekeza mucyumba cyo hasi muburyo bwa casade.Twabibutsa ko ibyumba bya casade bishobora kuzuzwa imiti itose cyangwa imiti yumye nka porojeri ya lyofili.Mu bushakashatsi bwakozwe ku gishushanyo cya 3a (i), wino itukura ivuye mu cyumba cyo hejuru iratemba hamwe nifu y irangi ryubururu (sulfate y'umuringa) mucyumba cya kabiri ihinduka ubururu bwijimye iyo igeze mu cyumba cyo hasi.Irerekana kandi umuvuduko wo kugenzura amazi arimo kuvomwa.Mu buryo nk'ubwo, iyo lever imwe ihujwe nibyumba bibiri, ihinduka uburyo bwo gutera inshinge icyarimwe, nkuko bigaragara mumitini.3a (ii), aho amazi ashobora gukwirakwizwa kimwe hejuru y'ibyumba bibiri cyangwa byinshi mugihe igitutu gishyizwe.Kubera ko igitutu gikomeye giterwa n'uburebure bwa leveri, uburebure bwa leveri burashobora guhinduka kugirango ugere ku buryo bwo gutera inshinge nkuko bigaragara ku mbuto.3a (iii).Ikibaho kirekire (hamwe nigitutu gikomeye Pc_long) cyahujwe nicyumba B naho leveri ngufi (hamwe nigitutu gikomeye Pc_short> Pc_long) yahujwe nicyumba A. Nkuko igitutu P1 (Pc_long <P1 <Pc_short) cyakoreshwaga, gusa amazi mumutuku Irashobora gutembera mucyumba B kandi mugihe igitutu cyariyongereye kuri P2 (> Pc_short), amazi yubururu arashobora gutemba mucyumba A. Ubu buryo bwo gutera inshinge zikurikizwa kumazi atandukanye yimurira mubyumba bifitanye isano bikurikiranye, bikaba ari ngombwa kugirango POCT igende neza igikoresho.Ikibaho kirekire (hamwe nigitutu gikomeye Pc_long) cyahujwe nicyumba B naho leveri ngufi (hamwe nigitutu gikomeye Pc_short> Pc_long) yahujwe nicyumba A. Nkuko igitutu P1 (Pc_long
Igishushanyo cyibikorwa byinshi, ni ukuvuga (i) gutondeka, (ii) icyarimwe, (iii) bikurikiranye, na (iv) umukoro watoranijwe.Imirongo yerekana akazi hamwe nibipimo byuburyo bune bwo gukwirakwiza.b Ibisubizo by'ibizamini byo kubika igihe kirekire mumazi ya deionion na Ethanol.n = Ubushakashatsi 5 bwigenga bwakozwe kandi amakuru yerekanwe nka ± sd c.Kwerekana ikizamini gihamye mugihe igikoresho cya FAST hamwe na capillary valve (CV) byari muri (i) bihagaze kandi (ii) bihindagurika.(iii) Umubumbe nigihe cyibikoresho bya FAST na CV kumurongo utandukanye.d Gutangaza ibisubizo byikizamini kubisabwa (i) igikoresho cyihuse na (ii) igikoresho cya CV.(iii) Isano iri hagati yubunini nigihe kubikoresho bya FAST na CV ukoresheje uburyo bwumuvuduko wigihe.Ibipimo byose bipima, cm 1.Amakuru yimibare atangwa nkamadosiye yibanze.
Kubika igihe kirekire reagent nikindi kintu cyingenzi kiranga igikoresho cyiza cya POCT kizafasha abakozi badahuguwe gukora reagent nyinshi.Mugihe tekinolojiya myinshi yerekanye ubushobozi bwayo bwo kubika igihe kirekire (urugero, microdispensers 35, paki 48 za blister, na paki 49), hasabwa icyumba cyabigenewe cyo kwakira paki, ibyo bikaba byongera ibiciro kandi bigoye;byongeye kandi, ubwo buryo bwo kubika ntibwemerera gutanga ibisabwa kandi bikaviramo gutakaza reagent kubera ibisigazwa mubipfunyika.Ubushobozi bwo kubika igihe kirekire bwagenzuwe no gukora ikizamini cyihuse cyubuzima ukoresheje ibikoresho bya PMMA byakozwe na CNC kubera ubukana bwacyo buke no kurwanya gaze (Ishusho yinyongera S5).Ibikoresho byo kwipimisha byuzuyemo amazi ya deyoniya (amazi ya deionisation) na 70% Ethanol (bigana reagent volatile) kuri 65 ° C muminsi 9.Amazi ya deionion na Ethanol yabitswe hakoreshejwe fayili ya aluminiyumu kugirango ibuze kwinjira hejuru.Ingano ya Arrhenius ningufu zo kwinjiza imbaraga zavuzwe mubitabo 50,51 byakoreshejwe mukubara igihe nyacyo gihwanye.Ku mutini.3b yerekana impuzandengo yo kugabanya ibiro kuburugero 5 rwabitswe kuri 65 ° C muminsi 9, bihwanye na 0,30% kumazi ya deionion na 0,72% kuri 70% Ethanol mumyaka 2 kuri 23 ° C.
Ku mutini.3c yerekana ikizamini cyo kunyeganyega.Kubera ko capillary valve (CV) aribwo buryo buzwi cyane bwo gukoresha amazi mu bikoresho bya POCT 28,29 bihari, igikoresho cya CV 300 µm z'ubugari na 200 µm z'uburebure bwakoreshejwe mu kugereranya.Birashobora kugaragara ko mugihe ibyo bikoresho byombi bigumye bihagaze, amazi yo muma platform ya FAST-POCT arafunga kandi amazi yo mubikoresho bya CV arafunga kubera kwaguka gutunguranye kwumuyoboro, bigabanya imbaraga za capillary.Ariko, uko orbital vibrator yumurongo wikurikiranya yiyongera, amazi yo murwego rwa FAST-POCT akomeza gufungwa, ariko amazi yo mubikoresho bya CV yinjira mubyumba byo hasi (reba na Filime yinyongera S3).Ibi birerekana ko impeta zihindagurika zumwanya wa FAST-POCT zishobora gukoresha imbaraga zumukanishi kuri module kugirango zifunge neza amazi mucyumba.Nyamara, mubikoresho bya CV, amazi aragumana kubera uburinganire hagati yicyiciro gikomeye, umwuka, namazi, bigatera ihungabana, hamwe no kunyeganyega bishobora guhungabanya uburinganire kandi bigatera imyitwarire itunguranye.Ibyiza bya platform FAST-POCT nuko itanga imikorere yizewe kandi ikirinda kunanirwa imbere yinyeganyeza zikunze kugaragara mugihe cyo gutanga no gukora.
Ikindi kintu cyingenzi kiranga urubuga FAST-POCT ni ukurekurwa kwayo, nicyo kintu cyingenzi gisabwa kugirango isesengura ryinshi.Ku mutini.3d igereranya kurekurwa kubisabwa bya FAST-POCT nibikoresho bya CV.Kuva ku mutini.3d (iii) tubona ko igikoresho cya FAST gisubiza vuba ibimenyetso byumuvuduko.Iyo igitutu cyashyizwe kumurongo FAST-POCT, amazi yatemba, mugihe igitutu cyarekuwe, umuvuduko uhita uhagarara (Ishusho 3d (i)).Iki gikorwa gishobora gusobanurwa nigaruka ryihuse rya hinge, kanda leveri inyuma yumwanya, gufunga icyumba.Nyamara, amazi yakomeje gutemba mubikoresho bya CV, amaherezo bivamo umuvuduko utunguranye wa 100 µl nyuma yumuvuduko urekuwe (Igishusho 3d (ii) na Sinema yinyongera S4).Ibi birashobora gusobanurwa no kubura kwa capillary pinning nyuma yo guhanagura burundu CV nyuma yo guterwa bwa mbere.
Ubushobozi bwo gutunganya ibintu bitandukanye byamazi nubushuhe mubikoresho bimwe bikomeje kuba ingorabahizi kubisabwa POCT.Ubushuhe bubi bushobora gutuma umuntu atemba cyangwa indi myitwarire itunguranye mu miyoboro, kandi ibikoresho bifasha nka vortex mixer, centrifuges na filteri akenshi birasabwa gutegura amazi meza cyane 52.Twagerageje isano iri hagati yumuvuduko ukabije nibintu byamazi (hamwe nurwego runini rwubushuhe nubwiza).Ibisubizo bigaragara mu mbonerahamwe ya 1 na Video S5.Birashobora kugaragara ko amazi yubushuhe butandukanye hamwe nubukonje arashobora gufungirwa murugereko, kandi mugihe ushizwemo igitutu, ndetse namazi afite ububobere bwa cP agera kuri 5500 cP ashobora kwimurirwa mubyumba byegeranye, bigatuma bishoboka gutahura ingero zifite hejuru viscosity (ni ukuvuga sputum, icyitegererezo cyiza cyane gikoreshwa mugupima indwara zubuhumekero).
Muguhuza ibikoresho byavuzwe haruguru bikora byinshi, ibikoresho byinshi bya FAST bishingiye kuri POCT birashobora gutezwa imbere.Urugero rwerekanwe ku gishushanyo cya 1. Igihingwa kirimo icyumba kibanziriza ububiko, icyumba kivanga, icyumba cyakira n’icyumba cy’imyanda.Reagents irashobora kubikwa mubyumba byabitswe mbere yigihe kinini hanyuma ikarekurwa mukivanga.Hamwe nigitutu gikwiye, reaction ivanze irashobora kwimurwa muburyo bwo gutoranya imyanda cyangwa icyumba cyakira.
Kuberako PCR itahura nicyizahabu cyo kumenya virusi nka H1N1 na COVID-19 kandi ikubiyemo intambwe nyinshi zo kubyitwaramo, twakoresheje urubuga FAST-POCT kugirango tumenye PCR nkibisabwa.Ku mutini.4 yerekana inzira yo gupima PCR ukoresheje urubuga FAST-POCT.Ubwa mbere, reagent ya reagent, magnetic microbead reagent, gukaraba A, hamwe no gukaraba W washyizwe mubyumba byabitswe mbere E, M, W1 na W2.Ibyiciro bya RNA adsorption byerekanwe mugitabo.4a kandi nibi bikurikira: (1) mugihe igitutu P1 (= 0.26 bar) gishyizwe mubikorwa, icyitegererezo cyimukira mubyumba M hanyuma gisohoka mubyumba bivanga..Nubwo uburyo bwinshi bwo kuvanga bwerekanye ubushobozi bwabo mukuvanga amazi kumurongo wa POCT (urugero: kuvanga inzoka 53, kuvanga 54 hamwe no kuvanga 55), uburyo bwo kuvanga no gukora neza ntiburashimishije.Ifata uburyo bwo kuvanga ibibyimba, aho umwuka winjizwa munsi yicyumba cyo kuvanga kugirango habeho ibibyimba mumazi, nyuma yumuyaga ukomeye urashobora kugera kuvanga byuzuye mumasegonda.Ubushakashatsi bwo kuvanga bubble bwakozwe kandi ibisubizo bitangwa mugice cyinyongera S6.Birashobora kugaragara ko iyo hashyizweho igitutu cya 0.10 bar, kuvanga byuzuye bifata amasegonda 8.Mugukomeza umuvuduko kuri 0.20 bar, kuvanga byuzuye bigerwaho mumasegonda 2.Uburyo bwo kubara kuvanga neza butangwa mugice cyuburyo..Ku mutini.4b, c yerekana intambwe yo gukaraba kugirango ikureho umwanda kurugero rukurikira: (1) Igisubizo cyo gukaraba A kuva mucyumba W1 gisohoka mu cyumba kivanga igitutu P1.(2) Noneho kora uburyo bwo kuvanga bubble..Gukaraba W (Ishusho 4c) byari bisa no gukaraba A (Ishusho 4b).Twabibutsa ko buri ntambwe yo gukaraba A na W byakozwe kabiri.Igicapo 4d cerekana intambwe zo gukuraho RNA kuva mumasaro;gukuraho no kuvanga intambwe yo gutangiza ni kimwe na RNA adsorption hamwe no gukaraba intambwe zasobanuwe haruguru.Mugihe reagent ya elution yimuriwe mubyumba byitwara bya PCR mukibazo cya P3 na P4 (= 0.23 bar), igitutu gikomeye kigerwaho kugirango ushireho ukuboko kwicyumba cya PCR.Mu buryo nk'ubwo, igitutu cya P4 nacyo gifasha gufunga inzira igana mucyumba cy’imyanda.Rero, reagent zose zo gukuraho zagabanijwe neza mubyumba bine bya PCR kugirango batangire reaction ya multiplex PCR.Inzira yavuzwe haruguru itangwa muri Sinema yinyongera S6.
Mu ntambwe ya RNA adsorption, icyitegererezo cyinjijwe muri inlet M hanyuma kigaterwa mucyumba cyo kuvanga hamwe nigisubizo cyabitswe mbere.Nyuma yo kuvanga no gukuraho granules, reagent zigabanywa mubyumba byimyanda.b na c gukaraba intambwe, menyesha ibintu bitandukanye byabitswe mbere yo kubitsa mubyumba bivanga, hanyuma nyuma yo kuvanga no kuvanaho amasaro, ohereza reagent mubyumba byamazi.d Intambwe yo Kurandura: Nyuma yo kumenyekanisha reagent ya elution, kuvanga no gukuramo amasaro, reagent yimurirwa mubyumba bya reaction ya PCR.Imirongo yerekana ibikorwa byakazi hamwe nibipimo bifitanye isano mubyiciro bitandukanye.Umuvuduko nigitutu gikoreshwa mubyumba byihariye.Ingano nubunini bwamazi mubyumba bivanga.Ibipimo byose bipima ni cm 1.Amakuru yimibare atangwa nkamadosiye yibanze.
Uburyo bwo gupima PCR bwakozwe kandi Ishusho yinyongera S7 yerekana imyirondoro yubushyuhe burimo iminota 20 yigihe cyo kwandukura hamwe niminota 60 yigihe cyo gusiganwa ku magare (95 na 60 ° C), hamwe nubushyuhe bumwe ni 90 s (Filime yinyongera S7)..FAST-POCT isaba igihe gito cyo kurangiza icyiciro kimwe cyumuriro (amasegonda 90) kuruta RT-PCR isanzwe (amasegonda 180 kumurongo umwe wubushyuhe).Ibi birashobora gusobanurwa nubuso burebure bwuburinganire bwikigereranyo hamwe nubushyuhe buke bwumuriro wa micro-PCR reaction.Ubuso bwa chambre ni mm 96,6 mm2 nubunini bwa chambre ni 25 mm3, bigatuma ubuso bugereranya hafi 3.86.Nkuko bigaragara mu gishushanyo mbonera cya S10, agace ka PCR kapima urubuga rwacu gafite umwobo kumwanya winyuma, bigatuma hepfo yicyumba cya PCR 200 µm.Ikariso yubushyuhe bwa elastike ihujwe nubushyuhe bwo kugenzura ubushyuhe, bigatuma habaho guhuza neza inyuma yisanduku yikizamini.Ibi bigabanya ubushyuhe bwumuriro wa platifomu kandi bitezimbere ubushyuhe / gukonjesha.Mugihe cyamagare yubushyuhe, paraffine yashyizwe kumurongo irashonga kandi ikinjira mucyumba cyitwara cya PCR, ikora nk'ikidodo kugirango ikumire imyuka ihumanya kandi yanduze ibidukikije (reba Filime y'inyongera S8).
Inzira zose zo gutahura PCR zasobanuwe haruguru zakozwe mu buryo bwuzuye hakoreshejwe igikoresho cyabigenewe cyakozwe na FAST-POCT, kigizwe n’ishami rishinzwe kugenzura umuvuduko ukabije, ishami rishinzwe gukuramo magneti, ishami rishinzwe kugenzura ubushyuhe, hamwe n’ishami rishinzwe gufata no gutunganya ibimenyetso bya fluorescent.Icyitonderwa, twakoresheje urubuga FAST-POCT rwo kwigunga kwa RNA hanyuma dukoresha ingero za RNA zavomwe kubitekerezo bya PCR dukoresheje sisitemu ya FAST-POCT na sisitemu ya desktop PCR kugirango tugereranye.Ibisubizo byari hafi nkuko bigaragara mu gishushanyo cya S8.Umukoresha akora umurimo woroshye: atangiza icyitegererezo muri M-chambre hanyuma yinjiza urubuga mubikoresho.Ibisubizo by'ibizamini biboneka mu minota igera kuri 82.Ibisobanuro birambuye kubyerekeye ibikoresho bya FAST-POCT murashobora kubisanga mumashusho yinyongera.C9, C10 na C11.
Ibicurane biterwa na grippe A (IAV), B (IBV), C (ICV) na D (IDV) virusi ni ibintu bisanzwe ku isi.Muri bo, IAV na IBV ni bo nyirabayazana w'indwara zikomeye n'ibyorezo by'ibihe, byanduza 5-15% by'abatuye isi, bitera abantu miliyoni 3-5 bikabije kandi bica abantu 290.000-650.000 buri mwaka.Indwara z'ubuhumekero 56,57.Kwipimisha hakiri kare IAV na IB ni ngombwa kugirango ugabanye uburwayi hamwe nuburemere bwubukungu.Muburyo bwo kwisuzumisha buboneka, reaction transcriptase polymerase urunigi (RT-PCR) ifatwa nkibintu byoroshye, byihariye, kandi byukuri (> 99%) 58,59.Muburyo bwo kwisuzumisha buboneka, reaction transcriptase polymerase urunigi (RT-PCR) ifatwa nkibintu byoroshye, byihariye, kandi byukuri (> 99%) 58,59.Скин доступныхкостостических полов полимеразная цепная бар с обратной зискриптазой (ОТ-ПЦР) считается наиболее чувствительной, специфичной и точной (> 99%) 58,59.Muburyo buboneka bwo kwisuzumisha, reaction transcriptase polymerase urunigi (RT-PCR) ifatwa nkibintu byoroshye, byihariye kandi byukuri (> 99%) 58,59. Из доступныхкостостических полов полимеразная цепная бар с обратной айскриптазой (ОТ-ПЦР) считается наиболее чувствительной, специфичной и точной (> 99%) 58,59.Muburyo bwo kwisuzumisha buboneka, reaction transcriptase polymerase urunigi (RT-PCR) ifatwa nkibintu byoroshye, byihariye kandi byukuri (> 99%) 58,59.Nyamara, uburyo gakondo bwa RT-PCR busaba gusubirwamo inshuro nyinshi, kuvanga, gutanga no guhererekanya amazi, bikagabanya imikoreshereze yabakozi babigize umwuga mugihe gito.Hano, urubuga FAST-POCT rwakoreshejwe mugutahura PCR ya IAV na IBV, kugirango babone imipaka yo hasi yo kumenya (LOD).Byongeye kandi, IAV na IBV byahinduwe byinshi kugira ngo bitandukane hagati y’imiterere itandukanye y’ibinyabuzima, bitanga urubuga rwiza rwo gusesengura amoko n’ubushobozi bwo kuvura neza indwara.
Ku mutini.5a yerekana ibisubizo byo kwipimisha HAV PCR ukoresheje 150 µl ya virusi ya RNA isukuye nkicyitegererezo.Ku mutini.5a (i) yerekana ko kuri HAV yibitseho kopi 106 / ml, ubukana bwa fluorescence (ΔRn) bushobora kugera kuri 0.830, kandi iyo intumbero igabanutse ikagera kuri kopi 102 / ml, ΔRn irashobora kugera kuri 0.365, ibyo bikaba bihuye nibirenze ibyo. y'itsinda rito ryigenzura (0.002), hejuru yinshuro 100.Kugereranya hashingiwe ku bushakashatsi butandatu bwigenga, umurongo wo guhinduranya umurongo watanzwe hagati y’ibiti byinjira hamwe n’ibizunguruka (Ct) bya IAV (Igishusho 5a (ii)), R2 = 0,993, kuva kuri kopi 102-106 / mL.ibisubizo bihuye neza nuburyo busanzwe bwa RT-PCR.Ku mutini.5a (iii) yerekana amashusho ya fluorescent yibisubizo byikizamini nyuma yinzinguzingo 40 za platform FAST-POCT.Twabonye ko urubuga FAST-POCT rushobora kumenya HAV munsi ya kopi 102 / mL.Nyamara, uburyo gakondo ntabwo bufite agaciro ka Ct kuri kopi 102 / mL, bigatuma LOD ya kopi zigera kuri 103 / mL.Twakekereje ko ibyo bishobora guterwa nubushobozi buhanitse bwo kuvanga bubble.Ubushakashatsi bwibizamini bya PCR bwakorewe kuri IAV RNA isukuye kugirango harebwe uburyo butandukanye bwo kuvanga, harimo kuvanga shake (uburyo bumwe bwo kuvanga nko mubikorwa bisanzwe bya RT-PCR), kuvanga vial (ubu buryo, 3 s kuri 0.12 bar) kandi nta kuvanga nkitsinda rishinzwe kugenzura ..Ibisubizo murashobora kubisanga mumashusho yinyongera S12.Birashobora kugaragara ko murwego rwo hejuru rwa RNA (kopi 106 / mL), indangagaciro za Ct zuburyo butandukanye bwo kuvanga zirasa nkizivanga bubble.Iyo kwibanda kwa RNA kwamanutse kuri kopi 102 / mL, kuvanga no kunyeganyega nta gaciro bya Ct, mugihe uburyo bwo kuvanga bubble bwakomeje gutanga Ct agaciro ka 36.9, kari munsi ya Ct ya 38. Ibisubizo byerekana kwerekana kuvanga ibintu byiganje imitsi, nayo yerekanwe mubindi bitabo, ishobora kandi gusobanura impamvu sensitivite ya platform ya FAST-POCT iri hejuru gato ugereranije na RT-PCR isanzwe.Ku mutini.5b yerekana ibisubizo bya PCR isesengura rya IBV RNA yatunganijwe neza kuva kuri 101 kugeza 106 kopi / ml.Ibisubizo byari bisa nikizamini cya IAV, kugera kuri R2 = 0,994 na LOD ya kopi 102 / mL.
isesengura rya PCR kuri virusi ya grippe A (IAV) hamwe na IAV yibanda kuri kopi 106 kugeza 101 kopi / mL ukoresheje TE buffer nkigenzura ribi (NC).(i) Igihe nyacyo fluorescence umurongo..(iii) IAV FAST-POCT ishusho ya fluorescent nyuma yizunguruka 40.b, PCR kumenya virusi ya grippe B (IBV) hamwe na (i) igihe nyacyo cya fluorescence..Umupaka wo hasi wo gutahura (LOD) kuri IAV na IBV ukoresheje urubuga FAST-POCT wari kopi 102 / mL, iri munsi yuburyo busanzwe (kopi 103 / mL).c Ibisubizo byinshi bya test ya IAV na IBV.GAPDH yakoreshejwe nkigenzura ryiza naho TE buffer yakoreshejwe nkigenzura ribi kugirango hirindwe kwanduza no kwiyongera kwinyuma.Ubwoko bune butandukanye bw'icyitegererezo bushobora gutandukanywa: (1) GAPDH-ingero mbi gusa (“IAV- / IBV-”);(2) Indwara ya IAV (“IAV + / IBV-”) hamwe na IAV na GAPDH;(3) Indwara ya IBV (“IAV- / IBV +”) hamwe na IBV na GAPDH;(4) Indwara ya IAV / IBV (“IAV + / IBV +”) hamwe na IAV, IBV na GAPDH.Umurongo utudomo ugereranya umurongo.n = Ubushakashatsi 6 bwigenga bwibinyabuzima bwakozwe, amakuru yerekanwe nka iation gutandukana bisanzwe.Ibyibanze bitangwa nkamadosiye yamakuru yibanze.
Ku mutini.5c yerekana ibisubizo byikizamini cyo kugwiza kuri IAV / IBV.Hano, virusi lysate yakoreshejwe nkigisubizo cyicyitegererezo mu mwanya wa RNA isukuye, kandi primers enye za IAV, IBV, GAPDH (kugenzura neza) na TE buffer (kugenzura nabi) zongerewe mubyumba bine bitandukanye byerekana urubuga rwa FAST-POCT.Igenzura ryiza kandi ribi rikoreshwa hano kugirango wirinde kwanduza no kuzamura iterambere.Ibizamini byagabanijwe mu matsinda ane: (1) GAPDH-ingero mbi (“IAV- / IBV-”);(2) Yanduye IAV (“IAV + / IBV-”) na IAV na GAPDH;(3) IBV-.yanduye (“IAV-”) - / IBV + ”) IBV na GAPDH;(4) Indwara ya IAV / IBV (“IAV + / IBV +”) yanduye IAV, IBV na GAPDH.Ku mutini.5c yerekana ko iyo ingero mbi zashyizwe mu bikorwa, ubukana bwa fluorescence ΔRn y'icyumba cyiza cyo kugenzura cyari 0.860, naho ΔRn ya IAV na IBV byari bisa no kugenzura nabi (0.002).Ku matsinda ya IAV + / IBV-, IAV- / IBV + na IAV + / IBV +, amatsinda ya IAV / GAPDH, IBV / GAPDH na IAV / IBV / GAPDH yerekanaga ubukana bwa fluorescence, mu gihe izindi kamera zerekanaga ubukana bwa fluorescence inyuma urwego rwa 40 nyuma yo gusiganwa ku magare.Duhereye ku bizamini byavuzwe haruguru, urubuga rwa FAST-POCT rwerekanye umwihariko kandi rutwemerera icyarimwe virusi ya grippe zitandukanye.
Kugirango twemeze amavuriro akoreshwa na FAST-POCT, twagerageje ingero 36 zamavuriro (izuru swab urugero) kubarwayi ba IB (n = 18) hamwe nubugenzuzi butari IB (n = 18) (Ishusho 6a).Amakuru y’abarwayi atangwa mu mbonerahamwe y’inyongera 3. Imiterere y’ubwandu bwa IB yemejwe mu bwigenge kandi protocole y’ubushakashatsi yemejwe n’ibitaro bya mbere by’ishami rya kaminuza ya Zhejiang (Hangzhou, Zhejiang).Buri cyitegererezo cy’abarwayi cyagabanyijwemo ibyiciro bibiri.Imwe yatunganijwe hakoreshejwe FAST-POCT indi itunganywa hakoreshejwe sisitemu ya desktop PCR (SLAN-96P, Ubushinwa).Isuzuma ryombi rikoresha ibikoresho bimwe byo kweza no gutahura.Ku mutini.6b yerekana ibisubizo bya FAST-POCT hamwe na transcript isanzwe ya PCR (RT-PCR).Twagereranije ubukana bwa fluorescence (FAST-POCT) na -log2 (Ct), aho Ct ni inzitizi yumuzingi kuri RT-PCR isanzwe.Hariho amasezerano meza hagati yuburyo bubiri.FAST-POCT na RT-PCR byagaragaje isano ikomeye hamwe nigipimo cya Pearson (r) agaciro ka 0.90 (Ishusho 6b).Twahise dusuzuma ukuri kwisuzumisha rya FAST-POCT.Ikwirakwizwa rya Fluorescence (FL) kubitekerezo byiza kandi bibi byatanzwe nkigipimo cyigenga cyo gusesengura (Ishusho 6c).Indangagaciro za FL zari hejuru cyane kubarwayi ba IB kuruta kugenzura (**** P = 3.31 × 10-19; t-umurizo wa t-test ebyiri) (Ishusho 6d).Ibikurikira, IBV yakira ibiranga imikorere (ROC) umurongo wateguwe.Twabonye ko kwisuzumisha neza byari byiza cyane, hamwe nigice kiri munsi yumurongo wa 1 (Ishusho 6e).Nyamuneka menya ko kubera gutumiza mask byateganijwe mubushinwa kubera COVID-19 guhera muri 2020, ntitwigeze tumenya abarwayi bafite IBD, kubwibyo byose byerekana ivuriro ryiza (urugero, izuru rya swab) byari ibya IBV gusa.
Igishushanyo mbonera cyo kwiga.Hasesenguwe ingero 36 zose, harimo 18 z’abarwayi n’ubushakashatsi 18 butari ibicurane, zasesenguwe hakoreshejwe urubuga rwa FAST-POCT hamwe na RT-PCR isanzwe.b Suzuma isesengura rihamye hagati ya FAST-POCT PCR na RT-PCR isanzwe.Ibisubizo byari bifitanye isano neza (Pearson r = 0.90).c Urwego rwa Fluorescence rwinshi mubarwayi 18 ba IB hamwe na 18 igenzura.d Mu barwayi ba IB (+), indangagaciro za FL zari hejuru cyane ugereranije nitsinda rishinzwe kugenzura (-) (**** P = 3.31 × 10-19; t-umurizo wa t-test ebyiri; n = 36).Kuri buri kare kare, ikimenyetso cyumukara hagati kigereranya median, naho epfo na ruguru hejuru yagasanduku kagereranya 25 na 75 kwijana.Whiskers igera kumurongo ntarengwa kandi ntarengwa wamakuru, udafatwa nkuwasohotse.e umurongo wa ROC.Umurongo utudomo d ugereranya agaciro kagereranijwe uhereye kubisesengura ROC.AUC kuri IBV ni 1. Amakuru yimibare yatanzwe nkamadosiye yibanze.
Muri iyi ngingo, turerekana FAST, ifite ibiranga bisabwa kuri POCT nziza.Ibyiza byikoranabuhanga ryacu birimo: (1) Kunywa inshuro nyinshi (caskade, icyarimwe, ikurikiranye kandi ihitamo), kurekura kubisabwa (kurekura byihuse kandi bigereranywa nigitutu cyashyizweho) hamwe nigikorwa cyizewe (vibrasiya kuri dogere 150) (2) kubika igihe kirekire (Imyaka 2 yo kwipimisha byihuse, kugabanya ibiro hafi 0.3%);..Muguhuza ibiyobora byinshi, urubuga rwa FAST-POCT rwashyizwe hamwe kugirango PCR igaragaze virusi ya grippe A na B yerekanwe kandi irakoreshwa.VUBA-POCT ifata iminota 82 gusa.Ibizamini byamavuriro hamwe na 36 byizuru byizuru byerekanaga uburinganire bwiza bwa fluorescence hamwe na RT-PCR isanzwe (Coefficients Pearson> 0.9).Ibizamini byamavuriro hamwe na 36 byizuru byizuru byerekanaga uburinganire bwiza bwa fluorescence hamwe na RT-PCR isanzwe (Coefficients Pearson> 0.9).Клинические Ты с 36 Обцами мазков из носа показали хорошее соответствие интенсности флуоресценции башной ОТ-ПЦР (коэффуш Пир Пираа> 0,9).Ibizamini bya Clinical hamwe na 36 byurugero rwizuru byerekanaga amasezerano meza nuburemere bwa fluorescence ya RT-PCR isanzwe (coefficient ya Pearson> 0.9) .RT-PCR Клинические испытания 36 обрцов мазков из носа показали хорошее совпадение интен инности флуоресценции со сдерной ОТ-ПЦР (коэфафентент Пирва> 0,9).Igeragezwa rya Clinical ya 36 yizuru rya swab ryerekanye amasezerano meza yuburemere bwa fluorescence hamwe na RT-PCR isanzwe (coefficient ya Pearson> 0.9).Mu buryo bubangikanye n'iki gikorwa, uburyo butandukanye bwibinyabuzima bugaragara (urugero, plasma yumuriro wa plasma yumuriro, immunoassays idafite amplification, hamwe na nanobody ikora) byagaragaje ubushobozi bwabo muri POCT.Ariko, kubera kubura urubuga rwuzuye kandi rukomeye rwa POCT, ubu buryo byanze bikunze busaba uburyo butandukanye bwo kubanza gutunganya (urugero, RNA kwigunga44, incubation45 no gukaraba 46), ibyo bikaba byuzuza imirimo iriho hamwe nuburyo bwo gushyira mubikorwa ibikorwa bya POCT bigezweho hamwe ibipimo bisabwa.kuzana-mu-gusubiza-gusohora imikorere.Muri iki gikorwa, nubwo pompe yumuyaga ikoreshwa mugukora valve ya FAST ni nto bihagije kugirango yinjizwe mubikoresho byo ku ntebe (Igicapo S9, S10), iracyakoresha imbaraga zikomeye kandi itera urusaku.Ihame, pompe pompe pompe pomatique irashobora gusimburwa nubundi buryo, nko gukoresha ingufu za electromagnetic cyangwa gukora urutoki.Ibindi byanonosorwa birashobora gushiramo, kurugero, guhuza ibikoresho kubikoresho bitandukanye kandi byihariye bya biohimiki, ukoresheje uburyo bushya bwo gutahura budasaba sisitemu yo gushyushya / gukonjesha, bityo bigatanga ibikoresho bidafite ibikoresho bya POCT kubikoresho bya PCR.Twizera ko urebye ko urubuga rwa FAST rutanga uburyo bwo gukoresha amazi, twizera ko tekinoroji yatanzwe ya FAST itanga ubushobozi bwo gushyiraho urubuga ruhuriweho atari ugupima ibinyabuzima gusa, ahubwo no kugenzura ibidukikije, gupima ubuziranenge bwibiribwa, ibikoresho hamwe na synthesis ..
Gukusanya no gukoresha ingero zamazuru yabantu byemejwe na komite ishinzwe imyitwarire yibitaro bya mbere bya kaminuza ya Zhejiang (IIT20220330B).Hakozwe icyegeranyo cya 36 cyizuru cya swab, kirimo abantu bakuru 16 <30 ans, 7 bakuru> imyaka 40, nabagabo 19, 17 b'igitsina gore.Hakozwe icyegeranyo cya 36 cyizuru cya swab, kirimo abantu bakuru 16 <30 ans, 7 bakuru> imyaka 40, nabagabo 19, 17 b'igitsina gore.Было собрано 36 обрцов мазков из носа, в кот ах кяли участие 16 взрослых <30 лет, 7 взрослых старше 40 лет, 19 мужчин и 17 женщин.Ibigereranyo 36 byizuru byakusanyirijwe hamwe kubantu 16 bakuze <30 ans, 30 bakuze barengeje imyaka 40, abagabo 19 nabagore 17.Imibare ya demokarasi itangwa mu mbonerahamwe yinyongera 3. Uruhushya rwamenyeshejwe rwabonetse kubitabiriye amahugurwa bose.Abitabiriye amahugurwa bose bakekwagaho kuba bafite ibicurane kandi bapimwe ku bushake nta ndishyi.
Urufatiro rwa FAST n'umupfundikizo bikozwe muri acide polylactique (PLA) kandi byacapishijwe na printer ya Ender 3 Pro 3D (Shenzhen Transcend 3D Technology Co., Ltd.).Kaseti ebyiri yaguzwe muri Adhesives Research, Inc. Model 90880. PET firime 100 µm yubugure yaguzwe na McMaster-Carr.Amashusho yombi hamwe na PET yaciwe hifashishijwe icyuma cya Silhouette Cameo 2 cyo muri Silhouette America, Inc. Filime ya elastique ikozwe mubikoresho bya PDMS hakoreshejwe inshinge.Ubwa mbere, ikarito ya 200 µm ya PET yaciwe ikoresheje sisitemu ya laser hanyuma ikomekwa ku rupapuro rwa mm 3 z'ubugari bwa PMMA ukoresheje 100 µm ebyiri zifata kaseti.PDMS ibanziriza (Sylgard 184; Igice A: Igice B = 10: 1, Dow Corning) noneho yasutswe mubibumbano hanyuma hakoreshwa inkoni yikirahure kugirango ikureho PDMS irenze.Nyuma yo gukira kuri 70 ° C. mumasaha 3, firime ya PDMS 300 mm irashobora gukurwa mubibumbano.
Amafoto yo gukwirakwiza ibintu byinshi, kubisaba gutangaza no gukora byizewe bifatwa hamwe na kamera yihuta (Sony AX700 1000 fps).Shitingi ya orbital yakoreshejwe mugupimisha kwizerwa yaguzwe muri SCILOGEX (SCI-O180).Umuvuduko wumwuka utangwa na compressor de air, kandi ibyuma byinshi bigenzura ibyuma bya digitale bikoreshwa muguhindura agaciro kumuvuduko.Uburyo bwo kugerageza imyitwarire yimyitwarire nuburyo bukurikira.Umubare wateganijwe wamazi yatewe mubikoresho byipimisha hanyuma kamera yihuta ikoreshwa kugirango yandike imyitwarire.Haracyariho amashusho yakuwe kuri videwo yimyitwarire yimyitwarire mugihe cyagenwe, hanyuma igice gisigaye kibarwa ukoresheje software-Pro Plus, hanyuma igwizwa nuburebure bwa kamera kugirango ibare amajwi.Ibisobanuro birambuye kuri sisitemu yo kugerageza imyitwarire irashobora kuboneka mugice cyinyongera S4.
Shyiramo 50 µl ya microbead na 100 µl y'amazi ya deionised mubikoresho bivanga vial.Amafoto avanze yimikorere yafashwe hamwe na kamera yihuta buri masegonda 0.1 kumuvuduko wa 0.1 bar, 0.15 bar na 0.2 bar.Amakuru ya Pixel mugihe cyo kuvanga urashobora kuboneka kuri aya mashusho ukoresheje software itunganya amafoto (Photoshop CS6).Kandi kuvanga imikorere birashobora kugerwaho hamwe na 53 ikurikira.
aho M ni kuvanga imikorere, N numubare rusange wicyitegererezo cya pigiseli, na ci na \ (\ bar {c} \) nibisanzwe kandi biteganijwe mubisanzwe.Kuvanga imikorere iri hagati ya 0 (0%, itavanze) kugeza 1 (100%, bivanze byuzuye).Ibisubizo byerekanwe mu gishushanyo cya S6.
Ibikoresho nyabyo RT-PCR kubikoresho bya IAV na IBV, harimo urugero rwa IAV na IBV RNA (injangwe no. RR-0051-02 / RR-0052-02, Liferiver, Ubushinwa), Tris-EDTA buffer (TE buffer no. B541019 , Sangon Biotech, Ubushinwa), Igikoresho cyiza cyo kugenzura RNA (Igice No Z-ME-0010, Liferiver, Ubushinwa) hamwe na GAPDH Solution (Igice No M591101, Sangon Biotech, Ubushinwa) iraboneka mubucuruzi.Ibikoresho byo kweza RNA birimo buffer ihuza, koza A, koza W, eluent, microbead ya magnetique, hamwe na transport ya acrylic.IAV na IBV mugihe nyacyo ibikoresho bya RT-PCR birimo IFVA nucleic aside PCR ivanga hamwe na enzyme ya RT-PCR.Ongeramo 6 µl ya AcrylCarrier na 20 µl yamasaro ya magneti kuri 500 µl yumuti uhuza ibisubizo, uzunguze neza hanyuma utegure igisubizo cyamasaro.Ongeramo ml 21 ya Ethanol kumesa A na W, kunyeganyeza neza kugirango ubone ibisubizo byogeswa A na W.Hanyuma, 18 µl ya fluorescent PCR ivanze na IFVA nucleic aside na 1 µl ya enzyme ya RT-PCR yongewe kuri 1 µl yumuti wa TE, iranyeganyezwa kandi yinjizwamo amasegonda menshi, ibona 20 µl ya primers ya IAV na IBV.
Kurikiza uburyo bukurikira bwo kweza RNA: (1) RNA adsorption.Pipette 526 µl yumuti wa pellet mumashanyarazi ya 1.5 ml centrifuge hanyuma wongereho 150 µl yicyitegererezo, hanyuma uzunguze intoki hejuru no hasi inshuro 10.Hindura 676 µl yuruvange kumurongo wa affinity na centrifuge kuri 1.88 x 104 g kumasegonda 60.Imiyoboro ikurikiraho irajugunywa.(2) Icyiciro cya mbere cyo gukaraba.Ongeramo 500 µl yo gukaraba igisubizo A kumurongo wa affinity, centrifuge kuri 1.88 x 104 g kuri 40 s, hanyuma ujugunye igisubizo cyakoreshejwe.Ubu buryo bwo gukaraba bwasubiwemo kabiri.(3) icyiciro cya kabiri cyo gukaraba.Ongeramo 500 µl yo gukaraba W W kumurongo wa affinity, centrifuge kuri 1.88 × 104 g kuri 15 s hanyuma ujugunye igisubizo cyakoreshejwe.Ubu buryo bwo gukaraba bwasubiwemo kabiri.(4) Gusiba.Ongeramo 200 µl ya eluate kumurongo wa affinity na centrifuge kuri 1.88 x 104 g kuri min 2..Igikorwa cyose cyo gutahura gifata iminota igera kuri 140 (iminota 20 yo kweza RNA niminota 120 yo kumenya PCR).
526 µl yumuti wamasaro, 1000 µl yumuti wo gukaraba A, 1000 µl yumuti wo gukaraba W, 200 µl ya eluate na 20 µl yumuti wa primer wongeyeho mbere ubikwa mubyumba M, W1, W2, E na PCR.Iteraniro.Hanyuma, 150 µl by'icyitegererezo cyinjijwe mu cyumba M hanyuma urubuga FAST-POCT rwinjizwa mu gikoresho cy'ibizamini cyerekanwe ku gishushanyo cya S9.Nyuma yiminota igera kuri 82, ibisubizo byikizamini byarabonetse.
Keretse niba byavuzwe ukundi, ibisubizo byose byikizamini bitangwa nkibisobanuro ± SD nyuma byibuze byibuze bitandatu byigana ukoresheje urubuga rwa FAST-POCT gusa hamwe nubushakashatsi bwigenga bwibinyabuzima.Nta makuru yakuwe mu isesengura.Ubushakashatsi ntabwo ari impanuka.Abashakashatsi ntibahumye amaso imirimo yitsinda mugihe cyubushakashatsi.
Kubindi bisobanuro kubijyanye nigishushanyo mbonera, reba Raporo yubushakashatsi bwibidukikije ifitanye isano niyi ngingo.
Amakuru ashyigikira ibisubizo byubu bushakashatsi araboneka mu makuru yinyongera.Iyi ngingo itanga amakuru yumwimerere.
Chagla, Z. & Madhukar, P. COVID-19 itera ibihugu bikize bizadindiza inkingo kuri bose.Chagla, Z. & Madhukar, P. COVID-19 itera ibihugu bikize bizadindiza inkingo kuri bose.Chagla, Z. na Madhukar, P. COVID-19 izamura mu bihugu bikize bizadindiza inkingo kuri buri wese.Chagla, Z. na Madhukar, P. COVID-19 gukingirwa mu bihugu bikize bizadindiza inkingo kuri buri wese.Ubuvuzi bw'igihugu.27, 1659–1665 (2021).
Faust, L. n'abandi.Ikizamini cya SARS-CoV-2 mu bihugu byinjiza amafaranga make kandi yo hagati: kuboneka no guhendwa mu bikorera ku giti cyabo.kwandura mikorobe.22, 511–514 (2020).
Umuryango mpuzamahanga wita ku buzima.Kwamamara kwisi yose hamwe no kwandura indwara zanduye zandurira mu mibonano mpuzabitsina: gusubiramo no kugereranya.Geneve: NINDE, NINDE / VIH_AIDS / 2 https://apps.wowe.int/iris/bitstream/handle/10665/66818/WHO_HIV_AIDS_2001.02.pdf (2001).
Fenton, EM n'abandi.Ibice byinshi 2D byashushanyije kuruhande rwibizamini.Porogaramu ASS.alma mater.Inter Milan.1, 124–129 (2009).
Schilling, KM n'abandi.Gufunga byuzuye microfluidic impapuro zishingiye kubisesengura.anus.Imiti.84, 1579–1585 (2012).
Lapenter, N. n'abandi.Impapuro zipiganwa zishingiye kuri immunochromatografiya hamwe na enzyme yahinduwe na electrode ituma habaho kugenzura bidasubirwaho no kumenya amashanyarazi ya cotinine.Sensors 21, 1659 (2021).
Zhu, X. n'abandi.Kugabanya indwara biomarkers hamwe na nanozyme ihuriweho na platifike ya flux ikoresheje glucometero.sensor biologiya.Bioelectronics.126, 690–696 (2019).
Boo, S. n'abandi.Ikizamini cyo gutwita kugirango hamenyekane bagiteri ziterwa na virusi ukoresheje concanavalin A-chorionic gonadotropin-Cu3 (PO4) 2 ya nanoflowers ya Hybrid, gutandukanya magneti no gusoma terefone ifite ubwenge.Microcomputer.Ikinyamakuru.185, 464 (2018).