Mugihe cyambere cyicyorezo, kubera iterambere ryihuse, gusuzuma byihuse abarwayi bakekwa nurufunguzo rwo kwirinda COVID-19.Bimwe mu byemezo bya nucleic aside byerekana reagent bifite igihe gito cyiterambere, kandi hariho ibibazo nko kwemeza imikorere byihuse, optimizasiyo idahagije, kandi itandukaniro rinini hagati yitsinda;Ibibazo bya laboratoire zitandukanye zamavuriro mubice bitandukanye byuburyo bwo gutahura aside nucleique birashobora no kugira ingaruka kubisubizo bya aside nucleique.Iyi ngingo izibanda ku masano y'ingenzi n'ingingo ziri muri SARS-CoV-2 ya nucleic acide iriho ubu, kandi isesengure ibibazo byo kubeshya no kongera gusuzuma isuzuma rya laboratoire nucleic aside itahura no kudahuza kwa muganga.

Amahame ya SARS-CoV-2 gutahura aside nucleic

SARS-CoV-2 ni virusi ya RNA ifite genome ikurikirana igera kuri 29 kb, hamwe na gen 10, zishobora kubika poroteyine 10.Virusi zigizwe na RNA na proteyine, naho igice cyo hanze ni igifuniko cyo hanze kigizwe na lipide na glycoproteine.Imbere, protein capsid ipfunyika RNA muri yo, bityo irinda RNA kwangirika byoroshye (P1).

P1 Imiterere ya SARS-COV-2

Virusi yibasira ingirabuzimafatizo zinyuze mu ngirabuzimafatizo zihariye zitera kwandura, kandi zigakoresha selile zo kwigana.

Ihame ryo kumenya aside nucleic virusi ni ukugaragaza virusi RNA ikoresheje lysate selile, hanyuma ugakoresha igihe nyacyo fluorescent revers transcription-polymerase reaction reaction (RT-PCR) kugirango tumenye.

Urufunguzo rw'ihame ryo gutahura ni ugukoresha primers na probe kugirango ugere kuri "guhuza intego" ya nucleic acide ikurikirana, ni ukuvuga gushakisha aside nucleique ikurikirana ya SARS-CoV-2 itandukanye nizindi virusi ziri mubirindiro bigera ku 30.000 (guhuza aside nucleique nizindi virusi) "Agace gato"), gushushanya primers na probe.

Primers na probe bihujwe cyane nakarere kihariye ka SARS-CoV-2 acide nucleic, ni ukuvuga umwihariko urakomeye cyane.Iyo igihe nyacyo fluorescent RT-PCR yongerewe ibisubizo byikitegererezo igomba gupimwa nibyiza, irerekana ko SARS-CoV-2 ihari murugero.Reba P2.

P2 Intambwe za SARS-CoV-2 nucleic aside igena (igihe nyacyo fluorescent RT-PCR)

Ibisabwa na laboratoire ya SARS-CoV-2 nucleic aside

Laboratoire yo gupima aside nucleique ninziza cyane kubidukikije bitameze neza, kandi bagomba kwitondera kugenzura umuvuduko, gukomeza umwuka, no gukuraho aerosole.Abakozi bapima aside nucleique bagomba kuba bafite impamyabumenyi ijyanye, bakakira amahugurwa ya polymerase yingirakamaro kandi bagatsinda isuzuma.Laboratoire igomba gucungwa neza, igashyirwa ahantu, kandi abakozi badafite aho babogamiye kwinjira.Ahantu hasukuye hagomba guhumeka no kwanduzwa ahantu.Ibintu bifitanye isano bishyirwa muri zone, bisukuye kandi byanduye biratandukana, bigasimburwa mugihe, kandi byanduye ahantu.Kwanduza inzira: Kwangiza Chlorine irimo disinfectant nigisubizo nyamukuru kubice binini, kandi 75% alcool irashobora gukoreshwa ahantu hato.Inzira nziza yo guhangana na aerosole nugukingura Windows kugirango uhumeke, kandi kwanduza ikirere nabyo birashobora gukorwa hakoreshejwe imirasire ya ultraviolet, kuyungurura, no kwanduza ikirere.

Ihuza ryingenzi nibipimo bya SARS-CoV-2 igena aside nucleic (igihe nyacyo fluorescent RT-PCR)

Nubwo muri rusange laboratoire zita cyane kuri acide nucleic “detection”, mubyukuri, aside nucleic “gukuramo” nayo ni imwe mu ntambwe zingenzi zo gutahura neza, bifitanye isano rya hafi no gukusanya no kubika ingero za virusi.

Kugeza ubu, ingero z’ubuhumekero zikoreshwa cyane, nka nasofaryngeal swabs, zikoresha uburyo bwa kabiri, aribwo buryo bwo kudakora (kubungabunga) bwateguwe bushingiye ku gukuramo aside nucleic hamwe nigisubizo cya lysis.Ku ruhande rumwe, iki gisubizo cyo kubungabunga virusi kirashobora kwamagana poroteyine ya virusi, gutakaza ibikorwa byayo kandi ntigishobora kwandura, kandi bikazamura umutekano w’icyiciro cyo gutwara no gutahura;kurundi ruhande, irashobora gutobora virusi kugirango irekure aside nucleic, ikureho aside nucleic aside yangirika, kandi ikingire virusi.RNA yangiritse.

Igisubizo cya virusi cyateguwe hashingiwe ku gukuramo aside nucleic aside.Ibice byingenzi ni umunyu uringaniye, Ethylenediaminetetraacetic acide chelating agent, umunyu wa guanidine (guanidine isothiocyanate, hydrochloride ya guanidine, nibindi), anionic surfactant (dodecane) Sodium sulfate), cactic surfactant (tetradecyl trimethyl ammonium oxalate, 8) byinshi cyangwa byinshi.Kugeza ubu, hari ubwoko bwinshi bwibikoresho byo gukuramo aside nucleic, kandi hakoreshwa aside nucleic aside itandukanye hamwe nogusukura.Nubwo hakoreshejwe aside nucleic imwe yo gukuramo no kweza reagent ikoreshwa, uburyo bwo kuvoma buri bikoresho buratandukanye.

Kugeza ubu, ibikoresho bya nucleic aside bitahura ibikoresho byemejwe n’ikigo cy’igihugu gishinzwe ubuvuzi byatoranijwe hashingiwe ku bwoko bwa ORF1ab, E na N muri gen SARS-CoV-2.Amahame yo gutahura ibicuruzwa bitandukanye mubyukuri arasa, ariko primers zabo hamwe nubushakashatsi bwa probe buratandukanye.Hariho ibice bimwe-bigenewe (ORF1ab), ibice bibiri-intego (ORF1ab, N cyangwa E), hamwe nibice bitatu (ORF1ab, N na E).Itandukaniro riri hagati yo gutahura no gusobanura, gukuramo aside nucleic na sisitemu nyayo ya fluorescent ya RT-PCR igomba kwifashisha amabwiriza agenga ibikoresho, kandi birasabwa ko abayikoresha bakurikiza byimazeyo uburyo bwo gusobanura bwerekanwe mumabwiriza ya kit kugirango basobanurwe.Uturere dusanzwe, primers hamwe na probe zikurikirana zongerewe nigihe-fluorescent RT-PCR irerekanwa muri P3.

P3 Ahantu intego ya SARS-CoV-2 amplicon kuri genome hamwe nurutonde rwa primers na probe

Gusobanura ibisubizo bya SARS-CoV-2 kugena aside nucleic (Real-Time fluorescent RT-PCR)

“Gahunda yo gukumira no kurwanya indwara y'umusonga yo kwandura SARS-CoV-2 (Edition ya kabiri)” ku nshuro ya mbere yasobanuye ibipimo ngenderwaho mu gusuzuma ibyavuye mu kongera gene imwe:

1. Nta Ct cyangwa Ct≥40 ni bibi;

2. Ct <37 ni nziza;

3. Ct agaciro ka 37-40 ni agace keza.Birasabwa gusubiramo igerageza.Niba ibisubizo byo kugabanya Ct <40 hamwe na amplification curve ifite impinga zigaragara, icyitegererezo gifatwa nkicyiza, naho ubundi kikaba ari kibi. ”

Igitabo cya gatatu cyuyobora hamwe ninshuro ya kane yubuyobozi byakomeje ibipimo byavuzwe haruguru.Ariko, kubera intego zitandukanye zikoreshwa mubikoresho byubucuruzi, igitabo cya 3 cyavuzwe haruguru nticyatanze ibipimo ngenderwaho kugirango hamenyekane intego, ushimangira Amabwiriza yatanzwe nuwabikoze azatsinda.Guhera ku ncuro ya gatanu y'amabwiriza, intego ebyiri zarasobanuwe, cyane cyane ibipimo byo guca urubanza ku ntego imwe igoye guca urubanza.Ni ukuvuga, niba laboratoire ishaka kwemeza ko urubanza ari rwiza kuri SARS-CoV-2 nucleic aside, hagomba gukurikira ibi bikurikira 1 kuri 2:

(1) Intego ebyiri za SARS-CoV-2 (ORF1ab, N) murugero rumwe zageragejwe neza na fluorescent RT-PCR.Niba intego imwe ari nziza, kongera gutoranya no kongera kugerageza.Niba ibisubizo byikizamini Niba intego imwe ikiri nziza, ifatwa nkibyiza.

.Nyamara, umurongo ngenderwaho ushimangira kandi ko ingaruka mbi zo gupima aside nucleique idashobora gukuraho ubwandu bwa SARS-CoV-2.Ibintu bishobora gutera ibibi bigomba gukurwaho, harimo ubuziranenge bwicyitegererezo (ingero zubuhumekero ziva muri oropharynx nibindi bice), gukusanya icyitegererezo hakiri kare cyangwa bitinze, Ingero ntizabitswe, zitwarwa, kandi zitunganywa neza, kandi ikoranabuhanga ubwaryo ryagize ibibazo (itandukaniro rya virusi, kubuza PCR), nibindi.

Impamvu zitera ibinyoma mubushakashatsi bwa SARS-CoV-2

Igitekerezo cya "ibinyoma bibi" mugupima aside nucleic kuri ubu bireba, akenshi bivuga "ibibi bibi" aho ibisubizo bya testi ya nucleic aside bidahuye nibigaragara kwa clinique, ni ukuvuga ibimenyetso byamavuriro nibisubizo byerekana amashusho bikekwa cyane kuri COVID-19, ariko ibizamini bya aside nucleique bihora "bibi" inshuro nyinshi.Ikigo cya Laboratwari ya Laboratwari ya Komisiyo y’ubuzima y’igihugu cyasobanuye ikizamini cya “ibinyoma bibi” SARS-CoV-2.

(1) Hariho virusi runaka mu ngirabuzimafatizo z'umuntu wanduye.Amakuru ariho yerekana ko umubiri umaze kwandura virusi, virusi yinjira mu muhogo binyuze mu mazuru no mu kanwa, hanyuma ikagera kuri trachea na bronchi, hanyuma ikagera kuri alveoli.Umuntu wanduye azahura nigihe cyo gukuramo, ibimenyetso byoroheje, hanyuma inzira yibimenyetso bikomeye, nibice bitandukanye byindwara.Kandi ingano ya virusi igaragara mu bice bitandukanye byumubiri iratandukanye.

Kubireba umutwaro wa virusi yubwoko bwakagari, selile ya alveolar epithelial selile (inzira yubuhumekero yo hepfo)> selile epithelial selile (inzira yubuhumekero yo hejuru)> fibroblast, selile endothelia, na macrophage, nibindi.;uhereye ku bwoko bw'icyitegererezo, alveolar lavage fluid (nziza cyane)> inkorora yimbitse> nasopharyngeal swab> oropharyngeal swab> maraso.Byongeye kandi, virusi irashobora no kugaragara mumyanda.Nyamara, urebye uburyo bworoshye bwo kubaga no kwakira abarwayi, gahunda isanzwe ikoreshwa mubuvuzi ni oropharyngeal swab> nasopharyngeal swab> bronchial lavage fluid (imikorere igoye) hamwe na spumumu yimbitse (mubisanzwe inkorora yumye, kuyibona biragoye).

Kubwibyo, ingano ya virusi mu ngirabuzimafatizo ya oropharynx cyangwa nasofarynx ya bamwe mu barwayi ni nto cyangwa ni nke cyane.Niba gusa ingero za oropharynx cyangwa nasofarynx zifashwe kugirango zipimishe, aside nucleic virusi ntishobora kuboneka.

(2) Nta selile zirimo virusi zegeranijwe mugihe cyo gukusanya icyitegererezo, cyangwa aside nucleic aside ntabwo yabitswe neza.

[Site Ikibanza cyo gukusanya kidakwiye, kurugero, mugihe cyo gukusanya ibibyimba bya oropharyngeal, ubujyakuzimu ntibuhagije, ibyegeranyo bya nasofaryngeal byegeranijwe ntibikusanyirizwa mu mwobo wizuru, nibindi. Byinshi mu ngirabuzimafatizo byakusanyirijwe hamwe bishobora kuba selile idafite virusi;

②Icyitegererezo cya swabs gikoreshwa nabi.Kurugero, fibre synthique nka PE fibre, fibre polyester na polypropilene fibre birasabwa kubintu byumutwe wa swab.Fibre naturel nka pamba ikoreshwa mubikorwa nyirizina (adsorption ikomeye ya poroteyine kandi ntibyoroshye koza) Na fibre ya nylon (gufata amazi nabi, biganisha ku bipimo by'icyitegererezo bidahagije);

Gukoresha nabi imiyoboro yo kubika virusi, nko gukoresha nabi polipropilene cyangwa polyethylene yububiko bwa plastike byoroshye gufata aside nucleic (ADN / RNA), bigatuma igabanuka rya acide nucleique mu gisubizo kibikwa.Mu myitozo, birasabwa gukoresha polyethylene-propylene polymer plastike hamwe nibikoresho bimwe na bimwe byakozwe na polipropilene ya plastike kugirango ubike aside nucleic aside.]

[Site Ikibanza cyo gukusanya kidakwiye, kurugero, mugihe cyo gukusanya ibibyimba bya oropharyngeal, ubujyakuzimu ntibuhagije, ibyegeranyo bya nasofaryngeal byegeranijwe ntibikusanyirizwa mu mwobo wizuru, nibindi. Byinshi mu ngirabuzimafatizo byakusanyirijwe hamwe bishobora kuba selile idafite virusi;

②Icyitegererezo cya swabs gikoreshwa nabi.Kurugero, fibre synthique nka PE fibre, fibre polyester na polypropilene fibre birasabwa kubintu byumutwe wa swab.Fibre naturel nka pamba ikoreshwa mubikorwa nyirizina (adsorption ikomeye ya poroteyine kandi ntibyoroshye koza) Na fibre ya nylon (gufata amazi nabi, biganisha ku bipimo by'icyitegererezo bidahagije);

Gukoresha nabi imiyoboro yo kubika virusi, nko gukoresha nabi polipropilene cyangwa polyethylene yububiko bwa plastike byoroshye gufata aside nucleic (ADN / RNA), bigatuma igabanuka rya acide nucleique mu gisubizo kibikwa.Mu myitozo, birasabwa gukoresha polyethylene-propylene polymer plastike hamwe nibikoresho bimwe na bimwe byakozwe na polipropilene ya plastike kugirango ubike aside nucleic aside.]

(4) Imikorere ya laboratoire ntabwo isanzwe.Icyitegererezo cyubwikorezi nububiko, imikorere isanzwe ya laboratoire yubuvuzi, gusobanura ibisubizo no kugenzura ubuziranenge nibintu byingenzi byerekana neza ibisubizo by ibizamini.Dukurikije ibyavuye mu isuzuma ry’ubuziranenge bwo hanze ryakozwe n’ikigo cya Laboratwari cya Laboratwari ya Komisiyo y’igihugu y’ubuzima ku ya 16-24 Werurwe 2020, muri laboratoire 844 zabonye ibisubizo bifatika, 701 (83.1%) zujuje ibisabwa, naho 143 (16.9%) ntabwo zari zujuje ibisabwa.Yujuje ibyangombwa, muri rusange ibizamini bya laboratoire nibyiza, ariko laboratoire zitandukanye ziracyafite itandukaniro mubushobozi bwabakozi, intego imwe nziza yo gusobanura neza, no kugenzura ubuziranenge.

Nigute ushobora kugabanya ibibi bya SARS-CoV-2 nucleic aside?

Kugabanya ibibi muburyo bwo kumenya aside nucleic bigomba gutezimbere uhereye kumpande enye zo kubyara ibibi.

(1) Hariho virusi runaka mu ngirabuzimafatizo z'umuntu wanduye.Ubwinshi bwa virusi mubice bitandukanye byumubiri wabakekwaho kwandura bizaba bitandukanye mubihe bitandukanye.Niba nta farynx ihari, irashobora kuba mumazi ya bronchial lavage cyangwa umwanda.Niba ubwoko bwinshi bwintangarugero bushobora gukusanyirizwa icyarimwe cyangwa mubyiciro bitandukanye byiterambere ryindwara kugirango bipimishe, Bizafasha kwirinda ibibi bibi.

(2) Ingirabuzimafatizo zirimo virusi zigomba gukusanywa mugihe cyo gukusanya icyitegererezo.Iki kibazo kirashobora gukemurwa murwego rwo gushimangira amahugurwa yabatoranya icyitegererezo.

(3) Reagent ya IVD yizewe.Mugukora ubushakashatsi kubijyanye no gusuzuma imikorere ya reagent kurwego rwigihugu, no kuganira kubibazo bihari, imikorere yo gutahura reagent irashobora kurushaho kunozwa kandi ibyiyumvo byisesengura birashobora kunozwa.

(4) Imikorere isanzwe ya laboratoire.Mu gushimangira amahugurwa y’abakozi ba laboratoire, gukomeza kunoza imikorere y’imicungire ya laboratoire, kugenzura amacakubiri ashyize mu gaciro, no kuzamura ubushobozi bw’abakozi bwo gutahura, birashoboka kugabanya ibibi biturutse ku bikorwa bya laboratoire bidakwiye.

Impamvu zo kongera kwipimisha icyiza cya SARS-CoV-2 nucleic aside yipimishije kubarwayi bakize kandi basezerewe

“COVID-19 yo gusuzuma no kuvura (Ikigeragezo cya karindwi)” isobanura neza ko kimwe mu bipimo by’abarwayi ba COVID-19 kugira ngo bakire kandi basohoke mu bitaro ari uko ingero ebyiri zikurikirana z'ubuhumekero zifite ibizamini bya aside nucleic (byibuze amasaha 24 atandukanye), ariko hari bike cyane abarwayi ba SARS-CoV-2 basuzumishijwe na virusi ya nucleique.

(1) SARS-CoV-2 ni virusi nshya.Birakenewe kurushaho gusobanukirwa nuburyo bwayo butera indwara, ishusho yuzuye yindwara yatewe nibiranga inzira yindwara.Kubwibyo, kuruhande rumwe, birakenewe gushimangira imiyoborere y’abarwayi basezerewe no gukora ubushakashatsi bw’iminsi 14.Kora gukurikirana, gukurikirana ubuzima no kuyobora ubuzima kugirango urusheho gusobanukirwa inzira zose zibaho, iterambere nibisubizo byindwara.

(2) Umurwayi ashobora kongera kwandura virusi.Umunyeshuri Zhong Nanshan yagize ati: Kubera ko abarwayi bakize bafite antibodi, SARS-CoV-2 irashobora gukurwaho na antibodi iyo bongeye gutera.Hariho impamvu nyinshi, zishobora kuba intandaro yumurwayi wakize, cyangwa birashobora kuba bifitanye isano na mutation ya virusi, cyangwa nimpamvu yo kwipimisha laboratoire.Niba ari virusi ubwayo, ihinduka rya SARS-CoV-2 rishobora gutuma antibody yakozwe n'umurwayi wakize idakora neza kuri virusi yahinduwe.Niba umurwayi yongeye kwandura virusi yahinduwe, kwipimisha aside nucleique birashobora kongera kuba byiza.

(3) Kubijyanye nuburyo bwo gupima laboratoire, buri buryo bwo gupima bufite aho bugarukira.Kumenya SARS-CoV-2 nucleic aside iterwa no guhitamo gene ikurikirana, ibigize reagent, sensitivite yuburyo nizindi mpamvu, biganisha kubikoresho bihari bifite aho bigarukira.Umurwayi amaze kuvurwa, virusi mu mubiri iragabanuka.Mugihe umutwaro wa virusi murugero ugomba gupimwa uri munsi yumupaka wo hasi wo gutahura, ibisubizo "bibi" bizagaragara.Ariko, ibisubizo ntabwo bivuze ko virusi mumubiri yazimye burundu.Virusi irashobora kuba nyuma yo kuvurwa.Kuzuka ”, komeza kwigana.Kubwibyo, birasabwa gusubiramo rimwe mu cyumweru mugihe cyibyumweru 2 kugeza kuri 4 nyuma yo gusohoka.

(4) Acide Nucleic ni ibikoresho bikomoka kuri virusi.Virusi yicwa nyuma yuko umurwayi amaze kwivuza virusi, ariko ibice bya virusi bya RNA bisigaye bikigumana mu mubiri w'umuntu, kandi ntibisohoka mu mubiri.Rimwe na rimwe, mubihe bimwe, birashobora kugumaho.Igihe kinini, kandi muriki gihe ikizamini cya acide nucleic kizaba "inzibacyuho" nziza.Hamwe no kwagura igihe cyo gukira k'umurwayi, nyuma y'ibice bya RNA bisigaye mu mubiri birangiye buhoro buhoro, ibisubizo bya aside nucleic aside bishobora guhinduka bibi.

.Birakenewe kwerekana niba umurwayi wongeye kwipimisha aside nucleic azongera kuba isoko yandura.Birakenewe gukora umuco wa virusi kurugero rwamavuriro no guhinga virusi "nzima" kugirango yerekane ko yanduye.

Incamake

Muri make, SARS-CoV-2 nucleic aside yipimisha ibibi, ibyiza byo kugerageza, nibindi bintu bidahuye nibigaragara kwa clinique ntibishobora kwirindwa rwose.Mu gusuzuma no kwipimisha nyirizina, birasabwa guhuza ibimenyetso byamavuriro, ibizamini byerekana amashusho (CT) hamwe nubushakashatsi Ikizamini cya Laboratoire (testi nucleic aside test + virusi yihariye ya antibody) ibisubizo byo kwisuzumisha byuzuye kugirango wirinde kwisuzumisha no kwisuzumisha nabi.Niba ibisubizo by'ibizamini bigaragara ko bidahuye n'ibigaragara ku mavuriro, birasabwa gukora isesengura ryuzuye ryerekana ibizamini byose (gukusanya icyitegererezo, kuzenguruka no gutunganya) kugirango wirinde virusi ya SARS-CoV-2 kwandura hakiri kare, kwandura kenshi cyangwa guhuzwa n'izindi virusi zandurira mu myanya y'ubuhumekero, n'ibindi birashoboka.Niba ibintu byemewe, birasabwa gukusanya ibyitegererezo byoroshye nka sputum cyangwa alveolar lavage fluid kugirango wongere usuzume.

Ibicuruzwa bifitanye isano ：

SARS-CoV-2 Nucleic Acide Detection Kit (Multiplex PCR Fluorescent Probe Method)