Ibicuruzwa
Lifecosm SARS-Cov-2-RT-PCR Kumenyekanisha ibikoresho bya 2019-nCoV
Biteganijwe gukoreshwa
Iki gikoresho gikoreshwa mugushakisha ubuziranenge bwa coronavirus nshya (2019-nCoV) ukoresheje umuhogo wo mu muhogo, nasopharyngeal swabs, bronchoalveolar lavage fluid, sputum.Ibisubizo byerekana iki gicuruzwa ni ibyerekeranye nubuvuzi gusa, kandi ntibigomba gukoreshwa nkibyonyine. ibimenyetso byo kwisuzumisha no kuvura.Isesengura ryuzuye ryimiterere irasabwa hamwe no kugaragara kwa muganga kwa muganga nibindi bizamini bya laboratoire.
Ihame ry'ubugenzuzi
Igikoresho gishingiye ku ntambwe imwe RT- PCR.Mubyukuri, coronavirus nshya ya 2019 (2019-nCoV) ORF1ab na N byatoranijwe nkuturere twibasiwe na amplification.Primers yihariye na fluorescente (N gene probe yanditseho FAM na ORF1ab probe yanditseho HEX) yagenewe kumenya 2019 ubwoko bushya bwa coronavirus RNA mubitegererezo.Igikoresho kirimo kandi sisitemu yo kugenzura imbere ya endogenous imbere (kugenzura imbere imbere ya gen probe yanditseho CY5) kugirango ikurikirane inzira yo gukusanya icyitegererezo, amplification ya RNA na PCR, bityo bigabanye ibisubizo bibi.
Ibice nyamukuru
| Ibigize | Umubumbe（48T / Kit） |
| Igisubizo cya RT-PCR | 96µl |
| nCOV primer TaqMan probemixture （ORF1ab, N Gene, RnaseP Gene） | 864µl |
| Kugenzura nabi | 1500µl |
| nCOV Impaka nziza OR l ORF1ab N Gene） | 1500µl |
Reagent nyirizina: gukuramo RNA cyangwa kweza.Igenzura ribi / ryiza: Igenzura ryiza ni RNA ikubiyemo igice cyagenewe, mugihe igenzura ribi ari amazi adafite aside nucleique.Mugihe cyo gukoresha, bagomba kugira uruhare mugukuramo kandi bagomba gufatwa nkuwanduye.Bagomba gukemurwa no kujugunywa hakurikijwe amabwiriza abigenga.
Imbere yimbere ni gene ya RnaseP.
Imiterere yo kubika nitariki izarangiriraho
-20 ± 5 ℃, irinde gukonjesha no gukonjesha inshuro zirenze 5, byemewe amezi 6.
Igikoresho gikoreshwa
Hamwe na FAM / HEX / CY5 nibindi bikoresho byinshi bya fluorescent PCR.
Kugaragaza ibisabwa
1.Ubwoko bwikigereranyo bwakoreshwa ： umuhogo wo mu muhogo, nasofaryngeal swabs, bronchoalveolar lavage fluid, sputum。
2.Icyegeranyo cyihariye (tekinike ya aseptic）
Pharyngeal swab: Ihanagura toni hamwe nurukuta rwinyuma rwa faryngeal hamwe na swabs ebyiri icyarimwe, hanyuma winjize umutwe wa swab mumiyoboro yikizamini irimo igisubizo cyicyitegererezo.
Sputum: Nyuma yuko umurwayi amaze gukorora cyane, kusanya spucum ikorora mumashanyarazi ya test ya screw irimo igisubizo cyicyitegererezo;bronchoalveolar lavage fluid ： Gutoranya ninzobere mubuvuzi.3.Kubika no gutwara ingero
Ibigereranyo byo gutandukanya virusi no gupima RNA bigomba gupimwa vuba bishoboka.Ibigereranyo bishobora kumenyekana mugihe cyamasaha 24 birashobora kubikwa kuri 4 ℃;ibidashobora kumenyekana muri 24
amasaha agomba kubikwa kuri -70 ℃ cyangwa munsi (niba nta bubiko bwa -70 ℃, bigomba kuba
bibitswe by'agateganyo kuri -20 ℃ firigo).Ingero zigomba kwirinda gukonjesha no gukonja mugihe cyo gutwara.Ibigereranyo bigomba koherezwa muri laboratoire vuba bishoboka nyuma yo gukusanya.Niba ingero zigomba gutwarwa kure, birasabwa kubika urubura rwumye.
Uburyo bwo Kwipimisha
1 Icyitegererezo cyo gutunganya no gukuramo RNA (icyitegererezo cyo gutunganya)
Birasabwa gufata 200μl yicyitegererezo cyamazi yo gukuramo RNA.Kubijyanye no gukuramo intambwe, reba amabwiriza yubucuruzi RNA ikuramo ibikoresho.Byombi nibibi nibibi
kugenzura muri iki gikoresho byagize uruhare mu gukuramo.
2 PCR itegura reagent (agace ko gutegura reagent)
2.1 Kuraho ibice byose mubikoresho hanyuma ushushe hanyuma uvange ubushyuhe bwicyumba.Centrifuge kuri 8000 rpm kumasegonda make mbere yo gukoresha;kubara umubare ukenewe wa reagent, kandi sisitemu yo kubyitwaramo yateguwe nkuko bigaragara mumeza akurikira:
| Ibigize | N gukorera (sisitemu 25µl) |
| nCOV primer TaqMan probemixture | 18 µl × N. |
| Igisubizo cya RT-PCR | 2 µl × N. |
| * N = umubare w'icyitegererezo cyageragejwe + 1 (kugenzura nabi) + 1 (nCOVkugenzura neza) | |
2.2 Nyuma yo kuvanga neza ibice, centrifuge mugihe gito kugirango ureke amazi yose kurukuta rwigituba agwe munsi yigituba, hanyuma agabanye sisitemu ya 20 µl amplification muri tube ya PCR.
3 Icyitegererezo (ahantu ho gutegura)
Ongeramo 5μl yubugenzuzi bubi nibyiza nyuma yo gukuramo.RNA yicyitegererezo igomba gupimwa yongewe kumuyoboro wa PCR.
Fata umuyoboro neza na centrifuge kuri 8000 rpm kumasegonda make mbere yo kuyimurira ahabigenewe.
4 Kwiyongera kwa PCR (ahantu hagaragara)
4.1 Shira umuyoboro wa reaction muri selile yicyitegererezo, hanyuma ushireho ibipimo bikurikira:
| icyiciro | Ukuzenguruka nimero | Ubushyuhe(° C) | Igihe | icyegeranyourubuga |
| Subiza inyumatranscript | 1 | 42 | 10min | - |
| Mbere yo gutandukanan | 1 | 95 | 1min | - |
| Ukuzenguruka | 45 | 95 | 15s | - |
| 60 | 30s | ikusanyamakuru |
Guhitamo ibikoresho byo gutoranya ibikoresho: Hitamo umuyoboro wa FAM 、 HEX 、 CY5 kubimenyetso bya fluorescence.Kubisobanuro bya fluorescent NTAWE, nyamuneka ntuhitemo ROX.
5 Isesengura ryibisubizo (Nyamuneka reba amabwiriza yubushakashatsi bwa buri gikoresho cyo gushiraho)
Nyuma yo kubyitwaramo, bika ibisubizo.Nyuma yisesengura, hindura agaciro ko gutangira, agaciro karangira, nigiciro cyibanze cyibanze ukurikije ishusho (uyikoresha arashobora guhindura ukurikije uko ibintu bimeze, agaciro ko gutangira gashobora gushirwa kuri 3 ~ 15, agaciro kanyuma gashobora gushyirwaho 5 ~ 20.
6 Igenzura rya Quauty control Igenzura ryikurikiranabikorwa rishyirwa mu kizamini control Igenzura ribi: Nta murongo ugaragara wa amplification umurongo wa FAM, HEX, CY5
COV igenzura neza: umurongo ugaragara wa amplification umurongo wa FAM na HEX yo gutahura, Ct agaciro ≤32, ariko ntagahunda yo gukwirakwiza umurongo wa CY5;
Ibisabwa haruguru bigomba kuba byujujwe icyarimwe mubigeragezo bimwe;bitabaye ibyo, ikigeragezo nticyemewe kandi gikeneye gusubirwamo.
7 Kumenya ibisubizo.
7.1 Niba nta amplification curve cyangwa Ct agaciro> 40 mumiyoboro ya FAM na HEX yicyitegererezo cyibizamini, kandi hariho umurongo wa amplification kumurongo wa CY5, hashobora kugaragara ko nta coronavirus nshya ya 2019 (2019-nCoV) RNA mu cyitegererezo;
.2 Niba icyitegererezo cyibizamini gifite umurongo ugaragara wa amplification mu miyoboro ya FAM na HEX, kandi agaciro ka Ct ni ≤40, dushobora kwemeza ko icyitegererezo ari cyiza kuri coronavirus nshya 2019 (2019-nCoV).
7.3.Niba ibisubizo byisubiramo bihuye, icyitegererezo gishobora kugaragara ko ari cyiza kubishya
coronavirus 2019 (2019-nCoV).Niba ibisubizo byisubiramo ari bibi, dushobora kwemeza ko icyitegererezo ari kibi kuri coronavirus nshya ya 2019 (2019-nCoV).
Agaciro keza k'urubanza
Uburyo bwa ROC bwo gutondeka bukoreshwa kugirango hamenyekane agaciro ka CT agaciro k'igikoresho naho igenzura ry'imbere ni 40.
Gusobanura ibisubizo by'ibizamini
1.Buri bushakashatsi bugomba kugeragezwa kubigenzura bibi kandi byiza.Ibisubizo by'ibizamini birashobora kugenwa gusa mugihe igenzura ryujuje ibisabwa kugenzura ubuziranenge
2.Iyo imiyoboro yo kumenya FAM na HEX ari nziza, ibisubizo biva kumuyoboro CY5 (umuyoboro wimbere) bishobora kuba bibi kubera guhatanira sisitemu.
3.Iyo ibisubizo byimbere byimbere ari bibi, niba imiyoboro ya test ya FAM na HEX yo kugenzura nayo itari mibi, bivuze ko sisitemu yahagaritswe cyangwa imikorere itariyo, t ikizamini nticyemewe.Kubwibyo, ibyitegererezo bigomba gusubirwamo.
