Ibicuruzwa birambuye
Ishami ry'Umuryango w'Abibumbye ryita ku Buzima (OMS) riyobora ko ibizamini byo gusuzuma byihuse (Ag-RDRs) bishobora gutanga uburyo bwihuse kandi buhenze bwo gusuzuma indwara zanduye SARS-CoV-2 kuruta ibizamini byo kongera aside nucleique (NAATs), kandi OMS nayo ikabisaba. ko Ag-RDTs zujuje ibyangombwa byibura zishobora gukoreshwa mugutahura ibibazo byambere, gushakisha amakuru, mugihe cyiperereza ryatangiye no gukurikirana imigendekere y’indwara mu baturage.
Ibiranga
Byuzuye:Intego eshatu zerekana gene mugupima kamwe
Guhuza:Guhuza nibikoresho bisanzwe hamwe na CY5, FAM, VIC / HEX.
Performance Imikorere myiza:Ibyiyumvo bihanitse kandi byihariye, LOD = 200 kopi / ml.
Ikigereranyo cya tekiniki
| Ibipimo byo gupakira | Ibizamini 50 / ibikoresho, ibizamini 100 / kit |
| Intego y'akarere | ORF1ab, N, E. |
| Icyitegererezo | Sputum, Oropharyngeal Swab |
| Imipaka ntarengwa | Kopi 200 / ml |
| Igipimo Cyuzuye Cyuzuye | 99.55% |
| Ct agaciro (CV,%) | ≤5.0% |
| Igipimo cyiza cyo guhura | 99,12% |
| Igipimo Cyimpanuka | 100% |
| Imiterere yo kubika nitariki izarangiriraho | Yabitswe kuri -20 ± 5 ℃, kandi ifite agaciro by'agateganyo amezi 12. |
| Igenzura ryimbere | Yego |
| Umubare wa Cataloge | A7793YF-50T, A7793YF-100T |
| Icyemezo | CE |
| Ingero | Nasopharyngeal swab, Oropharyngeal swab, Amazi ya Alveolar, Amacandwe na sputum |
| Igikoresho gikoreshwa | ABI 7500, Roche Light Cycler 480Ⅱ, Roche Cobas z 480, SLAN-96P Sisitemu nyayo-PCR |
Uburyo bwo Kwipimisha
1. Gukuramo Acide Nucleic
Igikorwa kigomba gukorwa ukurikije imfashanyigisho yo gukuramo ibikoresho.
2. Gutegura Sisitemu:
1) Kuramo reagent hanyuma ushishimure burundu.Hindura imvange na centrifuge ako kanya.N ibisubizo byikizamini (N = umubare wintangarugero ugomba kugeragezwa + kugenzura neza + kugenzura nabi + 1) byateguwe kuri sisitemu yogukora, nkuko bikurikira.
| Ibigize | Umubumbe wa sisitemu 1 reaction | Umubare wa N reaction ya sisitemu |
| Nucleic aside amplification reaction reaction ivanze (A7793YF) | 18 µL | 18 µL * N. |
| Enzyme ivanze | 2 µL | 2 µL * N. |
| Ingano yose | 20 µL | 20 µL * N. |
2) Gukwirakwiza reaction: Igisubizo cya reaction cyaravanze kandi gishyizwe hamwe, kandi buri muyoboro watanzwe muburyo bwa 20μL mumiyoboro ya PCR ikwiranye nibikoresho bya fluorescence PCR.
3. Kuremera
5μL ya acide nucleic sample yakuweho, acide nucleic aside igenzura neza hamwe na acide nucleic aside igenzura yongewe kuri sisitemu yo kubyitwaramo, hamwe nubunini bwa reaction ni 25μL.Komeza igifuniko cya tube hanyuma uyimure ahakorerwa ibizamini nyuma yamasegonda make ya centrifugation.
4. Isuzuma rya Amplification Assay
1) Shyira umuyoboro wa PCR mubikoresho bya fluorescent PCR kugirango ubone amplification.
2) Igenamiterere ryibizunguruka:
| Gahunda | Umubare wizunguruka | Ubushyuhe | Igihe cyo kubyitwaramo | |
| 1 | 1 | 50 ℃ | Imin. 10 | |
| 2 | 1 | 95 ℃ | 30 amasegonda | |
| 3 | 45 | 95 ℃ | 5 amasegonda | |
| 60 ℃ | 30 amasegonda | Icyegeranyo cya Fluorescence | ||
3) Igenamiterere ryo gutahura:
Imiyoboro yo gutahura yashyizwe kuri FAM, VIC, ROX na CY5, ihuye na ORF1ab, N gene, na E Gene, igenzura ryimbere rya RNase P."Irangi rya Quencher" na "Passive Reference" byashyizwe kuri "Ntayo" kubikoresho bya ABI 7500.Shiraho Igenzura ryiza, Igenzura ribi, hamwe nicyitegererezo (Utazwi) kugirango ukurikirane ingero zihuye, hanyuma ushireho izina ryicyitegererezo mumurongo "Izina ryicyitegererezo".
Kuri X-POCH16, imikorere na gahunda nibi bikurikira:
1) Nyuma yo kwipimisha ubwabyo birangiye, fungura umupfundikizo hanyuma ushyire imiyoboro ya PCR mumwanya wabigenewe.
2) Tangira uhitamo "Impuguke."ihitamo.Hitamo "Byose" cyangwa uhitemo intoki uhitamo reaction kuruhande rwibumoso bwa ecran.
3) Hitamo inzira "UMUYOBOZI";hitamo gahunda yikizamini;kanda "BIKOREWE" na "RUN".Porogaramu ifata 30min42s kugirango irangire.
Imiyoboro yo gutahura gahunda isanzwe yashyizwe kuri FAM, VIC, ROX na CY5, ihuye na ORF1ab, N gene, na E Gene, igenzura ryimbere rya RNase P.
Umuzenguruko wa cycle ya gahunda isanzwe niyi ikurikira:
| Gahunda | Umubare wa | Ubushyuhe | Igihe cyo kubyitwaramo |
| 1 | 1 | 50 ℃ | 2min |
| 2 | 1 | 95 ℃ | 30sec |
| 3 | 41 | 95 ℃ | 2sec |
| 60 ℃ | 13sec | Fluorescence |
5. Gushiraho imipaka
Ukurikije ishusho yasesenguwe, hindura agaciro ko Gutangira, Agaciro kanyuma ka Baseline na Threshold agaciro (Gutangira agaciro nagaciro kanyuma birasabwa gushyirwaho kuba 3 na 15 bikurikiranye, kandi amplification curve yo kugenzura nabi yahinduwe kugirango ibe igororotse cyangwa munsi yumurongo uteganijwe), kanda Isesengura kugirango uhite ubona isesengura icyitegererezo Ct agaciro.Reba ibisubizo muri Raporo Imigaragarire.
6. Igipimo cyo kugenzura ubuziranenge
Buri kugenzura ibikoresho bigomba kuba byujuje ibisabwa bikurikira hamwe na 'S' umurongo, bitabaye ibyo igeragezwa ntiremewe.
| Imiyoboro yo gutahura | Kugenzura nabi | Kugenzura neza |
| FAM (ORF1ab) | Oya Ct | Ct≤38 |
| VIC (N) | Oya Ct | Ct≤38 |
| ROX (E) | Oya Ct | Ct≤38 |
| CY5 (RP) | Oya Ct | Ct≤38 |
Agaciro Agaciro】
Ukurikije ibisubizo bya 100 ya oropharyngeal swab hamwe na 100 ya sputum, hamwe nuburyo bwa ROC bwo gutondeka, Agaciro-gaciro ka OFR1ab, N genes E Gene yiki gikoresho ni Ct = 38
Ibibazo
Muri iki gikoresho, primers hamwe nubushakashatsi bwibihe nyabyo bya tekinoroji ya PCR igenewe uturere twabitswe kandi twihariye twa ORF1ab, N na E gene ya 2019-nCoV.Mugihe cyo kongera PCR, iperereza rihuza inyandikorugero, kandi itsinda ryabanyamakuru 5'-barangije itsinda ryiperereza ryakozwe na enzyme ya Taq (5 '→ 3' exonuclease), bityo ikava mumatsinda yazimye kugirango itange ikimenyetso cya fluorescent .Igihe nyacyo cyo kwagura umurongo gihita gitegurwa hashingiwe ku kimenyetso cya fluorescence cyagaragaye, kandi icyitegererezo Ct kibarwa.Florophores ya FAM, VIC na ROX yanditseho gene ya ORF1ab, N gene na E.Ukoresheje ikizamini kimwe, kumenya neza genes eshatu zavuzwe haruguru za 2019-nCoV birashobora gukorwa icyarimwe.
Igikoresho gitangwa nigenzura ryimbere rigamije gene ya RNase P kugirango ikurikirane icyegeranyo cyamavuriro, ikorwa, ikururwa hamwe na RT-PCR kugirango birinde ibisubizo bibi-bibi.Igenzura ryimbere ryanditseho itsinda rya CY5 fluorescent.
1. Gusesengura no gusobanura ibisubizo byikizamini mugihe igikoresho gisanzwe, hamwe nigenzura ryiza, igenzura ribi hamwe nigisubizo cyimbere mugisubizo cyujuje ubuziranenge bwo kugenzura ubuziranenge.
2. Amplification curve yo kugenzura imbere (CY5) yerekana uburyo busanzwe bwa S umurongo na Ct ≤ 38, gusobanura ibisubizo bya genes bigenewe gukorerwa ibintu bikurikira.
| Imiyoboro yo gutahura | Gusobanura ibisubizo bya gen | ||
| FMA | VIC (N gene) | ROX (E gene) | |
| Ct≤38 | Ct≤38 | Ct≤38 | Hamwe na S amplification isanzwe ， Ct agaciro ni ≤38, intego ihuye na gen ni nziza. |
| 38 ＜ Ct ＜ 40 | 38 ＜ Ct ＜ 40 | 38 ＜ Ct ＜ 40 | Hamwe nibisanzwe S amplification curve ， ongera ugerageze intego ihuye na sample yongeye. Niba Ct agaciro ＜ 40 hamwe na S isanzwe ya amplification curve, intego ihuye na gen ni nziza;niba Ct agaciro≥40, intego ihuye na gen ni mbi |
| Ct≥40 | Ct≥40 | Ct≥40 | Intego ihuye nayo ni mbi |
Gusobanura ibisubizo bya 2019-nCoV:
Ukurikije ibisubizo bya ORF1ab, N gene na E gene, ibisobanuro bikurikira:
1) Niba genes ebyiri cyangwa GATATU za genes zagaragaye ari nziza, 2019-nCoV ni nziza;
2) Niba GUSA GUSA cyangwa NTAWE muri genes zagaragaye ari nziza, 2019-nCoV ni mbi.
Icyitonderwa: amplification curve ya positif nziza yicyitegererezo igomba kuba hamwe na S isanzwe.Ariko, niba intego yibandaho ari ndende cyane, igenzura ryimbere ntirishobora kongerwa kandi icyitegererezo gishobora kugaragara nkicyiza.Niba hari genes ebyiri zigenewe kubona Ct≤38, 2019-nCoV nibyiza.Niba hari genes ebyiri zigenewe kubona Ct≥40, 2019-nCoV ni mbi.Niba Ct≥40, cyangwa kwerekana nta gaciro, gusobanura ibisubizo bya gene igamije ni bibi.
3. Niba byose Ct indangagaciro za FAM, VIC, ROX na Cy5 zirenze 38 cyangwa ntabisanzwe bigaragara S amplification curve:
1) Hariho / nibintu (s) murugero rubuza PCR reaction.Birasabwa kugabanya icyitegererezo cyo kongera kugeragezwa.
2) Inzira yo gukuramo aside nucleique ntisanzwe, kubwibyo birasabwa kongera gukuramo
acide nucleic kugirango yongere yipimishe.
3) Iyi sample ntabwo yari icyitegererezo cyujuje ibisabwa mugihe cyo gutoranya, cyangwa guteshwa agaciro
mugihe cyo gutwara no kubika.
Nuburyo 2 dushobora kumenya ibi bihe: NAAT na Antigen.
(Bituruka kuri CDC ya Los Angeles)
