Ilaha warshadda Shiinaha Rt PCR Assay Reagent ee Virus-ka cusub ee 2019 (ORF1ab, N gene)
Qaado waajibaad buuxa si aad u qanciso dhammaan dalabaadka macaamiisheena;gaadhno horumar joogto ah annaga oo suuqgeynaynaa horumarka iibsadayaasheenna;u koraan si ay u noqdaan lamaanahaaga joogtada ah ee joogtada ah ee macaamiisha iyo sare u qaadida danaha macaamiisha ee Ilaha Warshadda China Rt PCR Assay Reagent ee 2019 Virus Cusub (ORF1ab, N hidda), Waxaan isku-kalsoonahay in aan abuurno guulo cajiib ah inta ay suurtagal tahay.Waxaan ugaadhsanaynay inaan noqono mid ka mid ah alaab-qeybiyeyaashaada ugu kalsoonida badan.
Qaado waajibaad buuxa si aad u qanciso dhammaan dalabaadka macaamiisheena;gaadhno horumar joogto ah annaga oo suuqgeynaynaa horumarka iibsadayaasheenna;u koraan si aad u noqoto lammaanaha ugu dambeeya ee iskaashiga joogtada ah ee macaamiisha oo aad u kordhiso danaha macaamiishaQalabka PCR ee Shiinaha, Qalabka Ogaanshaha PCr, Sugitaanka, waxaan la socon doonaa waqtiyada, sii wadida abuurista alaab cusub.Iyadoo kooxdayada cilmi-baarista xooggan, tas-hiilaadka wax-soo-saarka sare leh, maamulka sayniska iyo adeegyada ugu sarreeya, waxaan siin doonaa alaab tayo sare leh macaamiishayada adduunka oo dhan.Waxaan si daacad ah kuugu martiqaadeynaa inaad noqoto shuraakada ganacsi ee faa'iidooyinka wadaagga ah.
Sharaxaada
Xirmadani waxay adeegsataa nidaam u gaar ah oo lysis ah oo si dhakhso ah uga sii deyn kara RNA muunadaha unugyada dhaqameed ee falcelinta RT-qPCR, taas oo meesha ka saaraysa habka nadiifinta RNA waqti-qaadka iyo shaqada badan.Qaabka RNA waxa lagu heli karaa 7 daqiiqo oo kaliya.5× Toos ah RT Mix iyo 2× Toos ah qPCR Mix-SYBR reagents ay bixiso qalabku waxay si degdeg ah oo wax ku ool ah u heli karaan natiijooyinka PCR ee tirada dhabta ah.
5× Toos ah RT Mix iyo 2×QPCR Mix-SYBR toos ah waxay leeyihiin dulqaad adag oo ka hortag ah, iyo lysate ee shaybaarada waxaa loo isticmaali karaa qaab-dhismeedka RT-qPCR si toos ah.Qalabkani waxa uu ka kooban yahay RNA-ga gaarka ah ee xidhiidhka sare ee Foregene reverse transcriptase, iyo Hot D-Taq DNA polymerase, dNTPs, MgCl2, celiyaha falcelinta, PCR optimizer iyo stabilizer.
Tilmaamaha
200×20μl Rxns, 1000×20μl Rxns
Qaybaha kitka
Qaybta I | Buffer CL |
Foregene Protease Plus II | |
Buffer ST | |
Qaybta II | Tirtiraha DNA |
5× Toos ah RT Mix | |
2× Toos ah qPCR Mix-SYBR | |
50× ROX Tixraaca Midabka | |
RNase-free ddH2O | |
Tilmaamaha |
Tilmaamaha & faa'iidooyinka
■ Fudud oo wax ku ool ah: tignoolajiyada Cell Direct RT, muunadaha RNA waxaa lagu heli karaa 7 daqiiqo oo keliya.
■ Baahida muunada waa yar tahay, maadaama ay yar tahay 10 unug ayaa la tijaabin karaa.
■ Soo saarista sare: waxay si dhakhso ah u ogaan kartaa RNA ee unugyada lagu beeray 384, 96, 24, 12, 6-cells.
■ Tirtiraha DNA-da si dhakhso ah ayuu u saari karaa genome-yada la sii daayay, si weyn ayuu u yareynayaa saameynta natiijooyinka tijaabada dambe.
■ Nidaamka RT iyo qPCR ee la hagaajiyay ayaa ka dhigaya laba-tallaabo dib-u-celinta RT-PCR mid hufan iyo PCR mid gaar ah, oo aad u adkeysi badan ka-hortagga falcelinta RT-qPCR.
Codsiga qalabka
Baaxadda codsiga: unugyada dhaqanka.
- RNA lagu sii daayay muunad lysis: kaliya lagu dabaqi karo qaabka RT-qPCR ee xirmadan.
-Qalabka waxa loo isticmaali karaa ujeedooyinka soo socda: falanqaynta muujinta hidde-sidaha, xaqiijinta saamaynta aamusinta hidde-sidaha ee siRNA, baadhista daroogada, iwm.
Jaantuska
Kaydinta iyo nolosha shelf
Qaybta I ee xirmadan waa in lagu kaydiyaa 4℃;Qaybta II waa in lagu kaydiyaa at -20 ℃.
Foregene Protease Plus II waa in lagu kaydiyaa 4 ℃, ha ku barafaysan -20 ℃.
Reagent 2× Toos ah qPCR Mix-SYBR waa in lagu kaydiyaa -20℃ gudcurka;haddii si joogto ah loo isticmaalo, waxa kale oo lagu kaydin karaa 4℃ kaydinta muddada gaaban (isticmaal 10 maalmood gudahood)gaadhno horumar joogto ah annaga oo suuqgeynaynaa horumarka iibsadayaasheenna;u koraan si ay u noqdaan lamaanahaaga joogtada ah ee joogtada ah ee macaamiisha iyo sare u qaadida danaha macaamiisha ee Ilaha Warshadda China Rt PCR Assay Reagent ee 2019 Virus Cusub (ORF1ab, N hidda), Waxaan isku-kalsoonahay in aan abuurno guulo cajiib ah inta ay suurtagal tahay.Waxaan ugaadhsanaynay inaan noqono mid ka mid ah alaab-qeybiyeyaashaada ugu kalsoonida badan.
Isha warshaddaQalabka PCR ee Shiinaha, PCR Detection Kit, Sugitaanka, waxaan la socon doonaa waqtiyada, sii wadida abuurista alaab cusub.Iyadoo kooxdayada cilmi-baarista xooggan, tas-hiilaadka wax-soo-saarka sare leh, maamulka sayniska iyo adeegyada ugu sarreeya, waxaan siin doonaa alaab tayo sare leh macaamiishayada adduunka oo dhan.Waxaan si daacad ah kuugu martiqaadeynaa inaad noqoto shuraakada ganacsi ee faa'iidooyinka wadaagga ah.
Mabaadi'da naqshadaynta aasaasiga ah ee waqtiga dhabta ah PCR
Horudhac Horudhac iyo Horudhac Hordhac ah
PCR-da-waqtiga-dhabta ah, naqshadaynta asaasiga ahi aad bay muhiim u tahay.Furayaasha waxay la xiriiraan gaarnimada iyo hufnaanta kordhinta PCR, waxaana loo qaabayn karaa iyadoo la tixraacayo mabaadi'da soo socota:
Dhererka hore: 18-30bp.
Waxa ku jira GC: 40-60%.
Qiimaha Tm: Software-ka naqshadaynta aasaasiga ah, sida Primer 5, wuxuu ku siin karaa qiimaha Tm ee asaasiga ah.Qiimayaasha Tm ee hore iyo kuwa hoose waa inay ahaadaan kuwa ugu dhow ee suurtogalka ah.Qaaciddada xisaabinta Tm waxa kale oo la isticmaali karaa: Tm = 4 °C (G + C) + 2 °C (A + T).Marka la samaynayo PCR, heerkulka ka hooseeya qiimaha Tm ee aasaasiga ah ee 5 °C ayaa guud ahaan loo doortaa heer kulka soo jiidashada (kordhinta u dhiganta heerkulka xididada ayaa kordhin kara qeexida falcelinta PCR).
Qalabka aasaasiga ah iyo PCR:
Naqshadeynta asaasiga ah ee PCR dhererka alaabada ayaa la door bidaa 100-150bp.
Naqshadeynta aasaasiga ah ee aagga qaab dhismeedka sare ee template waa in laga fogaadaa sida ugu macquulsan.
Ka fogow samaynta 2 ama in ka badan oo saldhigyo dhammaystiran oo u dhexeeya 3′ daraf ee hore-u-socodyada sare iyo kuwa hoose.
Primer 3′ terminal saldhiga kuma jiri karo 3 dheeraad ah oo isku xigta G ama C.
Furayaasha laftoodu ma yeelan karaan dhismayaal is kaaba ah, haddii kale qaab-dhismeedka timaha ayaa la samayn doonaa, oo saamaynaya kor u qaadida PCR.
ATCG waa in loo qaybiyaa sida ugu siman ee suurtogalka ah ee isku xigxiga asaasiga ah, iyo 3′ saldhigga terminal waa in laga fogaadaa sida T.
Lifaaqa 1: Xidhmada kabka qaybta gacanta tooska ah ee RT-qPCR
1.Cell Lysis Solution
| |||
Qaybaha kitka (24-hab lysis / ceel) | DRT-01011-A1 | DRT-01011-A2 | |
100 T | 500 T | ||
QaybI | Buffer CL | 20 ml | 100 ml |
Foregene Protease Plus II | 400 μl | 1 ml × 2 | |
Buffer ST | 1 ml × 2 | 10 ml | |
QaybII | Tirtiraha DNA | 400 μl | 1 ml × 2 |
2.RT Isku darka
| |
Qaybaha kitka (Nidaamka falcelinta 20 μl) | DRT-01011-B1 |
200 T | |
5× Toos ah RT Mix | 800 μl |
RNase-free ddH2O | 1.7 ml × 2 |
| ||
Qaybaha kitka (Nidaamka falcelinta 20 μl) | DRT-01011-C1 | DRT-01011-C2 |
200 T | 1000 T | |
2× Toos ah qPCR Mix-SYBR | 1 ml × 2 | 1.7 ml × 6 |
50× ROX Tixraaca Midabka | 40 μl | 200 μl |
RNase-free ddH2O | 1.7 ml | 10 ml |