I-RT-qPCR Kit II eqondile
Incazelo yekhithi:
◮Ikhithi eqondile ye-RT-qPCR, isebenzisa i-Foreasy Reverse Transcriptase ne-Foreasy HS Taq DNA Polymerase eyakhiwe yi-Foregene, ene-react buffer eyingqayizivele, yenza le khithi ibe nokumelana okuqinile nokuhambisana, futhi ingahlola ngokuqondile ukusetshenziswa kwesistimu ye-Foregene Lysis njengesifanekiso.Le khithi ingasebenza ekutholeni ikhithi ye-COVID-19 nucleic acid kanye namanye amabhaktheriya e-pathogenic nucleic acid yokuthuthukiswa kwekhithi.
Incazelo
Ikhithi ye-RT-qPCR eqondile ihlinzeka ngebhafa ehlanganiswe ngokuhlukene ye-RT-qPCR, i-Foreasy HS Taq DNA Polymerase esebenza kahle kakhulu.kanye ne-Foreasy Reverse Transcriptase(MMLV),okwenza kube lula ekudaleni amakhithi asemuva kanye nokulungiswa kwesistimu, ngaphakathi kokuqinisekisa okulula.
Ikhithi eqondile ye-RT-qPCR, isebenzisa i-Foreasy Reverse Transcriptase ne-Foreasy HS Taq DNA Polymerase eyakhiwe yi-Foregene, ene-react buffer eyingqayizivele, yenza le khithi ibe nokumelana okuqinile nokuhambisana, futhi ingahlola ngokuqondile ukusetshenziswa kwesistimu ye-Foregene Lysis njengesifanekiso.Le khithi ingasebenza ekutholeni ikhithi ye-COVID-19 nucleic acid kanye namanye amabhaktheriya e-pathogenic nucleic acid yokuthuthukiswa kwekhithi.
Le khithi ingaba yingxenye yomkhiqizo we-IVD, isebenzisa kalula futhi ngokushesha.Idinga kuphela ukuqinisekiswa okulula, ngaphandle kokuthuthuka futhi.
Imininingwane
500T, 50,000T
Izingxenye zekhithi
| Okuqukethwe kwekhithi (Isistimu yokusabela engu-20 μL) | IM-05111-02 | IM-05112-02 |
| 500 T | 50,000 T | |
| I-Foreasy RI-Transcriptase ehlukile (200 U/μL) | 50 μL×1 | 1 mL×5 |
| I-Foreasy HS Taq DNA Polymerase (5 U/μL) | 100 μL×1 | 1 mL×10 |
| 2× I-RT-qPCR eqondileIbhafa | 1 mL × 5 | 500 mL × 1 |
| Isiyalezo | 1 | 1 |
Izinyathelo Zokusebenza
A: Lungiselela isifanekiso kanye ne-reagent
1. Lungiselela ithempulethi ye-RNA elungisiwe (Kutuswa ukusebenzisa ikhithi yochungechunge lwe-Foregene Total RNA Isolation Kit ukuze ukhiphe futhi uhlanze ithempulethi ye-RNA) noma isampula yomkhiqizo wokuqhekeka (Kutuswa ukusebenzisa uhlelo lwe-Foregene Lysis), ama-primers athile(10 μM) nezinye izinto ezisetshenziswayo ezifanele, ithuluzi.
2. Faka i-2× Direct RT-qPCR Buffer, RNase-Free ddH2O kanye ne-20× ROX Reference Dye(uma idingeka) ebhokisini elineqhwa, okwenza lezi zincibilike ngokwemvelo, futhi zixutshwe ngobumnene.
B: Lungiselela uhlelo lwe-RT-qPCR
Thola uhhafu wevolumu yebhafa yokusabela yesistimu yokusabela (isibonelo, uma ivolumu yesistimu ingu-20 μL, idinga ukuthola u-10 μL 2× Direct RT-qPCR Buffer). Ngobainani elilinganayo le-enzyme,siphakamisa ukuthiI-M-MLV: 10-30 U/20 μL uhlelo lokusabela,I-TaqI-DNA polymerase:1-2 U/20 μL uhlelo lokusabelalesi isiphakamiso sethu, kufanele usihlole futhi usilungise), engeza ithempulethi ye-RNA, iziqalo ezithile kanye nama-probe, bese wengeza i-ddH-RNase-Free2O kuya ku-20 μL.Ukulungiswa okuqondile kwesistimu yokusabela ye-RT-qPCR kungadluliselwa kuthebula elilandelayo 1.
Ithebula 1 : Lungiselela uhlelo lwe-RT-qPCR
| Isakhi | Ivolumu | Ukugxila kokugcina |
| 2× Ibhafa ye-RT-qPCR eqondile | 10 μL | 1× |
| I-Foreasy Reverse Transcriptase(MMLV) | 10-30 U | |
| I-Foreasy HS Taq DNA Polymerase | 1-2 U | |
| I-Primer Phambili(10 μM) | 0.8 μL | 50-900nM |
| I-Primer Ehlehlayo(10 μM) | 0.8 μL | 50-900nM |
| I-Probe(4 μM) | 1 μl | 200nM |
| Isifanekiso(i-RNA noma i-Lysate) | X μL | |
| I-20 × I-ROX Reference Dye | - | 1* |
| RNase-FreeddH2O | (6.4-X) μL | |
| Ivolumu ephelele | 20 μL |
Qaphela: I-Primer Phambili kanye ne-Reverse Primer ziyiziqalo ezithile zofuzo oluqondiwe.Uhlelo lwe-qPCR lungalungiswa ngokuya ngamamodeli asebenzayo wokuhlola kanye ne-PCR.Sincoma i-400nM ekugxiliseni kokugcina kwama-primers amaningi.Sicela ulungise ivolumu yama-primers athile kanye nama-probe ngokuya ngokugxila okulungisiwe kanye nokugxilisa okunconyiwe kokugcina, sicela.
I-1*: Khetha ukugxiliswa kokugcina okufanele kwe-ROX Reference Dye ngokuya ngamathuluzi ahlukahlukene we-PCR.Ukugxiliswa kwe-ROX Reference Dye okuphelele kwezinsimbi ezivamile ze-PCR kuboniswa kuthebula elilandelayo:
| Amathuluzi e-PCR amaningi | Ukuhlushwa kokugcina kwe-ROX Reference Dye |
| I-ABI PRISM 7000/7300/7700/7900HT/Isinyathelo sokuqala,njll. | 1× (isibonelo, engeza i-1μl 20×ROX Reference Daye ohlelweni lokusabela lwe-20 μL) |
| I-ABI 7500, 7500 Fast, Stratagene Mx3000P, Mx3005P ne-Mx4000, njll. | 0.5× (isibonelo, engeza u-0.5μl 20×ROX Reference Daye kusistimu yokusabela engu-20 μL) |
| Ithuluzi le-Roche PCR, ithuluzi le-Bio-Rad PCR, ithuluzi le-PCR le-Eppendorf quantitative PCR, njll. | Ngaphandle kwe-ROX Reference Dye |
Isitoreji kanye Nempilo yeshelufu
Ikhithi kufanele igcinwe ku -20 ± 5℃.Kufanele igcinwe ngokushesha ku -20 ℃ esiqandisini se-thermostatic.Isikhathi sokuqinisekisa iminyaka emi-2 uma sigcinwa sisesimweni esifanele.
