Direct RT-qPCR ngwa III
Nkọwa ngwa:
◮Kit Direct RT-qPCR, na-eji Foreasy Reverse Transcriptase na Foreasy HS Taq DNA Polymerase na-etolite site na Foregene, yana ihe nchekwa mmeghachi omume pụrụ iche, na-eme ngwa a na nguzogide siri ike na ndakọrịta, yana ọ nwere ike nwalee mmeghachi omume ozugbo iji sistemụ Foregene Lysis dị ka ndebiri.Ngwa a nwere ike itinye maka ngwa nchọpụta COVID-19 nucleic acid yana mmepe ngwa nchọpụta nje nje ndị ọzọ.
Nkọwa
Kit Direct RT-qPCR na-enye ihe nchekwa RT-qPCR ngwugwu dị iche iche (ya na Foreasy HS Taq DNA polymerase agwakọtara) yana ịrụ ọrụ nke ọma.Transcriptase ntụgharị ihu (MMLV),na-eme ka ọ dị mma maka ịmepụta ngwa azụ azụ na nhazi usoro, n'ime nkwenye dị mfe.
Kit Direct RT-qPCR, na-eji Foreasy Reverse Transcriptase na Foreasy HS Taq DNA Polymerase na-etolite site na Foregene, yana ihe nchekwa mmeghachi omume pụrụ iche, na-eme ngwa a na nguzogide siri ike na ndakọrịta, yana ọ nwere ike nwalee mmeghachi omume ozugbo iji sistemụ Foregene Lysis dị ka ndebiri.Ngwa a nwere ike itinye maka ngwa nchọpụta COVID-19 nucleic acid yana mmepe ngwa nchọpụta nje nje ndị ọzọ.
Ngwa a nwere ike bụrụ akụkụ nke ngwaahịa IVD ozugbo, na-eji ngwa ngwa na ngwa ngwa.Ọ chọrọ naanị nkwenye dị mfe, na-enweghị ịmalite ọzọ.
Nkọwapụta
500T, 50,000T
Ngwa ngwa
| Ngwa ọdịnaya (Sistemụ mmeghachi omume 20 μl) | IM-05111-03 | IM-05112-03 |
| 500 T | 50,000 T | |
| Amụma Rverse Transcriptase(200 U/μL) | 50 μl×1 | 1 ml×5 |
| 2 × Direct qPCRNgwakọta-Taqman | 1 ml ×5 | 500 ml×1 |
| Ntuziaka | 1 | 1 |
Nzọụkwụ arụ ọrụ
A: Kwadebe nke template na reagent
1. Kwadebe ndebiri RNA a kwadebere (A na-atụ aro ka iji Foregene Total RNA Isolation Kit series kit wepụ na sachaa RNA template) ma ọ bụ ngwaahịa cracking (A na-atụ aro ka iji usoro Foregene Lysis), primers kpọmkwem (10 μM) na ihe ndị ọzọ dị mkpa, ngwá ọrụ.
2. Tinye 2 × Direct RT-qPCR Buffer, RNase-Free ddH2O na 20 × ROX Reference Dye (ọ bụrụ na ọ dị mkpa) n'ime igbe na ice, na-eme ka ndị a gbazee n'ụzọ nkịtị, na-agwakọta nwayọọ.
B: Kwadebe usoro RT-qPCR
Nweta ọkara olu nke mmeghachi omume mmeghachi omume nke sistemu mmeghachi omume (dịka ọmụmaatụ, ọ bụrụ na olu sistemụ ahụ bụ 20 μL, ọ kwesịrị ịnweta 10 μL 2 × Direct RT-qPCR Buffer.). Makakwekọrọ na ego nke enzyme, anyị na-atụ aro naM-MLV: 10-30 U/20 μL usoro mmeghachi omume,TaqDNA polymerase:1-2 U/20 μL usoro mmeghachi omumeNke a bụ aro anyị, ị ga-anwale ma dozie ya), gbakwunye ndebiri RNA, ihe mbido na nyocha, wee tinye RNase-Free ddH2O ruo 20 μl.Enwere ike ịkọwa nkwadebe kpọmkwem nke sistemụ mmeghachi omume RT-qPCR na tebụl 1 na-esote.
Isiokwu 1: Kwadebe usoro RT-qPCR
| Akụkụ | Olu | Ntinye uche ikpeazụ |
| 2 × Direct RT-qPCR ihe nchekwa | 10 μl | 1× |
| Transcriptase ntụgharị ihu (MMLV) | 10-30 U | |
| Ihe atụ nke HS Taq DNA Polymerase | 1-2 U | |
| Ntugharị n'ihu (10 μM) | 0.8 μl | 50-900nM |
| Agbaghachi azụ (10 μM) | 0.8 μl | 50-900nM |
| Nyocha (4 μM) | 1 μl | 200nM |
| Template (RNA ma ọ bụ Lysate) | X μL | |
| 20× ROX ntụaka agbaji | - | 1* |
| RNase-FreeddH2O | (6.4-X) μL | |
| Mkpokọta olu | 20 μl |
Mara: Forward Primer na Reverse Primer bụ kpọmkwem ihe mmalite nke mkpụrụ ndụ ihe mgbaru ọsọ.Enwere ike ịhazi usoro nke qPCR dị ka nnwale bara uru na ụdị PCR.Anyị na-akwado 400nM maka ntinye ikpeazụ nke ọtụtụ primers.Gbanwee olu nke kpọmkwem primers na nyocha dị ka ntinye akwadoro na ntinye ikpeazụ akwadoro, biko.
1 *: Họrọ ntinye ikpeazụ nke ROX Reference Dye dị ka ngwa PCR dị iche iche si dị.Ntụle ROX Reference Dye kacha mma nke ngwa PCR ọnụ ọnụ ka egosiri na tebụl na-esote:
| Ngwa PCR ọnụ ọgụgụ | Ntinye ikpeazụ nke ROX Reference Dye |
| ABI PRISM 7000/7300/7700/7900HT/Nzọụkwụ Mbụ,wdg. | 1 × (dịka ọmụmaatụ, tinye 1μl 20 × ROX Reference Dye na sistemụ mmeghachi omume 20 μL) |
| ABI 7500, 7500 ngwa ngwa, Stratagene Mx3000P, Mx3005P na Mx4000, wdg. | 0.5 × (dịka ọmụmaatụ, tinye 0.5μl 20 × ROX Reference Dye na sistemụ mmeghachi omume 20 μL) |
| Ngwa Roche PCR, Ngwa Bio-Rad PCR, Eppendorf quantitative PCR instrument, wdg. | Na-enweghị ROX Reference Dye |
Nchekwa na Shelf ndụ
Ekwesịrị ịchekwa ngwa ahụ na -20 ± 5 ℃.Ekwesịrị ịchekwa ya ozugbo na friji -20 ℃ thermostatic.Oge nkwado bụ afọ 2 ma ọ bụrụ na echekwara ya na ọnọdụ kwesịrị ekwesị.
