Akwụkwọ akụkọ Coronavirus (SARS-CoV-2) Ngwa PCR Real-Time Multiplex
Ngwa PCR a dị njikere nke ukwuu dị n'ụdị lyophilized (usoro ịsa oyi) maka nchekwa ogologo oge.Enwere ike ibufe ngwa ahụ ma chekwaa ya na ụlọ okpomọkụ ma kwụsie ike maka otu afọ.Ọkpụkpọ premix ọ bụla nwere reagents niile achọrọ maka nkwalite PCR, gụnyere Reverse-transcriptase, Taq polymerase, primers, probes na dNTPs substrates.Ọ chọrọ ịgbakwunye 13ul distilled mmiri na 5ul amịpụtara RNA template, mgbe ahụ enwere ike ịgba ya ma gbasaa na ngwa PCR.
Igwe qPCR kwesịrị izute ihe ndị a:
1. dabara 8 warara PCR tube olu 0.2 ml
2. Nwee ọwa nchọpụta ihe karịrị anọ:
| Ọwa | Mmasị (nm) | Mgbapụta (nm) | Ejiji agbachagoro tupu oge eruo |
| 1. | 470 | 525 | FAM, SYBR Green I |
| 2 | 523 | 564 | VIC, HEX, TET, JOE |
| 3. | 571 | 621 | ROX, TEXAS-RED |
| 4 | 630 | 670 | CY5 |
PCR-ikpo okwu:
Sistemụ PCR 7500 Real-Time, Biorad CF96, iCycler iQ™ Sistemụ Nchọpụta PCR Real-Time, Stratagene Mx3000P, Mx3005P
Ihe isi ike nke ụgbọ njem yinye oyi nke Novel Coronavirus nucleic acid reagent nchọpụta
Mgbe a na-ebufe reagents nchọpụta nucleic acid a na-ahụkarị n'ebe dị anya, a chọrọ nchekwa (-20 ± 5) ℃ oyiri yinye nchekwa na njem iji hụ na bioactive nke enzyme na reagents ka na-arụ ọrụ.Iji jide n'aka na okpomọkụ ahụ ruru ọkọlọtọ, a chọrọ ọtụtụ kilogram nke ice akọrọ maka igbe ọ bụla nke nucleic acid test reagent ọbụna ihe na-erughị 50g, ma ọ nwere ike ịdịru naanị ụbọchị abụọ ma ọ bụ atọ.N'echiche nke omume ụlọ ọrụ, n'ezie arọ nke reagents nyere site emepụta bụ ihe na-erughị 10% (ma ọ bụ nnọọ ihe na-erughị nke a uru) nke akpa.Ọtụtụ n'ime ibu dị arọ na-abịa site na ice akọrọ, ngwugwu ice na igbe ụfụfụ, ya mere ọnụ ahịa njem dị oke elu.
Na Machị 2020, COVID-19 malitere itolite n'ọtụtụ mba ofesi, na ọchịchọ maka nchọpụta Nucleic acid Novel Coronavirus reagent mụbara nke ukwuu.N'agbanyeghị ọnụ ahịa dị elu nke mbupụ reagents na agbụ oyi, ọtụtụ ndị na-emepụta ihe ka nwere ike ịnakwere ya n'ihi nnukwu ego na nnukwu uru.
Otú ọ dị, na mmelite nke mba mbupụ atumatu maka mgbochi ọrịa na ngwaahịa, nakwa dị ka upgrading nke mba akara n'elu eruba nke ndị mmadụ na lọjistik, e nwere ndọtị na ejighị n'aka na njem oge nke reagents, nke mere ka a ma ama ngwaahịa nsogbu kpatara. site na njem njem.Ogologo oge njem (oge mbufe ihe dị ka ọkara ọnwa na-adịkarị) na-eduga na ọdịda ngwaahịa mgbe niile mgbe ngwaahịa ahụ ruru onye ahịa ahụ.Nke a akpatala nsogbu ọtụtụ ụlọ ọrụ nucleic acid reagents na-ebupụ.
Teknụzụ Lyophilized maka PCR reagent nyeere aka mbugharị nke ihe nchọpụta ihe nchọpụta novel Coronavirus nucleic acid n'ụwa niile
Enwere ike iburu PCR reagents lyophilized ma chekwaa ya n'ime ụlọ okpomọkụ, nke nwere ike ọ bụghị naanị ibelata ọnụ ahịa njem, kamakwa zere nsogbu dị mma nke usoro njem na-akpata.Ya mere, lyophilizing reagent bụ ụzọ kachasị mma isi dozie nsogbu nke njem mbupụ.
Lyophilization gụnyere ịsacha ihe ngwọta n'ime ọnọdụ siri ike, wee sublimate ma kewaa vapo mmiri n'okpuru ọnọdụ agụụ.Solute a mịrị amị na-anọgide n'ime akpa ahụ nwere otu ihe mejupụtara na ọrụ.N'iji ya na reagents mmiri mmiri a na-ahụkarị, ihe mejupụtara lyophilized Novel Coronavirus nucleic acid reagent nke Liming Bio mepụtara nwere njirimara ndị a:
Nkwụsi ike okpomọkụ siri ike:ọ nwere ike na-eguzobe ọgwụgwọ na 56 ℃ maka 60 ụbọchị, na morphology na arụmọrụ nke reagent na-agbanwe agbanwe.
Nchekwa okpomọkụ na njem ụgbọ njem nkịtị:ọ dịghị mkpa maka yinye oyi, ọ dịghị mkpa ka ịchekwaa na obere okpomọkụ tupu ịtọghe ya, hapụ ebe nchekwa oyi zuru oke.
Dị njikere iji:lyophilizing nke ihe niile, ọ dịghị mkpa maka nhazi usoro, na-ezere mfu nke ihe ndị nwere nnukwu viscosity dị ka enzyme.
Ebumnuche Multiplex na otu tube:ebumnuche nchọpụta ahụ na-ekpuchi akwụkwọ akụkọ coronavirus ORF1ab gene, N gene, S gene iji zere nje virus.Iji belata adịghị mma ụgha, a na-eji mkpụrụ ndụ RNase P mmadụ mee ihe dị ka njikwa ime, iji gboo mkpa ụlọ ọgwụ maka njikwa mma nlele.
