Lintlha tsa Sehlahisoa
Mokhatlo oa Lefatše oa Bophelo (WHO) o tataisa hore liteko tsa tlhahlobo tse potlakileng tsa antigen-detecting (Ag-RDRs) li ka fana ka mokhoa o potlakileng le o theko e tlase oa ho fumana ts'oaetso ea SARS-CoV-2 e sebetsang ho feta liteko tsa nucleic acid amplification (NAATs), mme WHO le eona e khothaletsa hore li-Ag-RDTs tse fihlelang bonyane litlhokahalo tsa ts'ebetso li ka sebelisoa bakeng sa ho lemoha linyeoe tsa mantlha, ho fumana batho bao ho ka batloang le bona, nakong ea liphuputso tsa ho qhoma le ho beha leihlo maemo a mafu metseng.
Likaroloana
● Kakaretso:Liphatsa tsa lefutso tse tharo li lemoha tekong e le 'ngoe
● e lumellana:E ikamahanya le lisebelisoa tse tloaelehileng tse nang le liteishene tsa CY5, FAM, VIC/HEX.
● Ts'ebetso e ntle haholo:Boikutlo bo phahameng le bo ikhethang, LOD = 200 likopi / ml.
Tekheniki Parameter
| Tlhaloso ea Pakete | 50 liteko / kit, 100 liteko / kit |
| Sebaka se reriloeng | ORF1ab, N, E |
| Mohlala o sebetsang | Sekhohlela, Oropharyngeal Swab |
| Moeli oa ho Fumana | 200 likopi / ml |
| Kakaretso ea Coincidence Rate | 99.55% |
| Ct value (CV,%) | ≤5.0% |
| Positive Coincidence Rate | 99.12% |
| Negative Coincidence Rate | 100% |
| Maemo a Polokelo le Letsatsi la ho Felloa ke Nako | E bolokiloe ho -20±5℃,' me e sebetsa nakoana bakeng sa likhoeli tse 12. |
| Taolo ea ka hare | Ee |
| Nomoro ea lethathamo | A7793YF-50T, A7793YF-100T |
| Setifikeiti | CE |
| Mehlala | Nasopharyngeal swab, Oropharyngeal swab, Alveolar lavage fluid, Mathe le sekhohlela. |
| Seletsa se Sebetsang | ABI 7500, Roche Light Cycler 480Ⅱ , Roche Cobas z 480, SLAN-96P Real-Time PCR System |
Mokhoa oa Teko
1. Nucleic Acid Extraction
Ts'ebetso e lokela ho etsoa ho latela bukana ea lisebelisoa tsa ho hula.
2. Tokiso ea Sisteme:
1) Ntša reagent 'me u qhibilihe reagent ka ho feletseng.Fetola motsoako le centrifuge hang-hang.Lits'ebetso tsa liteko tsa N (N = palo ea lisampole tse lokelang ho lekoa + taolo e nepahetseng + taolo e mpe + 1) e lokiselitsoe litsamaiso tsa karabelo, ka ho latellana, ka tsela e latelang.
| Li-Vomponents | Bolumo bakeng sa sistimi e le 'ngoe ea karabelo | Bolumo bakeng sa sistimi ea karabelo ea N |
| Nucleic acid amplification reaction solution Mix (A7793YF) | 18µL | 18µL * N |
| Motsoako oa enzyme | 2µL | 2µL * N |
| Kakaretso ea molumo | 20µL | 20µL * N |
2) Kabo ea karabelo: Tharollo ea karabelo e ne e tsoakane le centrifuged, 'me tube e' ngoe le e 'ngoe e ne e ajoa ka bongata ba 20μL ka har'a tube ea PCR e loketseng lisebelisoa tsa PCR tsa fluorescence.
3. Loading
5μL ea sampole e nkiloeng ea nucleic acid, taolo e ntle ea nucleic acid le taolo e mpe ea nucleic acid li eketsoa lits'ebetsong tsa karabelo, 'me molumo o felletseng oa karabelo ke 25μL.Tlama sekoahelo sa tube 'me u se ise sebakeng sa tlhahlobo ea amplification ka mor'a metsotsoana e seng mekae ea centrifugation.
4. PCR Amplification Assay
1) Kenya tube ea karabelo ea PCR ka har'a sesebelisoa sa amplification sa fluorescent PCR bakeng sa ho lemoha matlafatso.
2) Litlhophiso tsa parameter ea potoloho:
| Lenaneo | Palo ea lipotoloho | Mocheso | Nako ea ho arabela | |
| 1 | 1 | 50 ℃ | 10 mets | |
| 2 | 1 | 95℃ | 30 metsotsoana | |
| 3 | 45 | 95℃ | 5 metsotsoana | |
| 60 ℃ | 30 metsotsoana | Pokello ea Fluorescence | ||
3) Litlhophiso tsa ho fumana:
Mecha ea ho lemoha e behiloe ho FAM, VIC, ROX le CY5, e tsamaellanang le ORF1ab, N gene, le E Gene, RNase P taolo ea ka hare, ka ho latellana."Quencher Dye" le "Passive Reference" li behiloe ho "None" bakeng sa sesebelisoa sa ABI 7500.Beha Taolo e Ntle, Taolo e Negative, le Sample (E sa Tsejoeng) ka tatellano eo lisampole li lumellanang, 'me u behe lebitso la mohlala kholumong ea "Sample Name".
Bakeng sa X-POCH16, ts'ebetso le lenaneo ke tse latelang:
1) Kamora hore tlhahlobo ea hau e felile, bula sekwahelo 'me u kenye li-tubes tsa karabelo ea PCR libakeng tse khethiloeng ho sesebelisoa.
2) Qala ka ho khetha "Setsebi."kgetho.Khetha khetho ea "Bohle" kapa u ikhethele sebaka sa karabelo ka lehlakoreng le letšehali la skrini.
3) Khetha khetho ea "LOAD";khetha lenaneo la teko;tobetsa "ETSA" le "RUN".Lenaneo le nka 30min42s ho qeta.
Mecha ea ho lemoha ea lenaneo la Default e behiloe ho FAM, VIC, ROX le CY5, e lumellanang le ORF1ab, N gene, le E Gene, RNase P taolo ea ka hare, ka ho latellana.
Paramethara ea potoloho ea lenaneo la Default ke tse latelang:
| Lenaneo | Nomoro ea | Mocheso | Nako ea ho arabela |
| 1 | 1 | 50 ℃ | 2 mets |
| 2 | 1 | 95℃ | 30sec |
| 3 | 41 | 95℃ | 2mts |
| 60 ℃ | 13sec | Fluorescence |
5. Ho beha Mohaho
Ho ea ka setšoantšo se hlahlobiloeng, fetola boleng ba Qalo, Boleng ba Qetello ea Baseline le boleng ba Threshold (Boleng ba ho qala le boleng ba ho qetela bo khothaletsoa hore bo behoe ho 3 le 15 ka ho latellana, 'me sekhahla sa ho holisa sa taolo e fosahetseng se lokisoa hore se batalle kapa ka tlase ho moeli oa moeli), tobetsa Analysis ho fumana tlhahlobo ea sampole ea boleng ba Ct ka bo eona.Sheba liphetho ho sebopeho sa Tlaleho.
6. Maemo a Taolo ea Boleng
Taolo ka 'ngoe ea khiti e tlameha ho fihlela litlhokahalo tse latelang ka 'S' curve, ho seng joalo teko e fosahetse.
| Mecha ea ho lemoha | Taolo e mpe | Taolo e ntle |
| FAM(ORF1ab) | Ha ho Ct | Ct≤38 |
| VIC(N) | Ha ho Ct | Ct≤38 |
| ROX(E) | Ha ho Ct | Ct≤38 |
| CY5(RP) | Ha ho Ct | Ct≤38 |
【Boleng bo Khaotsoeng】
Ho ea ka liphetho tsa lisampole tse 100 tsa oropharyngeal swab le disampole tse 100 tsa sekhohlela, 'me ka mokhoa oa ROC curve, boleng ba Cut-off ea OFR1ab, N liphatsa tsa lefutso E Gene tsa kit ena ke Ct = 38.
LBH
Ka har'a kit ena, li-primers le probes tsa theknoloji ea PCR ea nako ea nnete ea fluorescent li etselitsoe libaka tse sirelelitsoeng le tse ikhethileng tsa mofuta oa ORF1ab, N le E tsa 2019-nCoV ka ho latellana.Nakong ea ntlafatso ea PCR, probe e tlamella template, 'me sehlopha sa motlalehi oa 5'-qetellong se phunyeletsoa ke enzyme ea Taq (5'→ 3' exonuclease mosebetsi), kahoo e tloha ho sehlopha sa quenching ho hlahisa lets'oao la fluorescent. .Sekhahla sa amplification sa nako ea sebele se reriloe ka boiketsetso ho ipapisitsoe le lets'oao la fluorescence le fumanoeng, 'me boleng ba sampole ea Ct bo baloa.FAM , VIC le ROX fluorophores li ngotsoe ho ORF1ab gene, N gene le E gene probes.Ka ho sebelisa tlhahlobo e le 'ngoe, tlhahlobo ea boleng ba mefuta e meraro e kaholimo ea 2019-nCoV e ka etsoa ka nako e le ngoe.
Setsi se fanoa ka taolo ea ka hare e lebisitseng mofuta oa RNase P ho lekola pokello ea lisampole tsa kliniki, ho ts'oaroa, ho ntšoa le ts'ebetso ea RT-PCR ho qoba liphetho tse fosahetseng.Taolo ea ka hare e ngotsoe ka sehlopha sa CY5 fluorescent.
1. Sekaseka le ho hlalosa liphetho tsa liteko ha sesebelisoa se tloaelehile, 'me taolo e nepahetseng, taolo e fosahetseng le sephetho sa tlhahlobo ea ka hare li finyella litekanyetso tsa taolo ea boleng.
2. Lekhalo la amplification la taolo ea ka hare (CY5) le bonts'a S curve e tloaelehileng le Ct ≤ 38, tlhaloso ea liphello tsa liphatsa tsa lefutso tse lebisitsoeng li tlas'a maemo a latelang.
| Mecha ea ho lemoha | Ho hlalosa liphello tsa liphatsa tsa lefutso tse lebisitsoeng | ||
| FMA | VIC (N gene) | ROX (E gene) | |
| Ct≤38 | Ct≤38 | Ct≤38 | Ka sekhahla se tloaelehileng sa S amplification, boleng ba Ct ke≤38, mofuta o lumellanang oa sepheo o nepahetse. |
| 38 ＜ Ct ＜ 40 | 38 ＜ Ct ＜ 40 | 38 ＜ Ct ＜ 40 | Ka curve e tloaelehileng ea S amplification, leka hape mofuta o lumellanang oa sampole hape. Haeba boleng ba Ct＜ 40 ka sekhahla se tloaelehileng sa amplification sa S, mofuta o lumellanang oa sepheo o nepahetse;haeba boleng ba Ct≥40, gene e tsamaisanang le eona e fosahetse |
| Ct≥40 | Ct≥40 | Ct≥40 | Lefutso le tsamaellanang le shebiloeng le mpe |
Tlhaloso ea liphetho tsa 2019-nCoV:
Ho ea ka liphetho tsa ORF1ab, N gene le E gene, tlhaloso ka tsela e latelang:
1) Haeba liphatsa tse PEDI kapa tse THARO tsa liphatsa tsa lefutso tse fumanoeng li le ntle, 2019-nCoV e ntle;
2) Haeba FEELA E LE FEELA kapa HONA E 'NGOE ea liphatsa tsa lefutso e fumanoeng e le ntle, 2019-nCoV ha e eo.
Tlhokomeliso: Mokokotlo oa amplification oa sampole e ntle e lokela ho ba le S curve e tloaelehileng.Leha ho le joalo, haeba sepheo sa sepheo se phahame haholo, taolo ea maemo a ka hare e ka 'na ea se ke ea ntlafatsoa' me sampuli e ka ahloloa ka ho toba e le ntle.Haeba mefuta e 'meli ea liphatsa tsa lefutso e fumana Ct≤38, 2019-nCoV e ntle.Haeba mefuta e 'meli ea liphatsa tsa lefutso e ka fumana Ct≥40, 2019-nCoV e mpe.Haeba Ct≥40, kapa e sa bontše boleng, tlhaloso ea liphetho tsa jini e reretsoeng e fosahetse.
3. Haeba litekanyetso tsohle tsa Ct tsa likanale tsa FAM, VIC, ROX le Cy5 li feta 38 kapa ha ho na mokhoa o totobetseng oa S amplification curve:
1) Ho na le/ho na le lintho (lintho) sampoleng tse thibelang karabelo ea PCR.Ho kgothaletswa ho hlapolla sampuli bakeng sa ho hlahlojoa hape.
2) Ts'ebetso ea ho ntšoa ha nucleic acid ha e tloaelehe, ka hona ho khothaletsoa ho ntša hape.
asiti ea nucleic bakeng sa tlhahlobo e ncha.
3) Mohlala ona e ne e SE sampuli e tšoanelehang nakong ea lisampole, kapa e senyehile
nakong ea lipalangoang le polokelo.
Ke litsela tse 2 tseo re ka lemohang maemo ana ka tsona: NAAT le Antigen.
(E tsoa ho CDC ea Los Angeles)
