Iinkcukacha zeMveliso
Umbutho wezeMpilo weHlabathi (i-WHO) ukhokela ukuba i-antigen-detecting tests quick diagnostic (Ag-RDRs) inokubonelela ngendlela ekhawulezayo nengabizi kakhulu yokuxilonga usulelo olusebenzayo lwe-SARS-CoV-2 kunovavanyo lwe-nucleic acid amplification (NAATs), kwaye i-WHO ikwacebisa. ukuba ii-Ag-RDTs zikhawulelana nezona mfuno zisezantsi zokwenziwa komsebenzi zinokusetyenziselwa ukufumanisa amatyala angundoqo, ukukhangelwa kwabafowunelwa, ngexesha lophando lokuqhambuka nokubeka iliso kwiintsingiselo zezifo eluntwini.
Iimbonakalo
● Banzi:Ufuzo oluthathu ekujoliswe kulo lubhaqa kuvavanyo olunye
● ehambelanayo:Ukulungelelanisa kwizixhobo eziqhelekileyo kunye ne-CY5, i-FAM, i-VIC / iziteshi ze-HEX.
● Ukusebenza okugqwesileyo:Uvakalelo oluphezulu kunye neenkcukacha, i-LOD = iikopi ze-200 / ml.
IParameter yobuGcisa
| Ukupakisha Specification | Iimvavanyo ezingama-50/ikhithi, iimvavanyo ezili-100/ikhithi |
| Ummandla ekujoliswe kuwo | ORF1ab, N, E |
| Isampulu esebenzayo | Isikhohlela, i-Oropharyngeal Swab |
| Umda wokuFumana | Iikopi ze-200 / ml |
| Ireyithi yeZihlo ezipheleleyo | 99.55% |
| Ct ixabiso (CV,%) | ≤5.0% |
| Ireyithi yokuNgenzelana okuhle | 99.12% |
| Ireyithi ye-Negative coincidence | 100% |
| Iimeko zoGcino kunye noMhla wokuphelelwa kwexesha | Igcinwe ku -20±5℃, kwaye isebenza okwethutyana iinyanga ezili-12. |
| Ulawulo lwangaphakathi | Ewe |
| Inombolo Yekhathalogu | A7793YF-50T, A7793YF-100T |
| Isiqinisekiso | CE |
| Imizekelo | Iswab yeNasopharyngeal, i-Oropharyngeal swab, i-Alveolar lavage fluid, amathe kunye nesikhohlela |
| Isixhobo Esisebenzayo | ABI 7500, Roche Light Cycler 480Ⅱ , Roche Cobas z 480, SLAN-96P Real-Time PCR System |
Inkqubo yoVavanyo
1. I-Nucleic Acid Extraction
Umsebenzi kufuneka wenziwe ngokungqinelana nencwadana yekiti yokutsala.
2. ULungiselelo lweNkqubo:
1) Thatha i-reagent kwaye unyibilikise i-reagent ngokupheleleyo.Guqula umxube kunye ne-centrifuge ngoko nangoko.Iimpendulo zovavanyo lwe-N (N = inani leesampuli eziza kuvavanywa + ulawulo olulungileyo + ulawulo olubi + 1) lulungiselelwe iinkqubo zokusabela, ngokulandelelanayo, ngolu hlobo lulandelayo.
| IiVomponents | Umthamo we-1 reaction system | Umthamo wenkqubo yokusabela kwe-N |
| I-Nucleic acid amplification reaction solution Mix (A7793YF) | 18µL | 18µL *N |
| Umxube we-enzyme | 2µL | 2µL *N |
| Umthamo opheleleyo | 20µL | 20µL *N |
2) Ukuhanjiswa kwe-reaction: Isisombululo sokusabela sasixutywe kwaye i-centrifuged, kwaye ityhubhu nganye yachithwa kwisixa se-20μL kwi-tube ye-PCR efanelekileyo kwi-fluorescence PCR apparatus.
3. Iyalayisha
I-5μL yesampulu ekhutshiweyo ye-nucleic acid, ulawulo oluhle lwe-nucleic acid kunye nolawulo olubi lwe-nucleic acid yongezwa kwiinkqubo zokuphendula, kwaye umthamo opheleleyo wokuphendula ngu-25μL.Qinisa isigqubuthelo setyhubhu kwaye uyihambise kwindawo yovavanyo lokukhulisa emva kwemizuzwana embalwa ye-centrifugation.
4. UVavanyo loKwandiswa kwePCR
1) Beka ityhubhu yokusabela ye-PCR kwisixhobo sokukhulisa i-PCR esinefluorescent ukuze sibonwe ukukhulisa.
2) Ukusetwa kweparamitha yomjikelo:
| Inkqubo | Inani lemijikelo | Ubushushu | Ixesha lokuphendula | |
| 1 | 1 | 50℃ | 10 imiz | |
| 2 | 1 | 95℃ | 30 imizuzwana | |
| 3 | 45 | 95℃ | 5 imizuzwana | |
| 60℃ | 30 imizuzwana | Iqoqo leFluorescence | ||
3) Useto lokubhaqa:
Imijelo yokufumanisa isetyenziswe kwi-FAM, VIC, ROX kunye ne-CY5, ehambelana ne-ORF1ab, i-N gene, kunye ne-E Gene, i-RNase P yolawulo lwangaphakathi, ngokulandelanayo."Idayi ye-Quencher" kunye ne "Passive Reference" zisetelwe "Akukho" kwisixhobo se-ABI 7500.Misela i-Positive Control, Control Negative, kunye neSampulu (engaziwa) ukwenzela ukuba iisampuli zihambelana, kwaye usethe igama lesampuli kwikholamu ethi "Sample Name".
Kwi-X-POCH16, ukusebenza kunye neprogram ngolu hlobo lulandelayo:
I-1) Emva kokuba uvavanyo lokuzihlola lugqityiwe, vula isiciko kwaye ubeke iityhubhu ze-PCR zokuphendula kwiindawo ezichongiweyo kwisixhobo.
2) Qala ngokukhetha i "Exper."ukhetho.Khetha "Konke" ukhetho okanye ukhethe ngesandla indawo yokusabela kwicala lasekhohlo lesikrini.
3) Khetha i "LOAD" ukhetho;khetha inkqubo yovavanyo;cofa u-"QEDILE" kunye no-"RUN".Inkqubo ithatha i-30min42s ukuyigqiba.
Imijelo yokufumanisa inkqubo ye-Default imiselwe kwi-FAM, i-VIC, i-ROX kunye ne-CY5, ehambelana ne-ORF1ab, i-N gene, kunye ne-E Gene, i-RNase P yolawulo lwangaphakathi, ngokulandelanayo.
Ipharamitha yomjikelo wenkqubo eMiselweyo zezi zilandelayo:
| Inkqubo | Inani le | Ubushushu | Ixesha lokuphendula |
| 1 | 1 | 50℃ | 2 min |
| 2 | 1 | 95℃ | 30sec |
| 3 | 41 | 95℃ | 2 imizuzwana |
| 60℃ | 13sec | I-Fluorescence |
5. Ukubeka iTreshold
Ngokomfanekiso ohlalutyiweyo, lungisa ixabiso lokuQala, ixabiso lokuphela kweBaseline kunye nexabiso leThreshold (Ixabiso lokuqala kunye nexabiso lokugqibela liyacetyiswa ukuba libekwe kwi-3 kunye ne-15 ngokulandelanayo, kwaye i-curve yokukhulisa ulawulo olubi luhlengahlengiswa ukuba lube flat okanye ngaphantsi kunomda we-threshold Line), cofa u-Analysis ukuze ufumane ngokuzenzekelayo uhlalutyo lwexabiso le-Ct lesampuli.Jonga iziphumo kujongano lweNgxelo.
6. Umgangatho woLawulo loMgangatho
Ulawulo ngalunye lwekhithi malufezekise ezi mfuno zilandelayo nge-'S' curve, kungenjalo umfuniselo awusebenzi.
| Amajelo okubona | Ulawulo olubi | Ulawulo oluhle |
| FAM(ORF1ab) | Akukho Ct | Ct≤38 |
| VIC(N) | Akukho Ct | Ct≤38 |
| ROX(E) | Akukho Ct | Ct≤38 |
| CY5(RP) | Akukho Ct | Ct≤38 |
【Ixabiso lokusikwa】
Ngokweziphumo ze-100 zeesampulu ze-oropharyngeal swab kunye neesampuli ze-sputum ezili-100, kunye ne-ROC curve method, ixabiso le-Cut-off ye-OFR1ab, i-N genes E Gene yale khithi yi-Ct = 38
FAQ
Kule khithi, iiprimers kunye neeprobe zetekhnoloji ye-PCR yexesha lokwenyani ziyilelwe imimandla egciniweyo kunye nekhethekileyo ye-ORF1ab, i-N kunye ne-E gene ka-2019-nCoV ngokulandelelanayo.Ngexesha lokwandiswa kwe-PCR, i-probe ibophelela kwi-template, kwaye iqela le-5'-end reporter ye-probe licandwa yi-enzyme ye-Taq (5'→ 3' umsebenzi we-exonuclease), ngaloo ndlela ihambela kude neqela lokucima ukuvelisa isignali ye-fluorescent. .I-curve ye-real-time amplification icwangciswe ngokuzenzekelayo ngokusekelwe kwisignali ye-fluorescence efunyenweyo, kwaye ixabiso lesampula le-Ct libalwa.I-FAM, i-VIC kunye ne-ROX fluorophores zibhalwe kwi-ORF1ab gene, i-N gene kunye ne-E gene probes.Ngokusebenzisa uvavanyo olunye, ukuchongwa okusemgangathweni kwezi ntlobo zintathu zofuzo zika-2019-nCoV kunokwenziwa ngaxeshanye.
Ikhithi ibonelelwa ngolawulo lwangaphakathi olujolise kwi-RNase P gene ukubeka iliso kwingqokelela yeesampulu zeklinikhi, ukuphatha, ukutsalwa kunye nenkqubo ye-RT-PCR ukuphepha iziphumo ezingalunganga.Ulawulo lwangaphakathi lubhalwe ngeqela le-CY5 fluorescent.
1. Hlalutya kwaye utolike iziphumo zovavanyo xa isixhobo siqhelekile, kunye nolawulo oluhle, ulawulo olubi kunye nesiphumo sovavanyo lwangaphakathi luhlangabezana nomgangatho wolawulo lomgangatho.
2. I-curve ye-amplification yolawulo lwangaphakathi (CY5) ibonisa i-curve ye-S eqhelekileyo kunye ne-Ct ≤ 38, ukutolikwa kweziphumo zezakhi zofuzo ezijoliswe kuzo zixhomekeke kule miqathango ilandelayo.
| Amajelo okubona | Ukutolikwa kweziphumo zemfuza ekujoliswe kuyo | ||
| I-FMA | VIC (N jini) | ROX (E gene) | |
| Ct≤38 | Ct≤38 | Ct≤38 | Nge-curve yokukhulisa i-S eqhelekileyo, ixabiso le-Ct ngu≤38, i-gene ekujoliswe kuyo ilungile. |
| 38 ＜ Ct ＜ 40 | 38 ＜ Ct ＜ 40 | 38 ＜ Ct ＜ 40 | Ngegophe eliqhelekileyo lokukhulisa i-S, phinda uvavanye uhlobo lofuzo oluhambelanayo lwesampulu kwakhona. Ukuba ixabiso le-Ct＜ 40 kunye ne-S curve ye-amplification eqhelekileyo, i-gene ekujoliswe kuyo ehambelana nayo ilungile;ukuba ixabiso le-Ct≥40, i-gene ekujoliswe kuyo ehambelanayo ayilunganga |
| Ct≥40 | Ct≥40 | Ct≥40 | Ijini ekujoliswe kuyo ayilunganga |
Ukutolikwa kweziphumo zika-2019-nCoV:
Ngokweziphumo ze-ORF1ab, N ijini kunye no-E, utoliko ngolu hlobo lulandelayo:
1) Ukuba iijini ezi-2 okanye ZINTATHU zofuzo ezichongiweyo zilungile, i-2019-nCoV ilungile;
2) Ukuba ENYE okanye AYIKHO enye yemfuza efunyenweyo, i-2019-nCoV ayinayo.
Qaphela: Igophe lokukhulisa lesampulu echanekileyo kufuneka ibe ne-S curve eqhelekileyo.Nangona kunjalo, ukuba ugxininiso olujoliswe kuyo luphezulu kakhulu, ulawulo lomgangatho wangaphakathi alunakwandiswa kwaye isampuli inokugwetywa ngokuthe ngqo njengelungileyo.Ukuba kukho naziphi na iigenes ezimbini ekujoliswe kuzo zifumana i-Ct≤38, i-2019-nCoV ilungile.Ukuba kukho naziphi na iijini ezimbini ekujoliswe kuzo zifumana i-Ct≥40, i-2019-nCoV ayinayo.Ukuba i-Ct≥40, okanye ayibonisi xabiso, utoliko lweziphumo zejini ekujoliswe kulo lubi.
3. Ukuba onke amaxabiso e-Ct e-FAM, i-VIC, i-ROX kunye ne-Cy5 amajelo angaphezulu kwe-38 okanye akukho nto icacileyo ye-curve yokukhulisa i-S:
I-1) Kukho / kukho izinto kwisampulu ethintela ukuphendula kwe-PCR.Kucetyiswa ukuba uhlambulule isampuli ukuze uphinde uvavanywe.
2) Inkqubo ye-nucleic acid extraction ayiqhelekanga, ngoko ke kucetyiswa ukuba uphinde ukhuphe
i-nucleic acid yokuvavanya kwakhona.
I-3) Le sampuli AYIKHO isampuli esifanelekileyo ngexesha lokulinganisa, okanye ukuthotywa
ngexesha lokuthutha kunye nokugcinwa.
Zii-2 iindlela esinokufumanisa ezi meko: i-NAAT kunye ne-Antigen.
(Ivela kwiCDC yaseLos Angeles)
