Imininingwane Yomkhiqizo
I-World Health Organization (WHO) iqondisa ukuthi ukuhlolwa kwe-antigen-detecting rapid diagnostic (Ag-RDRs) kunganikeza indlela esheshayo nengabizi kakhulu yokuxilonga ukutheleleka okusebenzayo kwe-SARS-CoV-2 kune-nucleic acid amplification test (NAATs), futhi i-WHO iphinde income. ukuthi i-Ag-RDTs ehlangabezana nezidingo eziyisisekelo zokusebenza ingase isetshenziselwe ukutholwa kwamacala ayinhloko, ukulandelela ukuthintana nabo, phakathi nophenyo lokuqubuka kwezifo kanye nokuqapha izitayela zezigameko zezifo emiphakathini.
Izici
● Okuphelele:Izakhi zofuzo eziqondiwe ezintathu zithola ekuhlolweni okukodwa
● ehambisanayo:Ivumelana nezisetshenziswa ezivamile ezineziteshi ze-CY5, FAM, VIC/HEX.
● Ukusebenza okuhle kakhulu:Ukuzwela okuphezulu nokucacisa, i-LOD = amakhophi angu-200/ml.
Ipharamitha Yezobuchwepheshe
| Ukupakisha Ukucaciswa | 50 test/kit, 100 test/kit |
| Isifunda okuhloswe ngaso | ORF1ab, N, E |
| Isampula Esebenzayo | Isikhwehlela, i-Oropharyngeal Swab |
| Umkhawulo Wokutholwa | 200 amakhophi/ml |
| Isilinganiso Esiphelele Sokuzenzakalela | 99.55% |
| Inani le-CT (CV,%) | ≤5.0% |
| Isilinganiso Sokuqondana Okuhle | 99.12% |
| Isilinganiso Esingesihle Sokuqondana | 100% |
| Izimo zesitoreji kanye nedethi yokuphelelwa yisikhathi | Igcinwe ku -20±5℃, futhi isebenza okwesikhashana izinyanga eziyi-12. |
| Ukulawula Kwangaphakathi | Yebo |
| Inombolo Yekhathalogi | I-A7793YF-50T, A7793YF-100T |
| Isitifiketi | CE |
| Izibonelo | I-Nasopharyngeal swab, i-Oropharyngeal swab, i-Alveolar lavage fluid, amathe nesikhwehlela |
| Ithuluzi Elisebenzayo | I-ABI 7500, Roche Light Cycler 480Ⅱ , Roche Cobas z 480, SLAN-96P Real-Time PCR System |
Inqubo Yokuhlola
1. I-Nucleic Acid Isizinda
Ukusebenza kufanele kwenziwe ngokuya ngebhukwana lekhithi yokukhipha.
2. Ukulungiselela Uhlelo:
1) Khipha i-reagent bese uncibilikisa i-reagent ngokuphelele.Guqula ingxube ne-centrifuge ngokushesha.Ukusabela kokuhlolwa kwe-N (N = inani lamasampula azohlolwa + ukulawula okuhle + ukulawulwa okungalungile + 1) kulungiselelwe amasistimu wokusabela, ngokulandelanayo, kanje.
| Ama-Vomponents | Ivolumu yesistimu yokusabela engu-1 | Ivolumu yesistimu yokusabela engu-N |
| I-Nucleic acid amplification reaction solution Mix (A7793YF) | 18µL | 18µL *N |
| Inhlanganisela ye-enzyme | 2µL | 2µL *N |
| Ivolumu ephelele | 20µL | 20µL *N |
2) Ukusatshalaliswa kokusabela: Isixazululo sokusabela sasixutshwe futhi safakwa i-centrifuged, futhi ishubhu ngalinye lakhishwa ngenani elingu-20μL kushubhu le-PCR elifanele izinto ze-fluorescence PCR.
3. Iyalayisha
I-5μL yesampula ye-nucleic acid ekhishiwe, ukulawula okuhle kwe-nucleic acid nokulawula okungalungile kwe-nucleic acid kwengezwa ezinhlelweni zokusabela, futhi ivolumu yokusabela iphelele ngu-25μL.Bopha ikhava yeshubhu bese uyihambisa endaweni yokuhlola yokukhulisa ukukhulisa ngemva kwemizuzwana embalwa ye-centrifugation.
4. PCR Amplification Assay
1) Faka ishubhu lokusabela le-PCR ensimbini yokukhulisa i-fluorescent PCR ukuze kutholwe ukukhulisa ivolumu.
2) Ukulungiselelwa kwepharamitha yomjikelezo:
| Uhlelo | Inombolo yemijikelezo | Izinga lokushisa | Isikhathi sokusabela | |
| 1 | 1 | 50℃ | 10 imiz | |
| 2 | 1 | 95℃ | 30 isekhondi | |
| 3 | 45 | 95℃ | 5 isekhondi | |
| 60℃ | 30 isekhondi | Iqoqo le-Fluorescence | ||
3) Izilungiselelo zokutholwa:
Iziteshi zokuthola zisethelwe ku-FAM, VIC ,ROX kanye ne-CY5, ehambisana ne-ORF1ab, i-N gene, ne-E Gene, ukulawula kwangaphakathi kwe-RNase P, ngokulandelanayo.I-"Quencher Dye" kanye ne-"Passive Reference" zisethwe ukuze zithi “None” kuthuluzi le-ABI 7500.Setha Ukulawula Okuhle, Ukulawula Okubi, kanye Nesampula (Akwaziwa) ukuze amasampuli ahambisane, bese usetha igama lesampula kukholamu "Igama Lesampula".
Ku-X-POCH16, ukusebenza nohlelo kumi kanje:
1) Ngemuva kokuthi ukuzihlola sekuqediwe, vula isivalo bese ufaka amashubhu okusabela e-PCR ezindaweni eziqokiwe entweni.
2) Qala ngokukhetha "Exper."inketho.Khetha inketho ethi "Konke" noma ukhethe mathupha indawo yokusabela ngakwesokunxele kwesikrini.
3) Khetha "LAISHA" inketho;khetha uhlelo lokuhlola;chofoza "KWENZIWE" kanye "RUN".Uhlelo luthatha ama-30min42s ukuqeda.
Iziteshi zokuthola zohlelo oluzenzakalelayo zisethelwe ku-FAM, VIC, ROX kanye ne-CY5, ehambisana ne-ORF1ab, i-N gene, kanye ne-E Gene, ukulawula kwangaphakathi kwe-RNase P, ngokulandelanayo.
Ipharamitha yomjikelezo wohlelo Oluzenzakalelayo imi kanje:
| Uhlelo | Inombolo ye | Izinga lokushisa | Isikhathi sokusabela |
| 1 | 1 | 50℃ | 2 imiz |
| 2 | 1 | 95℃ | 30 isekhondi |
| 3 | 41 | 95℃ | 2 isekhondi |
| 60℃ | 13 isekhondi | I-Fluorescence |
5. I-Threshold Setting
Ngokwesithombe esihlaziyiwe, lungisa inani Lokuqala, Inani Lokuphela Le-Baseline kanye nevelu ye-Threshold (inani lokuqala nenani lokugcina liyanconywa ukuthi limiswe libe ngu-3 no-15 ngokulandelana, futhi ijika lokukhulisa lokulawula okunegethivu liyalungiswa ukuze libe flat noma ngaphansi komugqa we-threshold), chofoza Ukuhlaziya ukuze uthole ngokuzenzakalelayo ukuhlaziya inani lesampula le-Ct.Buka imiphumela kusixhumi esibonakalayo sombiko.
6. Izinga Lokulawula Ikhwalithi
Ukulawula ngakunye kwekhithi kufanele kuhlangabezane nezimfuneko ezilandelayo ngejika elithi 'S', ngaphandle kwalokho ukuhlola akuvumelekile.
| Iziteshi zokutholwa | Ukulawula okungalungile | Ukulawula okuhle |
| I-FAM(ORF1ab) | Akekho u-Ct | Ct≤38 |
| VIC(N) | Akekho u-Ct | Ct≤38 |
| I-ROX(E) | Akekho u-Ct | Ct≤38 |
| I-CY5(RP) | Akekho u-Ct | Ct≤38 |
【Inani Elisikiwe】
Ngokwemiphumela yamasampula e-oropharyngeal swab angu-100 kanye namasampula esikhwehlela ayi-100, futhi ngendlela yejika le-ROC, inani lokusika le-OFR1ab, i-N gene E Gene yale khithi yi-Ct = 38.
FAQ
Kule kit, ama-primers nama-probe obuchwepheshe be-PCR besikhathi sangempela be-fluorescent yakhelwe izifunda ezilondoloziwe nezithize ze-ORF1ab, N kanye no-E gene ka-2019-nCoV ngokulandelanayo.Ngesikhathi sokukhulisa i-PCR, uphenyo lubophezela esifanekisweni, futhi iqembu lentatheli elingu-5'-ekupheleni lophenyo lihlukaniswa yi-enzyme ye-Taq (5'→3' exonuclease umsebenzi), ngaleyo ndlela isuka eqenjini lokucisha ukuze ikhiqize isignali ye-fluorescent. .Ijika lokukhulisa isikhathi sangempela lihlelwa ngokuzenzakalelayo ngokusekelwe kusiginali ye-fluorescence etholiwe, futhi inani lesampula le-Ct liyabalwa.I-FAM , VIC kanye ne-ROX fluorophores ibhalwe ku-ORF1ab gene , N gene kanye nama-E gene probes.Ngokusebenzisa ukuhlolwa okukodwa, ukutholwa kwekhwalithi kwezakhi zofuzo ezintathu ezingenhla ze-2019-nCoV kungenziwa kanyekanye.
Ikhithi ihlinzekwe ngokulawula kwangaphakathi okuqondise kufuzo lwe-RNase P ukuze kuqashwe ukuqoqwa kwamasampula omtholampilo, ukuphatha, ukukhishwa kanye nenqubo ye-RT-PCR ukuze kugwenywe imiphumela engemihle engamanga.Ukulawula kwangaphakathi kubhalwe ngeqembu le-CY5 fluorescent.
1. Hlaziya futhi uhumushe imiphumela yokuhlolwa lapho ithuluzi livamile, futhi ukulawula okuphozithivu, isilawuli esinegethivu kanye nomphumela wokuhlola wokulawula kwangaphakathi kuhlangabezana nezinga lokulawula ikhwalithi.
2. Ijika lokukhulisa lokulawula kwangaphakathi (CY5) libonisa ijika le-S elijwayelekile kanye ne-Ct ≤ 38, ukuhumusha imiphumela yezakhi zofuzo eziqondiwe kungaphansi kwezimo ezilandelayo.
| Iziteshi zokutholwa | Ukuhumusha imiphumela yezakhi zofuzo eziqondiwe | ||
| I-FMA | VIC (N gene) | I-ROX (E gene) | |
| Ct≤38 | Ct≤38 | Ct≤38 | Ngejika elijwayelekile lokukhulisa u-S, inani le-Ct ngu-≤38, ufuzo oluqondiwe oluhambisanayo lulungile. |
| 38 ＜ Ct ＜ 40 | 38 ＜ Ct ＜ 40 | 38 ＜ Ct ＜ 40 | Ngejika elijwayelekile lokukhulisa u-S, phinda uhlole uhlobo lofuzo oluhambisanayo lwesampula futhi. Uma inani le-Ct＜ 40 elinejika elijwayelekile lokukhulisa u-S, ufuzo oluqondiwe oluhambisanayo luyiphozithivu;uma inani le-Ct≥40, isakhi sofuzo esiqondiwe esihambisanayo sinegethivu |
| Ct≥40 | Ct≥40 | Ct≥40 | Ufuzo oluqondiwe oluhambisanayo lunegethivu |
Ukuhunyushwa kwemiphumela ka-2019-nCoV:
Ngokwemiphumela ye-ORF1ab, i-N gene ne-E gene, ukuhumusha ngale ndlela elandelayo:
1) Uma izakhi zofuzo EZIMBILI noma EZINTATHU zofuzo ezitholiwe zine-positive, i-2019-nCoV ithi positive;
2) Uma KUPHELA OKULODWA noma LONA LUTHO lwezakhi zofuzo olutholiwe, i-2019-nCoV inegethivu.
Qaphela: Ijika lokukhulisa lesampula eliphozithivu kufanele libe nejika lika-S elijwayelekile.Kodwa-ke, uma ukugxilisa okuqondiwe kuphezulu kakhulu, ukulawulwa okujwayelekile kwangaphakathi kungase kungakhuliswa futhi isampuli ingahlulelwa ngokuqondile njengephozithivu.Uma noma yiziphi ezimbili zezakhi zofuzo eziqondiwe zithola i-Ct≤38, i-2019-nCoV ivuma.Uma noma yiziphi ezimbili zezakhi zofuzo eziqondiwe zithola i-Ct≥40, i-2019-nCoV inegethivu.Uma i-Ct≥40, noma ingabonisi inani, ukutolika imiphumela yofuzo oluqondiwe kuba kunegethivu.
3. Uma wonke amanani e-Ct eziteshi ze-FAM, VIC, ROX kanye ne-Cy5 zingaphezu kuka-38 noma lingekho ijika elivamile lokukhulisa u-S:
1) Kukhona/izinto kusampula ezivimbela ukusabela kwe-PCR.Kunconywa ukuthi kuhlanjululwe isampula ukuze ihlolwe kabusha.
2) Inqubo yokukhipha i-nucleic acid ayijwayelekile, ngakho-ke kuphakanyiswa ukuthi kukhishwe kabusha
i-nucleic acid yokuhlolwa kabusha.
3) Le sampuli AKUYONA isampula efanelekayo ngesikhathi sokusampula, noma yehliswa
ngesikhathi sokuthutha nokugcinwa.
Ziyizindlela ezi-2 esingathola ngazo lezi zimo: i-NAAT ne-Antigen.
(Ivela ku-CDC yaseLos Angeles)
