Imikhiqizo
Ikhithi yokutholwa kwe-Lifecosm SARS-Cov-2-RT-PCR ka-2019-nCoV
Ukusetshenziswa okulindelekile
Le khithi isetshenziselwa ukutholwa kwekhwalithi ye-coronavirus entsha (2019-nCoV) kusetshenziswa ama-swabs omphimbo, i-nasopharyngeal swabs, i-bronchoalveolar lavage fluid, isikhwehlela. ubufakazi bokuxilongwa nokwelashwa komtholampilo.Ukuhlaziywa okuphelele kwesimo kunconywa ngokuhambisana nokubonakaliswa komtholampilo kwesiguli nokunye ukuhlolwa kwelabhorethri.
Umgomo wokuhlola
Ikhithi isuselwe kubuchwepheshe be-RT-PCR obuyisinyathelo esisodwa.Eqinisweni, i-coronavirus entsha ka-2019 (2019-nCoV) ORF1ab kanye nofuzo lwe-N zikhethwe njengezifunda eziqondiswe ku-amplification.Ama-primer akhethekile kanye nama-fluorescent probes (ama-N gene probe alebulwe nge-FAM futhi ama-ORF1ab probe alebulwe nge-HEX) aklanyelwe ukuthola uhlobo olusha lwe-coronavirus RNA ka-2019 kumasampuli.Ikhithi futhi ihlanganisa isistimu yokutholwa kokulawula kwangaphakathi engapheli (uphenyo lofuzo lokulawula lwangaphakathi olulebulwe ngokuthi CY5) ukuqapha inqubo yokuqoqwa kwesampula, ukukhulisa i-RNA ne-PCR, ngaleyo ndlela kuncishiswe imiphumela engemihle engamanga.
Izingxenye eziyinhloko
| Izingxenye | Ivolumu(48T/Kit） |
| Isixazululo sokusabela se-RT-PCR | 96µl |
| I-nCOV primer TaqMan probemixture (ORF1ab,N Gene,RnaseP Gene) | 864µl |
| Ukulawula okungalungile | 1500µl |
| I-nCOV Positive contro (l ORF1ab N Gene) | 1500µl |
Ama-reagents angowakho: Ukukhishwa kwe-RNA noma ama-reagents okuhlanza.Ukulawula okungekuhle/okuqondile: Ukulawula okuhle yi-RNA equkethe ucezu oluqondiwe, kuyilapho ukulawulwa okungekuhle kungamanzi angenayo i-nucleic acid.Ngesikhathi sokusetshenziswa, kufanele babambe iqhaza ekukhishweni futhi kufanele kubhekwe njengezifo ezithathelwanayo.Kufanele ziphathwe futhi zilahlwe ngokuhambisana nemithethonqubo efanele.
Isakhi sofuzo sangaphakathi esiyisethenjwa yisakhi sofuzo se-RnaseP yomuntu.
Izimo zesitoreji kanye nosuku lokuphelelwa yisikhathi
-20±5℃,gwema ukubanda okuphindaphindiwe nokuncibilika izikhathi ezingaphezu kwezi-5, kusebenza izinyanga eziyisi-6.
Ithuluzi elisebenzayo
Nge-FAM / HEX / CY5 nezinye izinsimbi ze-PCR ezineziteshi eziningi ze-fluorescent.
Izidingo zesifanekiso
1.Izinhlobo zesampula ezisebenzayo: amaswabs omphimbo, ama-nasopharyngeal swabs, bronchoalveolar lavage fluid, isikhwehlela.
2.Iqoqo lesampula (isu le-aseptic)
I-Pharyngeal swab: Sula amathoni nodonga lwangemuva lwepharyngeal ngamaswabhu amabili ngesikhathi esisodwa, bese ucwilisa ikhanda le-swab eshubhuni yokuhlola equkethe isisombululo sesampula.
Isikhwehlela: Ngemva kokuba isiguli sikhwehlele kakhulu, qoqa isikhwehlela esikhwehleleyo eshubhuni lokuhlola lesikulufa eliqukethe isixazululo sesampula;I-bronchoalveolar lavage fluid: Isampula ochwepheshe bezokwelapha.3.Ukugcinwa nokuthuthwa kwamasampula
Izibonelo zokuhlukaniswa kwegciwane kanye nokuhlolwa kwe-RNA kufanele zihlolwe ngokushesha okukhulu.Izibonelo ezingatholakala phakathi kwamahora angu-24 zingagcinwa ku-4℃;lezo ezingeke zibonwe phakathi kwama-24
amahora kufanele agcinwe ku -70 ℃ noma ngaphansi (uma singekho isimo sokugcina esingu -70 ℃, kufanele
igcinwe okwesikhashana ku -20 ℃ esiqandisini).Izibonelo kufanele zigweme ukuqhwa okuphindaphindiwe nokuncibilika ngesikhathi sokuthutha.Izibonelo kufanele zithunyelwe elabhorethri ngokushesha ngangokunokwenzeka ngemva kokuqoqwa.Uma amasampula edinga ukuthuthwa amabanga amade, ukugcinwa kweqhwa elomile kuyanconywa.
Izindlela Zokuhlola
1 Ukucubungula isampula kanye nokukhishwa kwe-RNA (indawo yokucubungula isampula)
Kunconywa ukuthatha u-200μl wesampula yoketshezi ukuze kukhishwe i-RNA.Ukuze uthole izinyathelo ezihlobene zokukhipha, bheka iziqondiso zemishini yokukhipha ye-RNA yezentengiso.Kokubili okubi nokubi
Izilawuli kule khithi bezihilelekile ekukhishweni.
2 Ukulungiswa kwe-PCR reagent (indawo yokulungiselela i-reagent)
2.1 Khipha zonke izingxenye zekhithi bese uncibilikisa bese uxuba ekamelweni lokushisa.I-Centrifuge ku-8,000 rpm imizuzwana embalwa ngaphambi kokusetshenziswa;bala inani elidingekayo lama-reagents, futhi uhlelo lokusabela lulungiswa njengoba kukhonjisiwe kuthebula elilandelayo:
| Izingxenye | I-N iseva (25µl system) |
| I-nCOV primer TaqMan probemixture | 18µl × N |
| Isixazululo sokusabela se-RT-PCR | 2µl × N |
| *N = inani lamasampula ahloliwe + 1 (ukulawula okungalungile) + 1 (nCOVukulawula okuhle) | |
2.2 Ngemva kokuxuba kahle izingxenye, i-centrifuge isikhathi esifushane ukuze uvumele lonke uketshezi olusodongeni lweshubhu luwele phansi kweshubhu, bese uliquot uhlelo lokukhulisa i-20 µl kushubhu ye-PCR.
3 Isampula (indawo yokulungiselela i-specimen)
Engeza u-5μl wezilawuli ezingezinhle nezihle ngemva kokukhipha.I-RNA yesampula ezohlolwa yengezwa kushubhu yokusabela ye-PCR.
Vala ishubhu ngokuqinile futhi i-centrifuge ku-8,000 rpm imizuzwana embalwa ngaphambi kokuyidlulisela endaweni yokuthola i-amplification.
I-4 PCR yokukhulisa (indawo yokutholwa ekhulisiwe)
4.1 Beka ishubhu yokusabela kuseli yesampula yensimbi, bese usetha amapharamitha kanje:
| esiteji | Umjikelezo inombolo | Izinga lokushisa(°C) | Isikhathi | iqoqoindawo |
| Reverseokulotshiweyo | 1 | 42 | 10 imiz | - |
| I-Pre-denaturationn | 1 | 95 | 1 imiz | - |
| Umjikelezo | 45 | 95 | 15s | - |
| 60 | 30s | ukuqoqwa kwedatha |
Ukukhetha isiteshi sokuthola insimbi: Khetha isiteshi se-FAM, HEX, CY5 sesignali ye-fluorescence.Ukuze uthole ireferensi ye-fluorescent NONE, sicela ungakhethi i-ROX.
5 Ukuhlaziywa kwemiphumela (Sicela ubhekisele emiyalweni yokuhlola yethuluzi ngalinye ukuze umiswe)
Ngemva kokusabela, gcina imiphumela.Ngemva kokuhlaziya, lungisa inani lokuqala, inani lokugcina, kanye nevelu yomkhawulo wesisekelo ngokuvumelana nesithombe (umsebenzisi angakwazi ukulungisa ngokuvumelana nesimo sangempela, inani lokuqala lingasethwa ku-3~15, inani lokugcina lingasethwa ukuze 5~20, ukulungiswa) kugrafu ye-logarithmic Embundwini wewindi, umugqa womkhawulo usesigabeni se-logarithmic, futhi ijika lokukhulisa lokulawula okunegethivu liwumugqa oqondile noma ngaphansi komugqa wonqenqema).
6 Ukulawulwa komthamo (Ukulawulwa kwenqubo kufakwe esivivinyweni) Ukulawula okungalungile: Alikho ijika elisobala lokukhulisa i-FAM, i-HEX, iziteshi zokuthola i-CY5n
Ukulawula okuhle kwe-COV: ijika elisobala lokukhulisa iziteshi ze-FAM ne-HEX, inani le-Ct≤32, kodwa alikho ijika lokukhulisa isiteshi se-CY5;
Izidingo ezingenhla kufanele zihlangatshezwe kanyekanye esivivinyweni esifanayo;ngaphandle kwalokho, ukuhlola akuvumelekile futhi kudinga ukuphindwa.
7 Ukunqunywa kwemiphumela.
7.1 Uma lingekho ijika lokukhulisa noma inani le-Ct> 40 eziteshini ze-FAM ne-HEX zesampula yokuhlola, futhi kukhona ijika lokukhulisa esiteshini se-CY5, kunganqunywa ukuthi ayikho i-coronavirus entsha ka-2019 (2019-nCoV) I-RNA kusampula;
.2 Uma isampula yokuhlola inamajika okukhulisa asobala eziteshini ze-FAM ne-HEX, futhi inani le-Ct lingu-≤40, kungase kubonakale sengathi isampula ivuma i-coronavirus entsha ka-2019 (2019-nCoV).
7.3 Uma isampula yokuhlola inejika elicacile lokukhulisa i-amplification esiteshini esisodwa se-FAM noma se-HEX, futhi inani le-Ct lingu-≤40, futhi lingekho ijika lokukhulisa kwesinye isiteshi, imiphumela idinga ukuphinda ihlolwe.Uma imiphumela yokuhlolwa kabusha ihambisana, isampuli ingathathwa njengephozithivu kwentsha
i-coronavirus 2019 (2019-nCoV).Uma umphumela wokuhlolwa kabusha unegethivu, kungahlulelwa ngokuthi isampula ayinalo i-coronavirus entsha ka-2019 (2019-nCoV).
Inani lokwahlulela elihle
Indlela yejika ye-ROC isetshenziselwa ukunquma inani eliyireferensi le-CT lekhithi kanye nevelu yereferensi yokulawula kwangaphakathi ingu-40.
Ukuhunyushwa kwemiphumela yokuhlolwa
1.Isivivinyo ngasinye kufanele sihlolelwe izilawuli ezingezinhle nezilungile.Imiphumela yokuhlolwa inganqunywa kuphela uma izilawuli zihlangabezana nezimfuneko zokulawula ikhwalithi
2.Lapho iziteshi zokuthola i-FAM ne-HEX zivuma, umphumela ovela esiteshini se-CY5 (umzila wokulawula wangaphakathi) ungase ube mubi ngenxa yokuncintisana kwesistimu.
3.Uma umphumela wokulawula kwangaphakathi uthi unegethivu, uma ishubhu lokuhlola le-FAM neziteshi ze-HEX nazo zinegethivu, , kusho ukuthi isistimu ivaliwe noma ukusebenza akulungile, t ukuhlola akuvumelekile.Ngakho-ke, amasampula adinga ukuphinda ahlolwe.
