Iimveliso
I-Lifecosm SARS-Cov-2-RT-PCR Ikhithi yokuFumana ka-2019-nCoV
Ukusetyenziswa okulindelekileyo
Le khithi isetyenziselwa ukufunyaniswa komgangatho we-coronavirus entsha (2019-nCoV) kusetyenziswa i-swabs yomqala, i-nasopharyngeal swabs, i-bronchoalveolar lavage fluid, isikhohlela. ubungqina bokuxilongwa kweklinikhi kunye nonyango.Uhlalutyo olunzulu lwemeko lunconywa ngokudibanisa nokubonakaliswa kwekliniki yesigulane kunye nezinye iimvavanyo zebhubhoratri.
Umgaqo wokuhlola
Ikhithi isekelwe kwitekhnoloji ye-RT-PCR enenyathelo elinye.Ngapha koko, i-coronavirus entsha ka-2019 (2019-nCoV) ORF1ab kunye nofuzo lwe-N ziye zakhethwa njengemimandla ekujoliswe kuyo yokukhulisa.Iiprimers ezithile kunye ne-fluorescent probes (i-N gene probes zibhalwe nge-FAM kunye ne-ORF1ab probes zibhalwe nge-HEX) ziyilelwe ukufumanisa i-coronavirus yohlobo olutsha luka-2019 kwiisampulu.Ikiti ikwabandakanya inkqubo yokukhangela yolawulo lwangaphakathi engapheliyo (iprobe yegene yolawulo lwangaphakathi ebhalwe ngeCY5) ukujonga inkqubo yokuqokelela isampulu, i-RNA kunye ne-PCR yokukhulisa, ngaloo ndlela inciphisa iziphumo ezingezizo zobuxoki.
Amacandelo aphambili
| Amacandelo | Umthamo(48T/Kit） |
| RT-PCR impendulo isisombululo | 96µl |
| I-nCOV primer TaqMan probemixture (ORF1ab,N Gene,RnaseP Gene) | 864µl |
| Ulawulo olubi | 1500µl |
| I-nCOV Positive contro (l ORF1ab N Gene) | 1500µl |
Ii-reagents ezizezakho: Ukukhutshwa kwe-RNA okanye ii-reagents zokucoca.Ulawulo olubi / olulungileyo: Ulawulo olulungileyo lune-RNA equkethe iqhekeza elijoliswe kuyo, ngelixa ulawulo olubi luyi-nucleic acid-free water.Ngethuba lokusetyenziswa, kufuneka bathathe inxaxheba kwi-extraction kwaye kufuneka bathathwe njengentsholongwane.Kufuneka ziphathwe kwaye zilahlwe ngokuhambelana nemimiselo efanelekileyo.
Ijene lereferensi yangaphakathi yiRnaseP yomntu.
Iimeko zokugcina kunye nomhla wokuphelelwa
-20±5℃,kunqande ukukhenkceza okuphindaphindiweyo kunye nokunyibilika ngaphezu kwamaxesha ama-5, kusebenze iinyanga ezi-6.
Isixhobo esisebenzayo
Nge-FAM / HEX / CY5 kunye nezinye izixhobo ezininzi ze-PCR ze-fluorescent.
Iimfuno zesampulu
1.Iintlobo zesampulu ezisebenzayo: iiswabs zomqala, iiswabs zenasopharyngeal, bronchoalveolar lavage fluid, isikhohlela.
2.Ukuqokelelwa komzekelo (ubuchule beaseptic)
I-Pharyngeal swab: Sula iitoni kunye nodonga lwangasemva lwe-pharyngeal ngeeswabhu ezimbini ngaxeshanye, emva koko untywilisele intloko ye-swab kwityhubhu yovavanyo enesisombululo sesampulu.
Isikhohlela: Emva kokuba isigulana sinokhohlokhohlo olunzulu, qokelela isikhohlela esikhohlelayo kwityhubhu yovavanyo enesikrufu esinesisombululo sesampulu;bronchoalveolar lavage fluid:Iisampulu ziingcali zonyango.3.Ukugcinwa kunye nokuthuthwa kweesampuli
Iisampulu zokwahlulwa kwentsholongwane kunye novavanyo lwe-RNA kufuneka zivavanywe ngokukhawuleza.Iisampulu ezinokuthi zibonwe kwiiyure ezingama-24 zingagcinwa kwi-4℃;ezo zingenakubonwa ngaphakathi kwe-24
iiyure kufuneka zigcinwe ku -70 ℃ okanye ngaphantsi (ukuba akukho meko yokugcina -70 ℃, kufuneka
igcinwe okwexeshana kwi -20℃efrijini).Iisampulu kufuneka zigweme ukukhenkceza okuphindaphindiweyo kunye nokunyibilika ngexesha lokuthutha.Iisampulu kufuneka zithunyelwe elabhoratri ngokukhawuleza emva kokuqokelela.Ukuba iisampulu kufuneka zihanjiswe kumgama omde, ukugcinwa komkhenkce owomileyo kuyacetyiswa.
Iindlela zoVavanyo
Ukusetyenzwa kweSampuli e-1 kunye nokutsalwa kwe-RNA (indawo yokusetyenzwa kwesampuli)
Kucetyiswa ukuba uthathe i-200μl yesampulu yolwelo yokutsalwa kwe-RNA.Ngamanyathelo okutsalwa anxulumeneyo, jonga kwimiyalelo yeekhithi zokutsalwa kweRNA zorhwebo.Zombini ezingalunganga nezibi
ulawulo kule khithi lubandakanyekile ekutsalweni.
I-2 PCR yokulungiswa kwe-reagent (indawo yokulungisa i-reagent)
2.1 Susa zonke iinxalenye kwikhithi kwaye unyibilike kwaye udibanise kwiqondo lokushisa.I-Centrifuge kwi-8,000 rpm imizuzwana embalwa ngaphambi kokusetyenziswa;bala inani elifunekayo le-reagents, kwaye inkqubo yokusabela ilungiswe njengoko kubonisiwe kwitheyibhile ilandelayo:
| Amacandelo | N ekhonza (25µl inkqubo) |
| I-nCOV primer TaqMan probemixture | 18µl × N |
| RT-PCR impendulo isisombululo | 2µl × N |
| *N = inani leesampuli ezivavanyiweyo + 1 (ulawulo olubi) + 1 (nCOVulawulo olulungileyo) | |
2.2 Emva kokuxuba ngokucokisekileyo amacandelo, i-centrifuge ixesha elifutshane ukuvumela lonke ulwelo kudonga lwetyhubhu luwele ezantsi kombhobho, kwaye ke aliquot inkqubo yokukhulisa i-20 µl kwityhubhu ye-PCR.
3 Iisampulu (indawo yokulungiselela iisampulu)
Yongeza i-5μl yolawulo olubi kunye nolulungileyo emva kokutsalwa.I-RNA yesampuli eza kuvavanywa yongezwa kwi-tube yokusabela ye-PCR.
Vala ityhubhu ngokuqinileyo kunye ne-centrifuge kwi-8,000 rpm imizuzwana embalwa ngaphambi kokuba uyidlulisele kwindawo yokufumanisa i-amplification.
Ukwandiswa kwe-PCR ye-4 (indawo yokubona eyandisiweyo)
4.1 Beka ityhubhu yokusabela kwisampuli yeseli yesixhobo, kwaye usete iiparamitha ngolu hlobo lulandelayo:
| iqonga | Umjikelo inani | Ubushushu(°C) | Ixesha | ingqokelelaindawo |
| Umvaushicilelo | 1 | 42 | 10min | - |
| I-Pre-denaturation | 1 | 95 | 1min | - |
| Umjikelo | 45 | 95 | 15s | - |
| 60 | 30s | ukuqokelelwa kwedatha |
Ukukhetha itshaneli yokuchongwa kwesixhobo: Khetha i-FAM, HEX, CY5 itshaneli yomqondiso we-fluorescence.Ukufumana ireferensi yefluorescent NONE, nceda ungakhethi ROX.
5 Uhlalutyo lweziphumo (Nceda ubhekisele kwimiyalelo yovavanyo yesixhobo ngasinye sokuseta)
Emva kokusabela, gcina iziphumo.Emva kokuhlalutya, lungisa ixabiso lokuqala, ixabiso lokuphela, kunye nexabiso lesiseko sesiseko ngokomfanekiso (umsebenzisi unokulungelelanisa ngokwemeko yangempela, ixabiso lokuqala lingamiselwa kwi-3 ~ 15, ixabiso lokuphela lingamiselwa I-5 ~ 20, ukulungiswa) kwigrafu ye-logarithmic Kumgubasi wefestile, umgca wecala ukwisigaba se-logarithmic, kwaye i-curve yokukhulisa yolawulo olubi ngumgca othe ngqo okanye ngaphantsi komgca wecala).
6 Ulawulo lobungakanani (Ulawulo lwenkqubo lubandakanyiwe kuvavanyo) Ulawulo olubi: Akukho jika licacileyo lokukhulisa i-FAM, i-HEX, i-CY5 yokuchongwa kwemijelo
Ulawulo olulungileyo lwe-COV: i-curve yokukhulisa ngokucacileyo ye-FAM kunye ne-HEX yokubona amajelo, ixabiso le-Ct≤32, kodwa akukho jiko lokukhulisa le-CY5 channel;
Ezi mfuno zingentla mazifikelelwe ngaxeshanye kuvavanyo olufanayo;ngenye indlela, uvavanyo alusebenzi kwaye lufuna ukuphindwa.
7 Ukumiselwa kweziphumo.
7.1 Ukuba akukho curve yokukhulisa okanye ixabiso le-Ct> 40 kwi-FAM kunye ne-HEX channels yesampula yovavanyo, kwaye kukho i-curve yokukhulisa kwi-channel ye-CY5, kunokugwetywa ukuba akukho 2019 entsha ye-coronavirus (2019-nCoV) I-RNA kwisampuli;
.2 Ukuba isampuli yovavanyo inee-curves zokukhulisa ngokucacileyo kwi-FAM kunye ne-HEX channels, kwaye ixabiso le-Ct ngu-≤40, kunokugwetywa ukuba isampuli ilungile kwi-coronavirus entsha ye-2019 (2019-nCoV).
7.3 Ukuba isampuli yovavanyo ine-curve yokukhulisa ecacileyo kuphela kwitshaneli enye ye-FAM okanye i-HEX, kwaye ixabiso le-Ct ngu-≤40, kwaye akukho ngqungquthela yokwandisa kwelinye ishaneli, iziphumo kufuneka zihlolwe kwakhona.Ukuba iziphumo zovavanyo kwakhona ziyahambelana, isampulu inokugqalwa njengelungileyo kwintsha
i-coronavirus 2019 (2019-nCoV).Ukuba iziphumo zovavanyo kwakhona zimbi, kunokugwetywa ukuba isampulu ayinayo i-coronavirus entsha ka-2019 (2019-nCoV).
Ixabiso lesigwebo esilungileyo
Indlela ye-curve ye-ROC isetyenziselwa ukumisela ixabiso le-CT yereferensi yekiti kunye nexabiso lereferensi yolawulo lwangaphakathi yi-40.
Ukutolikwa kweziphumo zovavanyo
1.Uvavanyo ngalunye kufuneka luvavanyelwe ulawulo olungalunganga nolulungileyo.Iziphumo zovavanyo zinokumiselwa kuphela xa ulawulo luhlangabezana neemfuno zolawulo lomgangatho
2.Xa i-FAM kunye ne-HEX yokubona iziteshi zilungile, umphumo ovela kwi-CY5 channel (ishaneli yokulawula yangaphakathi) ingaba yimbi ngenxa yokhuphiswano lwenkqubo.
3.Xa isiphumo solawulo lwangaphakathi sibi, ukuba ityhubhu yovavanyo ye-FAM kunye ne-HEX yokubona imijelo nayo ayilunganga, oko kuthetha ukuba inkqubo ivaliwe okanye umsebenzi awulunganga, uvavanyo alusebenzi.Ngoko ke, iisampulu kufuneka zivavanywe kwakhona.
