Zogulitsa
Lifecosm SARS-Cov-2-RT-PCR Detection kit ya 2019-nCoV
Kugwiritsa ntchito koyembekezeka
Chidachi chimagwiritsidwa ntchito pozindikira kuti pali coronavirus yatsopano (2019-nCoV) pogwiritsa ntchito swabs zapakhosi, swabs za nasopharyngeal, bronchoalveolar lavage fluid, sputum. umboni wa matenda matenda ndi mankhwala.Kusanthula mwatsatanetsatane za chikhalidwe tikulimbikitsidwa pamodzi ndi wodwala mawonetseredwe matenda ndi zina zasayansi mayesero.
Mfundo yoyendera
Zidazi zimatengera ukadaulo wa RT-PCR wa gawo limodzi.M'malo mwake, ma gene a 2019 a coronavirus (2019-nCoV) ORF1ab ndi N gene adasankhidwa kukhala zigawo zomwe akuyembekezeredwa.Zoyambira zenizeni ndi ma probes a fulorosenti (ma jini a N amalembedwa ndi FAM ndipo ma probe a ORF1ab amalembedwa ndi HEX) adapangidwa kuti azindikire mtundu watsopano wa coronavirus RNA wa 2019 mu zitsanzo.Chidacho chimaphatikizanso njira yodziwira zowongolera zamkati (zofufuza zamtundu wamkati zolembedwa ndi CY5) kuti ziwunikire njira yosonkhanitsira zitsanzo, kukulitsa kwa RNA ndi PCR, potero kuchepetsa zotsatira zabodza.
Zigawo zazikulu
| Zigawo | Voliyumu(48T/Kit） |
| RT-PCR reaction solution | 96l pa |
| nCOV primer TaqMan probemixture (ORF1ab,N Gene,RnaseP Gene) | 864µl |
| Kuwongolera kolakwika | 1500µl |
| nCOV Positive contro (l ORF1ab N Gene) | 1500µl |
Own reagents: RNA m'zigawo kapena kuyeretsa reagents.Kuwongolera koyipa / kwabwino: Kuwongolera kwabwino ndi RNA yokhala ndi chidutswa chandamale, pomwe kuwongolera koyipa ndi madzi opanda nucleic acid.Pogwiritsa ntchito, ayenera kutenga nawo mbali pochotsa ndipo ayenera kuonedwa ngati opatsirana.Ayenera kusamaliridwa ndikutayidwa motsatira malamulo ofunikira.
Jini lolozera mkati ndi jini ya RnaseP yamunthu.
Zosungirako komanso tsiku lotha ntchito
-20 ± 5 ℃, pewani kuzizira kobwerezabwereza ndi kusungunuka nthawi zoposa 5, zovomerezeka kwa miyezi isanu ndi umodzi.
Chida chogwiritsidwa ntchito
Ndi FAM / HEX / CY5 ndi zida zina zambiri zama fulorosenti PCR.
Zofunikira zachitsanzo
1. Mitundu yachitsanzo yogwiritsiridwa ntchito: swabs pakhosi, swabs nasopharyngeal, bronchoalveolar lavage fluid, sputum.
2.Kutolere kwa zitsanzo (njira yaaseptic)
Pharyngeal swab: Pukuta matani ndi khoma lakumbuyo la pharyngeal ndi swabs ziwiri nthawi imodzi, ndiye kumiza mutu wa swab mu chubu choyesera chomwe chili ndi yankho lachitsanzo.
Khohlela: Wodwala akakhosomola kwambiri, sonkhanitsani sputum mu chubu choyesera choyezera chomwe chili ndi njira yotsatsira;bronchoalveolar lavage fluid: Zitsanzo ndi akatswiri azachipatala.3.Kusungirako ndi kutumiza zitsanzo
Zitsanzo za kudzipatula kwa ma virus ndi kuyezetsa kwa RNA ziyenera kuyesedwa posachedwa.Zitsanzo zomwe zitha kuzindikirika mkati mwa maola 24 zitha kusungidwa pa 4 ℃;zomwe sizingawoneke mkati mwa 24
maola ayenera kusungidwa pa -70 ℃ kapena pansipa (ngati palibe chikhalidwe chosungira cha -70 ℃, ayenera kukhala
kusungidwa kwakanthawi pa -20 ℃firiji).Zitsanzo ziyenera kupewa kuzizira kobwerezabwereza ndi kusungunuka panthawi yoyendetsa.Zitsanzo ziyenera kutumizidwa ku labotale mwamsanga mukatha kusonkhanitsa.Ngati zitsanzo zikuyenera kunyamulidwa pamtunda wautali, kusungirako madzi oundana kumalimbikitsidwa.
Njira Zoyesera
1 Zitsanzo processing ndi RNA m'zigawo (chitsanzo processing m'dera)
Ndi bwino kutenga 200μl wa madzi chitsanzo kwa RNA m'zigawo.Kuti mudziwe njira zochotsera, onani malangizo a zida zamalonda za RNA.Zonse zoipa ndi zoipa
zowongolera mu zida izi zidakhudzidwa pakuchotsa.
Kukonzekera kwa 2 PCR reagent (malo okonzekera reagent)
2.1 Chotsani zinthu zonse mu kit ndikusungunula ndikusakaniza kutentha.Centrifuge pa 8,000 rpm kwa masekondi angapo musanagwiritse ntchito;kuwerengera kuchuluka kofunikira kwa ma reagents, ndipo machitidwe amakonzedwa monga momwe tawonetsera patebulo ili:
| Zigawo | N kutumikira (25µl system) |
| NCOV choyambirira cha TaqMan probemix | 18µl × N |
| RT-PCR reaction solution | 2 × N |
| *N = chiwerengero cha zitsanzo zoyesedwa + 1 (kuwongolera kolakwika) + 1 (nCOVkuwongolera kwabwino) | |
2.2 Mutatha kusakaniza bwino zigawozo, centrifuge kwa kanthawi kochepa kuti madzi onse pa chubu agwere pansi pa chubu, ndiyeno aliquot 20 µl amplification system mu PCR chubu.
3 Sampling (malo okonzera sampuli)
Onjezani 5μl ya zowongolera zoyipa ndi zabwino mutachotsa.RNA ya chitsanzo kuti iyesedwe imawonjezedwa ku PCR reaction chubu.
Valani chubu mwamphamvu ndi centrifuge pa 8,000 rpm kwa masekondi angapo musanasamutsire kumalo ozindikira matalikidwe.
Kukulitsa kwa 4 PCR (dera lodziwikiratu)
4.1 Ikani chubu chochitira mu cell yachitsanzo cha chidacho, ndikuyika magawo motere:
| siteji | Kuzungulira nambala | Kutentha(°C) | Nthawi | choperekamalo |
| M'mbuyozolemba | 1 | 42 | 10 min | - |
| Pre-denaturationn | 1 | 95 | 1 min | - |
| Kuzungulira | 45 | 95 | 15s | - |
| 60 | 30s | kusonkhanitsa deta |
Kusankha njira yodziwira zida: Sankhani njira ya FAM, HEX, CY5 ya siginecha ya fluorescence.Pazowunikira fulorosenti PALIBE, chonde musasankhe ROX.
Kusanthula kwa zotsatira za 5 (Chonde onani malangizo oyesera a chida chilichonse chokhazikitsa)
Zitachitika, sungani zotsatira.Pambuyo posanthula, sinthani mtengo woyambira, mtengo womalizira, ndi mtengo wapachiyambi molingana ndi chithunzicho (wogwiritsa ntchito amatha kusintha malinga ndi momwe zilili, mtengo woyambira ukhoza kukhazikitsidwa ku 3 ~ 15, mtengo womaliza ukhoza kukhazikitsidwa 5 ~ 20, kusintha) mu graph ya logarithmic Pakhomo la zenera, mzere wa pakhomo uli mu gawo la logarithmic, ndipo mzere wokulitsa wa kuwongolera kolakwika ndi mzere wowongoka kapena pansi pa mzere).
6 Quauty control (Kuwongolera mwadongosolo kumaphatikizidwa muyeso) Kuwongolera koyipa: Palibe njira yodziwikiratu yodziwikiratu ya FAM, HEX, CY5
Kuwongolera koyenera kwa COV: mayendedwe odziwikiratu a njira zodziwira za FAM ndi HEX, Ct value≤32, koma palibe njira yokulirapo ya CY5;
Zomwe zili pamwambazi ziyenera kukwaniritsidwa nthawi imodzi mukuyesera komweko;apo ayi, kuyesako ndi kosavomerezeka ndipo kumayenera kubwerezedwa.
7 Kutsimikiza kwa zotsatira.
7.1 Ngati palibe ma curve okulirapo kapena Ct value> 40 mu FAM ndi HEX channels of test sample, ndipo pali amplification curve mu CY5 channel, zitha kuganiziridwa kuti palibe coronavirus yatsopano ya 2019 (2019-nCoV) RNA mu chitsanzo;
.2 Ngati chitsanzocho chili ndi mapindikidwe odziwikiratu okulirapo mu tchanelo cha FAM ndi HEX, ndipo mtengo wa Ct ndi ≤40, tinganene kuti chitsanzocho ndi chabwino ku coronavirus yatsopano ya 2019 (2019-nCoV).
7.3 Ngati chitsanzo choyesera chili ndi mayendedwe omveka bwino a amplification mu njira imodzi yokha ya FAM kapena HEX, ndipo mtengo wa Ct ndi ≤40, ndipo palibe mphira wokulitsa mu njira ina, zotsatira ziyenera kuyesedwanso.Ngati zotsatira zoyesedwanso zikugwirizana, chitsanzocho chikhoza kuganiziridwa kuti ndi chabwino kwa chatsopano
coronavirus 2019 (2019-nCoV).Ngati zotsatira zoyesedwanso zilibe zolakwika, zitha kuganiziridwa kuti chitsanzocho ndi choyipa cha coronavirus yatsopano ya 2019 (2019-nCoV).
Mtengo woweruza wabwino
Njira yokhotakhota ya ROC imagwiritsidwa ntchito kudziwa mtengo wamtundu wa CT wa zida ndipo mtengo wowongolera wamkati ndi 40.
Kutanthauzira zotsatira za mayeso
1.Kuyesa kulikonse kuyenera kuyesedwa kwa zowongolera zoyipa komanso zabwino.Zotsatira zoyeserera zitha kudziwidwa pokhapokha zowongolera zikukwaniritsa zofunikira pakuwongolera
2.Pamene njira zowunikira za FAM ndi HEX zili zabwino, zotsatira zochokera ku CY5 channel (njira yoyendetsera mkati) zingakhale zoipa chifukwa cha mpikisano wa machitidwe.
3.Pamene zotsatira zoyendetsera mkati zimakhala zoipa, ngati tthe test tube's FAM ndi HEX zozindikiritsa njira zilinso zoipa, , zikutanthauza kuti dongosololi layimitsidwa kapena ntchitoyo ndi yolakwika, t kuyesako ndi kosavomerezeka.Choncho, zitsanzozo ziyenera kuyesedwanso.
