Cov khoom
Lifecosm SARS-Cov-2-RT-PCR Detection Kit for 2019-nCoV
Xav siv
Cov khoom siv no yog siv rau qhov zoo ntawm kev kuaj pom tus kabmob tshiab (2019-nCoV) siv lub caj pas swabs, nasopharyngeal swabs, bronchoalveolar lavage kua, hnoos qeev.Qhov kev kuaj pom ntawm cov khoom no tsuas yog siv rau kev soj ntsuam, thiab nws yuav tsum tsis txhob siv tib yam nkaus xwb. Cov ntaub ntawv pov thawj rau kev kuaj mob thiab kev kho mob.Ib qho kev ntsuam xyuas dav dav ntawm tus mob yog pom zoo ua ke nrog tus neeg mob cov tsos mob thiab lwm yam kev kuaj mob.
Kev tshuaj xyuas txoj cai
Cov khoom siv yog ua raws li ib-kauj ruam RT-PCR thev naus laus zis.Qhov tseeb, 2019 tus kabmob tshiab tshiab (2019-nCoV) ORF1ab thiab N noob tau raug xaiv los ua lub hom phiaj loj ntawm thaj chaw.Cov primers tshwj xeeb thiab fluorescent probes (N gene probes yog sau nrog FAM thiab ORF1ab probes yog sau nrog HEX) yog tsim los kuaj 2019 tshiab-type coronavirus RNA hauv cov qauv.Cov khoom siv kuj tseem suav nrog kev tshawb nrhiav endogenous internal control system (internal control gene probe labeled with CY5) los saib xyuas cov txheej txheem ntawm kev sau cov qauv, RNA thiab PCR amplification, yog li txo cov txiaj ntsig tsis zoo.
Cov khoom tseem ceeb
| Cheebtsam | Ntim(48 T/Kit) |
| RT-PCR tshuaj tiv thaiv | 96l ua |
| nCOV primer TaqMan probemixture (ORF1ab, N Gene, RnaseP Gene) | 864l ua |
| Kev tswj tsis zoo | 1500 l os |
| nCOV Positive contro (l ORF1ab N Gene) | 1500 l os |
Tus kheej reagents: RNA extraction lossis purification reagents.Kev tswj tsis zoo / qhov zoo: Kev tswj zoo yog RNA uas muaj lub hom phiaj tawg, thaum qhov kev tswj tsis zoo yog cov dej tsis muaj nucleic acid.Thaum siv, lawv yuav tsum koom nrog hauv kev rho tawm thiab yuav tsum suav tias yog kis tau.Lawv yuav tsum tau tuav thiab muab pov tseg raws li cov kev cai cuam tshuam.
Internal reference gene yog tib neeg RnaseP gene.
Cia tej yam kev mob thiab hnub tas sij hawm
-20 ± 5 ℃, tsis txhob rov khov thiab thawing ntau tshaj 5 zaug, siv tau rau 6 lub hlis.
Cov cuab yeej siv tau
Nrog FAM / HEX / CY5 thiab lwm yam khoom siv fluorescent PCR.
Cov qauv xav tau
1.Cov qauv siv tau: caj pas swabs, nasopharyngeal swabs, bronchoalveolar lavage kua, hnoos qeev.
2.Specimen collection (cov txheej txheem aseptic)
Pharyngeal swab: So cov tonsils thiab posterior pharyngeal phab ntsa nrog ob lub swabs tib lub sijhawm, tom qab ntawd muab lub taub hau swab tso rau hauv lub raj tshuaj uas muaj cov tshuaj sampling.
Sputum: Tom qab tus neeg mob hnoos tob, sau cov hnoos qeev rau hauv lub hau ntswj lub raj uas muaj cov tshuaj sampling;bronchoalveolar lavage kua: Qauv los ntawm cov kws kho mob.3. Kev khaws cia thiab kev thauj mus los ntawm cov qauv
Cov qauv rau kev cais tus kab mob thiab kuaj RNA yuav tsum tau sim sai li sai tau.Cov qauv uas tuaj yeem kuaj tau hauv 24 teev tuaj yeem khaws cia ntawm 4 ℃;cov uas tsis tuaj yeem kuaj pom hauv 24
cov xuab moos yuav tsum tau khaws cia ntawm -70 ℃ lossis qis dua (yog tias tsis muaj qhov chaw cia ntawm -70 ℃, lawv yuav tsum yog
khaws cia ib ntus ntawm -20 ℃ tub yees).Cov qauv yuav tsum tsis txhob rov khov thiab thawing thaum thauj.Cov qauv yuav tsum raug xa mus rau chav kuaj sai li sai tau tom qab sau.Yog tias cov qauv yuav tsum tau thauj mus los ntev, kev cia dej khov qhuav tau pom zoo.
Kev sim
1 Kev ua piv txwv thiab RNA rho tawm (piv txwv qhov chaw ua haujlwm)
Nws raug nquahu kom noj 200μl ntawm cov qauv ua kua rau RNA rho tawm.Rau cov txheej txheem rho tawm cuam tshuam, xa mus rau cov lus qhia ntawm cov khoom siv RNA rho tawm lag luam.Ob qhov tsis zoo thiab tsis zoo
tswj hauv cov khoom siv no tau koom nrog hauv kev rho tawm.
2 PCR reagent npaj (reagent npaj cheeb tsam)
2.1 Tshem tag nrho cov khoom ntawm cov khoom siv thiab thaw thiab sib tov ntawm chav tsev kub.centrifuge ntawm 8,000 rpm rau ob peb feeb ua ntej siv;xam qhov xav tau ntawm cov reagents, thiab cov tshuaj tiv thaiv yog npaj raws li qhia hauv qab no:
| Cheebtsam | N pab (25µl system) |
| nCOV primer TaqMan probemixture | 18 µl × N |
| RT-PCR tshuaj tiv thaiv | 2 µl × N |
| *N = number of samples tested + 1 (negative control) + 1 (nCOVzoo tswj) | |
2.2 Tom qab sib tov kom huv si, centrifuge rau ib lub sij hawm luv luv kom tag nrho cov kua ntawm lub raj phab ntsa poob mus rau hauv qab ntawm lub raj, thiab ces aliquot 20 µl amplification system rau hauv PCR raj.
3 Sampling (qhov chaw ua piv txwv)
Ntxiv 5μl ntawm qhov tsis zoo thiab kev tswj zoo tom qab rho tawm.RNA ntawm cov qauv yuav tsum tau kuaj yog ntxiv rau PCR cov tshuaj tiv thaiv raj.
Npog lub raj kom nruj thiab centrifuge ntawm 8,000 rpm rau ob peb feeb ua ntej xa mus rau qhov chaw nrhiav kom pom kev nthuav dav.
4 PCR amplification (ntau qhov chaw kuaj pom)
4.1 Muab cov tshuaj tiv thaiv raj rau hauv cov qauv ntawm tes ntawm qhov ntsuas, thiab teeb tsa cov ntsuas raws li hauv qab no:
| theem | Lub voj voog tus lej | Kub(°C) | Sijhawm | sauqhov chaw |
| Rov qabkev sau ntawv | 1 | 42 | 10 min | - |
| Ua ntej denaturationn | 1 | 95 | 1 min | - |
| Lub voj voog | 45 | 95 | 15s ib | - |
| 60 | 30s ib | sau cov ntaub ntawv |
Instrument detection channel xaiv: Xaiv FAM, HEX, CY5 channel rau cov teeb liab fluorescence.Rau kev siv fluorescent TSIS MUAJ, thov tsis txhob xaiv ROX.
5 Kev txheeb xyuas qhov tshwm sim (Thov xa mus rau cov lus qhia kev sim ntawm txhua lub cuab yeej rau kev teeb tsa)
Tom qab cov tshuaj tiv thaiv, txuag cov txiaj ntsig.Tom qab kev tsom xam, kho tus nqi pib, qhov kawg ntawm tus nqi, thiab tus nqi pib ntawm qhov pib raws li daim duab (tus neeg siv tuaj yeem kho raws li qhov xwm txheej tiag tiag, tus nqi pib tuaj yeem teem rau 3 ~ 15, tus nqi kawg tuaj yeem teem rau 5 ~ 20, kev hloov kho) nyob rau hauv lub logarithmic graph Nyob rau ntawm qhov chaw pib ntawm lub qhov rais, qhov pib kab yog nyob rau hauv lub logarithmic theem, thiab lub amplification nkhaus ntawm qhov kev tswj tsis zoo yog ib txoj kab ncaj nraim los yog hauv qab txoj kab pib).
6 Quauty tswj (Ib txheej txheem tswj tau suav nrog hauv kev sim) Kev tswj tsis zoo: Tsis muaj qhov pom tseeb ntawm kev nthuav dav rau FAM, HEX, CY5 nrhiav kom tau raws
COV kev tswj tau zoo: pom tseeb kev nthuav dav nkhaus ntawm FAM thiab HEX nrhiav kom tau raws, Ct tus nqi≤32, tab sis tsis muaj qhov nthuav dav ntawm CY5 channel;
Cov kev cai saum toj no yuav tsum tau ua tiav ib txhij hauv tib qhov kev sim;txwv tsis pub, qhov kev sim ua tsis raug thiab yuav tsum tau rov ua dua.
7 Kev txiav txim siab ntawm cov txiaj ntsig.
7.1 Yog tias tsis muaj qhov nkhaus nkhaus lossis Ct tus nqi> 40 hauv FAM thiab HEX raws ntawm cov qauv kuaj, thiab muaj qhov nthuav dav hauv CY5 channel, nws tuaj yeem txiav txim siab tias tsis muaj tus kabmob tshiab 2019 (2019-nCoV) RNA hauv tus qauv;
.2 Yog tias cov qauv kuaj pom muaj qhov ua kom pom tseeb qhov nkhaus hauv FAM thiab HEX raws, thiab tus nqi Ct yog ≤40, nws tuaj yeem txiav txim siab tias tus qauv zoo rau 2019 tus kabmob tshiab (2019-nCoV).
7.3 Yog hais tias tus qauv xeem muaj ib tug meej amplification nkhaus nkaus xwb nyob rau hauv ib tug channel ntawm FAM los yog HEX, thiab tus nqi Ct yog ≤40, thiab tsis muaj amplification nkhaus nyob rau hauv lwm yam channel, cov ntsiab lus yuav tsum tau rov kuaj dua.Yog tias cov txiaj ntsig rov qab zoo sib xws, cov qauv tuaj yeem txiav txim siab ua qhov zoo rau qhov tshiab
Coronavirus 2019 (2019-nCoV).Yog tias qhov ntsuas rov qab tsis zoo, nws tuaj yeem txiav txim siab tias tus qauv tsis zoo rau 2019 tus kabmob tshiab (2019-nCoV).
Kev txiav txim siab zoo
ROC txoj kev nkhaus yog siv los txiav txim qhov siv CT tus nqi ntawm cov khoom siv thiab tus nqi tswj sab hauv yog 40.
Kev txhais cov txiaj ntsig kev xeem
1.Txhua qhov kev sim yuav tsum tau kuaj xyuas qhov tsis zoo thiab kev tswj xyuas zoo.Cov txiaj ntsig kev ntsuas tsuas tuaj yeem txiav txim siab thaum tswj tau raws li kev tswj hwm zoo
2.Thaum FAM thiab HEX nrhiav tau raws li qhov zoo, qhov tshwm sim los ntawm CY5 channel (sab hauv tswj channel) tej zaum yuav tsis zoo vim yog kev sib tw system.
3.Thaum qhov kev tswj xyuas sab hauv yog qhov tsis zoo, yog tias qhov ntsuas lub raj FAM thiab HEX nrhiav tau qhov tsis zoo, , nws txhais tau hais tias lub kaw lus tsis ua haujlwm lossis kev ua haujlwm tsis raug, t qhov kev xeem tsis raug.Yog li ntawd, cov qauv yuav tsum tau rov kuaj dua.
