Lihlahisoa
Lifecosm SARS-Cov-2-RT-PCR Detection kit bakeng sa 2019-nCoV
Tšebeliso e lebeletsoeng
Setsi sena se sebelisetsoa ho lemoha boemo ba "coronavirus" e ncha (2019-nCoV) ho sebelisoa li-swabs tsa 'metso, nasopharyngeal swabs, bronchoalveolar lavage fluid, sputum. bopaki bakeng sa tlhahlobo ea bongaka le kalafo.Ho khothaletsoa tlhahlobo e felletseng ea boemo hammoho le lipontšo tsa kliniki tsa mokuli le liteko tse ling tsa laboratori.
Molao-motheo oa tlhahlobo
Thepa e ipapisitse le theknoloji ea RT-PCR ea mohato o le mong.Ebile, liphatsa tsa lefutso tse ncha tsa 2019 (2019-nCoV) ORF1ab le N li ile tsa khethoa e le libaka tseo ho lebeletsoeng ho tsona.Li-primers tse khethehileng le li-fluorescent probes (li-N gene probes li ngotsoe ka FAM le li-probe tsa ORF1ab li ngotsoe ka HEX) li etselitsoe ho bona mofuta o mocha oa coronavirus RNA ea 2019 ka lisampole.Setsi sena se boetse se kenyelletsa sistimi ea taolo ea kahare ea taolo ea kahare (probe ea gene e ngotsoeng ka CY5) ho lekola ts'ebetso ea pokello ea sampole, RNA le PCR amplification, ka hona ho fokotsa liphetho tse fosahetseng.
Likarolo tse ka sehloohong
| Likaroloana | Bolumo（48T/Kit） |
| Tharollo ea karabelo ea RT-PCR | 96µl |
| nCOV primer TaqMan probemixture (ORF1ab,N Gene,RnaseP Gene) | 864µl |
| Taolo e mpe | 1500µl |
| nCOV Positive contro (l ORF1ab N Gene) | 1500µl |
Li-reagents tsa hau: Ho ntšoa ha RNA kapa li-reagents tsa tlhoekiso.Taolo e mpe / e ntle: Taolo e ntle ke RNA e nang le sekhechana sa sepheo, athe taolo e mpe ke metsi a se nang nucleic acid.Nakong ea tšebeliso, ba lokela ho kenya letsoho ho ntšoa 'me ba lokela ho nkoa ba tšoaetsanoa.Li lokela ho tšoaroa le ho lahloa ho latela melaoana e amehang.
Mofuta oa ka hare oa referense ke mofuta oa RnaseP oa motho.
Maemo a polokelo le letsatsi la ho felloa ke nako
-20 ± 5 ℃, qoba ho qhoqhoa khafetsa le ho qhibiliha ho feta makhetlo a 5, e sebetsang bakeng sa likhoeli tse 6.
Sesebelisoa se sebetsang
Ka FAM / HEX / CY5 le lisebelisoa tse ling tsa PCR tsa fluorescent tse ngata.
Litlhoko tsa mohlala
1. Mefuta e sebetsang ea mohlala: likhohlela tsa 'metso, nasopharyngeal swabs, bronchoalveolar lavage fluid, sekhohlela.
2. Pokello ea mohlala (mokhoa oa aseptic)
Pharyngeal swab: Hlakola lithane le lebota le ka morao la pharyngeal ka li-swabs tse peli ka nako e le 'ngoe, ebe u qoelisa hlooho ka har'a tube ea teko e nang le tharollo ea sampole.
Sekhohlela: Ka mor'a hore mokuli a khohlele haholo, bokella sekhohlela se khohlela ka har'a sekoti sa ho hlahloba sekoahelo se nang le tharollo ea sampole;Bronchoalveolar lavage fluid: Mohlala ke litsebi tsa bongaka.3.Ho boloka le ho tsamaisa lisampole
Mehlala ea ho aroloa ha vaerase le tlhahlobo ea RNA e lokela ho hlahlojoa kapele kamoo ho ka khonehang.Mehlala e ka bonoang nakong ea lihora tse 24 e ka bolokoa ho 4 ℃;tse ke keng tsa bonoa nakong ea 24
lihora li lokela ho bolokoa ho -70 ℃ kapa ka tlase (haeba ho se na boemo ba polokelo ea -70 ℃, li lokela ho
bolokoa nakoana ka sehatsetsing -20℃).Mehlala e lokela ho qoba ho qhoqhoa khafetsa le ho qhibiliha nakong ea lipalangoang.Mehlala e lokela ho romelloa laboratoring kapele kamoo ho ka khonehang ka mor'a ho bokelloa.Haeba lisampole li hloka ho tsamaisoa libakeng tse telele, ho khothalletsoa ho boloka leqhoa le omileng.
Mekhoa ea Tlhahlobo
1 Mohlala oa ts'ebetso le tlhahiso ea RNA (sebaka sa ts'ebetso ea mohlala)
Ho khothalletsoa ho nka 200μl ea sampole ea metsi bakeng sa ho ntšoa ha RNA.Bakeng sa mehato e amanang le ho ntša, sheba litaelo tsa lisebelisoa tsa RNA tsa khoebo.Ka bobeli tse mpe le tse mpe
Litaolo ka har'a khithi ena li ne li ameha ho ntšeng.
Tokiso ea 2 PCR reagent (sebaka sa ho lokisa reagent)
2.1 Tlosa likarolo tsohle tsa kit 'me u qhibilihe ebe u kopanya mocheso oa kamore.Centrifuge ka 8,000 rpm metsotsoana e seng mekae pele e sebelisoa;bala palo e hlokahalang ea li-reagents, 'me tsamaiso ea karabelo e lokiselitsoe joalokaha ho bontšitsoe tafoleng e latelang:
| Likaroloana | N sebeletsa (25µl sistimi) |
| NCOV primer TaqMan probemixture | 18µl × N |
| Tharollo ea karabelo ea RT-PCR | 2 µl × N |
| *N = palo ea lisampole tse lekiloeng + 1 (taolo e mpe) + 1 (nCOVtaolo e ntle) | |
2.2 Kamora ho kopanya likarolo ka botlalo, centrifuge ka nako e khuts'oane ho tlohella mokelikeli o mong le o mong o leboteng la tube ho oela tlase ho thapo, ebe o kenya sistimi ea amplification ea 20 µl ka har'a tube ea PCR.
3 Sampling (sebaka sa ho lokisetsa mohlala)
Kenya 5μl ea taolo e mpe le e ntle ka mor'a ho ntšoa.RNA ea sampuli e lokelang ho lekoa e kenyelelitsoe ho tube ea karabelo ea PCR.
Koahela tube ka thata le centrifuge ho 8,000 rpm metsotsoana e seng mekae pele u e fetisetsa sebakeng sa ho lemoha matlafatso.
4 PCR amplification (sebaka se matlafalitsoeng sa ho lemoha)
4.1 Beha tube ea karabelo ka seleng ea mohlala ea sesebelisoa, 'me u behe li-parameter ka tsela e latelang:
| sethala | Baesekele palo | Mocheso(°C) | Nako | pokellosebaka |
| Kgutlasengoliloeng | 1 | 42 | 10 mets | - |
| Pele ho denaturationn | 1 | 95 | 1 mets | - |
| Baesekele | 45 | 95 | 15s | - |
| 60 | 30s | pokello ea lintlha |
Khetho ea mocha oa ho lemoha lisebelisoa: Khetha mocha oa FAM, HEX, CY5 bakeng sa lets'oao la fluorescence.Bakeng sa referense ea fluorescent NONE, ka kopo u se ke oa khetha ROX.
5 Sephetho sa tlhahlobo (Ka kopo, sheba litaelo tsa liteko tsa sesebelisoa ka seng bakeng sa ho seta)
Kamora karabelo, boloka liphetho.Ka mor'a tlhahlobo, fetola boleng ba ho qala, boleng ba ho qetela, le boleng ba moeli oa motheo ho ea ka setšoantšo (mosebelisi a ka fetola ho ea ka boemo ba sebele, boleng ba ho qala bo ka behoa ho 3 ~ 15, boleng ba ho qetela bo ka behoa ho 5 ~ 20, phetoho) ho graph ea logarithmic Moemong oa fensetere, moeli oa monyako o karolong ea logarithmic, 'me sekhahla sa amplification sa taolo e mpe ke mola o otlolohileng kapa ka tlas'a moeli oa moeli).
6 Taolo ea quauty (Taolo ea ts'ebetso e kenyellelitsoe tekong) Taolo e fosahetseng: Ha ho na lekhalo le hlakileng la amplification bakeng sa FAM, HEX, CY5 liteishene tsa ho lemoha
Taolo e ntle ea COV: curve e hlakileng ea amplification ea liteishene tsa ho lemoha tsa FAM le HEX, Ct value≤32, empa ha ho curve ea amplification ea CY5 channel;
Litlhoko tse ka holimo li tlameha ho finyelloa ka nako e le 'ngoe tekong e tšoanang;ho seng joalo, teko ha e sebetse 'me e hloka ho phetoa.
7 Boikemisetso ba liphetho.
7.1 Haeba ho se na sekhahla sa amplification kapa Ct value> 40 liteisheneng tsa FAM le tsa HEX tsa sampole ea tlhahlobo, 'me ho na le lekhalo la amplification mocha oa CY5, ho ka ahloloa hore ha ho na coronavirus e ncha ea 2019 (2019-nCoV) RNA ka mohlala;
.2 Haeba sampole ea tlhahlobo e na le li-curve tsa amplification tse hlakileng ho likanale tsa FAM le HEX, 'me boleng ba Ct ke ≤40, ho ka ahloloa hore sampole e ntle bakeng sa coronavirus e ncha ea 2019 (2019-nCoV).
7.3 Haeba sampole ea tlhahlobo e na le sekhahla se hlakileng sa amplification feela mocha o le mong oa FAM kapa HEX, 'me boleng ba Ct ke ≤40, 'me ha ho lekhalo la amplification mocha o mong, liphetho li hloka ho hlahlojoa hape.Haeba liphetho tsa tlhahlobo li lumellana, sampole e ka nkoa e le ntle bakeng sa e ncha
coronavirus 2019 (2019-nCoV).Haeba sephetho sa tlhahlobo hape se le mpe, ho ka ahloloa hore sampole e fosahetse bakeng sa coronavirus e ncha ea 2019 (2019-nCoV).
Boleng ba kahlolo e ntle
Mokhoa oa curve oa ROC o sebelisoa ho fumana boleng ba CT ea kit mme boleng ba litšupiso tsa taolo ea kahare ke 40.
Tlhaloso ea liphetho tsa liteko
1. Teko ka 'ngoe e lokela ho lekoa bakeng sa taolo e mpe le e ntle.Liphetho tsa liteko li ka khethoa feela ha litsamaiso li fihlela litlhoko tsa taolo ea boleng
2.Ha liteishene tsa ho lemoha tsa FAM le tsa HEX li le ntle, phello e tsoang ho CY5 channel (mocha oa taolo ea ka hare) e ka 'na ea e-ba mpe ka lebaka la tlhōlisano ea tsamaiso.
3.Ha sephetho sa taolo ea ka hare se le mpe, haeba tthe test tube's FAM le li-channel tsa HEX le tsona li le mpe, ho bolela hore sistimi e koetsoe kapa ts'ebetso e fosahetse, tlhahlobo ha e sebetse.Ka hona, lisampole li hloka ho hlahlojoa hape.
