Monkeypox Virus Nako ea 'Nete PCR Kit
Lintlha tsa sehlahisoa
Tšebeliso e reriloeng
E sebelisoa bakeng sa ho lemoha Monkeypox Virus ka serum ea motho kapa lisampole tsa lesion exudate ka ho sebelisa lisebelisoa tsa PCR tsa nako ea sebele.
Teko Molao-motheo
Sehlahisoa sena ke sistimi ea tlhahlobo ea PCR ea nako ea nnete ea Taqman® e thehiloeng ho fluorescent.Li-primers le li-probes tse khethehileng li etselitsoe ho lemoha mofuta oa F3L oa Monkeypox Virus.Taolo ea ka hare e shebaneng le mofuta o bolokiloeng oa motho e lekola pokello ea sampole, ts'ebetso ea sampole le ts'ebetso ea nako ea nnete ea PCR ho qoba liphetho tse fosahetseng.The kit ke full premix lyophilized system, e kenyeletsang lisebelisoa tse hlokahalang bakeng sa ho lemoha Monkeypox Virus: nucleic acid amplification enzyme, UDG enzyme, reaction buffer, primer e khethehileng le probe.
Litaba Tse ka Sehloohong
Likarolo tse fanoeng li thathamisitsoe tafoleng.
| Likaroloana | Sephutheloana | Motsoako |
| Kokoana ea MonkeypoxLyophilized Premix | Li-tubes tse 8 tsa PCR× 6 mekotla | Li-primers, probes, dNTP/dUTP Mix, Mg2+, Taq DNA polymerase, Enzyme ea UDG |
| Taolo e Ntle ea MPV | 400 μL × 1 tube | DNA tatelano e nang le Target gene |
| Taolo e Fosahetseng ea MPV | 400 μL × 1 tube | Tatelano ea DNA e nang le karolo ea lefutso la motho |
| Tharollo ea ho Qhala | 1 mL × 1 tube | Setsibotsi |
| Setifikeiti sa ho lumellana | 1 sengoathoana | / |
Phallo ea Ts'ebetso
1. MohlalaPokello:Setšoantšo se lokela ho bokelloa ka har'a li-tubes tse nyopa ho latela
ka litlhaloso tse tloaelehileng tsa tekheniki.
2. Tokiso ea Reagent (Sebaka sa Tokiso ea Reagent)
Ntša likarolo tsa kit, li leka-lekane mocheso oa kamore bakeng sa tšebeliso ea standby.
3. Ntlafatso ea Mohlala (Sebaka sa Ts'ebetso ea Mohlala)
3.1 Ho tlosoa ha Nucleic acid
E kgothaletswa ho nka 200μL mokelikeli disampole, Positive Control le Negative Taolo bakeng sa nucleic acid e ntšitsoeng, ho ea ka ditlhoko tse tsamaellanang le mekgwa ya tshebetso ya ho kokoana-hloko DNA extraction kits.
3.2 Ho qhibiliha ha phofo ea Lyophilized le ho eketsa template
Lokisetsa Monkeypox Virus Lyophilized premix ho latela palo ea lisampole.Mohlala o le mong o hloka tube e le 'ngoe ea PCR e nang le phofo ea Lyophilized premix.Taolo e mpe le taolo e nepahetseng e lokela ho nkuoa joalo ka mehlala e 'meli.
(1) Eketsa Tharollo ea ho Hlakola 15μL ka har'a tube e 'ngoe le e' ngoe ea PCR e nang le Lyophilized premix, ebe u eketsa 5μL e ntšitsoeng lisampole / Taolo e Negative / Taolo e Ntle ho tube e 'ngoe le e 'ngoe ea PCR ka ho latellana.
(2) Koahela li-tubes tsa PCR ka thata, fafatsa li-tubes tsa PCR ka letsoho ho fihlela phofo ea lyophilized e qhibiliha ka ho feletseng 'me e kopantsoe, bokella mokelikeli ho ea tlaase ho li-tubes tsa PCR ka centrifugation ea lebelo le tlaase hang-hang.
(3) Haeba u sebelisa sesebelisoa se tloaelehileng sa Real-time PCR bakeng sa ho lemoha, joale fetisetsa ka ho toba li-tubes tsa PCR sebakeng sa amplification;Haeba u sebelisa BTK-8 bakeng sa ho lemoha, joale u lokela ho etsa ts'ebetso e latelang: fetisetsa metsi a 10 μL ho tloha ho PCR tube ho ea ho reaction chip seliba sa BTK-8.Tube e le 'ngoe ea PCR e lumellana le seliba se le seng holim'a chip.Nakong ea ts'ebetso ea pipetting, etsa bonnete ba hore pipette e otlolohile ka likhato tse 90.Malebela a aerosol barrier pipette a lokela ho behoa bohareng ba seliba ka matla a itekanetseng 'me a khaotse ho sutumetsa pipette ha e fihla gear ea pele (ho qoba li-bubble).Ka mor'a hore liliba li tlatsoe, ntša lesela la reaction chip ho koahela liliba tsohle ebe chip e fetisetsoa sebakeng sa ho lemoha matlafatso.
4. Phatlalatso ea PCR (Sebaka sa ho Khetholla)
4.1 Kenya li-tubes/rection chip tsa PCR ka tankeng ea karabelo 'me u behe mabitso a karabelo e' ngoe le e 'ngoe hantle ka tatellano e lumellanang.
4.2 Litlhophiso tsa ho lemoha fluorescence: (1) Monkeypox virus (FAM);(2) Taolo ea ka hare (CY5).
4.3 Tsamaisa protocol e latelang ea libaesekele
Protocol ea ABI7500, Bio-Rad CFX96, SLAN-96S, QuantStudio:
| Mehato | Mocheso | Nako | Lipotoloho | |
| 1 | Pre-denaturation | 95℃ | 2 mets | 1 |
| 2 | Denaturation | 95℃ | 10 s | 45 |
| Anealing, katoloso, ho fumana fluorescence | 60 ℃ | 30 s | ||
Protocol ea BTK-8:
| Mehato | Mocheso | Nako | Lipotoloho | |
| 1 | Pre-denaturation | 95℃ | 2 mets | 1 |
| 2 | Denaturation | 95℃ | 5 s | 45 |
| Anealing, katoloso, ho fumana fluorescence | 60 ℃ | 14 s | ||
5. Tlhahlobo ea liphetho (ka kopo, sheba buka ea Instrument User Manual)
Kamora karabelo, liphetho li tla bolokeha ka bohona.Tobetsa "Analyze" ho sekaseka, 'me sesebelisoa se tla fetolela litekanyetso tsa Ct tsa sampole e ngoe le e ngoe kholomong ea sephetho.Liphetho tse mpe le tse ntle tsa taolo li tla lumellana le tse latelang "6. Taolo ea Boleng ".
6. Taolo ea Boleng
6.1 Taolo e Fosahetseng: Ha ho Ct kapa Ct> 40 mocheng oa FAM, Ct≤40 mocha oa CY5 o nang le sekhahla se tloaelehileng sa amplification.
6.2 Taolo e Ntle: Ct≤35 mocheng oa FAM o nang le sekhahla se tloaelehileng sa amplification, Ct≤40 ho CY5 channel e nang le sekhahla se tloaelehileng sa amplification.
6.3 Sephetho se nepahetse haeba lintlha tsohle tse ka holimo li fihletsoe.Ho seng joalo, sephetho ha se sebetse.
Tlhaloso ea Sephetho
Liphetho tse latelang lia khonahala:
| Ct boleng ba mocha oa FAM | Ct boleng ba CY5 channel | Tlhaloso | |
| 1# | Ha ho Ct kapa Ct>40 | ≤40 | Monkeypox virus negative |
| 2# | ≤40 | Liphetho life kapa life | Monkeypox virus positive |
| 3# | 40-45 | ≤40 | Teko hape;haeba e ntse e le 40 ~ 45, tlaleha e le 1# |
| 4# | Ha ho Ct kapa Ct>40 | Ha ho Ct kapa Ct>40 | Ha e sebetse |
HLOKOMELA: Haeba sephetho se fosahetseng se hlaha, sampole e hloka ho bokelloa le ho hlahlojoa hape.
Lintlha tsa Odara
| Lebitso la Sehlahiswa | Katse.Che | Boholo | Mohlala | Shelf Life | Trans.& Sto.Temp. |
| Monkeypox Virus Nako ea 'Nete PCR Kit | B001P-01 | 48 liteko/kit | Serum/ Ketsiso ya Lesion | Likhoeli tse 12 | -25℃-15℃ |
