I-Monkeypox Virus Isikhathi Sangempela PCR Kit
Imininingwane yomkhiqizo
Ukusetshenziswa Okuhlosiwe
Isetshenziselwa ukutholwa kwe-Monkeypox Virus ku-serum yomuntu noma amasampula e-lesion exudate ngokusebenzisa amasistimu e-PCR esikhathi sangempela.
Isimiso Sokuhlola
Lo mkhiqizo uwuhlelo lokuhlola lwe-PCR olusekelwe ku-fluorescent olusekelwe ku-fluorescent.Iziqalo ezithile kanye nama-probe akhelwe ukutholwa kofuzo lwe-F3L ye-Monkeypox Virus.Ukulawula kwangaphakathi okuqondise isakhi sofuzo esilondoloziwe somuntu siqapha ukuqoqwa kwesampula, ukuphathwa kwesampula kanye nenqubo ye-PCR yesikhathi sangempela ukuze kugwenywe imiphumela engemihle engamanga.Ikhithi iwuhlelo oluphelele lwe-lyophilized premix, oluhlanganisa izinto ezidingekayo ukuze kutholwe i-Monkeypox Virus: i-nucleic acid amplification enzyme, i-enzyme ye-UDG, i-react buffer, i-primer ethize kanye ne-probe.
Okuqukethwe Okuyinhloko
Izingxenye ezinikeziwe zibalwe kuthebula.
| Izingxenye | Iphakheji | Isithako |
| I-Monkeypox VirusI-Lyophilized Premix | 8 amashubhu e-PCR× 6 izikhwama | Iziqalo, ama-probes, i-dNTP/dUTP Mix, i-Mg2+, i-Taq DNA polymerase, i-UDG Enzyme |
| I-MPV Positive Control | 400 μL×1 ishubhu | Ukulandelana kwe-DNA equkethe isakhi sofuzo esiqondiwe |
| Ukulawula Okungalungile kwe-MPV | 400 μL×1 ishubhu | Ukulandelana kwe-DNA equkethe ingxenye yofuzo lomuntu |
| Ukuqedwa Isixazululo | 1 mL×1 ishubhu | Isiqinisi |
| Isitifiketi Sokuhambisana | 1 isiqephu | / |
Ukugeleza Kokusebenza
1. IsampulaIqoqo:Isampula kufanele iqoqwe kumashubhu oyinyumba ngokuhambisana
ngezincazelo ezijwayelekile zobuchwepheshe.
2. Ukulungiswa kwe-Reagent (Indawo Yokulungiselela I-Reagent)
Khipha izingxenye zekhithi, uzilinganise ekamelweni lokushisa ukuze zisetshenziswe ngokulinda.
3. Ukucutshungulwa Kwesampula (Indawo Yokucubungula Isifanekiso)
3.1 Ukukhishwa kwe-Nucleic acid
Kunconywa ukuthatha amasampula oketshezi angama-200μL, Ukulawula Okuhle kanye Nokulawula Okubi ukuze kukhishwe i-nucleic acid, ngokuya ngezidingo ezihambisanayo nezinqubo zamakhithi e-viral DNA extraction.
3.2 Ukuncibilika kwempushana ye-Lyophilized kanye nokwengezwa kwesifanekiso
Lungiselela i-Monkeypox Virus I-Lyophilized premix ngokwenani lamasampuli.Isampula eyodwa idinga ishubhu elilodwa le-PCR eliqukethe i-Lyophilized premix powder.Ukulawula okungalungile nokulawula okuhle kufanele kuthathwe njengamasampula amabili.
(1) Engeza Isixazululo Sokuhlakaza esingu-15μL kushubhu ngayinye ye-PCR equkethe i-Lyophilized premix, bese wengeza amasampula akhishiwe angu-5μL/ Ukulawula Okubi/Ukulawula Okuhle kushubhu ngayinye ye-PCR ngokulandelanayo.
(2) Mboza amashubhu e-PCR ngokuqinile, qhweba amashubhu e-PCR ngesandla kuze kube yilapho impushana e-lyophilized ichitheka ngokuphelele futhi ihlanganiswa, qoqa uketshezi uye phansi kwamashubhu e-PCR ngokuxhumanisa okunesivinini esiphansi ngokushesha.
(3) Uma usebenzisa ithuluzi elivamile le-PCR lesikhathi sangempela ukuze libonwe, bese udlulisela ngokuqondile amashubhu e-PCR endaweni yokukhulisa;uma usebenzisa i-BTK-8 ukuze uthole, kuzomele wenze imisebenzi elandelayo: dlulisa uketshezi oluyi-10 μL ukusuka kushubhu ye-PCR ukuya kumthombo we-chip wokusabela we-BTK-8.Ishubhu elilodwa le-PCR lihambisana nomthombo owodwa ku-chip.Ngesikhathi sokusebenza kwamapayipi, qiniseka ukuthi i-pipette iqondile ngama-degree angama-90.Amathiphu we-aerosol barrier pipette kufanele abekwe phakathi nendawo yomthombo ngamandla alinganiselwe futhi ayeke ukusunduza i-pipette lapho ifika egiyeni lokuqala (ukugwema ama-bubbles).Ngemva kokugcwaliswa kwemithombo, khipha ulwelwesi lwe-chip olusabelayo ukuze lumboze yonke imithombo bese i-chip idluliselwa endaweni yokuthola ukukhulisa i-amplification.
4. I-PCR Amplification (Indawo Yokuthola)
4.1 Faka amashubhu e-PCR/i-rection chip ethangini lokusabela bese usetha amagama okusabela ngakunye kahle ngokulandelana okuhambisanayo.
4.2 Izilungiselelo zokutholwa kwe-fluorescence: (1) Igciwane lemonkeypox (FAM);(2) Ukulawula Kwangaphakathi (CY5).
4.3 Qalisa iphrothokholi yokugibela ibhayisikili elandelayo
Iphrothokholi ye-ABI7500, Bio-Rad CFX96, SLAN-96S, QuantStudio:
| Izinyathelo | Izinga lokushisa | Isikhathi | Imijikelezo | |
| 1 | I-pre-denaturation | 95℃ | 2 imiz | 1 |
| 2 | I-Denaturation | 95℃ | 10 s | 45 |
| I-Annealing, isandiso, ukutholwa kwe-fluorescence | 60℃ | 30 s | ||
Iphrothokholi ye-BTK-8:
| Izinyathelo | Izinga lokushisa | Isikhathi | Imijikelezo | |
| 1 | I-pre-denaturation | 95℃ | 2 imiz | 1 |
| 2 | I-Denaturation | 95℃ | 5 s | 45 |
| I-Annealing, isandiso, ukutholwa kwe-fluorescence | 60℃ | 14 ik | ||
5. Ukuhlaziywa kwemiphumela (sicela ubhekisele ku-Instrument User Manual)
Ngemva kokusabela, imiphumela izosindiswa ngokuzenzakalelayo.Chofoza okuthi “Hlaziya” ukuze uhlaziye, futhi ithuluzi lizotolika ngokuzenzakalelayo amanani e-Ct wesampula ngayinye kukholomu yomphumela.Imiphumela yokulawula engemihle futhi emihle izohambisana nalokhu okulandelayo "6. Ukulawulwa Kwekhwalithi".
6. Ikhwalithi yokulawula
6.1 Ukulawula Okubi: Ayikho i-Ct noma i-Ct>40 esiteshini se-FAM, i-Ct≤40 esiteshini se-CY5 esinejika elivamile lokukhulisa.
I-6.2 Ukulawula Okuhle: I-Ct≤35 esiteshini se-FAM esinejika elijwayelekile lokukhulisa, i-Ct≤40 esiteshini se-CY5 esinejika elivamile lokukhulisa.
6.3 Umphumela uyasebenza uma zonke lezi zimo ezingenhla zifinyelelwe.Uma kungenjalo, umphumela awuvumelekile.
Ukuhunyushwa komphumela
Imiphumela elandelayo ingenzeka:
| Inani le-Ct lesiteshi se-FAM | Inani le-Ct lesiteshi se-CY5 | Ukuhunyushwa | |
| 1# | Akukho Ct noma Ct>40 | ≤40 | I-monkeypox virus negative |
| 2# | ≤40 | Noma yimiphi imiphumela | Igciwane lemonkeypox |
| 3# | 40; 45 | ≤40 | Hlola kabusha;uma kusengama-40~45, bika njengo-1# |
| 4# | Akukho Ct noma Ct>40 | Akukho Ct noma Ct>40 | Akuvumelekile |
QAPHELA: Uma umphumela ongavumelekile uvela, isampuli idinga ukuqoqwa futhi ihlolwe futhi.
Oda Ulwazi
| Igama Lomkhiqizo | Ikati.Cha | Usayizi | Isifanekiso | I-Shelf Life | Trans.& Sto.Temp. |
| I-Monkeypox Virus Isikhathi Sangempela PCR Kit | B001P-01 | 48 ukuhlolwa/ikhithi | I-Serum / Lesion Exudate | Izinyanga eziyi-12 | -25℃-15℃ |
