Monkeypox Virus Ixesha lokwenyani PCR Kit
Iinkcukacha zemveliso
Ukusetyenziswa okucetywayo
Isetyenziselwa ukubhaqa i-Monkeypox Virus kwi-serum yabantu okanye iisampuli ze-lesion exudate ngokusebenzisa iinkqubo ze-PCR zexesha lokwenyani.
Umgaqo woVavanyo
Le mveliso yinkqubo yovavanyo ye-PCR esekelwe kwi-fluorescent probe-based Taqman® real-time PCR.Iiprimers ezithile kunye neeprobe ziyilelwe ukuchongwa kweF3L gene yeMonkeypox Virus.Ulawulo lwangaphakathi olujolise kwimfuza egciniweyo yomntu ebeka iliso kwingqokelela yesampulu, ukuphathwa kwesampulu kunye nenkqubo yePCR yexesha langempela ukuphepha iziphumo ezingezizo zobuxoki.Ikhithi yinkqubo epheleleyo ye-premix lyophilized, equka izinto ezifunekayo kwiMonkeypox Virus ukubona: i-nucleic acid amplification enzyme, i-enzyme ye-UDG, i-reaction buffer, i-primer ethile kunye ne-probe.
Imixholo ephambili
Amacandelo anikiweyo adweliswe kwitheyibhile.
| Amacandelo | Iphakheji | Isithako |
| Intsholongwane yeMonkeypoxI-Lyophilized Premix | Iityhubhu ezi-8 zePCR× iipowutshi ezi-6 | Iiprimers, probes, dNTP/dUTP Mix, Mg2+, Taq DNA polymerase, UDG Enzyme |
| Ulawulo oluhle lweMPV | 400 μL×1 ityhubhu | Ulandelelwano lwe-DNA equlethe ijini ekujoliswe kuyo |
| Ulawulo olubi lweMPV | 400 μL×1 ityhubhu | Ulandelelwano lwe-DNA equlethe icandelo lemfuza yomntu |
| Ukunyibilikisa Isisombululo | 1 mL × 1 ityhubhu | Isizinzi |
| Isatifikethi sokuThobela | Iqhekeza eli-1 | / |
Operation Flow
1. IsampuliIngqokelela:Isampulu kufuneka iqokelelwe kwiityhubhu eziyinyumba ngokuhambelanayo
kunye neenkcukacha zobugcisa ezisemgangathweni.
2. ULungiselelo lwe-Reagent (iNdawo yokuLungiselela i-Reagent)
Thatha amacandelo ekhithi, uwalinganisele kwiqondo lobushushu begumbi ukwenzela ukusetyenziswa kokulinda.
3. Ukwenziwa koMfanekiso (iNdawo yokuLungiselela iSampuli)
3.1 Ukukhutshwa kwe-Nucleic acid
Kucetyiswa ukuba kuthathwe iisampulu zolwelo ezingama-200μL, Ulawulo olulungileyo kunye noLawulo olubi lwe-nucleic acid extraction, ngokweemfuno ezihambelanayo kunye neenkqubo zeekhithi zokutsalwa kwe-viral DNA.
3.2 I-Lyophilized powder yokunyibilika kunye nokudibanisa itemplate
Lungiselela iMonkeypox Virus Lyophilized premix ngokwenani leisampulu.Isampuli enye idinga ityhubhu ye-PCR ene-Lyophilized premix powder.Ulawulo olubi kunye nolawulo olulungileyo kufuneka luphathwe njengeesampuli ezimbini.
(1) Yongeza i-15μL Ukunyibilika kweSisombululo kwityhubhu nganye ye-PCR equlethe i-Lyophilized premix, uze udibanise i-5μL ekhutshiweyo iisampulu / Ulawulo olubi / uLawulo oluLungileyo kwityhubhu nganye ye-PCR ngokulandelelana.
(2) Gubungela iityhubhu ze-PCR ngokuqinileyo, nqakraza iityhubhu zePCR ngesandla de umgubo we-lyophilized unyibilike ngokupheleleyo kwaye udityaniswe, uqokelele ulwelo ukuya ezantsi kwiityhubhu zePCR ngesantya esisezantsi secentrifugation.
(3) Ukuba usebenzisa isixhobo esiqhelekileyo se-PCR yexesha eliqhelekileyo lokubona, ke udlulisele ngokuthe ngqo iityhubhu zePCR kwindawo yokukhulisa;ukuba usebenzisa i-BTK-8 ukuze ubhaqwe, ngoko kufuneka wenze le misebenzi ilandelayo: ukudlulisa i-10 μL yolwelo ukusuka kwityhubhu ye-PCR ukuya kwi-chip yokusabela kakuhle ye-BTK-8.Ityhubhu enye yePCR ihambelana nequla elinye kwitshiphu.Ngethuba lokusebenza kombhobho, qinisekisa ukuba i-pipette i-90 degrees nkqo.Iingcebiso ze-aerosol barrier pipette kufuneka zibekwe embindini womthombo ngamandla aphakathi kwaye ziyeke ukutyhala i-pipette xa ifikelela kwi-gear yokuqala (ukuphepha ama-bubbles).Emva kokuba iqula lizaliswe, thatha inwebu ye-chip yokusabela ukugquma onke amaqula kwaye itshiphu idluliselwe kwindawo yokufumanisa ukukhulisa.
4. Ukwandiswa kwePCR (iNdawo yoFundo)
4.1 Beka iityhubhu ze-PCR / i-reaction chip kwitanki yokusabela kwaye usete amagama empendulo nganye kakuhle ngokulandelelana okuhambelanayo.
4.2 Iisetingi zokufumanisa i-fluorescence: (1) iMonkeypox virus (FAM);(2) Ulawulo lwangaphakathi (CY5).
4.3 Qhuba le migaqo ilandelayo yokukhwela ibhayisekile
Iprothokholi ye-ABI7500, i-Bio-Rad CFX96, i-SLAN-96S, i-QuantStudio:
| Amanyathelo | Ubushushu | Ixesha | Imijikelo | |
| 1 | I-denaturation yangaphambili | 95℃ | 2 imiz | 1 |
| 2 | I-Denaturation | 95℃ | 10 s | 45 |
| I-Anealing, ukwandiswa, ukufumana i-fluorescence | 60℃ | 30 s | ||
Inkqubo ye-BTK-8:
| Amanyathelo | Ubushushu | Ixesha | Imijikelo | |
| 1 | I-denaturation yangaphambili | 95℃ | 2 imiz | 1 |
| 2 | I-Denaturation | 95℃ | 5 s | 45 |
| I-Anealing, ukwandiswa, ukufumana i-fluorescence | 60℃ | 14 s | ||
5. Uhlalutyo lweziphumo (nceda ubhekisele kwi-Instrument User Manual)
Emva kokusabela, iziphumo ziya kugcinwa ngokuzenzekelayo.Cofa "Hlaziya" ukuhlalutya, kwaye isixhobo siya kutolika ngokuzenzekelayo amaxabiso e-Ct yesampuli nganye kwikholamu yesiphumo.Iziphumo zolawulo ezingalunganga nezilungileyo ziyakuhambelana noku kulandelayo "6. Ulawulo loMgangatho".
6. Ulawulo lwemeko
6.1 Ulawulo olubi: Akukho Ct okanye i-Ct> 40 kwitshaneli ye-FAM, i-Ct≤40 kwi-CY5 channel ene-curve yokukhulisa eqhelekileyo.
6.2 Ulawulo oluLungileyo: I-Ct≤35 kwitshaneli ye-FAM ene-curve ye-amplification eqhelekileyo, i-Ct≤40 kwi-CY5 channel ene-curve ye-amplification eqhelekileyo.
6.3 Iziphumo zisebenza ngokusemthethweni ukuba zonke ezi khrayitheriya zifikelelwe.Ngaphandle koko, umphumo awusebenzi.
Isiphumo sokutolika
Ezi ziphumo zilandelayo zinokwenzeka:
| Ct ixabiso letshaneli yeFAM | Ixabiso le-CT ye-CY5 channel | Ukutolikwa | |
| 1# | Akukho Ct okanye Ct>40 | ≤40 | Intsholongwane yeMonkeypox ayinayo |
| 2# | ≤40 | Naziphi na iziphumo | Intsholongwane yemonkeypox |
| 3# | 40-45 | ≤40 | Uvavanyo kwakhona;ukuba isengama 40 ~ 45, xela njenge 1# |
| 4# | Akukho Ct okanye Ct>40 | Akukho Ct okanye Ct>40 | Ayisebenzi |
PHAWULA: Ukuba iziphumo ezingasebenziyo zenzekile, isampuli kufuneka iqokelelwe kwaye ivavanywe kwakhona.
Ulwazi lweodolo
| Igama lemveliso | Ikati.Hayi | Ubungakanani | Umzekelo | Beka ubomi kwishelufa | Trans.& Sto.Temp. |
| Monkeypox Virus Ixesha lokwenyani PCR Kit | B001P-01 | Iimvavanyo ezingama-48/ikhithi | ISerum / Lesion Exudate | Iinyanga ezili-12 | -25～-15℃ |
