Escherichia coli O157:H7 Nucleic Acid Detection Kit (Mokhoa oa PCR Fluorescent Probe)
Tlhaloso ea Kit:
Katse.No.FP102
E sebelisetsoa ho lemoha ka potlako le ho hlahloba E. coli O157: H7 lijong, lijong, lisampole tsa metsi le lisampole tsa tikoloho.
Litlhaloso
E sebelisetsoa ho lemoha ka potlako le ho hlahloba E. coli O157: H7 lijong, lijong, lisampole tsa metsi le lisampole tsa tikoloho .
[Molao-motheo oa teko]Ho ea ka molao-motheo oa theknoloji ea fluorescent PCR, li-primers tse khethehileng le li-probe tsa Taqman li etselitsoe mofuta o khethehileng oa Escherichia coli O157: H7, 'me e bonoa ke sesebelisoa sa fluorescent PCR, e le ho lemoha ho fumanoa ha Escherichia coli O157: H7 Ho lemoha boleng ba DNA.
Litaba tsa Kit
Tlhokomeliso: Probe ea mocha oa ROX ha e kenyelelitsoe.
| Cbahanyetsi | Tlhaloso | Qbonngoe |
| Buffer A | Tube | 1 |
| Buffer B | Tube | 1 |
| Taolo e ntle | Tube | 1 |
| Taolo e mpe | Tube | 1 |
Tšebeliso e Lebeletsoeng
E sebelisoa bakeng sa ho lemoha le ho hlahloba ka potlako E. coli O157: H7 lijong, fepa, lisampole tsa metsi le lisampole tsa tikoloho .
Maemo a polokelo le Letsatsi la ho Felloa ke Nako
Boloka ho -20 ℃ lefifing 'me u qobe serame khafetsa le ho qhibiliha.
Nako ea ho sebetsa ke 12likhoeli, 'me letsatsi la tlhahiso le bonts'oa sephutheloana sa kantle.
Lisebelisoa le Lisebelisoa
Sesebelisoa sa Fluorescent quantitative PCR, sethunya sa pipette le malebela a tšoanang, vortex shaker, mini centrifuge.
Tšebeliso
1. Mohlala oa ho sebetsa
1.1 Mofuta oa mohlala: Setsi sena se loketse lijo, phepo, lisampole tsa metsi le lisampole tse ling tseo ho belaelloang hore li silafalitsoe ke Escherichia coli O157:H7.Bakeng sa lihlahisoa tsa nama tse entsoeng ka botebo, lino le lintho tse ling tse nang le li-pigments, li hloka ho hlatsuoa ho qoba ho ama pokello ea matšoao a fluorescence.
1.2 Tšebetso ea sampuli: Sheba "GB 4789.10-2016 Food Safety National Standard Food Microbiological Examination of Escherichia coli O157: H7 Test" bakeng sa ho lokisetsa sampuli, setso sa ho ntlafatsa le ho itšehla thajana ea Escherichia coli O157: H7.
- Nho tlosoa ha ucleic acid
Nka 20 ml ea tharollo ea ho ntlafatsa ka har'a tube ea 1.5 mL ea centrifuge, eketsa 200 μL ea microbial lysate (ho hlokahala kit e eketsehileng), vortex bakeng sa metsotsoana e 30, centrifuge hakhutšoanyane, 'me u behelle ka thōko.
Tlhaloso: Ho ntšoa ha nucleic acid ho tloha lysate ho lokela ho phethoa ka hare ho metsotso ea 10, 'me e ke ke ea bolokoa nako e telele.
3. Nucleic Acid Amplification
3.1 Bulela sesebelisoa sa fluorescent quantitative PCR hore se sebelisoe.
Buffer A le Buffer B ho tsoa ho kit , li qhibilihe ka botlalo, 'me u li kenye ka bokhutšoane.Kenya 18 μL Buffer A le 2 μL Buffer B ho tube e 'ngoe le e 'ngoe ea PCR.Ebe u eketsa 5 mL e 'ngoe le e' ngoe ea taolo e mpe, e ntšitsoeng nucleic acid, le taolo e ntle ho PCR reaction tubes, cap the tubes, le centrifuge hakhutšoanyane.
3.3 Fetisetsa PCR reaction tube mochine oa fluorescent PCR , 'me u sebelise mekhoa e latelang ho etsa liteko tsa amplification : khetha 25 mL bakeng sa mokhoa oa ho arabela , bokella matšoao a fluorescence ho 60 ° C bakeng sa potoloho ka 'ngoe, 'me u khethe FAM bakeng sa mocha oa ho lemoha.
| Mohato | Lenaneo | Palo ea lipotoloho |
| 1 | 37℃ 5mins | 1 |
| 2 | 9 5 ℃ 3mins | 1 |
| 3 | 95°C 15s | 4 0 |
| 60 ℃ 30s (bokella fluorescence) |
Litaelo tsa tlhahlobo ea mathata
The following is an analysis of the problems that might be encountered in the extraction of viral RNA. We wish it would be helpful to your experiment. In addition, for other experimental or technical problems other than operating instructions and problem analysis, we have dedicated technical support to help you. Contact us if you need at : 028-83361257or E-mail：Tech@foregene.com。
Ha ho RNA e ka ntšoang kapa chai ea nucleic acid e tlase
Hangata ho na le lintlha tse ngata tse amang katleho ea ho hlaphoheloa, joalo ka: sampole ea litaba tsa RNA, mokhoa oa ts'ebetso, bophahamo ba molumo, joalo-joalo.
Tlhahlobo ea lisosa tse tloaelehileng:
1.Bate ea leqhoa kapa mocheso o tlase (4 ° C) centrifugation nakong ea ts'ebetso.
Tlhahiso: Mocheso oa kamore (15-25 ° C) o sebetsa, ha ho mohla ho hlatsoa leqhoa le mocheso o tlase oa centrifuge.
2. Polokelo e sa nepahalang ea mohlala kapa polokelo ea mohlala nako e telele haholo.
Tlhahiso: Boloka lisampole ho -80 ° C kapa u hatsele ka har'a metsi a naetrojene, 'me u qobe tšebeliso ea ho qhibiliha khafetsa;leka ho sebelisa lisampole tse sa tsoa bokelloa bakeng sa ho ntša RNA.
3.Sample e sa lekaneng ea lysis
Keletso: Ka kopo etsa bonnete ba hore sampole le tharollo e sebetsang (Linear Acrylamide) li tsoakoe ka botlalo 'me li kentsoe metsotso e 10 mocheso oa kamore (15-25 ° C)
4.The eluent e kentsoe ka phoso
Keletso: Etsa bonnete ba hore RNase-Free ddH2O e kentsoe bohareng ba lera la kholomo ea tlhoekiso.
5.Molumo o sa nepahalang oa ethanol ea anhydrous ho Buffer viRW2
Tlhahiso: Ka kopo latela litaelo, eketsa molumo o nepahetseng oa ethanol ea anhydrous ho Buffer viRW2 'me u li kopanye hantle pele u sebelisa kit.
6.Tsebeliso e fosahetseng ea sampole.
Tlhahiso: 200µl ea sampole ka 500μl ea Buffer viRL.Bophahamo bo feteletseng ba sampole bo tla fella ka ho fokotseha ha sekhahla sa ho ntšoa ha RNA.
7.Molumo o sa nepahalang oa tlhahiso kapa tlhahiso e sa fellang.
Tlhahiso: Molumo o hlakileng oa kholomo ea tlhoekiso ke 30-50μl;haeba phello ea elution e sa khotsofatse, ho kgothaletswa ho eketsa RNase-Free ddH e futhumetseng2O le ho eketsa nako ea ho beha mocheso oa kamore, joalo ka 5-10min
8.Kolumo ea tlhoekiso e na le masalla a ethanol ka mor'a ho hlatsoa Buffer viRW2.
Tlhahiso: Haeba ethanol e ntse e sala ka mor'a hore e hlatsoe ka Buffer viRW2 le centrifugation e se nang letho bakeng sa 2min, kholomo ea tlhoekiso e ka siuoa mocheso oa kamore bakeng sa 5min ka mor'a hore ho be le letho-tube centrifugation ho tlosa ethanol e setseng ka botlalo.
Ho senyeha ha limolek'hule tsa RNA tse hloekisitsoeng
Boleng ba RNA e hloekisitsoeng bo amana le lintlha tse kang polokelo ea sampuli, tšilafalo ea RNase, le ts'ebetso.
Tlhahlobo ea lisosa tse tloaelehileng:
1.Lisampole tse bokeletsoeng ha lia ka tsa bolokoa ka nako.
Tlhahiso: Haeba sampole e sa sebelisoe ka nako kamora ho bokelloa, ka kopo e boloke ho -80 ℃ kapa metsi a naetrojene hang-hang.Bakeng sa ho ntša limolek'hule tsa RNA, leka ho sebelisa lisampole tse sa tsoa bokelloa neng kapa neng ha ho khoneha.
2.Collected disampole ba ne ba hoama le ho qhibiliha khafetsa.
Tlhahiso: Qoba ho qhoqhoa khafetsa le ho qhibiliha (e seng ho feta hanngoe) nakong ea ho bokella le ho boloka sampole, ho seng joalo chai ea nucleic acid e tla fokotseha.
3.RNase e ile ea hlahisoa ka kamoreng ea ho buuoa kapa ho ne ho se na liatlana tse lahliloeng, limaske, joalo-joalo.
Tlhahiso: Ho ntšoa ha liteko tsa limolek'hule tsa RNA ho etsoa hantle ka phaposing e arohaneng ea tšebetso ea RNA, 'me tafole ea liteko e hloekisoa pele ho teko.Apara liatlana le limaske tse lahloang nakong ea liteko ho qoba ho senyeha ha RNA ho bakoang ke kenyelletso ea RNase.
4.The reagent e silafalitsoe ke RNase nakong ea tšebeliso.
Tlhahiso: Kenya sebaka ka Viral RNA Isolation Kit e ncha bakeng sa liteko tse amanang.
5. Tšilafalo ea RNase ea liphaephe tsa centrifuge, malebela a pipette, joalo-joalo. Tlhahiso: Etsa bonnete ba hore li-tubes tsa centrifuge, malebela a pipette, le lipeipi kaofela ha li na RNase.
Limolek'hule tsa RNA tse hloekisitsoeng li ile tsa ama liteko tse tlase
Limolek'hule tsa RNA tse hloekisitsoeng ke kholomo ea tlhoekiso li tla ama liteko tse tlase tsa metsi haeba ho na le li-ion tsa letsoai kapa liprotheine tse ngata haholo, joalo ka: reverse transcription, Northern Blot, joalo-joalo.
1.Ho na le li-ion tse setseng tsa letsoai ka har'a limolek'hule tsa eluted RNA.
Keletso: Etsa bonnete ba hore molumo o nepahetseng oa ethanol ea anhydrous e kentsoe Buffer viRW2, 'me u hlatsoe kholomo ea tlhoekiso habeli ho latela lebelo le nepahetseng la centrifugation litaelong tsa ts'ebetso; Haeba ho ntse ho e-na le li-ion tsa letsoai tse setseng, u ka eketsa Buffer viRW2 kholomong ea tlhoekiso, 'me u e tlohele mocheso oa kamore bakeng sa 5min.Ebe u etsa centrifugation ho tlosa silafatso ea ion ea letsoai ka tekanyo e kholo ka ho fetisisa
2.Ho na le ethanol e setseng ka har'a limolek'hule tsa RNA
Tlhahiso: hang ha o netefatsa hore litšiea tsa tlhoekiso li hlatsoitsoe ke Buffer viRW2, etsa centrifugation e se nang letho ho latela lebelo la centrifugal ho litaelo tsa ts'ebetso.Haeba ho ntse ho e-na le ethanol e setseng, e ka tloheloa metsotso e 5 mocheso oa kamore ka mor'a hore ho be le centrifugation e se nang letho ho tlosa ethanol e setseng ka tekanyo e kholo ka ho fetisisa.
Litaelo:
Viral RNA Isolation Kit Instruction Manual
