I-Escherichia Coli O157: I-H7 Nucleic Acid Detection Kit (I-PCR Fluorescent Probe Method/Lyophilization)
Incazelo yekhithi:
Ikati.No.FP103
Isetshenziselwa ukutholwa nokuhlolwa ngokushesha kwe-E. coli O157:H7 ekudleni, ekuphakeleni, kumasampula amanzi kanye namasampula emvelo.
Izincazelo
Isetshenziselwaukutholwa nokuhlolwa ngokushesha kwe-E. coli O157:H7 ekudleni, ekuphakeleni, kumasampula amanzi namasampula emvelo .
[Isimiso sokuhlola]
Ngokomgomo wobuchwepheshe be-fluorescent PCR, ama-primers athile nama-Taqman probe akhelwe uhlobo oluthile lwe-Escherichia coli O157: H7, futhi atholwa ithuluzi le-PCR elikhanyayo, ukuze kubonwe ukutholwa kwe-Escherichia coli O157: H7 Ukutholwa kwe-DNA.
Okuqukethwe Kwekhithi
Qaphela: Uphenyo lwesiteshi lwe-ROX alufakiwe.
| Cabaphikisayo | Ukucaciswa | Qubuthi |
| Buffer A | Ithubhu | 1 |
| I-Buffer B | Ithubhu | 1 |
| Ukulawula okuhle | Ithubhu | 1 |
| Ukulawula okungalungile | Ithubhu | 1 |
Ukusetshenziswa Okulindelekile
Isetshenziselwa ukutholwa nokuhlolwa ngokushesha kwe-E. coli O157:H7 ekudleni, ekuphakeleni, kumasampula amanzi namasampula emvelo .
Izimo Zesitoreji Nosuku Lokuphelelwa Isikhathi
Gcina ku -20 ℃ ebumnyameni futhi ugweme ukuqhwa okuphindaphindiwe nokuncibilika.
Isikhathi sokuqinisekisa siyi-12izinyanga, futhi usuku lokukhiqiza luboniswa ephaketheni langaphandle.
Izinsimbi Nezinto Ezisetshenziswayo
Ithuluzi le-PCR le-fluorescent quantitative, isibhamu sepipette namathiphu afanayo, i-vortex shaker, i-mini centrifuge.
Ukusetshenziswa
1. Ukucutshungulwa kwesampula
1.1 Uhlobo lwesampula: Le kit ifanele ukudla, okuphakelayo, amasampula amanzi kanye namanye amasampula okusolwa ukuthi angcoliswe i-Escherichia coli O157:H7.Ngemikhiqizo yenyama ejulile, iziphuzo nezinye izinto eziqukethe ama-pigments, zidinga ukuhlanzwa ukuze zigweme ukuthinta ukuqoqwa kwesignali ye-fluorescence.
1.2 Ukucutshungulwa kwesampula: Bheka "I-GB 4789.10-2016 Ukuphepha Kokudla Kukazwelonke Okujwayelekile Ukudla KweMicrobiological Examination ye-Escherichia coli O157: Ukuhlolwa kwe-H7" ukuze uthole ukulungiswa kwesampula, isiko lokunothisa kanye nokuhlukaniswa kwe-Escherichia coli O157: H7.
- Nukukhishwa kwe-ucleic acid
Thatha ama-mL angu-20 esixazululo sokunothisa kushubhu ye-centrifuge engu-1.5 mL, engeza u-200 μL we-microbial lysate (ikhithi eyengeziwe iyadingeka), i-vortex imizuzwana engu-30, i-centrifuge kafushane, bese uyibeka eceleni.
Amazwana: Ukukhishwa kwe-nucleic acid kusuka ku-lysate kufanele kuqedwe kungakapheli imizuzu eyi-10, futhi ayikwazi ukugcinwa isikhathi eside.
3. I-Nucleic Acid Amplification
3.1 Vula ithuluzi le-PCR le-fluorescent quantitative ukuze lisetshenziswe.
I-Buffer A kanye ne-Buffer B kusukela kukhithi, zincibilikise kahle, futhi zibe yi-centrifuge kafushane.Engeza i-18 μL Buffer A kanye no-2 μL Buffer B kushubhu ngayinye yokusabela ye-PCR.Bese wengeza u-5 ml ngayinye yokulawula okungalungile, ukukhishwa kwe-nucleic acid, nokulawula okuhle kumashubhu okusabela e-PCR, vala amashubhu, kanye ne-centrifuge kafushane.
3.3 Dlulisela ishubhu lokusabela le-PCR emshinini we-PCR wefluorescent , futhi usebenzise izinqubo ezilandelayo ukuze wenze izivivinyo zokukhulisa ukuphakama : khetha u-25 mL wesistimu yokusabela , qoqa amasignali e-fluorescence ku-60°C ngomjikelezo ngamunye, bese ukhetha i-FAM yesiteshi sokuthola.
| Isinyathelo | Uhlelo | Inombolo yemijikelezo |
| 1 | 37℃ 5 imiz | 1 |
| 2 | 9 5 ℃ 3 imiz | 1 |
| 3 | 95°C 15s | 4 0 |
| 60℃ 30s (qoqa i-fluorescence) |
Imihlahlandlela yokuhlaziya izinkinga
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。
Ayikho i-RNA engakhishwa noma isivuno se-nucleic acid siphansi
Ngokuvamile kuba nezinto eziningi ezithinta ukusebenza kahle kokululama, njengalezi: isampula yokuqukethwe kwe-RNA, indlela yokusebenza, ivolumu ye-elution, njll..
Ukuhlaziywa kwezimbangela ezivamile:
1.Ibhavu yeqhwa noma izinga lokushisa eliphansi (4 ° C) i-centrifugation ngesikhathi sokusebenza.
Ukusikisela: Izinga lokushisa legumbi (15-25 ° C), ungalokothi ugeze eqhweni kanye nezinga lokushisa eliphansi le-centrifuge.
2. Ukugcinwa kwesampula okungalungile noma isampula yokugcina isikhathi eside kakhulu.
Ukusikisela: Gcina amasampula ku- -80 ° C noma ufrize ku-nitrogen ewuketshezi, futhi ugweme ukusetshenziswa kokuqhwaza okuphindaphindiwe;zama ukusebenzisa amasampula asanda kuqoqwa ukuze kukhishwe i-RNA.
3.Isampula yesampula enganele
Okutuswayo: Sicela uqinisekise ukuthi isampula kanye nesixazululo sokusebenza (i-Linear Acrylamide) kuxutshwe kahle futhi kufakwe ekufukameleni imizuzu eyi-10 ekamelweni lokushisa (15-25 ° C)
4.I-eluent yengezwe ngokungalungile
Okutuswayo: Qiniseka ukuthi i-ddH2O ye-RNase-Free yengezwa phakathi nolwelwesi lwekholomu yokuhlanza.
5.Ivolumu engafanele ye-ethanol engenamanzi ku-Buffer viRW2
Ukusikisela: Sicela ulandele imiyalelo, wengeze umthamo olungile we-ethanol enganamanzi ku-Buffer viRW2 bese uyixuba kahle ngaphambi kokusebenzisa ikhithi.
6.Ukusetshenziswa kwesampula okungalungile.
Isiphakamiso: 200µl wesampula ngo-500μl ngamunye we-Buffer viRL.Ivolumu yesampula eyeqile izoholela ekwehliseni izinga lokukhishwa kwe-RNA.
7.Ivolumu ye-elution engafanele noma ukungezwani okuphelele.
Isiphakamiso: Umthamo ongacacile wekholomu yokuhlanza ngu-30-50μl;uma umphumela we-elution ungagculisi, kuyanconywa ukuthi wengeze i-ddH eshisiwe ngaphambilini ye-RNase-Free2O futhi wandise isikhathi obeka ekamelweni lokushisa, ezifana 5-10min
8.Ikholomu yokuhlanza inezinsalela ze-ethanol ngemva kokuyihlanza ku-Buffer viRW2.
Isiphakamiso: Uma i-ethanol isasele ngemva kokugeza ku-Buffer viRW2 kanye ne-empty-tube centrifugation imizuzu engu-2, ikholomu yokuhlanza ingashiywa ekamelweni lokushisa imizuzu emi-5 ngemva kokuhlanganisa ishubhu engenalutho ukuze ikhiphe ngokuphelele i-ethanol esele.
Ukuwohloka kwama-molecule e-RNA ahlanzekile
Ikhwalithi ye-RNA ehlanziwe ihlobene nezici ezifana nokugcinwa kwesampula, ukungcoliswa kwe-RNase, nokusebenza.
Ukuhlaziywa kwezimbangela ezivamile:
1.Amasampuli aqoqiwe awalondolozwanga ngesikhathi.
Isiphakamiso: Uma isampula lingasetshenziswa ngesikhathi ngemuva kokuqoqwa, sicela uyigcine ku- -80 ℃ noma i-nitrogen ewuketshezi ngokushesha.Ukuze ukhiphe ama-molecule e-RNA, zama ukusebenzisa amasampula asanda kuqoqwa noma nini lapho kungenzeka.
2.Amasampula aqoqiwe abeqhwa futhi ancibilika ngokuphindaphindiwe.
Isiphakamiso: Gwema ukuqhwaza okuphindaphindiwe nokuncibilika (okungaphezu kokukodwa) phakathi nokuqoqwa nokugcinwa kwesampula, ngaphandle kwalokho isivuno se-nucleic acid sizokwehla.
3.I-RNase yethulwa egunjini lokuhlinzela noma kwakungekho amagilavu alahlwayo, imaski, njll.
Isiphakamiso: Ukukhishwa kokuhlolwa kwama-molecule e-RNA kwenziwa kangcono ekamelweni lokusebenza elihlukile le-RNA, futhi ithebula lokuhlola liyahlanzwa ngaphambi kokuhlolwa.Gqoka amagilavu alahlwayo kanye nemaski ngesikhathi sokuhlolwa ukuze ugweme ukucekelwa phansi kwe-RNA okubangelwa ukwethulwa kwe-RNase.
4.I-reagent ingcoliswe yi-RNase ngesikhathi isetshenziswa.
Isiphakamiso: Faka esikhundleni se-Viral RNA Isolation Kit entsha ukuze uthole ukuhlolwa okuhlobene.
5.Ukungcoliswa kwe-RNase kwamashubhu e-centrifuge, amathiphu e-pipette, njll. Isiphakamiso: Qiniseka ukuthi amashubhu e-centrifuge, amathiphu e-pipette, namapayipi konke Akuna-RNAse.
Ama-molecule e-RNA ahlanziwe athinte ukuhlolwa okungaphansi komfula
Ama-molecule e-RNA ahlanzwe ikholomu yokuhlanza azothinta ukuhlolwa okuya phansi komfula uma kukhona ama-ion kasawoti amaningi noma amaprotheni, afana nalokhu: ukuloba okuhlanekezelwe, i-Northern Blot, njll..
1.Kukhona ama-ion kasawoti asele kuma-athomu e-RNA e-luted.
Okutuswayo: Qiniseka ukuthi ivolumu elungile ye-ethanol enganamanzi yengezwe ku-Buffer viRW2, futhi ugeze ikholomu yokuhlanza kabili ngokwejubane elifanele le-centrifugation emiyalweni yokusebenza;Uma kusekhona ama-ion kasawoti asele, ungakwazi ukwengeza i-Buffer viRW2 kukholamu yokuhlanza, futhi uyishiye ekamelweni lokushisa imizuzu emi-5.Bese wenza i-centrifugation ukuze ususe ukungcoliswa kwama-ion kasawoti ngezinga elikhulu kakhulu
2.Kune-ethanol esele kuma-athomu e-RNA akhishwe
Isiphakamiso: uma usuqinisekisa ukuthi amakholomu okuhlanza ahlanjululwe yi-Buffer viRW2, yenza i- empty-tube centrifugation ngokwesivinini se-centrifugal emiyalweni yokusebenza.Uma isekhona i-ethanol esele, ingashiywa imizuzu emi-5 ekamelweni lokushisa ngemva kwe-centrifugation yeshubhu engenalutho ukuze kukhishwe i-ethanol esele ngezinga elikhulu kakhulu.
Amamanuwali wemiyalo:
I-Viral RNA Isolation Kit Manual
