Escherichia coli O157:H7 Qalabka Lagu Ogaado Asiidhka Nucleic (PCR Fluorescent Probe Method)
Sharaxaada Qalabka:
Bisad.Maya.FP102
Waxa loo isticmaalaa ogaanshaha degdega ah iyo baadhista E.coli O157:H7 ee cuntada, quudinta, muunadaha biyaha iyo muunadaha deegaanka.
Sharaxaada
Waxa loo isticmaalaa ogaanshaha degdega ah iyo baadhista E.coli O157:H7 ee cuntada, quudinta, muunadaha biyaha iyo muunadaha deegaanka.
[Mabda'a Tijaabada]Marka loo eego mabda'a tignoolajiyada PCR ee fluorescent, curiyeyaasha gaarka ah iyo baaritaannada Taqman waxaa loogu talagalay hidda-wadaha gaarka ah ee Escherichia coli O157: H7, oo lagu ogaado aaladda PCR fluorescent, si loo xaqiijiyo ogaanshaha Escherichia coli O157: H7 Ogaanshaha tayada DNA.
Tusmada Kit
Xusuusin: Baaritaanka kanaalka ROX kuma jiro.
| Cdabadhilifyo | Tilmaamid | Qmidnimo |
| Buffer A | Tube | 1 |
| Buffer B | Tube | 1 |
| Xakamaynta togan | Tube | 1 |
| Xakamaynta xun | Tube | 1 |
Isticmaalka la filayo
Waxaa loo isticmaalaa ogaanshaha degdega ah iyo baaritaanka E. coli O157:H7 ee cuntada, quudinta, muunadaha biyaha iyo muunadaha deegaanka.
Shuruudaha Kaydinta iyo Taariikhda dhicitaanka
Ku kaydi meel u dhow -20℃ gudcurka oo iska ilaali baraf soo noqnoqda iyo dhalaalida.
Muddada ansaxnimadu waa 12bilaha, iyo taariikhda wax soo saarka ayaa lagu muujiyay baakadaha dibadda.
Qalabka iyo Alaabooyinka
Qalabka PCR ee cabirka fluorescent, qoriga pipette iyo talooyin ku habboon, ruxruxa vortex, mini centrifuge.
Isticmaalka
1. Sambalka Habaynta
1.1 Nooca Tusaalaha: Qalabkani waxa uu ku haboon yahay cuntada, quudinta, shaybaarada biyaha iyo muunado kale oo looga shakisan yahay in ay wasakhayso Escherichia coli O157:H7.Alaabooyinka hilibka ee si qoto dheer loo farsameeyay, cabitaanada iyo walxaha kale ee ay ku jiraan midabada, waxay u baahan yihiin in la dhaqo si looga fogaado inay saameeyaan ururinta ishaarada florescence.
1.2 Tusaalaha habaynta: Tixraac "GB 4789.10-2016 Badbaadada Cuntada Qaranka Heerka Badbaadada Cunnada Imtixaanka Microbiological Imtixaanka Escherichia coli O157: H7 Test" ee muunada diyaarinta, dhaqanka kobcinta iyo go'doominta Escherichia coli O157: H7.
- Nsoo saarista ucleic acid
Qaado 20 ml oo xal kobcin ah oo ku jira 1.5 mL tuubo sentifuge, ku dar 200 μL oo ah lysate microbial (xirmo dheeri ah ayaa loo baahan yahay), vortex 30 ilbiriqsi, sentifuge si kooban, oo dhinac dhig.
Xusuusin: Soo saarista aashitada nucleic ee lysate waa in lagu dhammeeyaa 10 daqiiqo gudahood, lamana kaydin karo waqti dheer.
3. Kordhinta Asiidhka Nukliyeerka
3.1 Daar qalabka PCR ee tirooyinka fluorescent si loo isticmaalo.
Buffer A iyo Buffer B ee xirmada, si fiican u dhalaali, oo si kooban u dhexji.Ku dar 18 μL Buffer A iyo 2 μL Buffer B tuubo kasta oo falcelinta PCR ah.Dabadeed ku dar 5 mL mid kasta oo ka mid ah kantaroolka taban, aashitada nucleic ee la soo saaray, iyo kontoroolka togan ee tuubooyinka falcelinta PCR, dabool tuubooyinka, oo centrifuge si kooban.
3.3 U wareeji tuubada falcelinta PCR mashiinka PCR fluorescent , oo isticmaal hababka soo socda si aad u sameyso tijaabooyinka xoojinta : dooro 25 mL habka falcelinta , soo ururi calaamadaha fluorescence ee 60 ° C wareeg kasta, oo dooro FAM kanaalka ogaanshaha.
| Talaabada | Barnaamijka | Tirada wareegyada |
| 1 | 37 ℃ 5 daqiiqo | 1 |
| 2 | 9 5 ℃ 3 daqiiqo | 1 |
| 3 | 95°C 15s | 40 |
| 60 ℃ 30s (Ururi fluorescence) |
Tilmaamaha falanqaynta dhibaatooyinka
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。
RNA lama soo saari karo ama wax soo saarka nucleic acid waa yar yahay
Caadi ahaan waxaa jira arrimo badan oo saameeya waxtarka soo kabashada, sida: muunadda RNA content, habka hawlgalka, mugga elution, iwm.
Falanqaynta sababaha caadiga ah:
1.Baraf baraf ama heerkul hooseeya (4 ° C) centrifugation inta lagu jiro hawlgalka.
Talo soo jeedin: Heerkulka qolka (15-25 ° C) qalliin, waligiis qubeyska baraf iyo heerkul hooseeya.
2. Kaydinta muunada aan habboonayn ama kaydinta muunada wakhti aad u dheer.
Talo-soo-jeedin: Ku kaydi shay-baarka heerkulka -80 ° C ama ku qabooji nitrogen dareeraha ah, kana fogow isticmaalka baraf-dhalashada ee soo noqnoqda;isku day inaad u isticmaasho muunado cusub oo la ururiyey si aad u soo saarto RNA.
3.Sida muunad ku filan
Talo soo jeedin: Fadlan hubi in muunada iyo xalalka shaqada (Linear Acrylamide) si fiican la isku qasay oo lagu duray 10 daqiiqo heerkulka qolka (15-25 ° C)
4.Eleent waxaa lagu daray si qaldan
Talo soo jeedin: Hubi in RNase-free ddH2O lagu daray dhexe ee xuubka tiirka sifaynta
5.Mugga aan habboonayn ee ethanol anhydrous ee ku jira Buffer viRW2
Soo jeedin: Fadlan raac tilmaamaha, ku dar mugga saxda ah ee ethanol anhydrous Buffer viRW2 oo si fiican u qas ka hor intaadan isticmaalin qalabka.
6.Isticmaalka muunad khaldan.
Soo jeedin: 200µl muunad halkii 500μl ee Buffer viRL.Mugga muunada xad dhaafka ah waxay keeni doontaa hoos u dhaca heerka soo saarista RNA.
7.Mugga sare-u-qaadka oo aan sax ahayn ama muuqaal aan dhammaystirnayn.
Soo jeedin: Mugga sare ee tiirka nadiifinta waa 30-50μl;haddii saamaynta sare-u-qaadista aysan ahayn mid lagu qanco, waxaa lagu talinayaa in lagu daro RNase-free ddH horay loo sii kululeeyay2O oo kordhi wakhtiga meelaynta heerkulka qolka, sida 5-10min
Tiirka nadiifinta ayaa leh hadhaaga ethanol ka dib ku luqluqashada Buffer viRW2.
Talo soo jeedin: Haddii ethanol weli sii jiro ka dib ku luqluqashada Buffer viRW2 iyo centrifugation-tuubo madhan 2min, tiirka sifaynta waxaa lagu dayn karaa heerkulka qolka 5min ka dib centrifugation-tuubo madhan si buuxda looga saaro ethanol haray.
Burburinta unugyada RNA la safeeyey
Tayada RNA la safeeyey waxay la xiriirtaa arrimo ay ka mid yihiin kaydinta muunada, faddaraynta RNase, iyo hawlgalka.
Falanqaynta sababaha caadiga ah:
1.Samballada la ururiyey laguma kaydin wakhtiga.
Soo jeedin: Haddii muunada aan la isticmaalin wakhti ka dib ururinta, fadlan ku kaydi -80 ℃ ama nitrogen dareere ah isla markiiba.Soo saarista unugyada RNA, isku day inaad isticmaasho muunado cusub oo la ururiyey mar kasta oo ay suurtogal tahay.
2.Samballada la ururiyey ayaa barafoobay oo si isdaba joog ah u dhalaalay.
Talo soo jeedin: Iska ilaali qaboojinta soo noqnoqda iyo dhalaalidda (wax aan ka badnayn hal mar) inta lagu jiro ururinta iyo kaydinta muunada, haddii kale wax-soosaarka nucleic acid wuu yaraan doonaa.
3.RNase waxaa lagu soo bandhigay qolka qalliinka ama lama gashan jirin galoofyada la tuuri karo, maaskaro, iwm.
Soo jeedin: Soo saarista tijaabada molecules RNA waxaa sida ugu fiican loogu sameeyaa qol qalliin RNA gaar ah, miiska tijaabada waa la nadiifiyaa ka hor tijaabada.Xiro galoofyada iyo maaskaro la tuuri karo inta lagu jiro tijaabada si aad uga fogaato xaalufka RNA ee ay keento hordhaca RNase.
4. Reagent-ku waxa ku wasakhoobay RNase inta la isticmaalayo.
Soo jeedin: Ku beddel Xirmada Go'doominta ee Viral RNA cusub ee tijaabooyinka la xidhiidha.
5. Wasakhda RNase ee tuubooyinka centrifuge, talooyinka pipette, iwm
Unugyada RNA ee la safeeyey ayaa saameeyay tijaabooyinka hoose
Unugyada RNA ee lagu sifeeyay tiirka sifaynta waxay saamayn doonaan tijaabooyinka hoose haddii ay jiraan ion milix badan ama borotiinno badan, sida: qoraal-celin, Northern Blot, iwm.
1. Waxaa ku haray ion milix ah oo ku jira molecules RNA eluted.
Talo soo jeedin: Hubi in mugga saxda ah ee ethanol anhydrous lagu daray Buffer viRW2, oo maydh tiirka nadiifinta laba jeer iyadoo loo eegayo xawaaraha centrifugation saxda ah ee tilmaamaha qalliinka;Haddii ay weli jiraan ion milix ah oo hadhay, waxaad ku dari kartaa Buffer viRW2 tiirka nadiifinta, oo ku dhaaf heerkulka qolka 5min.Ka dib samee centrifugation si aad uga saarto wasakhda ions milixda ilaa xadka ugu badan
2.Waxaa jira ethanol haray oo ku jira molecules RNA eluted
Soo jeedin: mar marka la xaqiijiyo in tiirarka sifaynta ay ku maydheen Buffer viRW2, samee centrifugation madhan tuubo si waafaqsan xawaaraha centrifugal ee tilmaamaha hawlgalka.Haddii ethanol uu weli ka harsan yahay, waxaa lagu dayn karaa 5 daqiiqo heerkulka qolka ka dib marka la dhigo centrifugation-tube madhan si looga saaro ethanol haray ilaa xadka ugu badan.
Buug-tilmaameedka:
Buugga Tilmaamaha Xirmada Go'doominta Viral RNA
