Escherichia Coli O157: H7 Nucleic Acid Detection Kit (PCR Fluorescent Probe Method/Lyophilization)
Fa'amatalaga o pusa:
Cat.Leai.FP103
E faʻaaogaina mo le vave suʻesuʻeina ma le suʻeina o le E. coli O157: H7 i meaʻai, fafaga, vai faʻataʻitaʻiga ma faʻataʻitaʻiga o le siosiomaga.
Fa'amatalaga
E fa'aaogaina movave su'esu'eina ma su'esu'eina o le E. coli O157:H7 i mea'ai, fafaga, vai fa'ata'ita'iga ma fa'ata'ita'iga o le si'osi'omaga.
[Faʻavae suʻega]
E tusa ai ma le mataupu faavae o tekinolosi PCR fluorescent, o primers faapitoa ma suʻesuʻega Taqman ua fuafuaina mo le kenera patino o Escherichia coli O157: H7, ma iloa e se meafaigaluega PCR fluorescent, ina ia iloa ai le suʻesuʻeina o Escherichia coli O157: H7 Qualitative suʻesuʻega o DNA.
Pusa i totonu
Manatua: ROX auala su'esu'e e le o aofia ai.
| Ctagata tetee | Fa'amatalaga | Quantity |
| Puipui A | Faila | 1 |
| Puipui B | Faila | 1 |
| Pulea lelei | Faila | 1 |
| Le pulea lelei | Faila | 1 |
Fa'amoemoega Fa'aoga
E fa'aaogaina mo vave su'esu'eina ma su'esu'eina o le E. coli O157:H7 i mea'ai, fafaga, vai fa'ata'ita'iga ma fa'ata'ita'iga o le si'osi'omaga.
Tulaga Teuteu ma Aso e muta ai
Teu i le -20 ℃ i le pogisa ma aloese mai le fa'a'aisa fa'asolo ma le liu.
Ole taimi ole aoga ole 12masina, ma o le aso gaosiga o loʻo faʻaalia i luga o le afifiina i fafo.
Meafaigaluega ma Mea'ai
Meafaifa'aili PCR fa'afuainumera, fana paipa ma fautuaga fa'afetaui, lulu vortex, centrifuge la'ititi.
Fa'aoga
1. Fa'ata'ita'iga Fa'agasologa
1.1 Faʻataʻitaʻiga ituaiga: O lenei pusa e fetaui lelei mo meaʻai, fafaga, vai faʻataʻitaʻiga ma isi faʻataʻitaʻiga masalomia e faʻaleagaina e Escherichia coli O157: H7.Mo mea'ai aano o manu, meainu ma isi mea o lo'o iai pigments, e mana'omia ona fufulu ina ia 'alofia le a'afiaina o le fa'aputuina o fa'ailo o le fluorescence.
1.2 Faʻataʻitaʻiga faʻataʻitaʻiga: Vaʻai ile "GB 4789.10-2016 Food Safety National Standard Food Microbiological Examination of Escherichia coli O157: H7 Test" mo sauniuniga faʻataʻitaʻiga, aganuu faʻatamaoaigaina ma le vavaeeseina o Escherichia coli O157: H7.
- Nucleic acid fa'amuta
Ave le 20 ml o le vaifofo fa'atamaoaigaina ile 1.5 mL centrifuge tube, fa'aopopo le 200 μL ole microbial lysate (faaopoopo pusa mana'omia), vortex mo le 30 sekone, centrifuge puupuu, ma tu'u ese.
Fa'amatalaga: O le aveeseina o le nucleic acid mai le lysate e tatau ona maeʻa i totonu ole 10 minute, ma e le mafai ona teuina mo se taimi umi.
3. Nucleic Acid Amplification
3.1 Fa'aola le meafaifa'aili PCR fa'a'ale'ale mo le fa'aoga.
Pa'u A ma le Pa'u B mai le pusa, fa'afefeteina mae'ae'a, ma fa'anofo fa'apuupuu.Fa'aopoopo le 18 μL Pa'u A ma le 2 μL Pa'u B i paipa fa'afoliga PCR ta'itasi.Ona fa'aopoopo lea o le 5 mL o le fa'atonuga leaga ta'itasi, o le nucleic acid ua maua mai, ma le fa'atonutonu lelei i totonu o paipa fa'agaoioiga PCR, tapuni fa'agaau, ma centrifuge fa'apuupuu.
3.3 Fa'afeiloa'i le paipa fa'afoliga PCR i se masini PCR fa'alelei, ma fa'aaoga faiga nei e fa'atino ai su'esu'ega fa'alautele: filifili le 25 mL mo le faiga fa'afoliga, aoina fa'ailo fa'amalama i le 60°C mo ta'amilosaga ta'itasi, ma filifili le FAM mo le auala e iloa ai.
| Laa | Polokalama | Numera o taamilosaga |
| 1 | 37℃ 5min | 1 |
| 2 | 9 5 ℃ 3min | 1 |
| 3 | 95°C 15s | 4 0 |
| 60 ℃ 30s (aoina mai le felanulanuaʻi) |
Ta'iala mo su'esu'ega fa'afitauli
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。
E leai se RNA e mafai ona maua mai pe maualalo le gaosiga o le nucleic acid
E masani ona tele mea e aʻafia ai le toe faʻaleleia lelei, e pei o le: faʻataʻitaʻiga RNA anotusi, auala o le faʻagaioiga, elution volume, etc.
Iloiloga o mafua'aga masani:
1. Ta'ele aisa po'o le maualalo o le vevela (4 ° C) centrifugation i le taimi o le taotoga.
Fautuaga: O le vevela o le potu (15-25 ° C) gaioiga, aua le ta'ele aisa ma le centrifuge maualalo.
2. Le lelei le teuina o faʻataʻitaʻiga poʻo le teuina o faʻataʻitaʻiga mo se taimi umi.
Fautuaga: Teu fa'ata'ita'iga ile -80°C po'o le fa'a'aisa ile fa'a'aisa sua, ma 'alofia le fa'aaogaina fa'a'aisa-liu;taumafai e faʻaaoga faʻataʻitaʻiga fou na aoina mo le faʻaaogaina o le RNA.
3. Le lava faʻataʻitaʻiga lysis
Fautuaga: Faʻamolemole faʻamautinoa o le faʻataʻitaʻiga ma le fofo galue (Linear Acrylamide) ua maeʻa faʻafefiloi ma faʻafefe mo le 10 min i le vevela o le potu (15-25 ° C)
4. Ua le sa'o le fa'aopoopoina o le eluent
Fautuaga: Ia mautinoa ua faaopoopo le RNase-Free ddH2O i le ogatotonu o le membrane o le koluma faʻamamaina.
5. Le talafeagai voluma o anhydrous ethanol i Buffer viRW2
Fautuaga: Fa'amolemole mulimuli i fa'atonuga, fa'aopoopo le voluma sa'o o le ethanol anhydrous ile Buffer viRW2 ma fa'afefiloi lelei a'o le'i fa'aogaina le pusa.
6. Fa'aoga fa'ata'ita'iga le talafeagai.
Fautuaga: 200μl o fa'ata'ita'iga ile 500μl ole Buffer viRL.O le tele o fa'ata'ita'iga voluma o le a fa'aitiitia ai le fua fa'atatau o le RNA.
7. le lelei o le elution volume po'o le le atoatoa o le elution.
Fautuaga: O le voluma eluent o le koluma faʻamamaina o le 30-50μl;afai e le faamalieina le aafiaga o le elution, e fautuaina e faʻaopoopo i ai le RNase-Free ddH muamua faʻavevela.2O ma faʻalautele le taimi tuʻu i le vevela o le potu, pei ole 5-10min
8. Fa'amama koluma ei ai ethanol toega ina ua uma ona fufulu i Buffer viRW2.
Fautuaga: Afai e tumau pea le ethanol pe a uma ona fufulu ile Buffer viRW2 ma centrifugation gaogao-tube mo le 2min, e mafai ona tuʻu le koluma faʻamamaina i le vevela o le potu mo le 5min pe a uma le centrifugation gaogao-tube e aveese atoa ai le ethanol totoe.
Le faʻaleagaina o mole RNA mama
O le lelei o le RNA faʻamamaina e fesoʻotaʻi ma mea taua e pei o le teuina o faʻataʻitaʻiga, faʻaleagaina o le RNase, ma le gaioiga.
Iloiloga o mafua'aga masani:
1. O faʻataʻitaʻiga na aoina e leʻi faʻasaoina i le taimi.
Fautuaga: Afai e le faʻaaogaina le faʻataʻitaʻiga i le taimi pe a maeʻa le aoina, faʻamolemole teu i le -80 ℃ poʻo le vai faʻamaʻi vave.Mo le su'eina o molela'au RNA, taumafai e fa'aoga fa'ata'ita'iga fou na aoina pe a mafai.
2. O fa'ata'ita'iga fa'aputuina sa fa'aaisa ma liusua fa'afia.
Fautuaga: Aloese mai le fa'aaisa ma fa'alili (ia le sili atu ma le tasi) i le taimi o le aoina ma le teuina o fa'ata'ita'iga, a leai o le a fa'aitiitia le fua o le nucleic acid.
3. Na fa'afeiloa'i le RNase i totonu o le potu tipitipi pe leai ni totini lima, matapulepule, ma isi mea na ofuina.
Fautuaga: Ole su'ega ole su'ega mole ole RNA e sili ona faia ile potu ta'i RNA, ma fa'amama le laulau fa'ata'ita'i a'o le'i faia le su'ega.Fa'aofu totigilima ma matapulepule i le taimi o le fa'ata'ita'iga e aloese ai mai le fa'aleagaina o le RNA e mafua mai i le fa'aofiina o le RNase.
4.O le reagent e faʻaleagaina e RNase i le taimi o le faʻaaogaina.
Fautuaga: Sui ile Viral RNA Isolation Kit mo su'ega fa'atatau.
5. O le RNase contamination o le centrifuge tubes, pipette tips, ma isi Fautuaga: Ia mautinoa o le centrifuge tubes, pipette tips, ma pipettes uma RNase-Free.
O molelaula RNA mama na a'afia i fa'ata'ita'iga i lalo
O mole RNA faʻamamaina e le koluma faʻamamaina o le a aʻafia ai faʻataʻitaʻiga i lalo pe afai e tele naua ion masima poʻo polotini, e pei ole: faʻaliliuga faʻaliliu, Northern Blot, ma isi.
1.O lo'o totoe ions masima i le eluted RNA molecules.
Fautuaga: Ia mautinoa ua faaopoopo le voluma sao o le ethanol anhydrous i le Buffer viRW2, ma fufulu le koluma faamama faalua e tusa ai ma le sao sao centrifugation i luga o faatonuga faagaioia; Afai o loʻo i ai pea ion masima o totoe, e mafai ona e faʻaopopo le Buffer viRW2 i le koluma faʻamamaina, ma tuʻu i le vevela o le potu mo le 5min.Ona faia lea o le centrifugation e aveese ai iona masima i se tulaga sili
2.O lo'o totoe le ethanol i le molelaula RNA eluted
Fautuaga: o le taimi lava e faʻamaonia ai o koluma faʻamamāina ua fufuluina e Buffer viRW2, fai le centrifugation gaogao-tube e tusa ai ma le saoasaoa centrifugal i luga o faatonuga faʻagaioiga.Afai o lo'o totoe pea le ethanol, e mafai ona tu'u mo le 5 minute i le vevela o le potu pe a mae'a le fa'amama gaogao e aveese ai le ethanol o totoe i le tele.
Tusi Taiala:
Viral RNA Isolation Kit Tusi Taiala
