Escherichia coli O157:H7 Nucleic Acid Detection Kit (PCR Fluorescent Probe Method)
Kit Description:
Katsi.No.FP102
Inoshandiswa kukurumidza kuona uye kuongororwa kweE. coli O157: H7 muzvokudya, zvokudya, sampuli dzemvura uye sampuli dzezvakatipoteredza.
Tsanangudzo
Inoshandiswa kukurumidza kuona uye kuongorora E. coli O157: H7 muzvokudya, zvokudya, zvidhori zvemvura uye sampuli dzezvakatipoteredza.
[Kuedza musimboti]Zvinoenderana nemusimboti weiyo fluorescent PCR tekinoroji, maprimers chaiwo uye Taqman probes akagadzirirwa iyo chaiyo gene yeEscherichia coli O157: H7, uye yakaonekwa nefluorescent PCR chiridzwa, kuitira kuti ione kuonekwa kweEscherichia coli O157: H7 Hunhu hwekuonekwa kweDNA.
Kit Zviri mukati
Ongorora: ROX chiteshi probe haina kubatanidzwa.
| Cvanopikisa | Tsanangudzo | Quantity |
| Buffer A | Tube | 1 |
| Buffer B | Tube | 1 |
| Positive control | Tube | 1 |
| Negative control | Tube | 1 |
Inotarisirwa Kushandiswa
Inoshandiswa kune kukurumidza kuona uye kuongororwa kweE. coli O157: H7 muzvokudya, zvokudya, zvidhori zvemvura uye sampuli dzezvakatipoteredza.
Mamiriro Ekuchengetedza Uye Zuva Rekupera
Chengetedza pa -20 ℃ murima uye dzivisa kudzokorora kutonhora nekunyunguduka.
Nguva yechokwadi ndeye 12mwedzi, uye zuva rekugadzira rinoratidzwa papakeji yekunze.
Zviridzwa Nezvinodyiwa
Fluorescent quantitative PCR chiridzwa, pipette pfuti uye matipi ekufananidza, vortex shaker, mini centrifuge.
Usage
1. Muenzaniso Processing
1.1 Sample type: Kiti iyi yakakodzera chikafu, chikafu, masampu emvura uye mamwe masampuli anofungidzirwa kuti akasvibiswa neEscherichia coli O157: H7.Kune zvigadzirwa zvenyama zvakadzika, zvinwiwa uye zvimwe zvinhu zvine pigment, zvinoda kugezwa kuti zvisakanganisa kuunganidzwa kwechiratidzo chefluorescence.
1.2 Sample processing: Tarisa ku "GB 4789.10-2016 Food Safety National Standard Food Microbiological Examination of Escherichia coli O157: H7 Test" yekugadzirira muenzaniso, kupfumisa tsika uye kuparadzaniswa kweEscherichia coli O157: H7.
- Nucleic acid kubviswa
Tora 20 mL yekupfumisa mhinduro mu 1.5 mL centrifuge chubhu, wedzera 200 μL ye microbial lysate (yakawedzerwa kit inodiwa), vortex kwemasekonzi makumi matatu, centrifuge muchidimbu, uye isa parutivi.
Tsanangudzo: Kubviswa kwe nucleic acid kubva ku lysate kunofanira kupedzwa mukati memaminitsi e10, uye haigoni kuchengetwa kwenguva yakareba.
3. Nucleic Acid Amplification
3.1 Batidza fluorescent quantitative PCR chiridzwa chekushandisa.
Buffer A uye Buffer B kubva pakiti , zvinyunguduse zvakakwana, uye centrifuge muchidimbu.Wedzera 18 μL Buffer A uye 2 μL Buffer B kune yega PCR reaction chubhu.Wobva wawedzera 5 mL imwe neimwe yekutonga kwakashata, yakabviswa nucleic acid, uye kutonga kwakanaka muPCR reaction tubes, cap the tubes, uye centrifuge muchidimbu.
3.3 Tumira PCR reaction chubhu kumushini wefluorescent PCR, uye shandisa nzira dzinotevera kuti uite zviedzo zvekusimudza : sarudza 25 mL yemaitiro ekugadzirisa, unganidza zviratidzo zvefluorescence pa 60 ° C pamutambo wega wega, uye sarudza FAM yenzira yekuona.
| Danho | Chirongwa | Nhamba yezvikamu |
| 1 | 37℃ 5 min | 1 |
| 2 | 9 5 ℃ 3 min | 1 |
| 3 | 95°C 15s | 4 0 |
| 60 ℃ 30s (unganidza fluorescence) |
Nhungamiro dzekuongorora matambudziko
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。
Hapana RNA inogona kutorwa kana goho re nucleic acid rakaderera
Pane kazhinji zvinhu zvakawanda zvinokanganisa kudzoreredza kushanda zvakanaka, senge: sampuli RNA yemukati, nzira yekushanda, elution vhoriyamu, nezvimwe..
Kuongorora kwezvikonzero zvinowanzoitika:
1.Ice kugeza kana kuderera-kupisa (4 ° C) centrifugation panguva yekushanda.
Zano: Tembiricha yekamuri (15-25 ° C) mashandiro, usambofa kugeza nechando uye yakaderera tembiricha centrifuge.
2. Zvisizvo sampuli yekuchengetedza kana sampuli yekuchengetedza kwenguva yakareba.
Zano: Chengetedza sampuli pa -80 ° C kana kuomesa munitrogen yemvura, uye dzivirira kudzokororwa kushandiswa kwechando-nyoro;edza kushandisa masampuli achangobva kuunganidzwa eRNA kutorwa.
3.Isina kukwana sampuli lysis
Kurudziro: Ndokumbira utarise kuti sampuli uye mhinduro yekushanda (Linear Acrylamide) yakanyatso kusanganiswa uye yakanamirwa kwemaminetsi gumi patembiricha yekamuri (15-25 ° C)
4.The eluent yakawedzerwa zvisizvo
Kurudziro: Ita shuwa kuti RNase-Yemahara ddH2O inowedzerwa pakati pe membrane yekoramu yekuchenesa.
5.Improper volume yeanhydrous ethanol muBuffer viRW2
Zano: Ndokumbira uteedzere mirairo, wedzera iyo chaiyo vhoriyamu yeanhydrous ethanol kuBuffer viRW2 wosanganisa zvakanaka usati washandisa kiti.
6.Improper sampuli kushandiswa.
Zano: 200µl yesampuli pa500μl yeBuffer viRL.Yakawandisa sampuli vhoriyamu inokonzeresa kuderedzwa kweRNA yekubvisa mwero.
7.Improper elution vhoriyamu kana kusakwana elution.
Zano: Huwandu hwakajeka hwekoramu yekuchenesa ndeye 30-50μl;kana iyo elution effect isingagutsi, zvinokurudzirwa kuwedzera pre-heated RNase-Mahara ddH.2O uye wedzera nguva yekuisa pane tembiricha yekamuri, senge 5-10min
8.Kunatsa koramu ine ethanol yasara mushure mekugezeswa muBuffer viRW2.
Zano: Kana ethanol ichiri kusara mushure mekugezeswa muBuffer viRW2 uye isina chinhu-chubhu centrifugation kwe2min, mbiru yekunatsa inogona kusiiwa patembiricha yekamuri kwemaminetsi mashanu mushure mekunge isina chinhu-chubhu centrifugation kubvisa zvachose ethanol yasara.
Kushatiswa kwemamorekuru eRNA akacheneswa
Hunhu hweRNA yakacheneswa hunoenderana nezvinhu zvakaita seyekuchengetedza sampuli, kusvibiswa kweRNase, uye kushanda.
Kuongorora kwezvikonzero zvinowanzoitika:
1.Masampuli akaunganidzwa haana kuchengetwa munguva.
Zano: Kana sampu ikasashandiswa nenguva mushure mekuunganidzwa, ndapota ichengetedze pa -80 ℃ kana mvura yenitrogen nekukasira.Pakubviswa kwemamorekuru eRNA, edza kushandisa masampuli achangobva kuunganidzwa pese pazvinogoneka.
2.Kuunganidza samples kwaive kutonhora uye kunyunguduka kakawanda.
Zano: Dzivisa kudzokorora kutonhora nekunyunguduka (kwete kanopfuura kamwe) panguva yekuunganidza nekuchengetedza sampuli, zvikasadaro goho re nucleic acid richadzikira.
3.RNase yakaunzwa muimba yekushanda kana pasina magurovhosi anoraswa, masiki, nezvimwe zvakapfekwa.
Zano: Kubviswa kweRNA mamorekuru kuyedza kunoitwa zvakanyanya mune yakaparadzana RNA kamuri yekuvhiya, uye tafura yekuedzwa inocheneswa isati yaedzwa.Pfeka magurovhosi anoraswa uye masiki panguva yekuyedza kudzivirira kusvibiswa kweRNA kunokonzerwa neRNase sumo.
4.Iyo reagent yakasvibiswa neRNase panguva yekushandiswa.
Zano: Tsiva neViral RNA Isolation Kit itsva yezviedzo zvinoenderana.
5.Kusvibiswa kweRNase kwemachubhu ecentrifuge, matipi epipette, etc. Zano: Iva nechokwadi chekuti machubhu ecentrifuge, matipi epipette, uye mapipette ese ari RNase-Mahara.
Iwo akacheneswa mamorekuru eRNA akakanganisa kuyedza kuzasi
Mamorekuru eRNA akacheneswa nekoramu yekuchenesa achakanganisa kuyedza kuzasi kana paine maion emunyu akawandisa kana mapuroteni, akadai se: reverse transcription, Northern Blot, nezvimwewo.
1.Pane asara ayoni emunyu mune eluted RNA mamorekuru.
Kurudziro: Ita shuwa kuti iyo chaiyo vhoriyamu yeanhydrous ethanol yawedzerwa kuBuffer viRW2, uye geza koramu yekuchenesa kaviri zvinoenderana neiyo chaiyo centrifugation kumhanya pamirayiridzo yekushanda; Kana pachine maion emunyu asara, unogona kuwedzera Buffer viRW2 kukoromo yekuchenesa, uye woisiya pakamuri tembiricha ye5min.Zvadaro ita centrifugation kubvisa salt ions kusvibiswa kusvika pamwero mukuru
2.Pane yasara ethanol mu eluted RNA mamorekuru
Zano: kana uchinge wasimbisa kuti mbiru dzekuchenesa dzakasukwa neBuffer viRW2, ita isina chinhu-chubhu centrifugation zvinoenderana nekumhanya kwecentrifugal pamirayiridzo yekushandisa.Kana kuchine ethanol yasara, inogona kusiiwa kwemaminetsi mashanu pakudziya kwekamuri mushure meisina-tube centrifugation kubvisa yasara ethanol kusvika pamwero mukuru.
Mirayiridzo yezvinyorwa:
Viral RNA Isolation Kit Instruction Manual
