Txhawm rau txhawm rau tiv thaiv kev sib kis thaum lub sijhawm ua haujlwm, lub ntsiab tseem ceeb rau nws tus qauv, kev tsim kho thiab kev ua haujlwm tswj hwm yog:
1. Kev tsim thiab kev sim ua qauv, kev nyab xeeb ua haujlwm lossis cov txheej txheem kev nyab xeeb kev ua haujlwm yuav tsum ua tau raws li qhov kev xav tau ntawm&cov lus qhia tam sim no; GB19489.
2. PCR tau muab faib ua plaub qhov chaw ua haujlwm raws li cov theem ua haujlwm, hu ua reagent cia thiab chaw npaj khoom, thaj chaw npaj, thaj chaw ua kom dav, thiab thaj chaw tsom cov khoom lag luam. Yog hais tias qhov ntsuas ntawm fluorescent PCR ntawm lub sijhawm tiag tiag, nruab nrog kev ua kom nrov thiab tsom cov khoom lag luam tuaj yeem ua ke. Txhua thaj chaw ua haujlwm yuav tsum raug cim kom meej meej, thiab cov cuab yeej thiab khoom hauv thaj chaw ua haujlwm sib txawv yuav tsum tsis txhob sib xyaw.
Cov huab cua khiav dej num ntawm PCR qhov chaw kuaj hluav taws xob tuaj yeem ua tiav raws li qhov chaw khaws cia thiab npaj chaw preparation thaj chaw thaj chaw npaj → qhov chaw ua kom pom tseeb → cov cheeb tsam tsom cov khoom lag luam kom tiv thaiv cov khoom lag luam kom nkag mus hauv thaj chaw ua ntej nrog rau cov cua ntws. Nws tuaj yeem nqa tawm ntawm qhov uas txo qis cua siab los ntawm kev rov qab khaws cia thiab thaj chaw npaj → thaj chaw thaj chaw npaj → qhov chaw ua kom nrov dua → cov chaw tsom kom tau khoom. Nws tuaj yeem ua tiav los ntawm kev txhim kho cov kiv cua tso pa tawm, cov cuab yeej hluav taws xob tsis zoo los yog lwm qhov kev ua tau.
3. Nkag mus rau thaj chaw ua haujlwm txhua tus yuav tsum ua raws nraim cov kev taw qhia ib qho, qhov ntawd yog, npaj chaw cia khoom thiab chaw npaj → thaj chaw thaj chaw npaj → thaj chaw ua kom muaj zog → qhov tsom cov khoom lag luam. Cov chaw ua haujlwm sib txawv (xws li xim sib txawv) yuav tsum tau siv. Cov neeg ua haujlwm yuav tsum tsis txhob hnav khaub ncaws ua haujlwm thaum lawv tawm ntawm thaj chaw ua haujlwm.
4. Kev tu lub chaw kuaj yuav tsum ua raws li qhov chaw khaws khoom cia thiab chaw npaj chaw → thaj chaw thaj chaw npaj → qhov chaw ua kom nrov dua → cov chaw tsom kom tau khoom. Cov chaw sib txawv sim sib txawv yuav tsum muaj lawv cov cuab yeej tu vaj tse kom tiv thaiv kev ua paug.
5. Tom qab ua haujlwm tiav, thaj chaw ua haujlwm yuav tsum tau muab ntxuav tam sim ntawd. Cov nplaim ntawm cov chaw sim hauv thaj chaw ua haujlwm yuav tsum muaj peev xwm tiv thaiv qhov kev tua kab mob thiab kev ua kom huv ntawm cov tshuaj xws li sodium hypochlorite. Cov xim xaim hluav taws xob tsis muaj xim nyob rau saum npoo chav kuaj yuav tsum yooj yim thiab ua haujlwm tau zoo. Txij li qhov kev ncua deb thiab lub zog ntawm ultraviolet irradiation yog qhov tseem ceeb heev rau qhov cuam tshuam txog kev tshem tawm, qhov txav ntawm ultraviolet teeb (254nm wavelength) tuaj yeem siv, thiab tom qab ua haujlwm tiav, nws tuaj yeem hloov kho rau irradiate tsis pub dhau 60-90cm ntawm lub rooj sim Cov. Txij li cov khoom lag luam nthuav dav tsuas yog ob peb puas lossis kaum lub hauv paus ntawm tus khub (bp), nws tsis rhiab rau UV puas, yog li UV-tsis muaj kev cuam tshuam nthuav dav yuav tsum tau irradiated rau lub sijhawm ntev dua, nyiam dua ib hmos.
Lub luag haujlwm ntawm cov khoom siv reagent cia thiab npaj thaj chaw yog: npaj rau cov khoom ntim cia, kev faib cov tshuaj reagents thiab kev npaj cov amplification cov tshuaj tiv thaiv kev sib xyaw, nrog rau kev khaws cia thiab kev npaj ntawm cov khoom siv xws li cov hlab centrifuge thiab cov lus qhia. Cov ntaub ntawv xws li cia cov tshuaj reagents thiab cov khoom siv tau siv rau cov qauv pov tseg yuav tsum tau thauj ncaj qha mus rau qhov chaw khaws khoom thiab npaj chaw, thiab tsis tuaj yeem hla thaj chaw nrhiav kom muaj zog. Cov cuab yeej tswj zoo thiab cov khoom tswj kom zoo hauv cov khoom siv yuav tsum tsis txhob muab khaws cia rau hauv thaj chaw no, tab sis yuav tsum khaws cia hauv thaj chaw no. Thaj tsam chaw ua.
Kev ua haujlwm ntawm lub nrws chaw hauv thaj chaw yog: nucleic acid (RNA, DNA) extraction, khaws thiab ntxiv rau lub raj tshuaj amplification. Rau kev ua haujlwm nrog cov qauv kuaj mob, cov tseev kom muaj rau kev tiv thaiv cov khoom tiv thaiv, kev tiv thaiv tus kheej, thiab kev qhia tshwj xeeb rau kev siv biosafety lub chaw soj ntsuam ob yuav tsum tau ntsib. Txij li kev paug los ntawm aerosols yuav tshwm sim thaum lub sijhawm sib xyaw ua qauv thiab kev ua kom huv nucleic acid, qhov kev mob siab tau tsim nyob hauv thaj chaw no kom tsis txhob muaj cov pa paug aerosol los ntawm thaj chaw ze. Txhawm rau kom tsis txhob sib kis ntawm cov qauv, tom qab ntxiv cov nucleic acid los kuaj, lub raj tshuaj tiv thaiv uas muaj cov tshuaj tiv thaiv yuav tsum tau npog. Rau cov khoom siv kis tau tus mob, lub hau yuav tsum qhib hauv lub txee kev nyab xeeb, thiab yuav tsum muaj cov txheej txheem qhia meej txog kev ua qauv thiab tuav tsis tau.
Cov kev ua haujlwm ntawm qhov ua kom pom tseeb yog: DNA synthesis, DNA amplification thiab kuaj pom. Txhawm rau kom tsis txhob muaj kuab paug tshwm sim los ntawm aerosols, taug kev hauv thaj chaw yuav tsum ua kom tsawg. Nws yuav tsum raug sau tseg tias txhua txoj kev ntsuas cov tshuaj tiv thaiv yuav tsum tsis txhob qhib rau hauv thaj chaw no.
Cov kev ua haujlwm ntawm qhov chaw hauv cov khoom lag luam loj ntxiv: kev txheeb xyuas ntxiv thiab kev txiav txim siab ntawm cov khoom tawg, xws li kev sib xyaw, enzyme plab zom mov electrophoresis, denaturing siab ua kua tshuaj tsom, ua ntu zus, thiab lwm yam. ntawm daim nyias nyias lossis ntawm cov microplates lossis chips (radionuclide labeling lossis non-radioactive nuclide labeling), ncaj qha lossis zom agarose gel electrophoresis, Polyacrylamide gel electrophoresis, Kev xa tawm sab qab teb, nucleic acid sib luag txoj kev, huab hwm coj spectrometry, thiab lwm yam.
Cov cuab yeej ua lag luam tsom hauv cheeb tsam yog qhov tseem ceeb ntawm cov khoom lag luam amplification hauv PCR qhov chaw kuaj, yog li kev saib xyuas yuav tsum ua kom tsis txhob nqa lub tshuab ua kom nrov zog los ntawm cov lus thiab ua haujlwm khaub ncaws hauv thaj chaw no. Thaum siv txheej txheem PCR-ELISA los kuaj cov khoom loj, ib lub tshuab ntxhua phaj yuav tsum siv ntxuav cov paib, thiab cov kua khib nyiab yuav tsum tau ntim rau hauv 1 mol / L HCl, thiab tsis tuaj yeem muab tso rau hauv chav kuaj, tab sis yuav tsum tau muab pov tseg los ntawm PCR kuaj. Tso Tseg. Qhov kev qhia siv yuav tsum tau muab tsau rau hauv 1 mol / L HCl thiab tom qab ntawd muab tso rau hauv lub hnab khib nyiab pov tseg raws li cov txheej txheem, xws li kev ua kom sov. Raws li thaj chaw no yuav siv qee yam kev hloov caj ces thiab tshuaj lom neeg xws li ethidium bromide, acrylamide, formaldehyde lossis radionuclides, thiab lwm yam, kev tiv thaiv kev nyab xeeb ntawm cov neeg sim yuav tsum tau them rau.