Txoj kev tshawb nrhiav molecular muaj peev xwm tsim tau ntau qhov ntim ntawm nucleic acid los ntawm kev nthuav dav ntawm cov kab ntau pom hauv cov qauv.Thaum qhov no muaj txiaj ntsig zoo rau kev ua kom pom kev nkag siab zoo, nws kuj qhia txog qhov muaj peev xwm ntawm kev sib kis los ntawm kev sib kis ntawm amplification aerosols hauv qhov chaw kuaj mob.Thaum ua cov kev sim, kev ntsuas yuav tsum tau ua kom tsis txhob muaj kev sib kis ntawm cov tshuaj reagents, cov khoom siv hauv chav kuaj thiab lub rooj ntev zaum, vim tias cov kab mob sib kis no tuaj yeem tsim cov txiaj ntsig tsis zoo (lossis tsis tseeb-tsis zoo).

Txhawm rau kom txo qis qhov tshwm sim ntawm kev sib kis, Kev Ua Haujlwm Zoo yuav tsum tau siv txhua lub sijhawm.Tshwj xeeb, yuav tsum tau ceev faj txog cov ntsiab lus hauv qab no:

1. Siv cov tshuaj reagents
2. Lub koom haum ntawm chaw ua haujlwm thiab khoom siv
3. Siv thiab ntxuav cov lus qhia rau qhov chaw molecular
4. General molecular biology tswv yim
5. Kev tswj hwm sab hauv
6. Cov ntawv sau cia

1. Siv cov tshuaj reagents

Luv luv centrifuge reagent raj ua ntej qhib kom tsis txhob tsim cov aerosols.Aliquot reagents kom tsis txhob muaj ntau yam khov-taws thiab kev sib kis ntawm cov khoom lag luam.Kom meej meej daim ntawv lo thiab hnub tim tag nrho cov reagent thiab cov tshuaj tiv thaiv raj thiab tuav cov cav ntawm reagent ntau thiab batch tooj siv nyob rau hauv tag nrho cov kev sim.Pipette tag nrho cov reagents thiab cov qauv siv cov lus qhia lim.Ua ntej yuav khoom, nws raug nquahu kom paub meej nrog cov chaw tsim khoom tias cov lus qhia lim haum rau cov hom pipette siv.

2. Lub koom haum ntawm chaw ua haujlwm thiab khoom siv

Qhov chaw ua haujlwm yuav tsum tau teeb tsa kom ntseeg tau tias kev khiav haujlwm ntawm kev ua haujlwm tshwm sim hauv ib qho kev coj, los ntawm thaj chaw huv (pre-PCR) mus rau qhov chaw qias neeg (post-PCR).Cov kev ceev faj dav dav hauv qab no yuav pab txo qis kev kis kab mob.Muaj cov chav cais, lossis tsawg kawg ntawm lub cev sib cais, rau: kev npaj mastermix, nucleic acid extraction thiab DNA template ntxiv, amplification thiab tuav cov khoom amplified, thiab cov khoom tsom xam, xws li gel electrophoresis.

Hauv qee qhov chaw, muaj 4 chav sib cais yog qhov nyuaj.Ib qho kev xaiv tau tab sis tsis xav tau yog ua qhov kev npaj mastermix nyob rau hauv qhov chaw ntim khoom, xws li lub txee laminar ntws.Nyob rau hauv cov ntaub ntawv ntawm nested PCR amplification, kev npaj ntawm mastermix rau lub thib ob puag ncig cov tshuaj tiv thaiv yuav tsum tau npaj nyob rau hauv lub 'huv' cheeb tsam rau mastermix npaj, tab sis cov inoculation nrog cov thawj PCR khoom yuav tsum tau ua nyob rau hauv lub amplification chav tsev, thiab yog hais tias ua tau. nyob rau hauv ib qho chaw tshwj xeeb ntim khoom (xws li laminar flow txee).

Txhua chav tsev / cheeb tsam xav tau ib qho kev sib cais ntawm cov ntawv sau kom meej meej, cov lus qhia lim, raj racks, vortexes, centrifuges (yog tias tsim nyog), cwj mem, generic lab reagents, lab coats thiab cov hnab looj tes uas yuav nyob twj ywm ntawm lawv qhov chaw ua haujlwm.Ob txhais tes yuav tsum tau ntxuav thiab hnab looj tes thiab cov tsho hauv chav kuaj tau hloov thaum tsiv ntawm thaj chaw uas tau xaiv.Cov tshuaj reagents thiab cov khoom siv yuav tsum tsis txhob txav ntawm qhov chaw qias neeg mus rau qhov chaw huv.Yog tias muaj qhov xwm txheej hnyav tshwm sim qhov twg cov khoom siv reagent lossis cov khoom yuav tsum tau txav rov qab, nws yuav tsum tau ua ntej decontaminated nrog 10% sodium hypochlorite, tom qab ntawd so nrog dej tsis muaj menyuam.

Nco tseg

Cov tshuaj 10% sodium hypochlorite yuav tsum tau ua tshiab txhua hnub.Thaum siv rau decontamination, lub sij hawm tiv tauj tsawg kawg yog 10 feeb yuav tsum tau ua raws li.
Tsis tas li ntawd, cov khoom lag luam muaj nyob rau hauv cov khoom lag luam uas tau lees paub raws li DNA-tua decontaminants decontaminants tuaj yeem siv yog tias cov lus pom zoo ntawm kev nyab xeeb hauv zos tsis tso cai rau kev siv sodium hypochlorite lossis sodium hypochlorite tsis haum rau decontaminating cov khoom siv hlau.

Qhov zoo tshaj plaws, cov neeg ua haujlwm yuav tsum ua raws li kev ua haujlwm ntawm kev ua haujlwm tsis ncaj ncees thiab tsis txhob mus ntawm qhov chaw qias neeg (post-PCR) rov qab mus rau thaj chaw huv (pre-PCR) nyob rau tib hnub.Txawm li cas los xij, tej zaum yuav muaj qee lub sijhawm thaum qhov no tsis tuaj yeem zam tau.Thaum muaj lub sijhawm zoo li no, cov neeg ua haujlwm yuav tsum tau saib xyuas kom huv si ntxuav tes, hloov hnab looj tes, siv lub tsho tiv thaiv kev sim thiab tsis qhia cov khoom siv uas lawv yuav xav nqa tawm hauv chav dua, xws li cov ntawv kuaj.Cov kev tswj xyuas zoo li no yuav tsum tau hais txog hauv kev cob qhia cov neeg ua haujlwm ntawm cov txheej txheem molecular.

Tom qab siv, lub rooj zaum yuav tsum tau ntxuav nrog 10% sodium hypochlorite (ua raws li cov dej tsis huv kom tshem tawm cov tshuaj bleach residual), 70% ethanol, lossis cov khoom lag luam muaj txiaj ntsig DNA-ua kom puas tsuaj.Qhov zoo tshaj plaws, ultra-violet (UV) teeb yuav tsum haum kom muaj kev sib kis los ntawm irradiation.Txawm li cas los xij, kev siv cov teeb ci UV yuav tsum tau txwv rau qhov chaw ua haujlwm kaw, xws li cov txee rau kev nyab xeeb, txhawm rau txwv cov neeg ua haujlwm hauv chav kuaj pom UV raug.Thov ua raws li cov chaw tsim khoom cov lus qhia rau kev saib xyuas UV teeb, qhov cua thiab ntxuav kom ntseeg tau tias cov roj teeb tseem siv tau.

Yog tias siv 70% ethanol es tsis txhob siv sodium hypochlorite, irradiation nrog UV teeb yuav tsum tau ua kom tiav cov kab mob.
Tsis txhob ntxuav lub vortex thiab centrifuge nrog sodium hypochlorite;Hloov chaw, so nrog 70% ethanol thiab nthuav tawm rau UV teeb, lossis siv cov khoom lag luam DNA-ua kom puas tsuaj.Rau kev nchuav, nrog rau cov chaw tsim khoom kom paub cov lus qhia ntxiv txog kev ntxuav.Yog tias cov chaw tsim khoom cov lus qhia tso cai rau nws, pipettes yuav tsum niaj hnub ua kom tsis muaj menyuam los ntawm autoclave.Yog tias cov pipettes tsis tuaj yeem ua rau autoclaved, nws yuav tsum txaus los ntxuav lawv nrog 10% sodium hypochlorite (ua raws li cov dej tsis huv) lossis nrog cov khoom lag luam DNA-ua kom puas tsuaj tom qab UV raug.

Kev ntxuav nrog cov feem pua ​​​​ntawm sodium hypochlorite thaum kawg tuaj yeem ua rau cov khoom siv yas thiab hlau puas tsuaj yog tias ua tsis tu ncua;xyuas cov lus pom zoo los ntawm cov chaw tsim khoom ua ntej.Txhua yam khoom siv yuav tsum tau calibrated tsis tu ncua raws li lub chaw tsim tshuaj paus-pom zoo lub sij hawm.Ib tus neeg raug xaiv yuav tsum yog tus saib xyuas kom ntseeg tau tias lub sijhawm ntsuas ntsuas tau ua raws, cov ntaub ntawv ntxaws ntxaws tau khaws cia, thiab cov ntawv qhia kev pabcuam tau pom meej meej ntawm cov khoom siv.

3. Siv thiab ntxuav cov lus qhia rau qhov chaw molecular

Pre-PCR: Reagent aliquoting / mastermix npaj: Qhov no yuav tsum yog qhov huv tshaj plaws ntawm txhua qhov chaw siv rau kev npaj cov kev sim tshuaj molecular thiab yuav tsum yog qhov tsim nyog rau lub laminar flow txee nruab nrog lub teeb UV.Cov qauv, muab rho tawm nucleic acid thiab amplified PCR cov khoom yuav tsum tsis txhob raug coj mus rau hauv cheeb tsam no.Amplification reagents yuav tsum tau khaws cia rau hauv lub freezer (los yog lub tub yees, raws li cov neeg tsim khoom pom zoo) nyob rau hauv tib qhov chaw, qhov zoo tshaj plaws ntawm lub laminar flow txee los yog pre-PCR cheeb tsam.Cov hnab looj tes yuav tsum tau hloov txhua zaus thaum nkag mus rau hauv qhov chaw ua ntej PCR lossis laminar flow txee.

Lub pre-PCR cheeb tsam los yog laminar flow txee yuav tsum tau muab ntxuav ua ntej thiab tom qab siv raws li nram no: So tag nrho cov khoom nyob rau hauv lub txee, xws li pipettes, tip boxes, vortex, centrifuge, raj racks, cwjmem, thiab lwm yam. nrog 70% ethanol los yog a lag luam DNA-ua kom puas decontaminant, thiab cia kom qhuav.Nyob rau hauv cov ntaub ntawv ntawm qhov chaw ua hauj lwm kaw, xws li lub laminar flow txee, tso lub hood rau UV teeb rau 30 feeb.

Nco tseg

Tsis txhob nthuav tawm cov reagent rau UV teeb;tsuas yog txav lawv mus rau hauv lub txee thaum nws huv.Yog tias ua haujlwm rov qab PCR, nws kuj tseem yuav pab tau los so cov chaw thiab cov khoom siv nrog kev daws teeb meem uas rhuav tshem RNases ntawm kev sib cuag.Qhov no tuaj yeem pab kom tsis txhob muaj qhov tsis zoo-tsis zoo los ntawm enzyme degradation ntawm RNA.Tom qab decontamination thiab ua ntej npaj cov mastermix, hnab looj tes yuav tsum tau hloov ib zaug ntxiv, thiab tom qab ntawd lub txee yog npaj siv.

Pre-PCR: Nucleic acid extraction / template ntxiv:

Nucleic acid yuav tsum tau muab rho tawm thiab ua haujlwm nyob rau hauv ib cheeb tsam thib ob, siv cov khoom sib cais ntawm cov pipettes, cov lus qhia lim, raj racks, hnab looj tes tshiab, cov tsho loj hauv lab thiab lwm yam khoom siv.Qhov no kuj yog rau kev sib ntxiv ntawm cov qauv, kev tswj hwm thiab trendlines rau lub mastermix tubes los yog daim hlau.Txhawm rau kom tsis txhob muaj kev sib kis ntawm cov qauv nucleic acid uas tau raug tshuaj xyuas, nws raug nquahu kom hloov cov hnab looj tes ua ntej tuav cov kev tswj xyuas zoo lossis cov qauv thiab siv cov txheej txheem cais.PCR reagents thiab amplified cov khoom yuav tsum tsis txhob pipetted nyob rau hauv lub cheeb tsam no.Cov qauv yuav tsum muab cia rau hauv lub fridges los yog lub freezers nyob rau tib cheeb tsam.Cov qauv ua haujlwm yuav tsum tau muab ntxuav kom zoo ib yam li qhov chaw mastermix.

Tom qab PCR: Amplification thiab tuav cov khoom amplified

Qhov chaw xaiv no yog rau cov txheej txheem tom qab kev nthuav dav thiab yuav tsum tau cais lub cev los ntawm qhov chaw ua ntej PCR.Nws feem ntau muaj cov thermocyclers thiab lub sijhawm tiag tiag platforms, thiab qhov zoo tshaj plaws yuav tsum muaj lub laminar flow txee rau ntxiv cov khoom siv PCR puag ncig 1 rau cov tshuaj tiv thaiv ib puag ncig 2, yog tias PCR tau ua tiav.PCR reagents thiab rho tawm nucleic acid yuav tsum tsis txhob ua haujlwm hauv cheeb tsam no vim tias muaj kev pheej hmoo ntawm kev kis kab mob siab.Cov cheeb tsam no yuav tsum muaj nyias cov hnab looj tes, cov tsho hauv tsev kawm ntawv, cov phaj thiab cov raj racks, pipettes, lim cov lus qhia, cov bins thiab lwm yam khoom siv.Cov raj yuav tsum tau centrifuged ua ntej qhib.Cov qauv ua haujlwm yuav tsum tau muab ntxuav kom zoo ib yam li qhov chaw mastermix.

Tom qab PCR: Kev txheeb xyuas cov khoom

Chav tsev no yog rau cov khoom siv tshawb nrhiav, xws li gel electrophoresis tso tsheb hlau luam, lub hwj chim pob, UV transilluminator thiab cov ntaub ntawv gel.Cov cheeb tsam no yuav tsum muaj nyias cov hnab looj tes, cov tsho hauv tsev kawm ntawv, cov phaj thiab cov raj racks, pipettes, lim tswv yim, bins thiab lwm yam khoom siv.Tsis muaj lwm cov tshuaj reagents tuaj yeem coj mus rau hauv thaj chaw no, tsis suav nrog kev thauj khoom dye, molecular marker thiab agarose gel, thiab cov khoom tsis sib xws.Cov qauv ua haujlwm yuav tsum tau muab ntxuav kom zoo ib yam li qhov chaw mastermix.

Lus ceeb toom

Qhov zoo tshaj plaws, cov chav ua ntej PCR yuav tsum tsis txhob nkag mus rau tib hnub yog tias ua haujlwm tau ua tiav hauv chav tom qab PCR.Yog tias qhov no tsis tuaj yeem zam tau, xyuas kom meej tias txhais tes yuav tsum tau ntxuav ua ntej kom huv si thiab cov tsho tiv thaiv tshwj xeeb tau hnav hauv chav.Cov ntawv kuaj thiab cov ntaub ntawv yuav tsum tsis txhob muab coj mus rau hauv chav ua ntej PCR yog tias lawv tau siv hauv chav tom qab PCR;yog tias tsim nyog, muab cov ntawv luam tawm ntawm cov txheej txheem / qauv ID, thiab lwm yam.

4. General molecular biology tswv yim

Siv cov hnab looj tes tsis muaj hmoov kom tsis txhob muaj cov tshuaj tiv thaiv.Cov txheej txheem pipetting kom raug yog qhov tseem ceeb los txo kev sib kis.Kev xa dej tsis raug tuaj yeem ua rau txaws thaum tso kua dej thiab tsim cov aerosols.Kev xyaum zoo kom raug pipetting muaj nyob rau ntawm cov kev sib txuas hauv qab no: Gilson phau ntawv qhia rau pipetting, Anachem pipetting txheej txheem yeeb yaj duab, centrifuge raj ua ntej qhib, thiab qhib lawv ua tib zoo kom tsis txhob txaws.Kaw cov raj tam sim tom qab siv kom tsis txhob muaj cov kab mob sib kis.

Thaum ua ntau yam tshuaj tiv thaiv, npaj ib tus mastermix uas muaj cov tshuaj reagents (xws li dej, dNTPs, tsis muaj, primers thiab enzyme) kom txo tau tus naj npawb ntawm cov reagent hloov thiab txo cov kev hem thawj ntawm kev sib kis.Nws raug nquahu kom teeb tsa tus mastermix ntawm cov dej khov lossis ib qho chaw txias.Kev siv Hot Start enzyme tuaj yeem pab txo qis kev tsim cov khoom lag luam uas tsis yog tshwj xeeb.Tiv thaiv cov reagents uas muaj fluorescent sojntsuam los ntawm lub teeb kom tsis txhob degradation.

5. Kev tswj hwm sab hauv

suav nrog cov yam ntxwv zoo, lees paub qhov zoo thiab qhov tsis zoo ntawm kev tswj hwm, nrog rau kev tswj tsis muaj tus qauv hauv txhua qhov kev cuam tshuam, thiab ntau lub ntsiab lus titrated trendline rau ntau qhov kev tawm tsam.Kev tswj xyuas qhov zoo yuav tsum tsis txhob muaj zog heev uas ua rau muaj kev pheej hmoo kis kab mob.suav nrog kev tswj xyuas qhov zoo thiab tsis zoo thaum ua haujlwm nucleic acid extraction.

Nws raug pom zoo kom muab cov lus qhia meej tso rau hauv txhua qhov chaw kom cov neeg siv paub txog cov cai ntawm kev coj ua.Cov chaw kuaj mob kuaj pom cov qib qis ntawm DNA lossis RNA hauv cov qauv kuaj mob yuav xav tau txais kev ntsuas kev nyab xeeb ntxiv ntawm kev muaj cov cua sib cais nrog cov huab cua zoo me ntsis hauv chav ua ntej PCR thiab huab cua tsis zoo me ntsis hauv chav tom qab PCR.

Thaum kawg, tsim kom muaj kev pov hwm zoo (QA) txoj kev npaj yuav pab tau.Cov phiaj xwm zoo li no yuav tsum suav nrog cov npe ntawm cov khoom lag luam reagent thiab cov khoom lag luam ua haujlwm, cov cai rau khaws cov khoom siv thiab cov tshuaj reagents, tshaj tawm cov txiaj ntsig ntawm kev tswj hwm, kev cob qhia cov neeg ua haujlwm, kev daws teeb meem algorithms, thiab kev kho thaum xav tau.

6. Cov ntawv sau cia

Aslan A, Kinzelman J, Dreelin E, Anan'eva T, Lavander J. Tshooj 3: Teeb tsa lub chaw kuaj qPCR.Daim ntawv qhia txog kev sim cov dej ua si uas siv USEPA qPCR txoj kev 1611. Lansing- Michigan State University.

Public Health England, NHS.UK cov qauv rau kev tshawb nrhiav microbiology: Kev Xyaum Ua Haujlwm Zoo thaum ua cov tshuaj ntsuam xyuas molecular amplification).Kev Qhia Zoo.2013; 4(4):1–15.

Mifflin T. Teeb tsa lub chaw kuaj PCR.Cold Spring Harb Protoc.2007; 7.

Schroeder S 2013. Routine txij nkawm ntawm centrifuges: tu, tu thiab tshuaj tua kab mob ntawm centrifuges, rotors thiab adapters (Dawb ntawv No. 14).Hamburg: Eppendorf;2013.

Viana RV, Wallis CL.Kev soj ntsuam kuaj pom zoo (GCLP) rau cov kev sim tshuaj molecular siv hauv cov chaw kuaj mob, Hauv: Akyar I, editor.Dav dav ntawm kev tswj kom zoo.Rijeka, Croatia: Intech;2011: 29–52.