Izindlela zokuthola amangqamuzana zinamandla okukhiqiza umthamo omkhulu we-nucleic acid ngokukhulisa amanani omkhondo atholakala kumasampula.Nakuba lokhu kunenzuzo ekuvumela ukutholwa okuzwelayo, kuphinde kwethule ithuba lokungcoliswa ngokusabalalisa ama-aerosol okukhulisayo endaweni yaselabhorethri.Lapho kwenziwa izivivinyo, izinyathelo zingenziwa ukuze kugwenywe ukungcoliswa kwama-reagents, amathuluzi aselabhorethri nendawo yebhentshi, njengoba lokho kungcola kungase kukhiqize imiphumela engemihle (noma engemihle) engamanga.

Ukusiza ukunciphisa amathuba okungcola, Ukuzijwayeza Kwelabhorethri Okuhle kufanele kwenziwe ngaso sonke isikhathi.Ngokukhethekile, kufanele kuthathwe izinyathelo zokuphepha mayelana namaphuzu alandelayo:

1. Ukuphatha ama-reagents
2. Ukuhleleka kwendawo yokusebenza kanye nemishini
3. Sebenzisa kanye nokuhlanza iseluleko sendawo yamangqamuzana emisiwe
4. Iseluleko esijwayelekile sebhayoloji yamangqamuzana
5. Izilawuli zangaphakathi
6. I-Bibliography

1. Ukuphatha ama-reagents

Kafushane amashubhu e-reagent centrifuge ngaphambi kokuvula ukugwema ukukhiqizwa kwama-aerosol.Ama-reagents e-Aliquot ukuze agweme ukuncibilika kweqhwa okuningi kanye nokungcoliswa kwezitoko ezinkulu.Faka ngokucacile ilebula futhi udethi wonke amashubhu asebenzayo kanye nokusabela futhi ulondoloze amalogu e-reagent lot nezinombolo zeqoqo ezisetshenziswa kukho konke ukuhlola.Pipette wonke ama-reagents namasampuli usebenzisa amathiphu okuhlunga.Ngaphambi kokuthenga, kunconywa ukuthi uqinisekise nomkhiqizi ukuthi amathiphu okuhlunga afanele uhlobo lwe-pipette oluzosetshenziswa.

2. Ukuhleleka kwendawo yokusebenza kanye nemishini

Indawo yokusebenza kufanele ihlelwe ukuze kuqinisekiswe ukuthi ukuhamba komsebenzi kwenzeka ohlangothini olulodwa, kusukela ezindaweni ezihlanzekile (ngaphambi kwe-PCR) kuya ezindaweni ezingcolile (i-post-PCR).Izindlela zokuphepha ezilandelayo ezijwayelekile zizosiza ekunciphiseni amathuba okutheleleka.Yiba namakamelo ahlukene akhethiwe, noma okungenani abe nezindawo ezihlukene ngokomzimba, zalokhu: ukulungiswa kwe-mastermix, ukukhipha i-nucleic acid kanye nokwengezwa kwesifanekiso se-DNA, ukukhuliswa nokuphathwa komkhiqizo okhulisiwe, nokuhlaziywa komkhiqizo, isb i-gel electrophoresis.

Kwezinye izilungiselelo, ukuba namakamelo angu-4 ahlukene kunzima.Inketho engenzeka kodwa engathandeki kakhulu ukwenza ukulungiswa kwe-mastermix endaweni yokugcina izinto, isb i-laminar flow cabinet.Endabeni yokukhulisa i-PCR esidlekeni, ukulungiswa kwe-mastermix yokusabela komzuliswano wesibili kufanele kulungiswe endaweni 'ehlanzekile' ukuze kulungiswe i-mastermix, kodwa ukujova ngomkhiqizo oyinhloko we-PCR kufanele kwenziwe ekamelweni lokukhulisa, futhi uma kungenzeka. endaweni yokuphatha ezinikele (isb. i-laminar flow cabinet).

Igumbi/indawo ngayinye idinga isethi ehlukile yamapayipi abhalwe ngokucacile, amathiphu okuhlunga, ama-tube racks, ama-vortexes, ama-centrifuges (uma kufanele), amapeni, ama-generic lab reagents, amajazi elebhu namabhokisi amagilavu ​​azohlala ezindaweni zawo zokusebenza.Izandla kufanele zigezwe futhi amagilavu ​​namajazi aselebhu ashintshwe lapho uhamba phakathi kwezindawo eziqokiwe.Ama-reagents nezinto zokusebenza akufanele zisuswe endaweni engcolile ziyiswe endaweni ehlanzekile.Uma kwenzeka kuvela isimo esibucayi lapho i-reagent noma ucezu lwesisetshenziswa ludinga ukuhlehliswa emuva, kufanele luqale lungcoliswe ngo-10% we-sodium hypochlorite, kulandele ukwesulwa phansi ngamanzi angenalutho.

Qaphela

Isixazululo se-sodium hypochlorite esingu-10% kufanele senziwe sisha nsuku zonke.Lapho isetshenziselwa ukususwa kokungcola, isikhathi esincane sokuxhumana semizuzu eyi-10 kufanele kulandelwe.
Okunye, imikhiqizo etholakalayo edayiswayo egunyazwe njengezingcolisi zangaphezulu ezicekela phansi i-DNA ingasetshenziswa uma izincomo zokuphepha kwendawo zingakuvumeli ukusetshenziswa kwe-sodium hypochlorite noma uma i-sodium hypochlorite ingafaneleki ukungcolisa izingxenye zensimbi zensimbi.

Okufanelekile, abasebenzi kufanele balandele i-unidirectional workflow ethos futhi bangahambi ezindaweni ezingcolile (post-PCR) babuyele ezindaweni ezihlanzekile (pre-PCR) ngosuku olufanayo.Nokho, kungase kube nezikhathi lapho lokhu kungenakugwenywa.Uma kwenzeka leso senzakalo, izisebenzi kufanele ziqikelele ukuthi zigeza izandla kahle, zishintsha amagilavu, zisebenzise ijazi lelebhu elikhethiwe futhi zingavezi noma yiziphi izinto ezizofuna ukuzikhipha ekamelweni futhi, njengezincwadi zelebhu.Izinyathelo ezinjalo zokulawula kufanele zigcizelelwe ekuqeqeshweni kwabasebenzi ngezindlela zamangqamuzana.

Ngemuva kokusetshenziswa, izikhala zamabhentshi kufanele zihlanzwe ngo-10% we-sodium hypochlorite (okulandelwa amanzi angenalutho ukuze kukhishwe i-bleach eyinsalela), i-ethanol engu-70%, noma isibulala-magciwane esitholakalayo esithengiswayo esicekela phansi i-DNA.Okufanelekile, amalambu e-Ultra-violet (UV) kufanele afakwe ukuze akwazi ukususwa kokungcola ngokukhanyisa.Kodwa-ke, ukusetshenziswa kwezibani ze-UV kufanele kukhawulelwe ezindaweni zokusebenza ezivaliwe, isb. amakhabethe okuphepha, ukuze kuncishiswe ukukhanya kwe-UV kwabasebenzi baselabhorethri.Sicela uthobele imiyalelo yomkhiqizi yokunakekelwa kwezibani ze-UV, ukungena komoya kanye nokuhlanza ukuze uqinisekise ukuthi izibani zihlala zisebenza.

Uma usebenzisa i-ethanol engu-70% esikhundleni se-sodium hypochlorite, ukukhanyiselwa ngemisebe ye-UV kuyodingeka ukuze kuqedelwe ukungcola.
Ungahlanzi i-vortex ne-centrifuge nge-sodium hypochlorite;esikhundleni salokho, sula phansi nge-ethanol engu-70% futhi uveze ukukhanya kwe-UV, noma usebenzise isibulala-magciwane esicekela phansi i-DNA.Mayelana nokuchitheka, hlola nomkhiqizi ukuze uthole iseluleko esengeziwe sokuhlanza.Uma iziqondiso zomkhiqizi zikuvumela, ama-pipette kufanele abulawe inzalo nge-autoclave.Uma ama-pipette engakwazi uku-autoclaved, kufanele anele ukuwahlanza ngo-10% we-sodium hypochlorite (okulandelwa ukusula phansi ngokuphelele ngamanzi oyinyumba) noma ngesinqamuleli esicekela phansi i-DNA esilandelwa ukuchayeka kwe-UV.

Ukuhlanza nge-sodium hypochlorite ephezulu yamaphesenti kungase kugcine kulimaze amapulasitiki e-pipette nezinsimbi uma kwenziwa njalo;hlola izincomo ezivela kumkhiqizi kuqala.Zonke izinto ezisetshenziswayo zidinga ukulinganiswa njalo ngokweshejuli enconywe ngumkhiqizi.Umuntu oqokiwe kufanele kube nguyena ophethe ukuqinisekisa ukuthi ishejuli yokulinganisa iyalandelwa, amalogi anemininingwane ayagcinwa, futhi amalebula esevisi abekwe ngokucacile emishinini.

3. Sebenzisa kanye nokuhlanza iseluleko sendawo yamangqamuzana emisiwe

I-Pre-PCR: Ukulungiswa kwe-Reagent aliquoting / mastermix: Lokhu kufanele kube indawo ehlanzekile kunazo zonke ezisetshenziselwa ukulungiswa kokuhlolwa kwamangqamuzana futhi kufanele kube yikhabhinethi ekhethiwe yokugeleza kwe-laminar efakwe isibani se-UV.Amasampula, i-nucleic acid ekhishiwe kanye nemikhiqizo ye-PCR ekhulisiwe akumele isingathwe kule ndawo.Ama-reagents okukhuliswa kufanele agcinwe efrijini (noma esiqandisini, ngokwezincomo zomkhiqizi) endaweni efanayo ekhethiwe, ngokufanelekile eduze kwekhabhinethi yokugeleza kwe-laminar noma indawo yangaphambi kwe-PCR.Amagilavu ​​kufanele ashintshwe isikhathi ngasinye lapho kungena endaweni yangaphambi kwe-PCR noma i-laminar flow cabinet.

Indawo yangaphambi kwe-PCR noma i-laminar flow cabinet kufanele ihlanzwe ngaphambi nangemva kokusetshenziswa ngale ndlela elandelayo: Sula zonke izinto ekhabetheni, isb. amapayipi, ama-tip box, i-vortex, i-centrifuge, ama-tube racks, amapeni, njll. nge-ethanol engu-70%. i-decontaminant ecekela phansi i-DNA, futhi ivumele ukuba yome.Endabeni yendawo yokusebenza evaliwe, isb. i-laminar flow cabinet, beka ihood ekukhanyeni kwe-UV imizuzu engu-30.

Qaphela

Ungavezi ama-reagents ekukhanyeni kwe-UV;uwafake ekhabetheni kuphela uma selihlanzekile.Uma wenza okulotshiweyo okuhlanekezelwe i-PCR, kungase futhi kusize ukusula indawo engaphezulu nokokusebenza ngesixazululo esihlukanisa ama-RNas lapho uthintana.Lokhu kungasiza ukugwema imiphumela engemihle-emibi evela ekonakaleni kwe-enzyme ye-RNA.Ngemuva kokuqeda ukungcola nangaphambi kokulungiselela i-mastermix, amagilavu ​​kufanele ashintshwe futhi, bese ikhabethe selilungele ukusetshenziswa.

I-Pre-PCR: Ukukhishwa kwe-Nucleic acid/ukwengezwa kwesifanekiso:

I-Nucleic acid kufanele ikhishwe futhi isingathwe endaweni yesibili eqokiwe, kusetshenziswa isethi ehlukile yamapayipi, amathiphu okuhlunga, amashubhu okubeka, amagilavu ​​amasha, amajazi aselebhu kanye nezinye izinto zokusebenza.Le ndawo futhi ingeyokwengezwa kwesifanekiso, izilawuli kanye nemigqa mastermix amashubhu noma amapuleti.Ukuze ugweme ukungcoliswa kwamasampula e-nucleic acid akhishiwe ahlaziywayo, kunconywa ukuthi ushintshe amagilavu ​​ngaphambi kokuphatha izilawuli ezinhle noma izindinganiso futhi usebenzise isethi ehlukile yamapayipi.Ama-PCR ama-reagents kanye nemikhiqizo ekhulisiwe akufanele ifakwe ngamapayipi kule ndawo.Amasampula kufanele agcinwe eziqandisini ezikhethiwe noma endaweni efanayo.Indawo yokusebenza yesampula kufanele ihlanzwe ngendlela efanayo nesikhala se-mastermix.

I-Post-PCR: Ukukhulisa nokuphathwa komkhiqizo okhulisiwe

Lesi sikhala esinqunyiwe esenzelwe izinqubo zangemuva kokukhulisa futhi kufanele sihluke ngokoqobo ezindaweni zangaphambi kwe-PCR.Ivamise ukuqukatha ama-thermocyclers namapulatifomu esikhathi sangempela, futhi kufanele ibe nekhabethe lokugeleza kwe-laminar yokwengeza umkhiqizo oyindilinga ongu-1 PCR ekuphenduleni okuyindilinga oku-2, uma i-PCR efakwe esidlekeni yenziwa.Ama-PCR reagents kanye ne-nucleic acid ekhishiwe akumele kuphathwe kule ndawo njengoba ubungozi bokungcola buphezulu.Le ndawo kufanele ibe nesethi ehlukile yamagilavu, amajazi elebhu, amapuleti namashubhu racks, amapayipi, amathiphu okuhlunga, imigqomo nezinye izinto zokusebenza.Amashubhu kufanele abekwe phakathi nendawo ngaphambi kokuvuleka.Indawo yokusebenza yesampula kufanele ihlanzwe ngendlela efanayo nesikhala se-mastermix.

I-Post-PCR: Ukuhlaziywa komkhiqizo

Leli gumbi elokuhlonza umkhiqizo, isb amathangi e-gel electrophoresis, amaphakethe kagesi, i-UV transilluminator kanye nesistimu yokubhala imibhalo yejeli.Le ndawo kufanele ibe namasethi ahlukene amagilavu, amajazi elebhu, amapuleti nama-tube racks, ama-pipette, amathiphu okuhlunga, imigqomo nezinye izinto zokusebenza.Awekho amanye ama-reagents angalethwa kule ndawo, ngaphandle kodayi wokulayisha, umaka wemolekyuli nejeli ye-agarose, nezingxenye zebhafa.Indawo yokusebenza yesampula kufanele ihlanzwe ngendlela efanayo nesikhala se-mastermix.

Inothi elibalulekile

Ngokufanelekile, amagumbi angaphambi kwe-PCR akufanele afakwe ngosuku olufanayo uma umsebenzi usuwenziwe kakade emakamelweni e-post-PCR.Uma lokhu kungenakugwenywa ngokuphelele, qiniseka ukuthi izandla zigezwa kahle kuqala nokuthi amajazi athile aselebhu ayagqokwa emakamelweni.Izincwadi zelebhu kanye namaphepha akumele kuyiswe emakamelweni angaphambi kwe-PCR uma kusetshenziswe emakamelweni angemva kwe-PCR;uma kunesidingo, thatha ukuphrinta okuyimpinda kwamaphrothokholi/amasampula omazisi, njll.

4. Iseluleko esijwayelekile sebhayoloji yamangqamuzana

Sebenzisa amagilavu ​​angenayo impushana ukuze ugweme ukuvinjelwa kokuhlolwa.Indlela efanele yokufaka amapayipi ibalulekile ekwehliseni ukungcola.Ukufakwa kwamapayipi okungalungile kungase kubangele ukufafaza lapho ukhipha uketshezi nokudala ama-aerosol.Umkhuba omuhle wokusebenzisa amapayipi okulungile ungatholakala kulezi zixhumanisi ezilandelayo: Umhlahlandlela ka-Gilson wamapayipi, amavidiyo we-Anachem pipetting, amashubhu e-Centrifuge ngaphambi kokuwavula, futhi uwavule ngokucophelela ukuze ugweme ukuchaphazeka.Vala amashubhu ngokushesha ngemva kokusetshenziswa ukuze ugweme ukwethulwa kokungcola.

Lapho wenza ukusabela okuningi, lungiselela ukuxuba okuyinhloko okukodwa okuqukethe ama-reagents avamile (isb amanzi, i-dNTPs, i-buffer, ama-primers kanye ne-enzyme) ukuze unciphise inani lokudluliswa kwe-reagent futhi unciphise usongo lokungcoliswa.Kunconywa ukusetha i-mastermix eqhweni noma endaweni ebandayo.Ukusetshenziswa kwe-Enzayimu Yokuqala Okushisayo kungasiza ekunciphiseni ukukhiqizwa kwemikhiqizo engaqondile.Vikela ama-reagents aqukethe ama-fluorescent probe ekukhanyeni ukuze ugweme ukuwohloka.

5. Izilawuli zangaphakathi

Bandakanya izilawuli ezinezimpawu ezinhle, eziqinisekisiwe ezivumayo nezingezinhle, kanye nokulawula okungekho isifanekiso kukho konke ukusabela, kanye nomugqa wethrendi onamaphuzu amaningi wokusabela komthamo.Ukulawula okuhle akufanele kube namandla kangangokuba kudale ingozi yokutheleleka.Faka phakathi izilawuli zokukhipha ezinhle nezingezinhle lapho wenza i-nucleic acid extraction.

Kunconywa ukuthi kufakwe imiyalelo ecacile endaweni ngayinye ukuze abasebenzisi bazi ngemithetho yokuziphatha.Amalebhu okuxilonga athola amazinga aphansi kakhulu e-DNA noma e-RNA kumasampula omtholampilo angase afune ukusebenzisa isilinganiso esingeziwe sokuphepha sokuba nezinhlelo zokubamba komoya ezihlukene ezinomfutho womoya omuhle kancane emakamelweni angaphambi kwe-PCR kanye nomfutho womoya omubi kancane emagumbini angemva kwe-PCR.

Okokugcina, ukwenza uhlelo lokuqinisekisa ikhwalithi (QA) kuyasiza.Icebo elinjalo kufanele lifake izinhlu zezitoko eziyinhloko ze-reagent kanye nezitokwe zokusebenza, imithetho yokugcina amakhithi nama-reagents, ukubika imiphumela yokulawula, izinhlelo zokuqeqesha abasebenzi, ama-algorithms okuxazulula inkinga, kanye nezinyathelo zokulungisa lapho kudingeka.

6. I-Bibliography

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I-Public Health England, NHS.Izindinganiso zase-UK zophenyo lwe-microbiology: Ukuzijwayeza Okuhle Kwelabhorethri lapho kwenziwa ukuhlolwa kokukhulisa amangqamuzana).Isiqondiso Sekhwalithi.2013;4(4):1–15.

I-Mifflin T. Isetha ilabhorethri ye-PCR.I-Cold Spring Harb Protoc.2007;7.

I-Schroeder S 2013. Ukugcinwa okujwayelekile kwama-centrifuges: ukuhlanza, ukugcinwa kanye nokubulala amagciwane ama-centrifuges, ama-rotor nama-adapter (Iphepha elimhlophe No. 14).E-Hamburg: Eppendorf;2013.

Viana RV, Wallis CL.I-Good Clinical Laboratory Practice (GCLP) yokuhlolwa okusekelwe kumangqamuzana asetshenziswa ezindaweni zokuxilonga, Ku: Akyar I, umhleli.I-spectra ebanzi yokulawula ikhwalithi.eRijeka, eCroatia: Intech;2011: 29-52.