Iindlela zokubona iimolekyuli ziyakwazi ukuvelisa umthamo omkhulu we-nucleic acid ngokukhulisa ubuninzi bomkhondo ofunyenwe kwiisampuli.Ngelixa oku kuluncedo ekwenzeni ubhaqo olubuthathaka, lukwazisa ukuba nokwenzeka kosulelo ngokusasazwa kwe-aerosols yokukhulisa kwindawo yelabhoratri.Xa kuqhutywa imifuniselo, amanyathelo anokuthathwa ukuze kuthintelwe ukungcoliseka kwee-reagents, izixhobo zelabhoratri kunye nendawo yebhentshi, njengoko ungcoliseko olunjalo lunokuvelisa iziphumo zobuxoki (okanye ezingezizo ezingezizo).

Ukunceda ukunciphisa amathuba okungcoliseka, ukuSebenzisa okuLungileyo kweLebhu kufuneka kwenziwe ngawo onke amaxesha.Ngokukodwa, amanyathelo okhuseleko kufuneka athathwe malunga nala manqaku alandelayo:

1. Ukuphatha ii-reagents
2. Ukulungelelaniswa kwendawo yokusebenza kunye nezixhobo
3. Ukusebenzisa kunye neengcebiso zokucoca kwindawo echongiweyo yemolekyuli
4. Ingcebiso ngokubanzi ngebhayoloji yemolekyuli
5. Ulawulo lwangaphakathi
6. Uluhlu lweencwadi

1. Ukuphatha ii-reagents

Ngokufutshane iityhubhu ze-reagent centrifuge phambi kokuvula ukunqanda ukuveliswa kwee-aerosols.I-Aliquot reagents ukunqanda ukunyibilika komkhenkce okuninzi kunye nokungcoliseka kwezitokhwe eziphambili.Ileyibheli ngokucacileyo kwaye ubhale umhla zonke ii-reagent kunye neetyhubhu zokusabela kwaye ugcine iilogi ze-reagent lot kunye namanani ebhetshi asetyenziswa kuyo yonke imifuniselo.I-Pipette zonke ii-reagents kunye neesampuli usebenzisa iingcebiso zokucoca.Ngaphambi kokuthenga, kuyacetyiswa ukuba uqinisekise kunye nomenzi ukuba iingcebiso zokucoca zihambelana ne-brand ye-pipette yokusetyenziswa.

2. Ukulungelelaniswa kwendawo yokusebenza kunye nezixhobo

Indawo yokusebenzela kufuneka ilungiselelwe ukuqinisekisa ukuba ukuhamba komsebenzi kwenzeka kwicala elinye, ukusuka kwiindawo ezicocekileyo (ngaphambi kwe-PCR) ukuya kwiindawo ezingcolileyo (i-post-PCR).La manyathelo okhuseleko alandelayo aya kunceda ukunciphisa amathuba osulelo.Yiba namagumbi akhethiweyo ahlukeneyo, okanye ubuncinane kwiindawo ezihlukeneyo ngokwasemzimbeni, ukulungiselela: ukulungiswa kwe-mastermix, i-nucleic acid extraction kunye ne-DNA template yokongeza, ukukhulisa kunye nokuphathwa kwemveliso ye-amplified, kunye nohlalutyo lwemveliso, umz. i-gel electrophoresis.

Kwezinye iimeko, ukuba namagumbi ama-4 ahlukeneyo kunzima.Inketho enokwenzeka kodwa enganqweneleki kangako kukwenza amalungiselelo omxube ophambili kwindawo yokuqulatha, umz. i-laminar flow cabinet.Kwimeko yokukhulisa i-PCR efakwe kwindlwane, ukulungiswa kwe-mastermix kwi-reaction ye-round round reaction kufuneka ilungiswe kwindawo 'ecocekileyo' ukulungiselela ukulungiswa kwe-mastermix, kodwa i-inoculation kunye ne-primary PCR imveliso kufuneka yenziwe kwigumbi lokukhulisa, kwaye ukuba kunokwenzeka. kwindawo yogcino olunikezelweyo (umz. i-laminar flow cabinet).

Igumbi/indawo nganye ifuna iseti eyahlukileyo ebhalwe ngokucacileyo, iingcebiso zokucoca, ii-tube racks, ii-vortexes, ii-centrifuges (ukuba zifanelekile), iipeni, ii-reagents zalebhu eziqhelekileyo, iidyasi zelebhu kunye neebhokisi zeeglavu eziya kuhlala kwiindawo zazo zokusebenza ngokwahlukeneyo.Izandla kufuneka zihlanjwe kwaye iiglavu kunye needyasi zaselebhu zitshintshwe xa zihamba phakathi kweendawo ezichongiweyo.Ii-reagents kunye nezixhobo akufanele zisuswe kwindawo engcolileyo ukuya kwindawo ecocekileyo.Ukuba kuvela imeko egqithisileyo apho i-reagent okanye intwana yesixhobo kufuneka ibuyiselwe umva, kufuneka kuqala ihlanjululwe nge-10% ye-sodium hypochlorite, ilandelwe kukusulwa ngamanzi angenazintsholongwane.

Phawula

Isisombululo se-10% ye-sodium hypochlorite kufuneka senziwe ngokutsha yonke imihla.Xa isetyenziselwa ukucoca, ubuncinci bexesha lokuqhagamshelana nemizuzu eyi-10 kufuneka ithotyelwe.
Ngenye indlela, iimveliso ezithengiswayo eziqinisekisiweyo njenge-DNA-etshabalalisa i-decontaminants yomhlaba ingasetyenziselwa ukuba iingcebiso zokhuseleko lwendawo azivumeli ukusetyenziswa kwe-sodium hypochlorite okanye ukuba i-sodium hypochlorite ayifanelekanga ukutshabalalisa iindawo zetsimbi zezixhobo.

Ngokufanelekileyo, abasebenzi kufuneka bahambisane ne-unidirectional workflow ethos kwaye bangahambi kwiindawo ezingcolileyo (i-post-PCR) babuyele kwiindawo ezicocekileyo (pre-PCR) ngosuku olufanayo.Nangona kunjalo, kusenokubakho amaxesha apho oku kungenakuthintelwa.Xa eso sihlandlo sivela, abasebenzi kufuneka balumke bazihlambe izandla ngocoselelo, batshintshe iiglavu, basebenzise idyasi yelebhu emiselweyo kwaye bangasazisi nasiphi na isixhobo abaya kufuna ukusikhupha kwakhona kwigumbi, njengeencwadi zelebhu.Amanyathelo olawulo anjalo kufuneka agxininiswe kuqeqesho lwabasebenzi kwiindlela zeemolekyuli.

Emva kokusetyenziswa, izithuba zebhentshi kufuneka zihlambuluke nge-10% ye-sodium hypochlorite (elandelwa ngamanzi angcolileyo ukususa i-bleach eseleyo), i-70% ye-ethanol, okanye i-decontaminant ye-DNA eqinisekisiweyo yokuthengisa.Ngokufanelekileyo, izibane ze-ultra-violet (UV) kufuneka zifakwe ukuze zikwazi ukukhupha ukungcola ngokukhanya.Nangona kunjalo, ukusetyenziswa kwezibane ze-UV kufuneka kuthintelwe kuphela kwiindawo zokusebenza ezivaliweyo, umz. iikhabhathi zokhuseleko, ukuze kuthintelwe ukukhuseleka kwe-UV yabasebenzi baselabhoratri.Nceda uthobele imiyalelo yomenzi yokhathalelo lwesibane se-UV, ukungena komoya kunye nokucoca ukuze uqinisekise ukuba izibane zihlala zisebenza.

Ukuba usebenzisa i-70% ye-ethanol endaweni ye-sodium hypochlorite, ukukhanya kwe-UV kuya kufuneka ukugqibezela ukungcola.
Musa ukucoca i-vortex kunye ne-centrifuge nge-sodium hypochlorite;endaweni yoko, sula phantsi nge-70% ye-ethanol kwaye ubonise ukukhanya kwe-UV, okanye usebenzise i-decontaminant ye-DNA yorhwebo.Ukufumana inkunkuma, jonga kumenzi ngengcebiso ezongezelelweyo zokucoca.Ukuba imiyalelo yomenzi iyayivumela, ii-pipettes kufuneka zicolwe rhoqo nge-autoclave.Ukuba ii-pipettes azikwazi ukuhlanjululwa ngokuzenzekelayo, kufuneka kwanele ukuzicoca nge-10% ye-sodium hypochlorite (elandelwa ngokucokisekileyo ukusula ngamanzi angenazintsholongwane) okanye nge-decontaminant ye-DNA yorhwebo elandelwa yi-UV exposure.

Ukucoca ngepesenti ephezulu ye-sodium hypochlorite ekugqibeleni kunokonakalisa iiplastiki zepipette kunye nesinyithi ukuba kwenziwa rhoqo;jonga iingcebiso kumenzi kuqala.Zonke izixhobo kufuneka zilinganiswe rhoqo ngokweshedyuli ecetyiswa ngumenzi.Umntu otyunjiweyo makabe noxanduva lokuqinisekisa ukuba kuthotyelwe ishedyuli yohlengahlengiso, iilogi ezineenkcukacha ezibanzi ziyagcinwa, kwaye iilebhile zenkonzo zixhonywe ngokucacileyo kwisixhobo.

3. Ukusebenzisa kunye neengcebiso zokucoca kwindawo echongiweyo yemolekyuli

I-PCR yangaphambili: I-Reagent aliquoting / ukulungiswa kwe-mastermix: Oku kufuneka kube yeyona ndawo icocekileyo kuzo zonke izithuba ezisetyenziselwa ukulungiswa kwemifuniselo ye-molekyuli kwaye kufanele ukuba ibe yikhabhathi ekhethiweyo yokuhamba kwe-laminar exhotyiswe ngokukhanya kwe-UV.Iisampulu, i-nucleic acid ekhutshiweyo kunye neemveliso ze-PCR ezikhulisiwe akufanele ziphathwe kule ndawo.I-reagents yokukhulisa i-amplification kufuneka igcinwe kwisikhenkcisi (okanye kwifriji, ngokwengcebiso zomenzi) kwindawo efanayo ekhethiweyo, ngokufanelekileyo kufuphi nekhabhinethi yokuhamba kwe-laminar okanye indawo ye-PCR yangaphambili.Iiglavu kufuneka zitshintshwe ixesha ngalinye xa ungena kwindawo ye-PCR yangaphambili okanye i-laminar flow cabinet.

Indawo yangaphambi kwePCR okanye i-laminar flow cabinet kufuneka icocwe phambi nasemva kokusetyenziswa ngolu hlobo lulandelayo: Sula zonke izinto ezikwikhabhathi, umz. iipipettes, tip boxes, vortex, centrifuge, tube racks, pen, njl. I-DNA yokuthengisa i-decontaminant, kwaye ivumele ukuba yome.Kwimeko yendawo yokusebenza evaliweyo, umz. ikhabhathi yokuhamba kwelaminar, beka ihood kwisibane se-UV imizuzu engama-30.

Phawula

Musa ukuveza i-reagents kwisibane se-UV;zizise kwikhabhathi kuphela xa sele icocekile.Ukuba wenza i-PCR yokukhuphela umva, kunokuba luncedo ukusula imiphezulu kunye nezixhobo ngesisombululo esaphula ii-RNases kuqhagamshelwano.Oku kunokunceda ukunqanda iziphumo ezingezizo ezingezizo ezisuka ekuthotyweni kwe-enzyme ye-RNA.Emva kokutshatyalaliswa kunye nangaphambi kokulungiselela i-mastermix, iiglavu kufuneka zitshintshwe kwakhona, kwaye ikhabhinethi ilungele ukusetyenziswa.

I-PCR yangaphambili: I-Nucleic acid extraction/itemplate yokongeza:

I-Nucleic acid kufuneka itsalwe kwaye iphathwe kwindawo yesibini echongiweyo, kusetyenziswa iseti eyahlukileyo yeepipettes, iingcebiso zokucoca, ii-tube racks, iiglavu ezintsha, iidyasi zelebhu kunye nezinye izixhobo. iityhubhu ze-mastermix okanye iipleyiti.Ukuze ugweme ukungcoliswa kweesampuli ze-nucleic acid ezicatshulwayo ezihlalutywayo, kucetyiswa ukuba utshintshe iiglavu ngaphambi kokuphatha ulawulo oluhle okanye imigangatho kunye nokusebenzisa isethi ehlukeneyo yeepipettes.Ii-reagents ze-PCR kunye neemveliso ezandisiweyo akufuneki zifakwe ngemibhobho kule ndawo.Iisampulu kufuneka zigcinwe kwiifriji ezichongiweyo okanye kwisikhenkcisi kwindawo enye.Indawo yokusebenzela isampula kufuneka icocwe ngendlela efanayo nendawo ye-mastermix.

I-Post-PCR: Ukwandiswa kunye nokuphathwa kwemveliso eyandisiweyo

Esi sithuba sichongiweyo senzelwe iinkqubo zasemva kokukhulisa kwaye kufuneka sahluke ngokwasemzimbeni kwiindawo zangaphambi kwe-PCR.Ngokuqhelekileyo iqulethe i-thermocyclers kunye namaqonga exesha langempela, kwaye ngokufanelekileyo kufuneka ibe nekhabhathi yokuhamba kwe-laminar yokongeza imveliso ye-PCR ejikelezayo kwi-reaction ye-2 ejikelezayo, ukuba i-PCR ene-nested iyenziwa.I-PCR reagents kunye ne-nucleic acid ekhutshweyo akufanele iphathwe kule ndawo ekubeni umngcipheko wokungcola uphezulu.Lo mmandla kufuneka ube neseti eyahlukileyo yeeglavu, iingubo zelebhu, ipleyiti kunye ne-tube racks, iipayipi, iingcebiso zokucoca, imigqomo kunye nezinye izixhobo.Iityhubhu kufuneka zifakwe kwindawo ephakathi phambi kokuba zivulwe.Indawo yokusebenzela isampula kufuneka icocwe ngendlela efanayo nendawo ye-mastermix.

I-Post-PCR: Uhlalutyo lwemveliso

Eli gumbi lelezixhobo zokubona imveliso, umz. itanki yejeli ye-electrophoresis, iipakethi zamandla, i-UV transilluminator kunye nenkqubo yamaxwebhu ejeli.Lo mmandla kufuneka ube neeseti ezihlukeneyo zeeglavu, iingubo zelebhu, ipleyiti kunye ne-tube racks, iipayipi, iingcebiso zokucoca, imigqomo kunye nezinye izixhobo.Azikho ezinye ii-reagents ezinokungeniswa kule ndawo, ngaphandle kwedayi yokulayisha, isiphawuli semolekyuli kunye nejeli ye-agarose, kunye nezixhobo ze-buffer.Indawo yokusebenzela isampula kufuneka icocwe ngendlela efanayo nendawo ye-mastermix.

Inqaku elibalulekileyo

Ngokufanelekileyo, amagumbi angaphambi kwe-PCR akufanele afakwe ngosuku olufanayo ukuba umsebenzi sele wenziwe kumagumbi asemva kwe-PCR.Ukuba oku akunakuthintelwa ngokupheleleyo, qinisekisa ukuba izandla zihlanjwa ngocoselelo kuqala kwaye iidyasi ezithile zaselebhu ziyanxitywa kumagumbi.Iincwadi zeLebhu kunye namaphepha mazingangeniswa kumagumbi angaphambi kwePCR ukuba ziye zasetyenziswa kumagumbi asemva kwePCR;ukuba kuyimfuneko, thatha ushicilelo oluphindwe kabini lweprotocol/isampulu ye-ID, njl.

4. Ingcebiso ngokubanzi ngebhayoloji yemolekyuli

Sebenzisa iiglavu ezingenamgubo ukunqanda ukuthintela uvavanyo.Indlela echanekileyo yokufaka imibhobho ibaluleke kakhulu ekunciphiseni ungcoliseko.Ukufakwa kwemibhobho engafanelekanga kunokubangela ukutshiza xa kuhanjiswa ulwelo kunye nokudalwa kwee-aerosols.Indlela elungileyo yokufaka imibhobho echanekileyo inokufumaneka kula makhonkco alandelayo: Isikhokelo sikaGilson sokufakela imibhobho, iividiyo zobuchule bemibhobho ye-Anachem, iityhubhu ze-Centrifuge phambi kokuvula, kwaye uzivule ngononophelo ukuphepha ukutshiza.Vala iibhubhu ngokukhawuleza emva kokusetyenziswa ukuze ugweme ukufakwa kwezinto ezingcolileyo.

Xa usenza ii-reactions ezininzi, lungiselela i-mastermix enye equlethe ii-reagents eziqhelekileyo (umz. amanzi, i-dNTPs, i-buffer, ii-primers kunye ne-enzyme) ukunciphisa inani lokudluliselwa kwe-reagent kunye nokunciphisa isoyikiso songcoliseko.Kunconywa ukuseta i-mastermix kwiqhwa okanye ibhloko ebandayo.Ukusetyenziswa kwe-enzayimi ye-Hot Start kunokunceda ukunciphisa ukuveliswa kweemveliso ezingezizo ezodwa.Khusela ii-reagents ezine-fluorescent probes ekukhanyeni ukuze ugweme ukuthotywa.

5. Ulawulo lwangaphakathi

Bandakanya ulawulo oluphawulwe kakuhle, oluqinisekisiweyo oluqinisekisayo kunye nolungalunganga, kunye nolawulo olungena-template kuzo zonke iimpendulo, kunye ne-multi-point titrated trendline ye-quantitative reactions.Ulawulo olulungileyo akufanele lube namandla kangangokuba lubeka umngcipheko wosulelo.Bandakanya ulawulo olulungileyo kunye olubi xa usenza i-nucleic acid extraction.

Kucetyiswa ukuba kufakwe imiyalelo ecacileyo kwindawo nganye ukuze abasebenzisi bazi imithetho yokuziphatha.Iilebhu zokuxilonga ezifumanisa amanqanaba asezantsi kakhulu e-DNA okanye i-RNA kwiisampulu zeklinikhi zinokufuna ukwamkela umlinganiselo wokhuseleko owongezelelweyo wokuba neenkqubo zokuphatha umoya ezahlukeneyo ezinoxinzelelo lomoya oluthe kratya kumagumbi angaphambi kwe-PCR kunye noxinzelelo lomoya olubi kancinci kumagumbi asemva kwe-PCR.

Ekugqibeleni, ukuphuhlisa isicwangciso sokuqinisekisa umgangatho (QA) luncedo.Isicwangciso esinjalo kufuneka siquke uluhlu lwe-reagent master stocks kunye ne-workstocks, imithetho yokugcina iikiti kunye nee-reagents, ingxelo yeziphumo zolawulo, iinkqubo zoqeqesho lwabasebenzi, i-algorithms yokuxazulula iingxaki, kunye nezenzo zokulungisa xa kufuneka.

6. Uluhlu lweencwadi

U-Aslan A, uKinzelman J, uDreelin E, u-Anan'eva T, uLavander J. Isahluko sesi-3: Ukumisela ilabhoratri ye-qPCR.Uxwebhu lwesikhokelo sokuvavanya amanzi okuzonwabisa kusetyenziswa indlela ye-USEPA qPCR 1611. Lansing- Michigan State University.

Impilo yoLuntu eNgilani, i-NHS.Imigangatho yase-UK yophando lwe-microbiology: Ukuziqhelanisa neLabhoratri eLungileyo xa usenza iimvavanyo zokwandisa imolekyuli).IsiKhokelo soMgangatho.2013;4(4):1–15.

Mifflin T. Ukumisela ilabhoratri yePCR.Cold Spring Harb Protoc.2007;7.

I-Schroeder S 2013. Ukugcinwa rhoqo kwee-centrifuges: ukucocwa, ukugcinwa kunye nokutshatyalaliswa kwee-centrifuges, i-rotors kunye ne-adapters (iphepha elimhlophe le-14).EHamburg: Eppendorf;2013.

Viana RV, Wallis CL.I-Good Clinical Laboratory Practice (GCLP) kwiimvavanyo ezisekelwe kwimolekyuli ezisetyenziswa kwiilebhu zokuxilonga, Ku: Akyar I, umhleli.Uluhlu olubanzi lolawulo lomgangatho.eRijeka, eCroatia: Intech;2011: 29–52.