Nzira dzekuongorora mamorekuru dzine kugona kugadzira huwandu hwakawanda hwe nucleic acid kuburikidza nekuwedzera huwandu hwe trace hunowanikwa mumasampuli. Kunyange zvazvo izvi zvichibatsira pakugonesa kuonekwa zviri nyore, zvinounzawo mukana wekusvibiswa kuburikidza nekupararira kwe aerosols ye amplification munzvimbo yerabhoritari. Pakuita kuyedza, matanho anogona kuitwa kudzivirira kusvibiswa kwe reagents, zvishandiso zverabhoritari nenzvimbo yebhenji, sezvo kusvibiswa kwakadaro kunogona kuunza mhedzisiro yenhema (kana yenhema-negative).
Kuti kuderedze mukana wekusvibiswa, Good Laboratory Practice inofanira kuitwa nguva dzese. Zvikuru sei, matanho ekudzivirira anofanira kutorwa maererano nepfungwa dzinotevera:
1. Kubata ma reagents
2. Kurongeka kwenzvimbo yebasa nemidziyo
3. Mazano ekushandisa nekuchenesa nzvimbo yakatarwa yemamorekuru
4. Mazano ezvebhayoloji yemamorekuru
5. Kudzora kwemukati
6. Mabhuku emabhuku
1. Kubata ma reagents
Isa machubhu e centrifuge reagent kwenguva pfupi usati wavhura kudzivirira kugadzirwa kweaerosols. Isa ma reagents kuti usanyanya kuomeswa nechando uye kusvibiswa kwe master stocks. Isa mazita uye nyora zuva remachubhu ese e reagent ne reaction uye chengeta magwaro e reagent lot ne batch numbers anoshandiswa mukuyedza kwese. Isa ma reagents ese ne samples uchishandisa filter tips. Usati watenga, zvinokurudzirwa kuti usimbise nemugadziri kuti filter tips dzacho dzinokodzera rudzi rwepipette ichashandiswa.
2. Kurongeka kwenzvimbo yebasa nemidziyo
Nzvimbo yebasa inofanira kurongwa kuti ive nechokwadi chekuti kufamba kwebasa kunoitika nenzira imwe chete, kubva kunzvimbo dzakachena (pre-PCR) kusvika kunzvimbo dzakasviba (post-PCR). Nzira dzinotevera dzekudzivirira dzichabatsira kuderedza mukana wekusvibiswa. Iva nemakamuri akasiyana, kana kuti nzvimbo dzakasiyana, dze: kugadzirira mastermix, kubvisa nucleic acid uye kuwedzera DNA template, kuwedzera uye kubata chigadzirwa chakasimudzwa, uye kuongorora chigadzirwa, semuenzaniso gel electrophoresis.
Mune dzimwe nzvimbo, kuva nemakamuri mana akasiyana kwakaoma. Sarudzo inogoneka asi isingadikanwi ndeyekugadzira mastermix munzvimbo ine containment, semuenzaniso kabhineti ye laminar flow. Kana paine nested PCR amplification, kugadzirira mastermix ye second round reaction kunofanirwa kugadzirwa munzvimbo 'yakachena' yekugadzirira mastermix, asi kubaiwa ne primary PCR product kunofanirwa kuitwa mu amplification room, uye kana zvichibvira munzvimbo yakatsaurirwa containment (semuenzaniso kabhineti ye laminar flow).
Kamuri/nzvimbo imwe neimwe inoda seti yakasiyana yemapipetti akanyorwa zvakajeka, mafirita, machubhu ekuisa, mavortexes, macentrifuge (kana zvichikodzera), mapeni, mageneric lab reagents, majasi elabhoritari nemabhokisi emagirovhosi acharamba ari munzvimbo dzavo dzekushandira. Maoko anofanira kugezwa uye magirovhosi nemajasi elabhoritari anochinjwa kana uchifamba pakati penzvimbo dzakatarwa. Mareagents nemidziyo hazvifanirwe kutamiswa kubva panzvimbo yakasviba kuenda kunzvimbo yakachena. Kana pakaitika nyaya yakakura apo reagent kana chidimbu chemuchina chinoda kutamiswa kumashure, chinofanira kutanga chabviswa tsvina ne10% sodium hypochlorite, chozoteverwa nekupukuta nemvura isina tsvina.
Cherechedza
Mushonga we10% sodium hypochlorite unofanira kugadzirwa uri mutsva zuva nezuva. Kana ukashandiswa kubvisa tsvina, nguva shoma yekubata nayo inofanira kuteverwa nemaminitsi gumi.
Neimwe nzira, zvigadzirwa zvinowanikwa kutengeswa zvinosimbiswa sezvinhu zvinoparadza DNA zvinogona kushandiswa kana mazano ekuchengetedza emunharaunda asingabvumidze kushandiswa kwe sodium hypochlorite kana kana sodium hypochlorite isina kukodzera kubvisa kusvibiswa kwezvikamu zvesimbi zvemidziyo.
Zvakakodzera kuti vashandi vatevedzere mitemo yekushanda nenzira imwe chete uye vasadzokere kunzvimbo dzakasviba (post-PCR) kunzvimbo dzakachena (pre-PCR) pazuva rimwe chete. Zvisinei, panogona kunge paine nguva apo izvi zvisingadzivisike. Kana chiitiko chakadaro chikaitika, vashandi vanofanira kungwarira kugeza maoko zvakanaka, kuchinja magirovhosi, kushandisa jira rekurabhoritari rakasarudzwa uye kusaunza chero michina yavanoda kutora mukamuri zvakare, senge mabhuku ekurabhoritari. Matanho akadaro ekudzora anofanira kusimbiswa mukudzidziswa kwevashandi nezvenzira dzemamorekuru.
Mushure mekushandisa, nzvimbo dzemabhenji dzinofanira kucheneswa ne10% sodium hypochlorite (inoteverwa nemvura isina tsvina kubvisa bleach yasara), 70% ethanol, kana mushonga unoparadza DNA unowanikwa kumusika. Zvakakodzera, marambi e-ultra-violet (UV) anofanira kuiswa kuti abvise kusvibiswa nemwaranzi. Zvisinei, kushandiswa kwemarambi e-UV kunofanira kungoshandiswa munzvimbo dzakavharwa dzekushanda, semuenzaniso makabati ekuchengetedza, kuitira kuderedza kusvibiswa kweUV nevashandi vemurabhoritari. Ndapota tevedzerai mirairo yemugadziri wekutarisira marambi e-UV, kufefetera uye kuchenesa kuitira kuti marambi arambe achishanda.
Kana ukashandisa 70% ethanol pachinzvimbo chesodium hypochlorite, kupiswa nemwaranzi yeUV kuchadiwa kuti upedze kubvisa tsvina.
Usachenese nzvimbo ine mhepo inoyerera mvura ne sodium hypochlorite; panzvimbo pezvo, pukuta ne 70% ethanol woisa pachiedza che UV, kana kushandisa mushonga unoparadza DNA. Kana painenge yadonha, bvunza mugadziri kuti uwane mamwe mazano ekuchenesa. Kana mirairo yemugadziri ichibvumira, mapipettes anofanira kugara achicheneswa ne autoclave. Kana mapipettes asingakwanise kucheneswa ne autoclave, zvinokwana kuachenesa ne 10% sodium hypochlorite (inoteverwa nekupukuta nemvura isina tsvina) kana ne decotaminant inoparadza DNA inoteverwa nekubuda mu UV.
Kuchenesa ne sodium hypochlorite ine huwandu hwakanyanya kunogona kukanganisa mapurasitiki nesimbi kana zvikaitwa nguva dzose; tanga watarisa mazano kubva kumugadziri. Midziyo yese inofanira kugadzikiswa nguva nenguva zvichienderana nehurongwa hunokurudzirwa nemugadziri. Munhu akasarudzwa anofanira kuve nebasa rekuona kuti hurongwa hwekugadzirisa hwatevedzwa, magwaro akazara akachengetedzwa, uye mazita ebasa anoratidzwa zvakajeka pamidziyo.
3. Mazano ekushandisa nekuchenesa nzvimbo yakatarwa yemamorekuru
Pre-PCR: Kugadzira Reagent aliquoting / mastermix: Iyi inofanira kunge iri nzvimbo yakachena kupfuura dzimwe dzese dzinoshandiswa pakugadzirira kuyedza kwemamorekuru uye inofanira kunge iri kabhineti yekuyerera kwelaminar yakagadzirirwa chiedza cheUV. Samples, nucleic acid yakabviswa uye zvigadzirwa zvePCR zvakasimudzwa hazvifanirwe kubatwa munzvimbo iyi. Ma reagent ekuwedzera anofanirwa kuchengetwa mufiriji (kana firiji, sezvakataurwa nemugadziri) munzvimbo imwe chete yakatarwa, zviri nani padyo nekabhineti yekuyerera kwelaminar kana nzvimbo yepre-PCR. Magirovhosi anofanirwa kuchinjwa nguva imwe neimwe paunopinda munzvimbo yepre-PCR kana kabhineti yekuyerera kwelaminar.
Nzvimbo ye pre-PCR kana laminar flow cabinet inofanira kucheneswa usati washandisa uye mushure mekushandisa seizvi: Pukuta zvinhu zvese zviri mukabati, semuenzaniso mapipettes, matip boxes, vortex, centrifuge, tube racks, pens, nezvimwewo ne 70% ethanol kana commercial DNA-destroying detaminant, worega iome. Kana iri nzvimbo yekushanda yakavharwa, semuenzaniso laminar flow cabinet, isa hood kuchiedza cheUV kwemaminitsi makumi matatu.
Cherechedza
Usaise ma reagents muchiedza cheUV; ingoaisa mukabati kana achena. Kana uchiita reverse transcription PCR, zvinogonawo kubatsira kupukuta nzvimbo nemidziyo uchishandisa mushonga unopwanya maRNases kana abatwa. Izvi zvinogona kubatsira kudzivirira mhedzisiro yenhema-negative kubva mukuora kweRNA nema enzyme. Mushure mekubvisa utachiona uye usati wagadzira mastermix, magirovhosi anofanira kuchinjwa zvakare, uye kabati yagadzirira kushandiswa.
Pre-PCR: Kubviswa kweNucleic acid/kuwedzera template:
Nucleic acid inofanira kutorwa uye kubatwa munzvimbo yechipiri yakatarwa, uchishandisa seti yakasiyana yemapipettes, mafirita, machubhu ekuisa, magirovhosi matsva, majasi ekurabhoritari nezvimwe zvishandiso. Nzvimbo iyi ndeyekuwedzerawo template, macontrol uye trendlines kumachubhu emastermix kana maplate. Kuti udzivise kusvibiswa kwemasampuli enucleic acid akabviswa ari kuongororwa, zvinokurudzirwa kuchinja magirovhosi usati wabata macontrol akanaka kana zviyero uye kushandisa seti yakasiyana yemapipettes. MaPCR reagents uye zvigadzirwa zvakasimudzwa hazvifanirwe kuiswa mumapipetti munzvimbo iyi. Masampure anofanira kuchengetwa mumafiriji kana mafiriji akasarudzwa munzvimbo imwe chete. Nzvimbo yekushandira yesampure inofanira kucheneswa nenzira imwecheteyo nenzvimbo yemastermix.
Mushure mePCR: Kuwedzera uye kubata kwechigadzirwa chakakwidziridzwa
Nzvimbo iyi yakatarwa ndeyekushandisa mushure mekuwedzera simba uye inofanira kunge yakasiyana nenzvimbo dze pre-PCR. Inowanzova ne thermocyclers uye mapuratifomu enguva chaiyo, uye zvakanaka kuti ive ne laminar flow cabinet yekuwedzera chigadzirwa che round 1 PCR kune round 2 reaction, kana PCR ine nested iri kuitwa. PCR reagents uye extracted nucleic acid hazvifanirwe kubatwa munzvimbo iyi sezvo njodzi yekusvibiswa yakakura. Nzvimbo iyi inofanira kunge iine seti yakasiyana yemagirovhosi, ma lab coats, plate and tube racks, ma pipettes, ma filter tips, ma bin nezvimwe zvishandiso. Ma tubes anofanira ku centrifuged asati avhurwa. Nzvimbo yekushandira yemuenzaniso inofanira kucheneswa nenzira imwecheteyo nenzvimbo ye mastermix.
Post-PCR: Kuongorora chigadzirwa
Imba iyi ndeyemidziyo yekuona zvigadzirwa, semuenzaniso matangi e gel electrophoresis, ma power packs, UV transilluminator uye system yekunyora gel. Nzvimbo iyi inofanira kunge iine magirovhosi akasiyana, ma lab coats, ma plate ne tube racks, ma pipettes, ma filter tips, ma bin nezvimwe zvishandiso. Hapana zvimwe zvinogadzirisa zvinogona kuunzwa munzvimbo iyi, kusasanganisa loading dye, molecular marker ne agarose gel, uye buffer components. Nzvimbo yekushandira yemuenzaniso inofanira kucheneswa nenzira imwecheteyo nenzvimbo ye mastermix.
Chinyorwa chakakosha
Zvakanaka kuti makamuri e pre-PCR asapindwe pazuva rimwe chete kana basa rakatoitwa mumakamuri e post-PCR. Kana izvi zvisingadzivisike zvachose, iva nechokwadi chekuti maoko anyatsogezwa uye kuti majasi chaiwo e lab apfekwe mumakamuri. Mabhuku e lab nemapepa hazvifanirwe kuendeswa mumakamuri e pre-PCR kana akamboshandiswa mumakamuri e post-PCR; kana zvichidikanwa, tora makopi akadhindwa e protocols/sample IDs, nezvimwewo.
4. Mazano ezvebhayoloji yemamorekuru
Shandisa magirovhosi asina hupfu kudzivirira kudziviswa kwekuyedzwa. Nzira chaiyo yekuisa mapaipi ndiyo inonyanya kukosha mukuderedza kusvibiswa. Kuisa mapaipi zvisirizvo kunogona kukonzera kupfapfaidzwa kwemvura paunenge uchiisa mvura uye kugadzirwa kweaerosols. Maitiro akanaka ekuisa mapaipi zvakanaka anogona kuwanikwa pane zvinotevera: Gilson guide to piping, Anachem piping technique mavhidhiyo, Centrifuge tubes usati wavhura, uye vhura zvinyoro nyoro kudzivirira kupfapfaidzwa kwemvura. Vhara machubhu nekukurumidza mushure mekushandisa kudzivirira kusvibiswa.
Paunenge uchiita ma "reactions" akawanda, gadzira "mastermix" imwe chete ine ma "common reagents" (semuenzaniso mvura, dNTPs, buffer, primers uye enzyme) kuderedza huwandu hwe "reagent transfers" uye kuderedza njodzi yekusvibiswa. Zvinokurudzirwa kuisa "mastermix" iyi pachando kana pa "cold block". Kushandisa "Hot Start enzyme" kunogona kubatsira kuderedza kugadzirwa kwezvinhu zvisiri izvo. Dzivirira ma "reagents" ane "fluorescent probes" kubva kuchiedza kudzivirira kuora.
5. Kudzora kwemukati
Isa zvinhu zvinodzora zvakanaka nezvakaipa zvine hunhu hwakanaka, zvakasimbiswa, pamwe chete nekudzora kusina template mukuita kwese, uye trendline ine mapoinzi akawanda yekuita kwehuwandu. Kudzora kwakanaka hakufanirwe kunge kwakasimba zvekuti kunogona kukonzera njodzi yekusvibiswa. Isa zvinhu zvinodzora zvakanaka nezvakaipa pakuita nucleic acid extraction.
Zvinokurudzirwa kuti mirairo yakajeka inyorwe munzvimbo imwe neimwe kuitira kuti vashandisi vazive mitemo yekuzvibata. Marabhoritari ekuongorora anoona huwandu hwakaderera hweDNA kana RNA mumienzaniso yekiriniki angada kushandisa imwe nzira yekuchengetedza yekuva nehurongwa hwakasiyana hwekubata mweya hune kumanikidzwa kwemhepo kwakanaka zvishoma mumakamuri e pre-PCR uye kumanikidzwa kwemhepo kusina kunaka zvishoma mumakamuri e post-PCR.
Chekupedzisira, kugadzira hurongwa hwekusimbisa mhando (QA) kunobatsira. Hurongwa hwakadaro hunofanira kusanganisira runyorwa rwemasheya makuru e reagent uye masheya anoshanda, mitemo yekuchengetedza makit uye ma reagent, mishumo yemhedzisiro yekudzora, mapurogiramu ekudzidzisa vashandi, ma algorithms ekugadzirisa matambudziko, uye matanho ekugadzirisa kana zvichidikanwa.
6. Mabhuku emabhuku
Aslan A, Kinzelman J, Dreelin E, Anan'eva T, Lavander J. Chitsauko 3: Kugadzira rabhoritari yeqPCR. Gwaro rekutungamira rekuyedza mvura dzekuvaraidza uchishandisa nzira yeUSEPA qPCR 1611. Lansing- Michigan State University.
Public Health England, NHS. Mitemo yeUK yekuferefeta kwe microbiology: Maitiro Akanaka eLaboratory pakuita bvunzo dzekuwedzera mamorekuru). Guidance yeHunhu. 2013;4(4):1–15.
Mifflin T. Kugadzira rabhoritari yePCR. Cold Spring Harb Protoc. 2007;7.
Schroeder S 2013. Kugadziriswa kwema centrifuge nguva dzose: kuchenesa, kugadzirisa uye kubvisa utachiona muma centrifuge, ma rotor nema adapter (White bepa Nhamba 14). Hamburg: Eppendorf; 2013.
Viana RV, Wallis CL. Good Clinical Laboratory Practice (GCLP) yekuyedzwa kwakavakirwa pamorekuru kunoshandiswa mumarabhoritari ekuongorora, Muna: Akyar I, mupepeti. Kudzora mhando dzakasiyana-siyana. Rijeka, Croatia: Intech; 2011: 29–52.
Nguva yekutumira: Chikunguru-16-2020
