Maitiro ekuona mamorekuru ane kugona kuburitsa huwandu hukuru hwenyucleic acid kuburikidza nekukwidziridzwa kwehuwandu hwekutsvaga hunowanikwa mumasampuli.Kunyangwe izvi zvichibatsira pakugonesa kutarisisa, zvinounza zvakare mukana wekusvibiswa kuburikidza nekupararira kwemaaerosols ekusimudzira munzvimbo yerabhoritari.Paunenge uchiita kuyedza, matanho anogona kuitwa kudzivirira kusvibiswa kwemareagents, michina yemurabhoritari uye nzvimbo yebhenji, sezvo kusvibiswa kwakadaro kunogona kuunza mhedzisiro yenhema (kana yenhema-negative).

Kubatsira kudzikisira mukana wekusvibiswa, Yakanaka Laboratory Kudzidzira inofanirwa kushandiswa nguva dzese.Kunyanya, kuchenjerera kunofanira kutorwa maererano nemapoinzi anotevera:

1. Kubata reagents
2. Kurongeka kwenzvimbo yekushanda uye midziyo
3. Shandisa uye kuchenesa mazano kune yakatarwa molecular space
4. General molecular biology mazano
5. Kudzora mukati
6. Bibliography

1. Kubata reagents

Muchidimbu centrifuge reagent chubhu isati yavhurwa kudzivirira chizvarwa cheaerosols.Aliquot reagents kudzivirira akawanda chando-thaws uye kusvibiswa kwemaster stocks.Nyoresa nyora uye date ese reagent uye reaction machubhu uye chengetedza matanda eiyo reagent yakawanda uye batch nhamba dzinoshandiswa mune zvese zviedzo.Pipette ese mareagents uye samples uchishandisa mafirita matipi.Usati watenga, zvinokurudzirwa kuti usimbise nemugadziri kuti mazano ekusefa anokodzera mhando yepipette inoshandiswa.

2. Kurongeka kwenzvimbo yekushanda uye midziyo

Nzvimbo yekushanda inofanira kurongeka kuti ione kuti kufamba kwebasa kunoitika mune imwe nzira, kubva munzvimbo dzakachena (pre-PCR) kune nzvimbo dzine tsvina (post-PCR).Zvinotevera zvinodzivirira zvakajairika zvichabatsira kuderedza mukana wekusvibiswa.Iva nemakamuri akasarudzwa akaparadzana, kana padiki nzvimbo dzakapatsanurwa mumuviri, dze: mastermix kugadzirira, nucleic acid kutorwa uye DNA template yekuwedzera, kukwidziridzwa uye kubata kwechigadzirwa chakasimudzirwa, uye kuongororwa kwechigadzirwa, semuenzaniso gel electrophoresis.

Mune mamwe marongero, kuva nemakamuri mana akasiyana kwakaoma.Imwe nzira inogoneka asi isingadikanwi ndeyekuita iyo mastermix kugadzirira munzvimbo inochengeterwa, semuenzaniso laminar flow cabinet.Kana iri dendere rePCR amplification, kugadzirira kwe mastermix yechipiri round reaction kunofanirwa kugadzirirwa munzvimbo 'yakachena' yekugadzirira mastermix, asi inoculation nePrimary PCR chigadzirwa inofanira kuitwa mukamuri yekusimudza, uye kana zvichibvira. munzvimbo yakatsaurirwa yekuchengetedza (semuenzaniso laminar flow cabinet).

Kamuri yega yega / nzvimbo inoda seti yakaparadzana yakanyorwa zvakajeka mapipetti, matipi esefa, machubhu racks, vortexes, centrifuges (kana zvichikodzera), mapeni, generic lab reagents, majasi erabhoritari uye mabhokisi emagurovhosi acharamba ari panzvimbo dzavo dzekushandira.Maoko anofanirwa kugezwa uye magirovhosi nemajasi erabhoritari achichinjwa kana achifamba pakati penzvimbo dzakatarwa.Reagents uye zvishandiso hazvifanirwe kutamiswa kubva munzvimbo ine tsvina kuenda kunzvimbo yakachena.Kana nyaya yakanyanyisa ikamuka apo reagent kana chidimbu chemudziyo chinoda kudzoserwa kumashure, chinofanira kutanga chasvibiswa ne10% sodium hypochlorite, ichiteverwa nekupusika pasi nemvura isina utachiona.

Cherechedza

Iyo 10% sodium hypochlorite mhinduro inofanirwa kugadzirwa nyowani zuva nezuva.Kana ichishandiswa pakusvibisa, nguva shoma yekubata yemaminitsi gumi inofanira kutevedzerwa.
Neimwe nzira, zvigadzirwa zvinotengeswa zvakasimbiswa seDNA-inoparadza pamusoro pezvinosvibisa zvinogona kushandiswa kana kurudziro yekuchengetedzwa kwenzvimbo isingabvumire kushandiswa kwesodium hypochlorite kana sodium hypochlorite isingakodzere kusvibisa zvikamu zvesimbi zvemidziyo.

Nenzira yakanaka, vashandi vanofanira kutevedzera unidirectional work flow ethos uye kwete kubva munzvimbo dzine tsvina (post-PCR) kudzokera kunzvimbo dzakachena (pre-PCR) pazuva rimwe chete.Zvisinei, panogona kuva nenguva apo izvi zvisingadzivisiki.Kana chiitiko chakadai chamuka, vashandi vanofanira kungwarira kugeza maoko zvakakwana, kuchinja magirovhosi, kushandisa jasi rerabhu rakasarudzwa uye kusasuma chero mudziyo wavanozoda kuburitsa mukamuri zvakare, semabhuku emurabhoritari.Matanho akadaro ekudzora anofanirwa kusimbiswa mukudzidziswa kwevashandi pane nzira dzemamorekuru.

Mushure mekushandiswa, nzvimbo dzemabhenji dzinofanira kucheneswa ne10% sodium hypochlorite (inoteverwa nemvura isina utachiona kubvisa yakasara bleach), 70% ethanol, kana inotenderwa inotengeswa DNA-inoparadza decontaminant.Sezvineiwo, marambi e-ultra-violet (UV) anofanira kuiswa kuti agone kusvibiswa nekuvhenekerwa.Nekudaro, kushandiswa kwemarambi eUV kunofanirwa kungoitwa munzvimbo dzakavharwa dzekushanda, semuenzaniso makabati ekuchengetedza, kuitira kuti vashandi vemurabhoritari vasaonekwe neUV.Ndokumbira uteerere mirairo yemugadziri yekutarisira mwenje weUV, kufefetedza uye kuchenesa kuitira kuti mwenje urambe uchishanda.

Kana ukashandisa 70% ethanol pachinzvimbo chesodium hypochlorite, irradiation ine UV mwenje inozodiwa kupedzisa kusvibisa.
Usachenesa vortex uye centrifuge ne sodium hypochlorite;pachinzvimbo, pukuta pasi ne 70% ethanol uye kufumura kune UV mwenje, kana shandisa yekushambadzira DNA-inoparadza decontaminant.Pakurasika, bvunza mugadziri kuti uwane mamwe mazano ekuchenesa.Kana mirairo yemugadziri ichibvumira, pipettes inofanira kugara yakasvibiswa ne autoclave.Kana mapipette asingakwanisi kuvharwa, zvinofanirwa kukwana kuachenesa ne10% sodium hypochlorite (inoteverwa nekupukuta pasi nemvura ine utachiona) kana nekutengesa DNA-inoparadza decontaminant inoteverwa neUV exposure.

Kuchenesa ne-high-percentage sodium hypochlorite kunogona kupedzisira kukuvadza pipette mapurasitiki nesimbi kana zvichiitwa nguva dzose;tarisa mazano kubva kumugadziri kutanga.Yese michina inoda kuverengerwa nguva nenguva zvichienderana neyakakurudzirwa mugadziri.Munhu akasarudzwa anofanirwa kunge achitarisira kuona kuti hurongwa hwekugadzirisa hwatevedzwa, matanda akadzama anochengetedzwa, uye maregi ebasa anoratidzwa zvakajeka pamidziyo.

3. Shandisa uye kuchenesa mazano kune yakatarwa molecular space

Pre-PCR: Reagent aliquoting / mastermix kugadzirira: Iyi inofanirwa kuve yakachena pane dzese nzvimbo dzinoshandiswa kugadzirira bvunzo dzemamorekuru uye inofanirwa kuve yakasarudzika laminar inoyerera kabati ine UV mwenje.Samples, yakabviswa nucleic acid uye yakawedzera PCR zvigadzirwa hazvifanirwe kubatwa munzvimbo ino.Amplification reagents anofanirwa kuchengetwa mufiriji (kana mufiriji, sekurudziro yemugadziri) munzvimbo imwechete yakatarwa, padyo nelaminar flow cabinet kana pre-PCR nharaunda.Magirovhosi anofanirwa kuchinjwa nguva yega yega paunopinda pre-PCR nharaunda kana laminar kuyerera kabati.

Iyo pre-PCR nzvimbo kana laminar flow cabinet inofanira kucheneswa isati yashandiswa uye mushure mekushandisa sezvinotevera: Pukuta pasi zvinhu zvose zviri mukhabhineti, eg pipettes, tip boxes, vortex, centrifuge, tube racks, pen, etc. ne 70% ethanol kana a yekutengesa DNA-inoparadza decontaminant, uye bvumira kuoma.Kana iri nzvimbo yakavharwa yekushanda, semuenzaniso laminar flow cabinet, ratidza hood kuUV chiedza kwemaminitsi makumi matatu.

Cherechedza

Usafumure reagents kune UV mwenje;ingodzipinza mukabati kana yachena.Kana uchiita reverse transcription PCR, zvinogona zvakare kubatsira kupukuta pasi pasi nemidziyo ine mhinduro inopwanya RNases pakubata.Izvi zvinogona kubatsira kudzivirira manyepo-negative mhedzisiro kubva kune enzyme degradation yeRNA.Mushure mekusvibiswa uye musati magadzirira mastermix, magurovhosi anofanira kuchinjwa zvakare, uye ipapo kabati yakagadzirira kushandiswa.

Pre-PCR: Nucleic acid kubviswa / template yekuwedzera:

Nucleic acid inofanira kubviswa uye kubatwa munzvimbo yechipiri yakasarudzwa, uchishandisa imwe seti yepipettes, mafirita matipi, machubhu racks, magirovhosi matsva, majasi erabhoritari nemimwe midziyo. mastermix machubhu kana ndiro.Kuti udzivise kusvibiswa kweiyo yakabviswa nucleic acid samples iri kuongororwa, zvinokurudzirwa kushandura magirovhosi usati wabata kudzora kwakanaka kana zviyero uye kushandisa seti yakaparadzana yepipette.PCR reagents uye zvigadzirwa zvakasimudzwa hazvifanirwe kupinzwa munzvimbo ino.Samples dzinofanirwa kuchengetwa mumafiriji akasarudzwa kana mafiriza munzvimbo imwe chete.Sample workspace inofanira kucheneswa nenzira imwechete neye mastermix space.

Post-PCR: Kukwidziridzwa uye kubata kwechigadzirwa chakawedzerwa

Iyi nzvimbo yakasarudzwa ndeye post-amplification maitiro uye inofanira kunge yakaparadzana nemuviri kubva kune pre-PCR nzvimbo.Iyo inowanzo ine thermocyclers uye chaiyo-nguva mapuratifomu, uye zvine hungwaru inofanirwa kuve nelaminar inoyerera kabati yekuwedzera yakatenderedza 1 PCR chigadzirwa kune yakatenderedza 2 maitiro, kana nested PCR iri kuitwa.PCR reagents uye yakabviswa nucleic acid haifanirwe kubatwa munzvimbo ino sezvo njodzi yekusvibiswa yakakwira.Iyi nzvimbo inofanirwa kunge iine seti yakaparadzana yegurovhosi, majasi erabhoritari, ndiro uye chubhu racks, pipettes, mafirita matipi, mabhini nemimwe michina.Machubhu anofanirwa kuiswa mukati asati avhurwa.Sample workspace inofanira kucheneswa nenzira imwechete neye mastermix space.

Post-PCR: Kuongororwa kwechigadzirwa

Iri kamuri nderemidziyo yekuona zvigadzirwa, semuenzaniso matangi emagetsi emagetsi, mapaketi emagetsi, UV transilluminator uye gel zvinyorwa zvinyorwa.Iyi nzvimbo inofanirwa kunge iine seti dzakaparadzana dzemagurovhosi, majasi erabhoritari, ndiro uye chubhu racks, pipettes, mafirita matipi, mabhini nemimwe michina.Hapana mamwe mareagents anogona kuunzwa munzvimbo ino, kusasanganisa kurodha dhayi, molecular marker uye agarose gel, uye buffer zvikamu.Sample workspace inofanira kucheneswa nenzira imwechete neye mastermix space.

Chinyorwa chakakosha

Zvakanaka, makamuri e-pre-PCR haafaniri kupindwa pazuva rimwe chete kana basa rakatoitwa mumakamuri e-post-PCR.Kana izvi zvisingadzivisiki zvachose, iva nechokwadi chokuti maoko anotanga kugezwa zvakakwana uye kuti mabhachi chaiwo anoshandiswa mumakamuri.Mabhuku eLab uye mapepa haafaniri kutorwa mumakamuri e-pre-PCR kana akashandiswa mumakamuri e-post-PCR;kana zvichidikanwa, tora kudhindwa-kubuda kweprotocol/sample ID, nezvimwe.

4. General molecular biology mazano

Shandisa magurovhosi ehupfu kuti udzivise assay inhibition.Nzira yakarurama yepipetting yakakosha pakuderedza kusvibiswa.Pipetting isiriyo inogona kukonzera kupfachuka kana uchipa zvinwiwa uye kugadzira aerosols.Tsika yakanaka yekukwenenzvera pombi inogona kuwanikwa pane anotevera zvinongedzo: Gilson gwairira kune pombi, Anachem pipetting matekiniki mavhidhiyo, Centrifuge machubhu asati avhura, uye avhure nekungwarira kudzivirira kupfapfaidza.Vhara machubhu pakarepo mushure mekushandisa kudzivirira kuiswa kwezvinosvibisa.

Paunenge uchiita maitiro akawanda, gadzirira imwe mastermix ine zvakajairika reagents (semuenzaniso mvura, dNTPs, buffer, primers uye enzyme) kudzikisa huwandu hwekuchinjisa reagent uye kuderedza kutyisidzira kwekusvibiswa.Inokurudzirwa kugadzirisa mastermix pachando kana chando chechando.Kushandiswa kweHot Start enzyme kunogona kubatsira kudzikisira kugadzirwa kwezvinhu zvisiri-chaiyo.Chengetedza ma reagents ane fluorescent probes kubva kuchiedza kuitira kudzivirira kudzikira.

5. Kudzora mukati

Sanganisira ane hunhu hwakanaka, akasimbiswa akanaka uye asina kunaka zvidzoreso, pamwe neasina-template kutonga mune zvese maitiro, uye akawanda-point titrated trendline yehuwandu hwekuita.Kudzora kwakanaka hakufanirwe kuve kwakasimba zvekuti kunoisa njodzi yekusvibiswa.Sanganisira zvakanaka uye zvisina kunaka zvidzoreso zvekubvisa paunenge uchiita nucleic acid yekubvisa.

Zvinokurudzirwa kuti mirairo yakajeka inotumirwa mune imwe neimwe yenzvimbo kuitira kuti vashandisi vazive mitemo yekuzvibata.Diagnostic Labs anoona akaderera kwazvo eDNA kana RNA mumakiriniki samples angangoda kutora imwe chiyero chekuchengetedza chekuve nemhepo yakaparadzana masisitimu ane mhepo yakanaka zvishoma mumakamuri pre-PCR uye kushomeka kwemhepo kushoma mumakamuri ePCR.

Chekupedzisira, kugadzira vimbiso yemhando (QA) chirongwa chinobatsira.Urongwa hwakadai hunofanirwa kusanganisira rondedzero yeagent master stocks uye masheya ekushanda, mitemo yekuchengeta kits uye reagents, kushuma yezvidzoreso zvabuda, zvirongwa zvekudzidzisa vashandi, kugadzirisa matambudziko, uye kugadzirisa zviito pazvinenge zvichidikanwa.

6. Bibliography

Aslan A, Kinzelman J, Dreelin E, Anan'eva T, Lavander J. Chitsauko 3: Kugadzira rabhoritari yeqPCR.Gwaro rekutungamira rekuyedza mvura yekuvaraidza uchishandisa USEPA qPCR nzira 1611. Lansing- Michigan State University.

Hutano Hwevanhu England, NHS.UK miyero yekuferefeta microbiology: Yakanaka Laboratory Dzidzira paunenge uchiita mamorekuru amplification assays).Hunhu Hwakanaka.2013;4(4):1–15.

Mifflin T. Kumisikidza PCR rabhoritari.Cold Spring Harb Protoc.2007;7.

Schroeder S 2013. Kuchengetedza nguva dzose ye centrifuges: kuchenesa, kuchengetedza uye disinfection ye centrifuges, rotors uye adapters (White paper No. 14).Hamburg: Eppendorf;2013.

Viana RV, Wallis CL.Yakanaka Clinical Laboratory Practice (GCLP) yemamorekuru akavakirwa bvunzo anoshandiswa mukuongorora marabhoritari, Mu: Akyar I, mupepeti.Wide spectra of quality control.Rijeka, Croatia: Intech;2011: 29–52.