I-Direct PCR iwukusabela okusebenzisa ngokuqondile izicubu zezilwane noma zezitshalo ukuze kukhuliswe ngaphandle kokukhipha i-nucleic acid.Ngezindlela eziningi, i-PCR eqondile isebenza njenge-PCR evamile

Umehluko omkhulu i-buffer yangokwezifiso esetshenziswa ku-PCR eqondile, isampula ingaba ngaphansi kokusabela kwe-PCR ngokuqondile ngaphandle kokukhipha i-nucleic acid, kodwa kunezidingo ezihambisanayo zokubekezelela ama-enzyme kanye nokuhambisana kwe-buffer ehilelekile ekuphenduleni okuqondile kwe-PCR.

Nakuba kukhona ama-PCR inhibitors amaningi noma ngaphansi kumasampuli avamile, i-PCR eqondile isengakwazi ukuzuza ukukhulisa okuthembekile ngaphansi kwesenzo sama-enzyme namabhafa.Ukusabela okuvamile kwe-PCR kudinga i-nucleic acid yekhwalithi ephezulu njengesifanekiso, esingavimbela ukuqhubeka okushelelayo kokusabela kwe-PCR uma isifanekiso siqukethe amaprotheni nokunye ukungcola.I-Direct PCR njengamanje ingobunye bobuchwepheshe obudume kakhulu emkhakheni wokuxilongwa kwamangqamuzana.

01 I-Direct PCR ekuqaleni yayisetshenziselwa izilwane nezitshalo

Ukusetshenziswa kwasekuqaleni kwe-PCR eqondile kusensimini yezilwane nezitshalo, njengegazi, izicubu kanye noboya begundane, ikati, inkukhu, unogwaja, izimvu, izinkomo, njll., amaqabunga ezitshalo nembewu, njll., esetshenziselwa ukutadisha i-genotyping, i-transgenic, ukutholwa kwe-plasmid, ukuhlaziya i-Gene knockout, ukuhlonza umthombo we-DNA, ukuhlonza izinhlobo, ukuhlaziywa kwe-SNP neminye imikhakha.

Lezi zinkambu zinezici ezithile ezivamile, okungukuthi, okuqukethwe kofuzo okuhlosiwe kuphakeme kakhulu futhi ukukhishwa kwe-nucleic acid kuyinkinga, ngakho-ke i-PCR eqondile ayikwazi nje ukugcina isikhathi futhi ibe nomthelela omncane emiphumeleni, kodwa futhi yonga izindleko.

I-PCR eqondile esetshenziselwa ukutholwa kwe-pathogen yindaba yeminyaka yakamuva, abanye abakhiqizi be-PCR reagent benze imizamo eminingi kulokhu lapho benza izinto ezintsha.Ikakhulukazi kulolu bhubhane lwe-COVID-19, imikhiqizo eminingi enjalo yokuhlonza isivele emakethe, njenge-SARS-CoV-2 Nucleic Acid Detection Kit (Multiplex PCR Fluorescent Probe Method) ecwaningwe futhi yathuthukiswa yi-Foregene, esebenzisa ubuchwepheshe be-Real-time RT PCR (rRT-PCR) ukuthola i-qualitative human-Cophary2 ye-SARS-Cophary2 acid. amasampula we-ngeal swab.

I-Foregene ingenye yezinkampani ezisebenzisa ubuchwepheshe be-Direct PCR, ukuthola i-ORF1ab, N, E, kanyeokuhlukile ihlukanisa ama-nucleic acid kumasampula e-nasopharyngeal noma e-oropharyngeal swab afana ne-SARS-CoV-2 B.1.1.7 lineage (UK), B.1.351 lineage (ZA), B.1.617 (IND) kanye nohlu lozalo lwe-P.1 (BR).

02  Ama-reagents adingekayo ku-PCR eqondile

Isibonelo se-Lysate

I-lysate yesampula ingalungiswa uwena noma ithengwe.Umehluko ekubunjweni kwemikhiqizo ehlukene ye-lysate uzokwenza ikhono lokuqamba amanga lihluke, bese isikhathi sokuqamba amanga sizohluka kancane.Isibonelo, ukulungiswa kwamasampula ezicubu zezilwane, imizuzu engama-30 noma i-lysis yasebusuku ngokuvamile iyanconywa, futhi isisombululo se-lysis samagciwane sisuka kumizuzu emi-3-10.

PCR master mix

Kunconywa ukusebenzisa i-hot-start DNA polymerase ukuze kuthuthukiswe i-amplification ethize futhi kwandiswe ikhono lokukhulisa.Umnyombo we-PCR eqondile iyi-polymerase ebekezelela kakhulu.

Khipha noma vimbela izingxenye kusampula ezithinta ukukhuliswa kwe-DNA

Ngemuva kokuthi isampula isicutshungulwe nge-lysate, amaprotheni, ama-lipids nezinye izinsalela zamaseli zizokhishwa, lezi zinto zizovimbela ukusabela kwe-PCR.Ngakho-ke, i-PCR eqondile idinga ukungezwa kokususwa okuhambisanayo noma ama-inhibitors ukunciphisa umthelela walezi zici.

03  Iqoqo lamaphuzu amahlanu wolwazi we-PCR eqondile

Okokuqala, ubuchwepheshe be-Direct PCR ubuchwepheshe be-PCR obuqondile bamasampula ezinto eziphilayo.Ngaphansi kwalesi simo sobuchwepheshe, asikho isidingo sokuhlukanisa nokukhipha i-nucleic acid, sebenzisa ngokuqondile isampula yethishu njengento, bese wengeza ama-primer gene okuhloswe ngawo ukwenza ukusabela kwe-PCR.

Okwesibili, ubuchwepheshe be-Direct PCR akubona nje kuphela ubuchwepheshe bokukhulisa isifanekiso se-DNA, kodwa futhi buhlanganisa i-RNA template reverse transcription PCR.

Okwesithathu, ubuchwepheshe be-PCR obuqondile abenzi kuphela ngokuqondile ukusabela kwekhwalithi ye-PCR kumasampula ezicubu, kodwa futhi buhlanganisa ukusabela kwe-qPCR Kwesikhathi Sangempela, okudinga isistimu yokusabela ukuthi ibe nekhono eliqinile lokuphazanyiswa kwe-fluorescence elimelene nengemuva kanye ne-endogenous fluorescence icisha ikhono lokuphikisa.

Okwesine, amasampula aqondiswe ubuchwepheshe be-Direct PCR adinga kuphela ukukhishwa kwezifanekiso ze-nucleic acid, futhi angasusi amaprotheni, ama-polysaccharides, ama-ion kasawoti, njll. aphazamisa ukusabela kwe-PCR.Okudinga i-nucleic acid polymerase kanye ne-PCR Mix ohlelweni lokusabela ukuze kube nokumelana okuhle kakhulu nokuzivumelanisa nezimo ukuze kuqinisekiswe umsebenzi we-enzyme kanye nokunemba kokuphindaphinda ngaphansi kwezimo eziyinkimbinkimbi.

Okwesihlanu, isampula yethishu eqondiswe ubuchwepheshe be-Direct PCR ngaphandle kokwelashwa okunothisa i-nucleic acid kanye nenani lesifanekiso lincane kakhulu, okudinga ukuthi uhlelo lokusabela lube nokuzwela okuphezulu kakhulu nokusebenza kahle kokukhulisa.