I-PCR ethe ngqo yindlela yokusabela esebenzisa ngokuthe ngqo izicubu zezilwanyana okanye zezityalo zokukhulisa ngaphandle kokutsalwa kwe-nucleic acid.Ngeendlela ezininzi, i-PCR ngqo isebenza njenge-PCR eqhelekileyo

Umahluko oyintloko yi-buffer yesiko elisetyenziswe kwi-PCR ethe ngqo, isampuli inokulawulwa ngokuthe ngqo kwi-PCR reaction ngaphandle kwe-nucleic acid extraction, kodwa kukho iimfuno ezihambelanayo zokunyamezela i-enzymes kunye nokuhambelana kwe-buffer echaphazelekayo kwi-reaction ye-PCR ngqo.

Nangona kukho ngaphezulu okanye ngaphantsi kwe-PCR inhibitors kwiisampulu eziqhelekileyo, i-PCR ethe ngqo isenokuzuza ulwandiso oluthembekileyo phantsi kwesenzo se-enzymes kunye ne-buffers.Ukusabela kwe-PCR yendabuko kufuna umgangatho ophezulu we-nucleic acid njenge template, enokuthintela inkqubela phambili egudileyo ye-PCR reaction ukuba ithemplate iqulethe iiprotheni kunye nezinye izinto ezingcolileyo.I-PCR ethe ngqo ngoku yenye yetekhnoloji ethandwa kakhulu kwicandelo loxilongo lwemolekyuli.

01 I-PCR ethe ngqo yayisetyenziselwa izilwanyana nezityalo

Ukusetyenziswa kwangaphambili kwe-PCR ngqo kwintsimi yezilwanyana kunye nezityalo, ezifana negazi, izicubu kunye neenwele zegundane, ikati, inkukhu, umvundla, iigusha, iinkomo, njl., amagqabi ezityalo kunye nembewu, njl., esetyenziselwa ukufunda i-genotyping, i-transgenic, ukufumanisa i-plasmid, uhlalutyo lwe-Gene knockout, ukuchongwa komthombo we-DNA, ukuchongwa kweentlobo, uhlalutyo lwe-SNP kunye nezinye iinkalo.

Le mimandla ineempawu eziqhelekileyo, oko kukuthi, umxholo we-gene ekujoliswe kuwo uphezulu kwaye i-nucleic acid extraction iyinkathazo, ngoko i-PCR ethe ngqo ayikwazi ukugcina ixesha kwaye ibe nempembelelo encinci kwiziphumo, kodwa iphinde igcine iindleko.

I-PCR ethe ngqo esetyenziselwa ukufumanisa i-pathogen ngumcimbi weminyaka yakutshanje, abanye abavelisi be-PCR baye benza imizamo emininzi kweli cala xa besenza izinto ezintsha.Ngokukodwa kolu bhubhani lwe-COVID-19, uninzi lweemveliso zobhaqo ezinje zivele kwintengiso, ezinje nge-SARS-CoV-2 Nucleic Acid Detection Kit (Multiplex PCR Fluorescent Probe Method) ephandwe kwaye yaphuhliswa yiForegene, esebenzisa itekhnoloji yexesha langempela le-RT PCR (rRT-PCR) ukufunyaniswa kwe-nucleic-Cov-PCR ye-nucleic ye-nucleic ye-SARS okanye i-opharysophary2 yomgangatho we-SARS. iisampulu swab ngeal.

I-Foregene yenye yeenkampani ezisebenzisa itekhnoloji ye-Direct PCR, ukubhaqa i-ORF1ab eqhelekileyo, i-N, i-E, kunyeukwahluka imigca ye-nucleic acids kwi-nasopharyngeal yabantu okanye iisampulu ze-oropharyngeal swab ezifana ne-SARS-CoV-2 B.1.1.7 yomgca (UK), B.1.351 umnombo (ZA), B.1.617 umnombo (IND) kunye ne-P.1 umnombo (BR).

02  Ii-reagents ezifunekayo kwi-PCR ngqo

Umzekelo weLysate

Isampuli ye-lysate inokulungiswa nguwe okanye ithengwe.Umahluko ekubunjweni kweempawu ezahlukeneyo ze-lysate ziya kwenza ukuba amandla okuxoka ahluke, kwaye ke ixesha lokulala liya kuba lihluke kancinane.Ngokomzekelo, ukulungiselela iisampulu zenyama yezilwanyana, imizuzu engama-30 okanye i-lysis yasebusuku iyacetyiswa ngokubanzi, kwaye isisombululo se-lysis kwiintsholongwane sisuka kwi-3-10 imizuzu.

PCR master mix

Kuyacetyiswa ukuba kusetyenziswe i-polymerase ye-DNA eshushu ukuqala ukukhulisa ulwandiso oluthile kunye nokwandisa isakhono sokukhulisa.Undoqo wePCR ngqo yipolymerase enonyamezelo kakhulu.

Ukuphelisa okanye ukuthintela amacandelo kwisampulu echaphazela i-DNA amplification

Emva kokuba isampuli iqhutywe nge-lysate, iiprotheni, i-lipids kunye nezinye i-debris zeseli ziya kukhutshwa, ezi zinto ziya kuthintela ukuphendula kwe-PCR.Ngoko ke, i-PCR ngqo idinga ukongezwa kokususwa okuhambelanayo okanye i-inhibitors ukunciphisa impembelelo yale miba.

03  Ingqokelela yamanqaku amahlanu olwazi lwePCR ngqo

Okokuqala, ubuchwepheshe be-PCR bubuchwephesha be-PCR obuthe ngqo kwiisampulu zebhayoloji ezahlukeneyo.Ngaphantsi kwesi simo sobugcisa, akukho mfuneko yokwahlula kunye nokukhupha i-nucleic acid, sebenzisa ngokuthe ngqo isampuli yezicubu njengento, kwaye ungeze i-primers ye-gene ejoliswe kuyo ukwenza i-PCR reaction.

Okwesibini, itekhnoloji ye-PCR ethe ngqo ayisiyotekhnoloji yokukhulisa itemplate ye-DNA, kodwa ikwabandakanya i-RNA template reverse transcription PCR.

Okwesithathu, itekhnoloji ye-PCR ethe ngqo ayenzi ngokuthe ngqo kuphela iimpendulo zesiqhelo ezisemgangathweni ze-PCR kwiisampulu zethishu, kodwa ikwabandakanya ukusabela kweXesha lokwenyani le-qPCR, efuna inkqubo yokusabela ibe namandla okuphazamisana ne-anti-background fluorescence kunye ne-endogenous fluorescence icima isakhono esichasayo.

Okwesine, iisampuli ezijoliswe kwi-teknoloji ye-PCR echanekileyo zifuna kuphela ukukhululwa kweetemplates ze-nucleic acid, kwaye musa ukususa iiprotheni, i-polysaccharides, i-ion yetyuwa, njl.Oko kufuna i-nucleic acid polymerase kunye ne-PCR Mix kwinkqubo yokusabela ukuba ibe nokuchasana okugqwesileyo kunye nokulungelelaniswa kokuqinisekisa umsebenzi we-enzyme kunye nokuphindaphinda ukuchaneka phantsi kweemeko ezinzima.

Okwesihlanu, isampuli yezicubu ezijoliswe kwithekhnoloji ye-PCR ethe ngqo ngaphandle konyango lokutyebisa i-nucleic acid kunye nesixa setemplate sincinci kakhulu, esifuna inkqubo yokusabela ukuba ibe novakalelo oluphezulu kakhulu kunye nokusebenza kakuhle kokukhulisa.