Direct PCR ndikuchita komwe kumagwiritsa ntchito minofu ya nyama kapena zomera kuti ikulitse popanda nucleic acid.Munjira zambiri, PCR yolunjika imagwira ntchito ngati PCR wamba

Kusiyanitsa kwakukulu ndi chizolowezi chotchinga chomwe chimagwiritsidwa ntchito mwachindunji PCR, chitsanzocho chikhoza kuchitidwa mwachindunji ndi PCR popanda nucleic acid m'zigawo, koma pali zofunikira zogwirizana ndi kulolerana kwa michere ndi kuyanjana kwa bafa yomwe ikukhudzidwa mwachindunji ndi PCR.

Ngakhale pali ma PCR inhibitors ochulukirapo kapena ocheperako pamasampulo wamba, PCR yolunjika imathabe kukulitsa zodalirika potengera ma enzyme ndi ma buffers.Zomwe zimachitika pa PCR zimafuna nucleic acid yapamwamba kwambiri ngati template, yomwe ingalepheretse kuyenda bwino kwa PCR ngati template ili ndi mapuloteni ndi zonyansa zina.Direct PCR pakadali pano ndi imodzi mwamatekinoloje odziwika bwino pazambiri zama cell.

01 Direct PCR idagwiritsidwa ntchito popangira nyama ndi zomera

Kugwiritsa ntchito koyambirira kwa PCR mwachindunji kuli m'munda wa nyama ndi zomera, monga magazi, minofu ndi tsitsi la makoswe, mphaka, nkhuku, kalulu, nkhosa, ng'ombe, ndi zina zotero, masamba a zomera ndi mbewu, ndi zina zotero, zomwe zimagwiritsidwa ntchito pophunzira genotyping, transgenic, plasmid kuzindikira, Gene knockout analysis, DNA source identification, mitundu yodziwika, SNP kusanthula ndi zina.

Minda imeneyi ili ndi zinthu zina zomwe zimafanana, ndiko kuti, zomwe zili mu jini ndizokwera kwambiri ndipo kutulutsa kwa nucleic acid kumakhala kovuta, kotero PCR yolunjika sikungopulumutsa nthawi ndikukhala ndi zotsatira zochepa, komanso kupulumutsa mtengo.

Direct PCR yogwiritsidwa ntchito pozindikira tizilombo toyambitsa matenda ndi nkhani yazaka zaposachedwa, opanga ena a PCR reagent ayesetsa kwambiri kuchita izi popanga zatsopano.Makamaka mu mliri wa COVID-19, zinthu zambiri zodziwikiratu zapezeka pamsika, monga SARS-CoV-2 Nucleic Acid Detection Kit (Multiplex PCR Fluorescent Probe Method) yofufuzidwa ndikupangidwa ndi Foregene, yomwe imagwiritsa ntchito ukadaulo wa Real-time RT PCR (rRT-PCR) pozindikira nucleic-Cophary2 yaumunthu ya SARS-Cophary2 zitsanzo za swab.

Foregene ndi imodzi mwamakampani omwe amagwiritsa ntchito ukadaulo wa Direct PCR, pozindikira za ORF1ab, N, E, ndizosiyana mzere wa ma nucleic acid mu zitsanzo za swab za nasopharyngeal kapena oropharyngeal monga SARS-CoV-2 B.1.1.7 mzere (UK), B.1.351 mzere (ZA), B.1.617 mzere (IND) ndi P.1 mzere (BR).

02  Ma reagents ofunikira pa PCR yolunjika

Chitsanzo cha Lysate

Chitsanzo cha lysate chikhoza kukhazikitsidwa ndi inu nokha kapena kugula.Kusiyana kwa mapangidwe amitundu yosiyanasiyana ya lysate kumapangitsa kuti luso la kunama likhale losiyana, ndiyeno nthawi yabodza idzakhala yosiyana pang'ono.Mwachitsanzo, pokonzekera zitsanzo za minofu ya nyama, nthawi zambiri amalimbikitsidwa kwa mphindi 30 kapena usiku wonse, ndipo yankho la lysis la ma virus limachokera ku 3-10 mphindi.

PCR master mix

Ndikofunikira kugwiritsa ntchito DNA polymerase yotentha kuti ipititse patsogolo kakulidwe kake ndikuwonjezera luso lokulitsa.Pakatikati pa PCR yolunjika ndi polymerase yololera kwambiri.

Chotsani kapena kuletsa zigawo zachitsanzo zomwe zimakhudza kukulitsa kwa DNA

Zitsanzozo zitakonzedwa ndi lysate, mapuloteni, lipids ndi zinyalala zina zama cell zidzatulutsidwa, zinthu izi zidzalepheretsa kuchita kwa PCR.Chifukwa chake, PCR yolunjika imafuna kuwonjezera kuchotsedwa kofananira kapena zoletsa kuti muchepetse kukopa kwazinthu izi.

03  Kutolere kwa mfundo zisanu za chidziwitso cha PCR yolunjika

Choyamba, ukadaulo wa Direct PCR ndiukadaulo wachindunji wa PCR wamitundu yosiyanasiyana yazachilengedwe.Pansi pa luso ili, palibe chifukwa cholekanitsa ndi kuchotsa nucleic acid, kugwiritsa ntchito mwachindunji chitsanzo cha minofu monga chinthucho, ndikuwonjezera jini yomwe imayang'ana kuti ipange PCR.

Chachiwiri, ukadaulo wa Direct PCR siukadaulo wokulirapo wa template wa DNA, komanso umaphatikizansopo RNA template reverse transcription PCR.

Chachitatu, ukadaulo wa Direct PCR sikuti umangochita zomwe zimachitika pafupipafupi pa PCR pazitsanzo za minofu, komanso umaphatikizanso zochitika za Real-Time qPCR, zomwe zimafuna kuti kachitidwe kachitidwe kake kakhale ndi mphamvu zotsutsana ndi maziko a fluorescence kusokoneza komanso kutha kwa fulorosenti kumathetsa luso lotsutsa.

Chachinayi, zitsanzo zomwe zimayang'aniridwa ndi teknoloji ya Direct PCR zimangofuna kutulutsa ma templates a nucleic acid, ndipo musachotse mapuloteni, ma polysaccharides, ayoni amchere, ndi zina zotero zomwe zimasokoneza machitidwe a PCR.Zomwe zimafunikira nucleic acid polymerase ndi PCR Mix mumayendedwe kachitidwe kuti zikhale zolimba komanso zosinthika kuti zitsimikizire zochita za enzyme ndi kubwereza kulondola pamikhalidwe yovuta.

Chachisanu, chitsanzo cha minofu chomwe chimayang'aniridwa ndi teknoloji ya Direct PCR popanda chithandizo chowonjezera cha nucleic acid ndipo kuchuluka kwa template ndi kochepa kwambiri, komwe kumafuna kuti machitidwewa akhale okhudzidwa kwambiri ndi kukulitsa mphamvu.