Direct PCR maitiro anoshandisa zvakananga mhuka kana matishu echirimwa kuti awedzere pasina nucleic acid kutorwa.Nenzira dzakawanda, yakananga PCR inoshanda seyakajairwa PCR

Musiyano mukuru ndeye tsika buffer inoshandiswa muPCR yakananga, sampuli inogona kuiswa zvakananga kuPCR reaction pasina nucleic acid kutorwa, asi pane zvinoenderana nezvinodiwa zvekushivirira kwema enzymes uye kuenderana kweiyo buffer inobatanidzwa mune yakananga PCR reaction.

Kunyangwe paine akawanda kana mashoma PCR inhibitors mumasamples akajairwa, yakananga PCR ichiri kugona kuwana yakavimbika amplification pasi pechiito che enzymes uye buffers.Iyo yechinyakare PCR maitiro inoda yakakwirira-mhando nucleic acid se template, iyo inogona kutadzisa kufambira mberi kwakanaka kwePCR reaction kana template ine mapuroteni uye kumwe kusvibiswa.Direct PCR parizvino ndeimwe yeanonyanya kufarirwa matekinoroji mumunda wekuongororwa kwema molecular.

01 Direct PCR yakatanga kushandiswa kumhuka uye chirimwa

Kushandiswa kwekutanga kwePCR kwakananga mumunda wemhuka nezvirimwa, zvakaita seropa, matishu uye mvere dzegonzo, katsi, huku, tsuro, hwai, mombe, nezvimwewo, mashizha ezvirimwa nembeu, nezvimwewo, yakashandiswa kudzidza genotyping, transgenic, plasmid yekuona, Gene knockout analysis, DNA source identification, kuzivikanwa kwemarudzi, SNP kuongorora uye mamwe minda.

Iyi minda ine zvimwe zvakajairika maficha, kureva, iyo inotarirwa gene yemukati yakakwira uye nucleic acid kudhirowa kunonetsa, saka yakananga PCR haingogone kuchengetedza nguva chete uye kuve nekukanganisa kudiki pamhedzisiro, asiwo kuchengetedza mutengo.

Yakananga PCR inoshandiswa kuona pathogen inyaya yemakore achangopfuura, vamwe vagadziri vePCR reagent vakaita kwakawanda kuedza munzira iyi pakuita hunyanzvi.Kunyanya mudenda iri reCCIDID-19, zvigadzirwa zvakawanda zvakadai zvekuona zvakabuda pamusika, zvakaita seSARS-CoV-2 Nucleic Acid Detection Kit (Multiplex PCR Fluorescent Probe Method) yakatsvagwa uye yakagadziridzwa neForegene, inoshandisa Real-nguva RT PCR tekinoroji (rRT-PCR) yehunhu hweSARS-CoV-PCR) yemhando yeNucleic-Cophary2 yeSARS-Cophary2 ngeal swab samples.

Foregene ndeimwe yemakambani anoshandisa Direct PCR tekinoroji, yekuona yakajairika ORF1ab, N, E, uye.variant lineages nucleic acids mumunhu nasopharyngeal kana oropharyngeal swab samples dzakadai SARS-CoV-2 B.1.1.7 lineage (UK), B.1.351 mutsara (ZA), B.1.617 mutsara (IND) uye P.1 mutsara (BR).

02  Reagents inodiwa kune yakananga PCR

Muenzaniso Lysate

Sample lysate inogona kugadzirwa iwe pachako kana kutengwa.Misiyano mukuumbwa kwemhando dzakasiyana dze lysate ichaita kuti kugona kwenhema kuve kwakasiyana, uye ipapo nguva yekunyepa ichave yakasiyana zvishoma.Semuyenzaniso, pakugadzirira matishu emhuka samples, maminetsi makumi matatu kana husiku hwese lysis inokurudzirwa, uye mhinduro ye lysis yemavhairasi inotangira pa3-10 maminetsi.

PCR master mix

Zvinokurudzirwa kushandisa inopisa-kutanga DNA polymerase kusimudzira chaiyo amplification uye kuwedzera kugona kwekusimudzira.Iyo yakakosha yePCR yakananga inoshivirira zvakanyanya polymerase.

Bvisa kana kurambidza zvikamu mumuenzaniso zvinokanganisa DNA amplification

Mushure mekunge sampu yagadziriswa neiyo lysate, mapuroteni, lipids uye mamwe maseru marara anoburitswa, zvinhu izvi zvinodzivirira kuita kwePCR.Naizvozvo, yakananga PCR inoda kuwedzererwa kwekubvisa kunoenderana kana inhibitors kudzikisa pesvedzero yezvinhu izvi.

03  Muunganidzwa wemapoinzi mashanu eruzivo rweyakananga PCR

Chekutanga, Direct PCR tekinoroji ndiyo yakananga PCR tekinoroji kune akasiyana biological samples.Pasi peiyi tekinoroji mamiriro, hapana chikonzero chekuparadzanisa uye kubvisa iyo nucleic acid, shandisa zvakananga sampuli yetishu sechinhu, uye kuwedzera chinangwa chejini primers ndechekuita PCR reaction.

Chechipiri, Direct PCR tekinoroji haingori yechinyakare DNA template amplification tekinoroji, asi zvakare inosanganisira RNA template reverse transcription PCR.

Chetatu, Direct PCR tekinoroji haingoite zvakanangana nemaitiro ePCR maitiro pamasampuni ematishu, asi zvakare inosanganisira Real-Nguva qPCR maitiro, ayo anoda kuti reaction system ive yakasimba anti-background fluorescence kupindira kugona uye endogenous fluorescence inodzima kugona kweanopikisa.

Chechina, masampuli anotarirwa neDirect PCR teknolojia inongoda kusunungurwa kwe nucleic acid templates, uye usabvisa mapuroteni, polysaccharides, salt ions, nezvimwewo zvinokanganisa maitiro ePCR.Izvo zvinoda iyo nucleic acid polymerase uye PCR Sanganisa mune rekuita system kuti ive nekupokana kwakanyanya uye kuchinjika kuti ive nechokwadi chekuita enzyme uye kudzokorodza kwechokwadi pasi pemamiriro akaomarara.

Chechishanu, sampuli yetishu yakanangidzirwa neDirect PCR tekinoroji isina chero nucleic acid inopfumisa kurapwa uye huwandu hwetemplate idiki kwazvo, izvo zvinoda kuti maitiro ekuita ave nekunyanya kunzwisiswa uye kukwidziridza kunyatsoita.