PCR kai tsaye wani martani ne wanda ke amfani da kyallen dabba ko shuka kai tsaye don haɓakawa ba tare da cirewar acid nucleic ba.A hanyoyi da yawa, PCR kai tsaye yana aiki kamar PCR na yau da kullun

Babban bambanci shine buffer na al'ada da aka yi amfani da shi a cikin PCR kai tsaye, ana iya ƙaddamar da samfurin kai tsaye ga amsawar PCR ba tare da cirewar acid nucleic ba, amma akwai buƙatu masu dacewa don jurewar enzymes da kuma dacewa da buffer da ke cikin kai tsaye na PCR.

Kodayake akwai fiye ko žasa masu hana PCR a cikin samfuran gama gari, PCR kai tsaye har yanzu na iya samun ingantaccen haɓakawa a ƙarƙashin aikin enzymes da buffers.Halin PCR na al'ada yana buƙatar acid nucleic mai inganci a matsayin samfuri, wanda zai iya hana ingantaccen ci gaba na amsawar PCR idan samfurin ya ƙunshi sunadarai da sauran ƙazanta.PCR kai tsaye a halin yanzu ɗaya ce daga cikin fitattun fasahohin fasaha a fagen binciken ƙwayoyin cuta.

01 Direct PCR an fara amfani dashi don dabba da shuka

Aikace-aikacen farko na PCR kai tsaye shine a fagen dabbobi da shuke-shuke, irin su jini, nama da gashin bera, cat, kaza, zomo, tumaki, shanu, da dai sauransu, ganye da tsaba, da dai sauransu, ana amfani da su don nazarin genotyping, transgenic, plasmid detection, Gene knockout analysis, DNA source ganewa, ganewar jinsin, nazarin SNP da sauran filayen.

Wadannan filayen suna da wasu fasalulluka na yau da kullun, wato, abubuwan da aka yi niyya ga abubuwan da ke cikin abubuwan da aka yi niyya suna da inganci kuma haɓakar acid nucleic yana da wahala, don haka PCR kai tsaye ba zai iya adana lokaci kawai ba kuma yana da ɗan ƙaramin tasiri akan sakamakon, amma kuma yana adana farashi.

PCR kai tsaye da aka yi amfani da shi don gano ƙwayoyin cuta al'amari ne na 'yan shekarun nan, wasu masana'antun PCR reagent sun yi ƙoƙarce-ƙoƙarce a cikin wannan jagorar yayin yin sabbin abubuwa.Musamman a cikin wannan annoba ta COVID-19, yawancin irin waɗannan samfuran ganowa sun bayyana akan kasuwa, irin su SARS-CoV-2 Nucleic Acid Detection Kit (Tsarin Bincike na Multiplex PCR Fluorescent Probe) wanda Foregene yayi bincike kuma ya haɓaka, wanda ke amfani da fasahar RT PCR ta Real-time (rRT-PCR) don gano ƙimar ingancin SARS-CoV-2 a cikin samfurin nassophan acid na mutum.

Foregene ɗaya ne daga cikin kamfanoni masu amfani da fasahar PCR Direct, don gano ORF1ab, N, E, da na al'ada.bambancin lineages nucleic acid a cikin mutum nasopharyngeal ko oropharyngeal swab samfurori irin su SARS-CoV-2 B.1.1.7 lineage (UK), B.1.351 lineage (ZA), B.1.617 lineage (IND) da P.1 lineage (BR).

02  Reagents da ake buƙata don PCR kai tsaye

Misalin Lysate

Za'a iya saita samfurin lysate da kanka ko siya.Bambance-bambance a cikin nau'ikan nau'ikan nau'ikan nau'ikan lysate daban-daban zai sa ikon yin lysing ya bambanta, sannan lokacin lysing zai ɗan bambanta.Misali, don shirye-shiryen samfuran nama na dabba, ana ba da shawarar mintuna 30 ko lysis na dare gabaɗaya, kuma maganin lysis na ƙwayoyin cuta yana daga mintuna 3-10.

Farashin PCR

Ana ba da shawarar yin amfani da DNA polymerase mai zafi don haɓaka takamaiman haɓakawa da haɓaka ƙarfin haɓakawa.Jigon PCR kai tsaye shine polymerase mai jurewa.

Cire ko hana abubuwan da ke cikin samfurin da ke shafar haɓakar DNA

Bayan an sarrafa samfurin tare da lysate, sunadarai, lipids da sauran tarkace tantanin halitta za a saki, waɗannan abubuwa zasu hana aikin PCR.Sabili da haka, PCR kai tsaye yana buƙatar ƙara daidai cirewa ko masu hanawa don rage tasirin waɗannan abubuwan.

03  Tarin abubuwan ilimi guda biyar na PCR kai tsaye

Na farko, fasahar PCR kai tsaye fasaha ce ta PCR kai tsaye don samfuran halitta iri-iri.A ƙarƙashin wannan yanayin fasaha, babu buƙatar rabuwa da cire acid nucleic, yi amfani da samfurin nama kai tsaye a matsayin abu, kuma ƙara abubuwan da aka yi niyya don yin aikin PCR.

Na biyu, Fasahar PCR kai tsaye ba fasahar haɓaka samfurin DNA ta gargajiya ce kawai ba, har ma ya haɗa da samfurin RNA na juyar da PCR.

Na uku, fasahar PCR kai tsaye ba kai tsaye tana aiwatar da halayen PCR na yau da kullun akan samfuran nama ba, har ma ya haɗa da halayen qPCR na Real-Time, wanda ke buƙatar tsarin amsawa don samun ƙarfin tsangwama mai ƙarfi na baya-baya da kyalli na ƙarshe yana kashe ƙarfin adawa.

Na hudu, samfuran da aka yi niyya ta hanyar fasahar PCR kai tsaye kawai suna buƙatar sakin samfuran acid nucleic, kuma kada su cire sunadaran, polysaccharides, ions gishiri, da sauransu waɗanda ke tsoma baki tare da amsawar PCR.Wanne yana buƙatar polymerase acid nucleic da PCR Mix a cikin tsarin amsawa don samun kyakkyawan juriya da daidaitawa don tabbatar da aikin enzyme da daidaiton kwafi a ƙarƙashin yanayi masu rikitarwa.

Na biyar, samfurin nama wanda aka yi niyya ta hanyar fasaha ta PCR kai tsaye ba tare da wani magani na wadatar nucleic acid ba kuma adadin samfurin yana da ƙanƙanta, wanda ke buƙatar tsarin amsawa don samun haɓakar haɓakawa sosai da haɓaka haɓakawa.