COVID-19 ni indwara yandura iterwa na Syndrome ikabije y'ubuhumekero Coronavirus Ubwoko bwa 2. Iyo umuntu yanduye, ibimenyetso bikunze kugaragara harimo umuriro, inkorora, no guhumeka neza.

Ingero zikoreshwa mugupima zishobora gukusanywa na nasopharyngeal swabs cyangwa oropharyngeal swabs.

PCR ni iki?

Uburyo busanzwe bwo kumenya coronavirus ni polymerase yumunyururu, PCR.Ubu ni uburyo bukoreshwa cyane muri biologiya ya biologiya.Irashobora gukoporora vuba miriyoni kugeza kuri miliyari z'ibice byihariye bya ADN.

Coronavirus nshya irimo genome ndende ya RNA imwe.Kugirango hamenyekane izo virusi na PCR, molekile ya RNA igomba guhindurwa muburyo bwa ADN bwuzuzanya hifashishijwe inyandiko zinyuranye, hanyuma ADN nshya ikomatanyirizwa hamwe ishobora kongerwa nuburyo busanzwe bwa PCR, busanzwe buzwi nka RT-PCR.

Inzira ya RT-PCR

Gukuramo RNA

Kugirango ukore ubu buryo, virusi RNA igomba gukurwamo.Ibikoresho bitandukanye byo kweza RNA birashobora gukoreshwa muburyo bworoshye, bwihuse kandi bwiza.

Gukuramo virusi ya RNA ukoresheje ibikoresho byubucuruzi, banza wongere icyitegererezo kuri microcentrifuge hanyuma ubivange na lysis buffer.Iyi buffer yamaganwe cyane kandi mubisanzwe igizwe na fenol na guanidine isothiocyanate.Byongeye kandi, inzitizi za RNase zisanzwe ziboneka muri lysis buffer kugirango harebwe virusi ya RNA idakira.

Nyuma yo kongeramo buffer ya lysis, vortex ivanga umuyoboro na pulse hanyuma ube mubushyuhe bwicyumba.Virusi ihita yanduzwa mubihe bitangwa cyane na lysis buffer.

Icyitegererezo kimaze guterwa, umuyoboro wa centrifuge ukoreshwa muburyo bwo kweza.Icyitegererezo gipakirwa muri centrifuge hanyuma igashyirwa muri centrifuged.

Ubu buryo nuburyo bukomeye bwo gukuramo icyiciro aho icyiciro gihagaze kigizwe na materix ya silika.

Mugihe cyumunyu mwiza na pH, molekile ya RNA ihuza na silika membrane.

Muri icyo gihe, poroteyine n’ibindi bihumanya bikurwaho.

Nyuma ya centrifugation, shyira umuyoboro wa centrifuge mumiyoboro isukuye neza, ujugunye akayunguruzo, hanyuma wongereho gukaraba.

Shira umuyoboro muri centrifuge ongera uhatire kumesa yoza unyuze muri membrane.Ibi bizakuraho umwanda wose usigaye muri membrane, hasigara RNA gusa ihambiriye kuri silika gel.

Icyitegererezo kimaze gukaraba, shyira umuyoboro muri microcentrifuge isukuye hanyuma wongereho buffer.

Icyo gihe ni centrifuged kugirango ihatire elfer buffer binyuze muri membrane.Buffer ya elution ikuraho virusi ya RNA mu nkingi ya spin hanyuma ikabona RNA isukuye idafite proteyine, inhibitor, nibindi byanduza.

INTAMBWE 2

Kwivanga

Nyuma yo gukuramo virusi RNA, intambwe ikurikiraho ni ugutegura imvange ya reaction yo kongera PCR.Muri iyi ntambwe, kwibandaho birakoreshwa.Igisubizo cyibanze nigisubizo cyibanze cyibanze kigizwe na premix, reverse transcriptase, nucleotide, primer primer, revers primer, TaqMan probe na polymerase ya ADN.

Hanyuma, kugirango urangize iyi reaction ivanze, inyandikorugero ya RNA yongeyeho.Imiyoboro ivangwa na pulse vortexing, hanyuma imvange ya reaction yapakiwe mubisahani bya PCR.Isahani ya PCR isanzwe irimo amariba 96 kandi irashobora gusesengura icyarimwe icyarimwe.

INTAMBWE 3

Kwiyongera kwa PCR

Ibikurikira, shyira isahani mumashini ya PCR, mubyukuri ni cycler yumuriro.

Igihe nyacyo RT-PCR ikoreshwa mugutahura igitabo gishya cya coronavirus ya 2019 muguhuza intego zikurikirana muri gen RdrRP, E gene na N.Guhitamo intego ya gene biterwa na primer na probe ikurikirana.

Intambwe yambere ya RT-PCR ni transcript transcript.Umugozi wambere wa ADN yuzuzanya urahuzwa, utangizwa na PCR revers primer, ihuza igice cyuzuzanya cya virusi ya RNA.Noneho reverse transcriptase yongeramo nucleotide ya ADN kuri 3′end ya primer kugirango ihuze ADN yuzuzanya na virusi RNA.Ubushyuhe nigihe cyintambwe biterwa na primers, intego RNA, hamwe na transcriptase ikoreshwa.

Ibikurikira, intambwe yambere yo gutandukana irakurikizwa, bikavamo gutandukanya imvange ya RNA-ADN.Iyi ntambwe irakenewe kugirango ukore ADN polymerase.Mugihe kimwe, reaction transcriptase idakora.

PCR igizwe nurukurikirane rw'ibizunguruka.Buri cyiciro kigizwe no gutandukana, guhuza no kwagura intambwe.

Intambwe yo gutandukana ikubiyemo gushyushya icyumba cya reaction kuri dogere selisiyusi 95 no kuyikoresha mugutandukanya icyitegererezo cya ADN ebyiri.

Mu ntambwe ikurikiraho, ubushyuhe bwa reaction bwaragabanutse kugera kuri dogere selisiyusi 58, bituma primer yimbere yerekeza kuri anneal igice cyuzuzanya cyicyitegererezo cya ADN imwe.Ubushyuhe bwa annealing buterwa nuburebure nibigize primer.

Mu ntambwe yo kwaguka, polymerase ya ADN ihuza umurongo mushya wa ADN wuzuzanya nu murongo wa ADN.Mugushyiramo nuclei yubusa yuzuza inyandikorugero muri 5′to 3′kuyobora kuva reaction ivanze.Ubushyuhe bwiyi ntambwe buterwa na polymerase ya ADN yakoreshejwe.

Nyuma yizunguruka ya mbere, intego ya ADN igizwe kabiri.

Noneho, andika uruziga rwa kabiri.ADN ifite imirongo ibiri yamaganwe kugirango ikore molekile ebyiri za ADN imwe.

Mu ntambwe ikurikiraho, ubushyuhe bwa reaction buragabanuka, primers zomekwa kuri buri cyitegererezo cya ADN imwe, kandi iperereza rya Taq-man ryometse ku gice cyuzuzanya cya ADN.

Ubushakashatsi bwa TaqMan bugizwe na fluorophore ihujwe na 5′end ya oligonucleotide.Iyo ushimishijwe nisoko yumucyo wamagare, fluorophore isohora fluorescence.Mubyongeyeho, iperereza rigizwe no kuzimya kuri 3′end.Kuba hafi yumunyamakuru gene kumurwango birinda kumenya fluorescence.

Muntambwe yo kwaguka, ADN polymerase ihuza umurongo mushya.Iyo polymerase igeze kuri TaqMan probe, ibikorwa byayo bya endogenous 5′nuclease ikora iperereza, itandukanya irangi nu kuzimya.

Hamwe na buri cyiciro cya PCR, harekurwa molekile nyinshi zirangi, bikavamo kwiyongera kwingufu za fluorescence ugereranije numubare wa amplicons synthesize.

Ubu buryo butuma ugereranya umubare wurutonde rwatanzwe rugaragara murugero.Umubare wibice bibiri bya ADN byikubye kabiri muri buri cyiciro.Kubwibyo, PCR irashobora gukoreshwa mugusesengura ingero nto cyane.

Mugupima ibimenyetso bya fluorescent, itara rya tungsten halogen, akayunguruzo gashimishije, indangururamajwi, lens, iyungurura imyuka hamwe nogukoresha ibikoresho bifashishije kamera ya CCD.

INTAMBWE 4 Menya

Mugupima ibimenyetso bya fluorescent, itara rya tungsten halogen, akayunguruzo gashimishije, indangururamajwi, lens, iyungurura imyuka hamwe nogukoresha ibikoresho bifashishije kamera ya CCD.

Itara ryungurujwe riva mumatara rigaragazwa nicyuma kimurika, kinyura mumurongo wa kondenseri, kandi cyerekejwe hagati ya buri mwobo.Noneho fluorescence isohoka mu mwobo igaragarira mu ndorerwamo, ikanyura muyungurura imyuka, kandi ikamenyekana na kamera ya CCD.Muri buri cyiciro cya PCR, urumuri rwa fluorophore rwonyine rushobora gutahurwa na CCD.

Ihindura urumuri rwafashwe mumibare yamakuru.Ubu buryo bwitwa PCR-nyayo, kandi itanga igihe nyacyo cyo gukurikirana iterambere rya PCR.