COVID-19 chirwere chinotapuriranwa chinokonzerwa neSevere Acute Respiratory Syndrome Coronavirus Type 2. Kana munhu aine utachiona, zviratidzo zvinonyanyozivikanwa zvinosanganisira fivha, kukosora, uye kutadza kufema zvakanaka.

Iwo masampula anoshandiswa pakuyedza anogona kuunganidzwa ne nasopharyngeal swabs kana oropharyngeal swabs.

Chii chinonzi PCR?

Iyo yakajairwa nzira yekuona coronavirus ndeye polymerase chain reaction, PCR.Iyi inzira inoshandiswa zvakanyanya mumolecular biology.Inogona kukurumidza kukopa mamiriyoni kumabhiriyoni ezvimedu zveDNA.

Iyo coronavirus nyowani ine yakarebesa imwe-yakasungwa RNA genome.Kuti uone mavhairasi aya nePCR, mamorekuru eRNA anofanirwa kuchinjirwa kuita iwo anowirirana eDNA kutevedzana nereverse transcriptase, uyezve iyo ichangogadzirwa DNA inogona kukwidziridzwa neyakajairwa PCR maitiro, ayo anozivikanwa seRT-PCR.

RT-PCR maitiro

RNA kubviswa

Kuita iyi nzira, hutachiona RNA hunofanira kubviswa.Yakasiyana-siyana yeRNA yekuchenesa kits inogona kushandiswa kune nyore, nekukurumidza uye kunoshanda kupatsanura.

Kuti ubvise hutachiona hweRNA uchishandisa kit yekutengeserana, tanga wawedzera sample kune microcentrifuge chubhu wozoisanganisa ne lysis buffer.Iyi buffer ine denatured zvakanyanya uye kazhinji ine phenol uye guanidine isothiocyanate.Mukuwedzera, RNase inhibitors inowanzovapo mu lysis buffer kuti ive nechokwadi chekuparadzaniswa kwehutachiona hweRNA.

Mushure mekuwedzera iyo lysis buffer, vortex iyo yekusanganisa chubhu nekupuruzira uye incubate pakamuri tembiricha.Hutachiona hunobva hwanyudzwa pasi pemamiriro akanyanya dhinaturing anopihwa neiyo lysis buffer.

Mushure mekunge sampu yaiswa lysed, chubhu yecentrifuge inoshandiswa pakunatswa.Sample inotakurwa mu centrifuge chubhu uye ipapo centrifuged.

Iyi maitiro inzira yakasimba yekubvisa chikamu umo iyo yakamira chikamu ine silica gel matrix.

Pasi peyakanyanya munyu uye pH mamiriro, mamorekuru eRNA anosunga kune silica membrane.

Panguva imwecheteyo, mapuroteni nezvimwe zvinosvibisa zvinobviswa.

Mushure mekuita centrifugation, isa chubhu yecentrifuge muchubhu yekuunganidza yakachena, rasa iyo filtrate, wobva wawedzera washing buffer.

Isa chubhu mucentrifuge zvakare kuti umanikidzire kugeza buffer kuburikidza ne membrane.Izvi zvinobvisa zvese zvakasara tsvina kubva mu membrane, zvichisiya chete RNA yakasungwa kune silica gel.

Mushure mekunge sampu yasukwa, isa chubhu muchubhu yakachena microcentrifuge uye wedzera elution buffer.

Inobva yaiswa centrifused kumanikidza elution buffer kuburikidza ne membrane.Iyo elution buffer inobvisa viral RNA kubva ku spin column uye inowana yakacheneswa RNA isina mapuroteni, inhibitors, uye zvimwe zvinosvibisa.

STEP 2

Mixed concentrate

Mushure mekutora hutachiona RNA, danho rinotevera nderekugadzirira musanganiswa wekuita wePCR amplification.Padanho iri, kuisa pfungwa kunoshandiswa.Iyi yakagadziriswa mhinduro ndeye premixed concentrated solution inosanganisira premix, reverse transcriptase, nucleotides, forward primer, reverse primer, TaqMan probe uye DNA polymerase.

Chekupedzisira, kuti upedze iyi reaction musanganiswa, iyo RNA template inowedzerwa.Machubhu anosanganiswa ne pulse vortexing, uye ipapo musanganiswa wekuita unoiswa muPCR ndiro.Iyo PCR ndiro inowanzo ine 96 matsime uye inogona kuongorora akawanda samples panguva imwe chete.

STEP 3

PCR kuwedzera

Tevere, isa ndiro yacho mumushini wePCR, unova unopisa bhasikoro.

Chaiyo-nguva RT-PCR inoshandiswa kuona iyo 2019 novel coronavirus nekusimudzira inoteedzana inoteedzana muRdrRP gene, E gene uye N gene.Sarudzo yejini rinotangwa zvinoenderana nekutanga uye probe kutevedzana.

Nhanho yekutanga yeRT-PCR ndeye reverse transcription.Rudzi rwekutanga rwe DNA inopindirana inogadzirwa, iyo inotangwa nePCR reverse primer, iyo inosungira kune inopindirana chikamu chehutachiona RNA genome.Zvadaro reverse transcriptase inowedzera DNA nucleotides kune 3′ yekupedzisira yeprimer kuti igadzirise DNA inoenderana nehutachiona RNA.Tembiricha uye nguva yedanho iri zvinoenderana nemaprimers, chinangwa RNA, uye reverse transcriptase inoshandiswa.

Tevere, danho rekutanga rekudhirowa rinoiswa, izvo zvinozokonzera denaturation yeRNA-DNA hybrid.Iyi nhanho inofanirwa kumisa iyo DNA polymerase.Panguva imwecheteyo, reverse transcriptase inoregedzwa.

PCR ine nhevedzano yemafuta ekutenderera.Kutenderera kwega kwega kunosanganisira denaturation, annealing uye matanho ekuwedzera.

Denaturation nhanho inosanganisira kudziyisa mukamuri yekupindura kusvika ku95 madhigirii uye kuishandisa kuita denaturation yekaviri tambo yeDNA template.

Mudanho rinotevera, tembiricha yekuita inodzikiswa kusvika pa58 degrees Celsius, zvichibvumira iyo yekutanga primer kuti iende kune inopindirana chikamu cheiyo-stranded stranded DNA template.Iyo annealing tembiricha zvakananga zvinoenderana nehurefu uye kuumbwa kweiyo primer.

Mudanho rekuwedzera, iyo DNA polymerase inogadzira nyowani DNA strand inopindirana neDNA template strand.Nekuwedzera nuclei yemahara inopindirana kune template mu5'to 3'direction kubva pamusanganiswa wekuita.Kupisa kwedanho iri kunoenderana neDNA polymerase inoshandiswa.

Mushure mekutenderera kwekutanga, chinangwa cheDNA chine tambo mbiri chinowanikwa.

Zvadaro, pinda chikamu chechipiri.Iyo ine tambo mbiri DNA inoshongedzwa kuti igadzire mamorekuru maviri eDNA ane tambo imwe chete.

Muchinhanho chinotevera, tembiricha yekuita inodzikiswa, maprimers anonamirwa kune imwe neimwe-stranded DNA template, uye Taq-man probe inonamirwa kune inopindirana chikamu chechinangwa cheDNA.

Iyo TaqMan probe ine fluorophore yakabatana yakabatana kune iyo 5′ yekupedzisira yeiyo oligonucleotide probe.Kana uchifadzwa nechiedza chemutenderedzwa, iyo fluorophore inoburitsa fluorescence.Mukuwedzera, iyo probe inoumbwa neanodzima pa 3′end.Kuswedera kwemutori wenhau gene kune quencher kunodzivirira kuonekwa kwefluorescence.

Mudanho rekuwedzera, iyo DNA polymerase inogadzira tambo nyowani.Kana iyo polymerase ichisvika paTaqMan probe, endogenous 5′nuclease chiitiko inotsemura probe, ichiparadzanisa dhayi kubva kune inodzima.

Nekutenderera kwega kwega kwePCR, mamwe mamorekuru edhayi anoburitswa, zvichikonzera kuwedzera kwefluorescence intensity inoenderana nehuwandu hwemaamplicon akagadzirwa.

Iyi nzira inobvumira kufungidzira nhamba yenhevedzano yakapihwa iripo mumuenzaniso.Nhamba yezvimedu zveDNA zvakakoshwa kaviri inopetwa kaviri mukutenderera kwega kwega.Naizvozvo, PCR inogona kushandiswa kuongorora diki sampuli.

Yekuyera siginecha yefluorescent, tungsten halogen mwenje, excitation sefa, reflector, lens, emission sefa uye kuchaja yakasanganiswa mudziyo-shandisa CCD kamera.

STEP 4 Ziva

Yekuyera siginecha yefluorescent, tungsten halogen mwenje, excitation sefa, reflector, lens, emission sefa uye kuchaja yakasanganiswa mudziyo-shandisa CCD kamera.

Chiedza chakanatswa kubva pamwenje chinoratidzwa nereflector, chinopfuura nepacondenser lens, uye chinotariswa pakati pegomba rega rega.Zvadaro fluorescence inobudiswa kubva mugomba inoratidzwa kubva pagirazi, inopfuura nepasefa emission, uye inowanikwa neCCD kamera.Muchikamu chimwe nechimwe chePCR, chiedza chekuzvifadza chefluorophore chinogona kuonekwa neCCD.

Inoshandura chiedza chakatorwa kuita data yedhijitari.Iyi nzira inonzi chaiyo-nguva PCR, uye inobvumira chaiyo-nguva yekutarisa kufambira mberi kwePCR reaction.