I-COVID-19 isifo esithathelwanayo esibangelwa i-Severe Acute Respiratory Syndrome Coronavirus Type 2. Uma umuntu ethelelekile, izimpawu ezivame kakhulu zihlanganisa imfiva, ukukhwehlela, nokuphefumula.

Amasampula asetshenziselwa ukuhlolwa angaqoqwa ngama-nasopharyngeal swabs noma ama-oropharyngeal swabs.

Iyini i-PCR?

Indlela ejwayelekile yokutholwa kwe-coronavirus i-polymerase chain reaction, i-PCR.Lena indlela esetshenziswa kakhulu kubhayoloji yamangqamuzana.Ingakwazi ukukopisha ngokushesha izigidi ezigidini zezingcezu ezithile ze-DNA.

I-coronavirus entsha iqukethe i-RNA genome ende enomucu owodwa.Ukuze kutholwe lawa magciwane nge-PCR, ama-molecule e-RNA kufanele aguqulelwe ekulandeleni kwawo kwe-DNA ehambisanayo nge-reverse transcriptase, bese i-DNA esanda kuhlanganiswa ingakhuliswa ngezinqubo ezijwayelekile ze-PCR, ngokuvamile ezaziwa ngokuthi i-RT-PCR.

Inqubo ye-RT-PCR

Ukukhishwa kwe-RNA

Ukwenza le ndlela, i-RNA yegciwane kufanele ikhishwe.Izinhlobonhlobo zamakhithi okuhlanza e-RNA angasetshenziselwa ukwahlukanisa okulula, okusheshayo nokuphumelelayo.

Ukuze ukhiphe i-RNA yegciwane usebenzisa ikhithi yezohwebo, qala wengeze isampula kushubhu encane ye-microcentrifuge bese uyixuba ne-lysis buffer.Le buffer ine-denatured ephezulu futhi ngokuvamile iqukethe i-phenol ne-guanidine isothiocyanate.Ukwengeza, ama-RNase inhibitors avamise ukuba khona ku-lysis buffer ukuze kuqinisekiswe ukuhlukaniswa kwe-RNA yegciwane eqinile.

Ngemva kokwengeza i-lysis buffer, vunguza ishubhu lokuxuba ngokushaya kwe-pulse futhi ufukamele ekamelweni lokushisa.Igciwane libe selihlaliswa ngaphansi kwezimo ezishintshashintshayo kakhulu ezihlinzekwa yi-lysis buffer.

Ngemuva kokuthi isampula i-lysed, ithubhu ye-centrifuge isetshenziselwa inqubo yokuhlanza.Isampula ilayishwa kushubhu ye-centrifuge bese i-centrifuge.

Le nqubo iyindlela eqinile yokukhipha isigaba lapho isigaba esimile siqukethe i-matrix yejeli ye-silica.

Ngaphansi kwezimo ezifanele zikasawoti kanye ne-pH, ama-molecule e-RNA abophezela kulwelwesi lwe-silica.

Ngesikhathi esifanayo, amaprotheni nezinye izinto ezingcolile ziyasuswa.

Ngemuva kwe-centrifugation, faka ishubhu le-centrifuge eshubhuni leqoqo elihlanzekile, ulahle i-filtrate, bese wengeza isivimbeli sokuwasha.

Faka ishubhu ku-centrifuge futhi ukuze uphoqe isigcinalwazi sokugeza ngolwelwesi.Lokhu kuzosusa konke ukungcola okusele kulwelwesi, kushiye i-RNA kuphela eboshwe kujeli yesilika.

Ngemva kokuba isampula seligeziwe, faka ishubhu eshubhuni elihlanzekile le-microcentrifuge bese wengeza isivimbeli se-elution.

Bese ifakwa i-centrifuged ukuze iphoqe isibari se-elution kulwelwesi.I-elution buffer isusa i-RNA yegciwane kukholomu ejikelezayo futhi ithola i-RNA ehlanzekile engenawo amaprotheni, ama-inhibitor, nokunye ukungcola.

ISINYATHELO 2

I-concentrate ehlanganisiwe

Ngemva kokukhipha i-RNA yegciwane, isinyathelo esilandelayo ukulungisa ingxube yokusabela yokukhulisa i-PCR.Kulesi sinyathelo, kusetshenziswa ukugxilisa ingqondo.Lesi sixazululo esigxilisiwe siyisixazululo esixutshwe ngaphambili esihlanganisa i-premix, i-reverse transcriptase, ama-nucleotides, i-forward primer, i-reverse primer, i-TaqMan probe kanye ne-DNA polymerase.

Ekugcineni, ukuqedela le ngxube yokusabela, isifanekiso se-RNA siyengezwa.Amashubhu axutshwa yi-pulse vortexing, bese ingxube yokusabela ilayishwa epuleti le-PCR.Ipuleti le-PCR ngokuvamile liqukethe imithombo engama-96 futhi lingahlaziya amasampula amaningi ngesikhathi esisodwa.

ISINYATHELO 3

Ukukhulisa i-PCR

Okulandelayo, beka ipuleti emshinini we-PCR, okuwumjikelezo oshisayo.

I-RT-PCR yesikhathi sangempela isetshenziselwa ukuthola i-coronavirus yenoveli ka-2019 ngokukhulisa ukulandelana okuhlosiwe ohlotsheni lwe-RdrRP, isakhi sofuzo sika-E kanye nofuzo lwe-N.Ukukhetha isakhi sofuzo esiqondiwe kuncike ekulandeleni kwe-primer kanye ne-probe.

Isinyathelo sokuqala se-RT-PCR ukuloba okuhlanekezelwe.Umucu wokuqala we-DNA ehambisanayo uyahlanganiswa, oqalwa yi-PCR reverse primer, ebophezela engxenyeni ehambisanayo ye-viral RNA genome.Bese i-reverse transcriptase yengeza ama-nucleotide e-DNA ku-3′end ye-primer ukuze kuhlanganiswe i-DNA ehambisana ne-RNA yegciwane.Izinga lokushisa nobude balesi sinyathelo kuncike kuma-primer, i-RNA eqondiwe, kanye ne-reverse transcriptase esetshenzisiwe.

Okulandelayo, isinyathelo sokuqala sokuguqula i-denaturation, esiholela ekuguqulweni kwe-RNA-DNA hybrid.Lesi sinyathelo siyadingeka ukuze kusebenze i-DNA polymerase.Ngesikhathi esifanayo, i-reverse transcriptase ayisebenzi.

I-PCR iqukethe uchungechunge lwemijikelezo eshisayo.Umjikelezo ngamunye uqukethe izinyathelo ze-denaturation, annealing kanye nesandiso.

Isinyathelo se-denaturation sifaka ukushisisa igumbi lokusabela libe ngu-95 degrees Celsius futhi ulisebenzisele ukuguqula isifanekiso se-DNA esinemicu ekabili.

Esinyathelweni esilandelayo, izinga lokushisa lokusabela liyehliswa libe ngu-58 degrees Celsius, okuvumela i-primer eya phambili ukuthi ihlangane nengxenye ehambisanayo yesifanekiso sayo esinomucu owodwa we-DNA.Izinga lokushisa le-annealing lincike ngokuqondile kubude nokubunjwa kwe-primer.

Esinyathelweni sokunwetshwa, i-DNA polymerase ihlanganisa umucu omusha we-DNA ohambisana nomucu wesifanekiso se-DNA.Ngokungeza ama-nuclei amahhala ahambisanayo kusifanekiso esiku-5′to 3′direction kusukela kungxube yokusabela.Izinga lokushisa lalesi sinyathelo lincike ku-DNA polymerase esetshenzisiwe.

Ngemuva komjikelezo wokuqala, i-DNA enemicu ephindwe kabili iyatholakala.

Bese, faka umjikelezo wesibili.I-DNA enemicu ephindwe kabili yenziwa i-denatured ukuze ikhiqize ama-molecule amabili e-DNA anomucu owodwa.

Esinyathelweni esilandelayo, izinga lokushisa lokusabela liyehliswa, ama-primer axhunywe kusifanekiso ngasinye esinomucu owodwa we-DNA, futhi uphenyo lwe-Taq-man luxhunywe engxenyeni ehambisanayo ye-DNA eqondiwe.

Uphenyo lwe-TaqMan luqukethe i-fluorophore exhunywe ngokuhlanganyela ekugcineni kwe-oligonucleotide probe.Uma ijatshuliswa umthombo wokukhanya womjikelezo, i-fluorophore ikhipha i-fluorescence.Ukwengeza, i-probe yenziwe nge-quencher ekupheleni kwe-3.Ukusondela kofuzo lwentatheli kusicisha kuvimbela ukutholwa kwe-fluorescence.

Esinyathelweni sokunwetshwa, i-DNA polymerase ihlanganisa umucu omusha.Lapho i-polymerase ifinyelela kuphenyo lwe-TaqMan, umsebenzi wayo ongapheli we-5′nuclease uhlukanisa uphenyo, uhlukanise udayi nesicishi.

Ngomjikelezo ngamunye we-PCR, ama-molecule kadayi amaningi akhululwa, okuholela ekwandeni kokuqina kwe-fluorescence ngokulingana nenani lama-amplicon ahlanganisiwe.

Le ndlela ivumela ukulinganisa inombolo yokulandelana okunikeziwe ekhona kusampula.Inani lezingcezu ze-DNA ezinemicu emibili liphindeka kabili emjikelezweni ngamunye.Ngakho-ke, i-PCR ingasetshenziswa ukuhlaziya amasampula amancane kakhulu.

Ngokulinganisa isignali ye-fluorescent, isibani se-tungsten halogen, isihlungi esivusa amadlingozi, isibonisi, ilensi, isihlungi se-emission kanye nokushaja ikhamera ye-CCD ehambisana nedivayisi.

ISINYATHELO 4 Thola

Ngokulinganisa isignali ye-fluorescent, isibani se-tungsten halogen, isihlungi esivusa amadlingozi, isibonisi, ilensi, isihlungi se-emission kanye nokushaja ikhamera ye-CCD ehambisana nedivayisi.

Ukukhanya okuhlungiwe kwesibani kuboniswa yi-reflector, kudlula i-condenser lens, futhi kugxilwe phakathi nendawo yembobo ngayinye.Khona-ke i-fluorescence ephuma emgodini ibonakala esibukweni, idlula isihlungi sokukhishwa, futhi itholwe yikhamera ye-CCD.Kumjikelezo we-PCR ngamunye, ukukhanya kwe-fluorophore okuzijabulisa kungatholwa yi-CCD.

Iguqula ukukhanya okuthunjiwe kube idatha yedijithali.Le ndlela ibizwa nge-PCR yesikhathi sangempela, futhi ivumela ukuqapha kwesikhathi sangempela kokuqhubeka kokusabela kwe-PCR.