I-COVID-19 sisifo esosulelayo esibangelwa yiSevere Acute Respiratory Syndrome Coronavirus Type 2. Xa umntu osulelekile, ezona mpawu zixhaphakileyo ziquka umkhuhlane, ukhohlokhohlo, kunye nokuphefumla nzima.

Iisampuli ezisetyenziselwa ukuvavanya zingaqokelelwa nge-nasopharyngeal swabs okanye i-oropharyngeal swabs.

Yintoni i-PCR?

Indlela eqhelekileyo yokufunyaniswa kwe-coronavirus yipolymerase chain reaction, PCR.Le yindlela esetyenziswa kakhulu kwibhayoloji yemolekyuli.Iyakwazi ukukhuphela ngokukhawuleza izigidi ukuya kwiibhiliyoni zamaqhekeza athile eDNA.

I-coronavirus entsha iqulethe i-RNA genome enemisonto enye.Ukuze kubonwe ezi ntsholongwane ngePCR, iimolekyuli ze-RNA kufuneka ziguqulwe zibe kulandelelwano lwe-DNA ehambelanayo nge-reverse transcriptase, kwaye ke i-DNA esanda kwenziwa inokwandiswa ngeenkqubo ze-PCR eziqhelekileyo, ezaziwa ngokuba yi-RT-PCR.

Inkqubo ye-RT-PCR

Ukukhutshwa kwe-RNA

Ukwenza le ndlela, i-RNA yentsholongwane kufuneka ikhutshwe.Iintlobo ngeentlobo zeekiti zokuhlanjululwa kwe-RNA zingasetyenziselwa ukwahlukana okulula, okukhawulezayo kunye nokusebenzayo.

Ukukhupha i-RNA yentsholongwane usebenzisa ikiti yorhwebo, faka kuqala isampuli kwi-tube ye-microcentrifuge uze uyixube kunye ne-lysis buffer.Esi sithinteli sine-denatured ephezulu kwaye idla ngokuba ne-phenol kunye ne-guanidine isothiocyanate.Ukongeza, i-RNase inhibitors ihlala ikhona kwi-lysis buffer ukuqinisekisa ukwahlukaniswa kwe-RNA yentsholongwane engaguqukiyo.

Emva kokongeza i-lysis buffer, vortex ityhubhu yokuxuba nge-pulse kwaye ufukamele kwiqondo lokushisa.Intsholongwane ke iye ihlaliswe phantsi kweemeko ze-denaturing ezibonelelwa yi-lysis buffer.

Emva kokuba isampuli i-lysed, ityhubhu ye-centrifuge isetyenziselwa inkqubo yokucoca.Isampuli ilayishwe kwityhubhu ye-centrifuge kwaye emva koko i-centrifuged.

Le nkqubo yindlela eyomeleleyo yokukhutshwa kwesigaba apho isigaba sokumisa sibandakanya i-silica gel matrix.

Ngaphantsi kwetyuwa efanelekileyo kunye neemeko ze-pH, iimolekyuli ze-RNA zibophelela kwi-silica membrane.

Ngelo xesha, iiprotheyini kunye nezinye izinto ezingcolileyo ziyasuswa.

Emva kwe-centrifugation, faka ityhubhu ye-centrifuge kwi-tube yokuqokelela ecocekileyo, ulahle i-filtrate, kwaye wongeze i-buffer yokuhlamba.

Beka ityhubhu kwi-centrifuge kwakhona ukunyanzela isithinteli sokuhlamba kwi-membrane.Oku kuya kususa konke ukungcola okuseleyo kwi-membrane, kushiya kuphela i-RNA ebophelelwe kwi-silica gel.

Emva kokuba isampulu ihlanjiwe, faka ityhubhu kwityhubhu ecocekileyo ye-microcentrifuge kwaye wongeze isithinteli elution.

Emva koko i-centrifuged ukunyanzela isithinteli elution kwi-membrane.I-elution buffer isusa i-RNA yentsholongwane kwikholamu ye-spin kwaye ifumana i-RNA ecocekileyo ngaphandle kweeprotheni, i-inhibitors, kunye nezinye izinto ezingcolileyo.

INYATHELO 2

Umxhesho oxutyiweyo

Emva kokukhupha i-RNA yentsholongwane, inyathelo elilandelayo kukulungiselela umxube wokusabela kwi-PCR yokukhulisa.Kweli nyathelo, kugxininise kusetyenziswa.Esi sisombululo sigxininisekile sisisombululo esixutyiweyo esine-premix, i-reverse transcriptase, i-nucleotides, i-primer yangaphambili, i-reverse primer, i-TaqMan probe kunye ne-DNA polymerase.

Ekugqibeleni, ukugqiba lo mxube wokusabela, itemplate ye-RNA yongezwa.Iityhubhu zixutywe yi-pulse vortexing, kwaye ke umxube wokusabela ulayishwe kwipleyiti yePCR.Ipleyiti yePCR idla ngokuqulatha amaqula angama-96 kwaye inokuhlalutya iisampuli ezininzi ngaxeshanye.

INYATHELO 3

Ukwandiswa kwePCR

Okulandelayo, beka ipleyiti kumatshini wePCR, eyona nto ingumjikelo oshushu.

I-RT-PCR yexesha langempela isetyenziselwa ukufumanisa i-coronavirus yenoveli ka-2019 ngokwandisa ulandelelwano olujoliswe kuyo kwi-RdrRP gene, E gene kunye ne-N gene.Ukukhethwa kwejini ekujoliswe kuyo kuxhomekeke kwi-primer kunye nokulandelelana kweprobe.

Inyathelo lokuqala le-RT-PCR kukukhutshelwa umva.I-strand yokuqala ye-DNA encedisayo idityanisiwe, eqaliswe yi-PCR reverse primer, ebophelela kwi-complementary part ye-viral RNA genome.Emva koko i-reverse transcriptase yongeza i-nucleotides ye-DNA kwi-3′end ye-primer ukudibanisa i-DNA ehambelana ne-RNA yentsholongwane.Iqondo lokushisa kunye nobude beli nyathelo lixhomekeke kwii-primers, i-RNA ekujoliswe kuyo, kunye ne-reverse transcriptase esetyenzisiweyo.

Emva koko, inyathelo lokuqala lokuguqula i-denaturation lisetyenziswa, elibangela ukuchithwa kwe-RNA-DNA hybrid.Eli nyathelo liyimfuneko ukuze kusebenze i-DNA polymerase.Kwangaxeshanye, i-reverse transcriptase ayisebenzi.

I-PCR iqulethe uluhlu lwemijikelezo ye-thermal.Umjikelo ngamnye uqulathe amanyathelo e-denaturation, i-annealing kunye nokwandiswa.

Inyathelo le-denaturation libandakanya ukufudumeza igumbi lokuphendula ukuya kuma-95 degrees Celsius kwaye lisebenzise ukudenaturation yetemplate enemisonto emibini yeDNA.

Kwinqanaba elilandelayo, iqondo lobushushu reaction lincitshiswe ukuya 58 degrees Celsius, ivumela primer phambili ukuba anneal ukuya inxalenye ehambelanayo template yayo enemisonto enye DNA.Ukushisa kwe-annealing ngokuthe ngqo kuxhomekeke kubude kunye nokubunjwa kwe-primer.

Kwinqanaba lokwandiswa, i-DNA polymerase idibanisa i-DNA strand entsha ehambelana ne-DNA template strand.Ngokudibanisa ii-nuclei zasimahla ezihambelana netemplate kwi-5′to 3′direction ukusuka kumxube wokusabela.Ubushushu beli nyathelo buxhomekeke kwiDNA polymerase esetyenziswayo.

Emva komjikelezo wokuqala, i-DNA ephindwe kabini ekujoliswe kuyo ifunyenwe.

Emva koko, faka umjikelo wesibini.I-DNA enemisonto emibini yenziwe ukuba ivelise iimolekyuli zeDNA ezinemisonto enye.

Kwinqanaba elilandelayo, iqondo lobushushu reaction lithotyiwe, iiprimers annealed kwi-template nganye enemisonto enye DNA, kwaye Taq-man probe annealed ukuya inxalenye ehambelanayo ekujoliswe DNA.

I-TaqMan probe iqulathe i-fluorophore edityaniswe ngokudibeneyo ne-5′end ye-oligonucleotide probe.Xa uvuyiswa ngumthombo wokukhanya we-cycler, i-fluorophore ikhupha i-fluorescence.Ukongezelela, i-probe iqulunqwe ngumcimi kwi-3′end.Ukusondela kofuzo lwentatheli kwi-quencher kuthintela ukubonwa kwe-fluorescence.

Kwinqanaba lokwandiswa, i-DNA polymerase idibanisa i-strand entsha.Xa i-polymerase ifikelela kwiprobe ye-TaqMan, umsebenzi wayo ongapheliyo we-5′nuclease uqhekeza i-probe, yahlula idayi kwi-quencher.

Ngomjikelezo ngamnye we-PCR, iimolekyuli zedayi ezininzi zikhutshwa, okukhokelela ekunyukeni kokuqina kwe-fluorescence ngokulingana nenani le-amplicon edibeneyo.

Le ndlela ivumela ukuqikelela inani lolandelelwano olunikiweyo olukhoyo kwisampulu.Inani leenxalenye zeDNA ezinemisonto ephindwe kabini liphinda kabini kumjikelo ngamnye.Ngoko ke, i-PCR ingasetyenziselwa ukuhlalutya iisampuli ezincinci kakhulu.

Ukulinganisa isignali yefluorescent, isibane se-tungsten halogen, isihluzo esivuselelayo, isibonisi, ilensi, isihluzo esikhutshwayo kunye nentlawulo edityanisiweyo yekhamera yeCCD.

INYATHELO LESI-4 Khangela

Ukulinganisa isignali yefluorescent, isibane se-tungsten halogen, isihluzo esivuselelayo, isibonisi, ilensi, isihluzo esikhutshwayo kunye nentlawulo edityanisiweyo yekhamera yeCCD.

Ukukhanya okuhluziweyo okuvela kwisibane kubonakaliswa ngumbonakalisi, kudlula kwi-lens ye-condenser, kwaye kugxininiswe kumbindi womngxuma ngamnye.Emva koko i-fluorescence ephuma emngxunyeni ibonakala kwisibuko, idlula kwi-emission filter, kwaye ifunyenwe yikhamera yeCCD.Kumjikelo ngamnye we-PCR, ukukhanya kwe-fluorophore okuzithandayo kunokubonwa yiCCD.

Iguqula ukukhanya okufakiweyo kwidatha yedijithali.Le ndlela ibizwa ngokuba yi-PCR yexesha langempela, kwaye ivumela ukujonga ixesha lokwenyani inkqubela ye-PCR reaction.