COVID-19 ke lefu le tšoaetsanoang le bakoang ke Severe Acute Respiratory Syndrome Coronavirus Type 2. Ha motho a tšoaelitsoe, matšoao a tloaelehileng a kenyelletsa feberu, ho khohlela, le ho hema ka thata.

Mehlala e sebelisetsoang tlhahlobo e ka bokelloa ka li-swabs tsa nasopharyngeal kapa li-oropharyngeal swabs.

PCR ke eng?

Mokhoa o tloaelehileng oa ho lemoha coronavirus ke polymerase chain reaction, PCR.Ona ke mokhoa o sebelisoang haholo ho baeloji ea limolek'hule.E ka khona ho kopitsa ka potlako likotoana tse itseng tsa DNA tse limilione ho libilione.

Coronavirus e ncha e na le genome e telele haholo ea RNA e nang le likhoele tse le 'ngoe.E le ho lemoha livaerase tsena ka PCR, limolek'hule tsa RNA li tlameha ho fetoloa tatellano ea tsona ea DNA ka reverse transcriptase, ebe DNA e sa tsoa etsoa e ka holisoa ka mekhoa e tloaelehileng ea PCR, eo hangata e tsejoang e le RT-PCR.

Mokhoa oa RT-PCR

Tlhahiso ea RNA

Ho etsa mokhoa ona, vaerase ea RNA e lokela ho ntšoa.Mefuta e fapaneng ea lisebelisoa tsa tlhoekiso ea RNA e ka sebelisoa bakeng sa karohano e bonolo, e potlakileng le e sebetsang.

Ho ntša RNA ea kokoana-hloko ka ho sebelisa lisebelisoa tsa khoebo, qala ka ho eketsa sampole ho microcentrifuge tube ebe u e kopanya le lysis buffer.Buffer ena e na le denatured haholo 'me hangata e na le phenol le guanidine isothiocyanate.Ho feta moo, li-inhibitors tsa RNase hangata li teng ka har'a lysis buffer ho netefatsa ho itšehla thajana ha RNA e sa fetoheng ea vaerase.

Ka mor'a hore u kenye lysis buffer, vortex mixing tube by pulse and incubate incubate the roomine.Kokoana-hloko e ntan'o lysed tlas'a maemo a thata haholo a fanoeng ke lysis buffer.

Ka mor'a hore sampuli e sebelisoe, ho sebelisoa tube ea centrifuge bakeng sa mokhoa oa ho hloekisa.Mohlala o kenngoa ka har'a tube ea centrifuge ebe e kenngoa ka centrifuge.

Mokhoa ona ke mokhoa o tiileng oa ho ntša mokhahlelo oo ho oona karolo e emeng e nang le matrix a silica gel.

Tlas'a maemo a nepahetseng a letsoai le pH, limolek'hule tsa RNA li tlama lera la silika.

Ka nako e ts'oanang, liprotheine le litšila tse ling li tlosoa.

Ka mor'a centrifugation, kenya tube ea centrifuge ka har'a tube e hloekileng ea pokello, lahla filtrate, ebe u eketsa buffer ea ho hlatsoa.

Beha tube ka har'a centrifuge hape ho qobella "buffer" ea ho hlatsoa ka lera.Sena se tla tlosa litšila tsohle tse setseng ho lera, ho siea RNA feela e tlameletsoe ho gel ea silica.

Ka mor'a hore sampole e hlatsuoe, kenya tube ka har'a tube e hloekileng ea microcentrifuge 'me u kenye buffer ea elution.

E ntan'o kenngoa bohareng ho qobella "lution buffer" ka har'a lera.The elution buffer e tlosa RNA ea kokoana-hloko ho tloha mokokotlong mme e fumana RNA e hloekisitsoeng e se nang liprotheine, li-inhibitors, le litšila tse ling.

MOHATO 2

Motsoako oa ho tsepamisa maikutlo

Kamora ho ntša RNA ea kokoana-hloko, mohato o latelang ke ho lokisa motsoako oa karabelo bakeng sa ho holisa PCR.Mothating ona, ho sebelisoa concentrate.Tharollo ena e tsepamisitsoeng ke tharollo e kopantsoeng ea premixed e nang le premix, reverse transcriptase, nucleotides, foremer primer, reverse primer, TaqMan probe le DNA polymerase.

Qetellong, ho phethela motsoako ona oa karabelo, template ea RNA e eketsoa.Li-tubes li kopantsoe ke pulse vortexing, ebe motsoako oa karabelo o kenngoa ka har'a poleiti ea PCR.PCR poleiti hangata e na le liliba tse 96 mme e ka sekaseka lisampole tse ngata ka nako e le 'ngoe.

MOHATO 3

PCR e ntlafatsa

Ka mor'a moo, beha poleiti mochineng oa PCR, oo ha e le hantle e leng sebaesekele se futhumatsang.

RT-PCR ea nako ea 'nete e sebelisetsoa ho bona "coronavirus" ea 2019 ka ho holisa tatellano ea sepheo ho mofuta oa RdrRP, mofuta oa E le mofuta oa N.Khetho ea liphatsa tsa lefutso e itšetlehile ka tatellano ea li-primer le probe.

Mohato oa pele oa RT-PCR ke ho fetolela ka morao.Karolo ea pele ea DNA e tlatsetsang e entsoe, e qalisoa ke PCR reverse primer, e kopanyang karolo e tlatsetsang ea kokoana ea RNA genome.Ebe reverse transcriptase e eketsa li-nucleotide tsa DNA ho 3′end ea primer ho kopanya DNA e tlatsetsang ho RNA ea kokoana-hloko.Thempereichara le nako ea mohato ona li itšetlehile ka li-primers, sepheo sa RNA, le reverse transcriptase e sebelisitsoeng.

Ka mor'a moo, ho sebelisoa mohato oa pele oa denaturation, o fellang ka hore lebasetere la RNA-DNA le fetohe.Mohato ona oa hlokahala ho kenya tšebetsong DNA polymerase.Ka nako e ts'oanang, reverse transcriptase ha e sebetse.

PCR e na le letoto la lipotoloho tsa mocheso.Potoloho e 'ngoe le e 'ngoe e na le mehato ea denaturation, annealing le katoloso.

Bohato ba denaturation bo kenyelletsa ho futhumatsa kamore ea karabelo ho likhato tse 95 tsa Celsius le ho e sebelisa ho hlahisa template ea DNA e nang le likhoele tse peli.

Mohatong o latelang, mocheso oa karabelo o fokotsehile ho likhato tse 58 tsa Celsius, e leng se lumellang hore primer e fetelletseng e kenelle karolong e tlatselletsang ea thempleite ea eona ea DNA e nang le khoele e le 'ngoe.Thempereichara ea annealing ka ho toba e itšetlehile ka bolelele le sebopeho sa primer.

Mohatong oa katoloso, DNA polymerase e kopanya DNA strand e ncha e tlatselletsang DNA template strand.Ka ho kenyelletsa li-nuclei tsa mahala tse tlatselletsang template ho 5'to 3'direction ho tloha motsoako oa karabelo.Mocheso oa mohato ona o itšetlehile ka DNA polymerase e sebelisitsoeng.

Ka mor'a potoloho ea pele, sepheo sa DNA se nang le likarolo tse peli se fumanoa.

Ebe, kenya potoloho ea bobeli.DNA e meloho e 'meli e fetotsoe ho hlahisa limolek'hule tse peli tsa DNA tse nang le khoele e le 'ngoe.

Mohato o latelang, mocheso oa karabelo o theoleloa, li-primers li kenngoa ho template e 'ngoe le e' ngoe ea DNA e nang le mefuta e le 'ngoe,' me lipatlisiso tsa Taq-man li kenngoa karolong e tlatsetsang ea DNA e lebisitsoeng.

Tlhahlobo ea TaqMan e na le fluorophore e hokahaneng hantle le pheletsong ea 5 ea oligonucleotide.Ha e thabisoa ke mohloli o khanyang oa cycler, fluorophore e ntša fluorescence.Ho phaella moo, probe e entsoe ka quencher qetellong ea 3′.Ho ba haufi le moqolotsi oa liphatsa tsa lefutso ho quencher ho thibela ho fumanoa ha fluorescence.

Mohatong oa katoloso, DNA polymerase e kopanya khoele e ncha.Ha polymerase e fihla phuputsong ea TaqMan, ts'ebetso ea eona ea 5′nuclease ea endo native e phunya probe, e arola dae ho quencher.

Ka potoloho e 'ngoe le e' ngoe ea PCR, ho lokolloa limolek'hule tse ngata tsa dae, e leng se bakang keketseho ea matla a fluorescence ho lekana le palo ea li-amplicon tse entsoeng.

Mokhoa ona o lumella ho hakanya palo ea tatellano e fanoeng e teng sampoleng.Palo ea likhechana tsa DNA tse nang le mela e 'meli e imena habeli potolohong ka 'ngoe.Ka hona, PCR e ka sebelisoa ho sekaseka lisampole tse nyane haholo.

Bakeng sa ho metha lets'oao la fluorescent, lebone la tungsten halogen, filthara ea ho hlasimolla, sebonela-sebonela, lense, kh'amera ea emission le kh'amera ea CCD e sebelisanang le lisebelisoa.

MOHATO 4 Fumana

Bakeng sa ho metha lets'oao la fluorescent, lebone la tungsten halogen, filthara ea ho hlasimolla, sebonela-sebonela, lense, kh'amera ea emission le kh'amera ea CCD e sebelisanang le lisebelisoa.

Leseli le tlhotliloeng le tsoang leboneng le bonahatsoa ke seipone, le feta ka lense ea condenser, 'me le lebisitsoe bohareng ba lesoba le leng le le leng.Joale fluorescence e hlahisoang ka sekoting e bontšoa ho tloha seiponeng, e feta ka har'a sefahla sa emission, 'me e fumanoa ke khamera ea CCD.Ka potoloho e 'ngoe le e' ngoe ea PCR, khanya ea fluorophore e ithabisang e ka bonoa ke CCD.

E fetolela leseli le hapiloeng hore e be data ea dijithale.Mokhoa ona o bitsoa PCR ea nako ea sebele, 'me o lumella ho shebella ka nako ea sebele tsoelo-pele ea karabelo ea PCR.