COVID-19 ndi matenda opatsirana omwe amayamba chifukwa cha Severe Acute Respiratory Syndrome Coronavirus Type 2. Munthu akadwala, zizindikiro zofala kwambiri ndi kutentha thupi, chifuwa, komanso kupuma movutikira.

Zitsanzo zomwe zimagwiritsidwa ntchito poyesa zimatha kusonkhanitsidwa ndi nasopharyngeal swabs kapena oropharyngeal swabs.

Kodi PCR ndi chiyani?

Njira yodziwika bwino yodziwira coronavirus ndi polymerase chain reaction, PCR.Iyi ndi njira yomwe imagwiritsidwa ntchito kwambiri mu biology ya molekyulu.Imatha kukopera mwachangu mamiliyoni mpaka mabiliyoni a tizidutswa tapadera ta DNA.

Coronavirus yatsopanoyo ili ndi mtundu umodzi wautali kwambiri wa RNA.Kuti azindikire ma virus ndi PCR, mamolekyu a RNA ayenera kusinthidwa kukhala ma DNA awo otsatizana ndi reverse transcriptase, ndiyeno DNA yongopangidwa kumene imatha kukulitsidwa ndi njira za PCR, zomwe zimadziwika kuti RT-PCR.

RT-PCR ndondomeko

Kusintha kwa RNA

Kuti muchite izi, ma virus a RNA ayenera kuchotsedwa.Mitundu yosiyanasiyana ya zida zoyeretsera za RNA zitha kugwiritsidwa ntchito kupatukana kosavuta, mwachangu komanso kothandiza.

Kuti mutenge kachilombo ka RNA pogwiritsa ntchito zida zamalonda, choyamba yonjezerani chitsanzo ku chubu cha microcentrifuge ndikusakaniza ndi lysis buffer.Chotchinga ichi chimakhala chopindika kwambiri ndipo nthawi zambiri chimakhala ndi phenol ndi guanidine isothiocyanate.Kuphatikiza apo, ma RNase inhibitors nthawi zambiri amapezeka mu lysis buffer kuti atsimikizire kudzipatula kwa ma virus a RNA.

Pambuyo powonjezera chotchinga cha lysis, tembenuzani chubu chosakaniza ndi kugunda ndi kuumitsa kutentha.Kachilombo kameneka kamayikidwa pansi pazikhalidwe zotsika kwambiri zoperekedwa ndi lysis buffer.

Pambuyo poti lysed, chubu cha centrifuge chimagwiritsidwa ntchito poyeretsa.Chitsanzocho chimayikidwa mu chubu cha centrifuge ndiyeno centrifuged.

Njirayi ndi njira yolimba yochotsa gawo lomwe gawo loyima limapangidwa ndi matrix a gel osakaniza.

Pansi pa mchere wabwino kwambiri ndi pH, mamolekyu a RNA amamangiriza ku nembanemba ya silika.

Panthawi imodzimodziyo, mapuloteni ndi zonyansa zina zimachotsedwa.

Pambuyo pa centrifugation, ikani chubu cha centrifuge mu chubu chosonkhanitsira choyera, chotsani kusefera, kenaka yikani bafa yotsuka.

Ikani chubu mu centrifuge kachiwiri kukakamiza kutsuka chotchinga kudzera mu nembanemba.Izi zidzachotsa zonyansa zonse zomwe zatsala ku nembanemba, ndikusiya RNA yokha yomangidwa ku gel osakaniza.

Zitsanzo zikatsukidwa, ikani chubu mu chubu choyera cha microcentrifuge ndikuwonjezera chotchinga cha elution.

Kenako imayikidwa pakati kuti ikakamize buffer ya elution kudzera mu nembanemba.Buffer ya elution imachotsa ma virus a RNA kuchokera pamzere wozungulira ndikupeza RNA yoyeretsedwa yopanda mapuloteni, zoletsa, ndi zonyansa zina.

CHOCHITA 2

Kuphatikizika kosakanikirana

Pambuyo pochotsa kachilombo ka RNA, chotsatira ndicho kukonzekera kusakaniza kwa PCR.Mu sitepe iyi, kuika maganizo kumagwiritsidwa ntchito.Yankho loyikirali ndi yankho losakanikirana lopangidwa ndi premix, reverse transcriptase, nucleotides, forward primer, reverse primer, TaqMan probe ndi DNA polymerase.

Pomaliza, kuti mumalize kusakaniza kumeneku, template ya RNA imawonjezedwa.Machubu amasakanizidwa ndi pulse vortexing, ndiyeno kusakaniza kwake kumayikidwa mu mbale ya PCR.Mbale ya PCR nthawi zambiri imakhala ndi zitsime 96 ndipo imatha kusanthula zitsanzo zingapo nthawi imodzi.

CHOCHITA 3

Kuchulukitsa kwa PCR

Kenako, ikani mbaleyo pamakina a PCR, omwe kwenikweni amakhala oyendetsa matenthedwe.

Real-time RT-PCR imagwiritsidwa ntchito kuzindikira coronavirus yatsopano ya 2019 pokulitsa zomwe mukufuna mu jini ya RdrRP, E gene ndi N jini.Kusankhidwa kwa jini yomwe mukufuna kutsata kumadalira pamayendedwe oyambira ndi kafukufuku.

Gawo loyamba la RT-PCR ndikulemba mosintha.Chingwe choyamba cha DNA chothandizira chimapangidwa, chomwe chimayambitsidwa ndi PCR reverse primer, yomwe imamangiriza ku gawo lothandizira la ma virus a RNA genome.Kenako reverse transcriptase imawonjezera ma DNA nucleotides ku 3′mapeto a primer kuti apange DNA yothandizirana ndi ma virus a RNA.Kutentha ndi kutalika kwa sitepeyi kumadalira zoyambira, chandamale cha RNA, ndi reverse transcriptase yomwe imagwiritsidwa ntchito.

Kenako, sitepe yoyamba yosinthira imayikidwa, zomwe zimapangitsa kuti RNA-DNA hybrid isinthe.Izi ndizofunikira kuti muyambitse DNA polymerase.Nthawi yomweyo, reverse transcriptase imachotsedwa.

PCR imakhala ndi matenthedwe angapo.Kuzungulira kulikonse kumakhala ndi ma denaturation, annealing ndi masitepe owonjezera.

Dongosolo la denaturation limaphatikizapo kutentha chipinda chochitirapo kanthu mpaka madigiri 95 Celsius ndikuchigwiritsa ntchito kuwonetsa template ya DNA yamitundu iwiri.

Mu sitepe yotsatira, kutentha kumachepetsedwa kufika pa 58 digiri Celsius, kulola kuti choyambira kutsogolo kuti chigwirizane ndi gawo lothandizira la template ya DNA ya chingwe chimodzi.Kutentha kwa annealing mwachindunji kumadalira kutalika ndi mawonekedwe a primer.

Powonjezerapo, DNA polymerase imapanga chingwe chatsopano cha DNA chomwe chimagwirizana ndi DNA template strand.Powonjezera ma nuclei aulere owonjezera ku template mu 5'to 3'direction kuchokera kusakaniza kosakaniza.Kutentha kwa sitepeyi kumadalira DNA polymerase yomwe imagwiritsidwa ntchito.

Pambuyo pa kuzungulira koyamba, chandamale cha DNA yazingwe ziwiri chimapezedwa.

Kenako, lowetsani mkombero wachiwiri.DNA yamitundu iwiri imapangidwa kuti ipange mamolekyu awiri a DNA a chingwe chimodzi.

Mu sitepe yotsatira, kutentha kumatsitsidwa, zoyambira zimayikidwa pa template ya DNA yokhala ndi chingwe chimodzi, ndipo kafukufuku wa Taq-man amalowetsedwa ku gawo lothandizira la DNA.

Kufufuza kwa TaqMan kumakhala ndi fluorophore yolumikizidwa bwino ndi 5'mapeto a kafukufuku wa oligonucleotide.Ikasangalala ndi gwero la kuwala kwa woyendetsa, fluorophore imatulutsa fluorescence.Kuphatikiza apo, kafukufukuyu amapangidwa ndi chozimitsa pa 3′mapeto.Kuyandikira kwa jini ya mtolankhani ku quencher kumalepheretsa kuzindikira kwa fluorescence.

Mu sitepe yowonjezera, DNA polymerase imapanga chingwe chatsopano.Polymerase ikafika pa kafukufuku wa TaqMan, ntchito yake yokhazikika ya 5′nuclease imadula kafukufukuyo, kulekanitsa utoto ndi chozimitsira.

Pakuzungulira kulikonse kwa PCR, mamolekyu ambiri a utoto amatulutsidwa, zomwe zimapangitsa kuti mphamvu ya fluorescence ichuluke molingana ndi kuchuluka kwa amplicon opangidwa.

Njirayi imalola kuyerekezera chiwerengero cha mndandanda woperekedwa womwe ulipo mu chitsanzo.Chiwerengero cha tiziduswa ta DNA tokhala ndi zingwe ziwiri chimaŵirikiza kaŵiri m’njira iliyonse.Choncho, PCR ingagwiritsidwe ntchito kusanthula zitsanzo zazing'ono kwambiri.

Poyezera chizindikiro cha fulorosenti, nyali ya tungsten halogen, fyuluta yosangalatsa, chowonetsera, lens, fyuluta yotulutsa mpweya ndi chojambulira chophatikizana ndi kamera ya CCD.

CHOCHITA 4 Dziwani

Poyezera chizindikiro cha fulorosenti, nyali ya tungsten halogen, fyuluta yosangalatsa, chowonetsera, lens, fyuluta yotulutsa mpweya ndi chojambulira chophatikizana ndi kamera ya CCD.

Kuwala kosefedwa kuchokera ku nyali kumawonetsedwa ndi chowunikira, kumadutsa mu lens ya condenser, ndipo kumayang'ana pakati pa dzenje lililonse.Ndiye fluorescence yotuluka mu dzenje ikuwonekera kuchokera pagalasi, imadutsa mu fyuluta yotulutsa mpweya, ndipo imadziwika ndi kamera ya CCD.Pamkombero uliwonse wa PCR, kuwala kodzisangalatsa kwa fluorophore kumatha kuzindikirika ndi CCD.

Imasintha kuwala kojambulidwa kukhala deta ya digito.Njirayi imatchedwa PCR yeniyeni, ndipo imalola kuyang'anitsitsa nthawi yeniyeni ya momwe PCR ikuyendera.