Ko te COVID-19 he mate hopuhopu na te Severe Acute Respiratory Syndrome Coronavirus Type 2. Ki te pangia te tangata, ko nga tohu tino kitea ko te kirikaa, te maremare, me te poto o te manawa.

Ko nga tauira e whakamahia ana mo te whakamatautau ka taea te kohia e te nasopharyngeal swabs or oropharyngeal swabs.

He aha te PCR?

Ko te tikanga paerewa mo te kitenga coronavirus ko te tauhohenga mekameka polymerase, PCR.He tikanga tenei e whakamahia nuitia ana i roto i te koiora ngota.Ka taea e ia te kape tere i nga miriona ki nga piriona o nga waahanga DNA motuhake.

Kei roto i te coronavirus hou he ira ira RNA tino roa.Hei kite i enei huaketo ma te PCR, me huri nga ngota ngota RNA ki o raatau raupapa DNA taapiri ma te whakamuri transcriptase, katahi ka taea te whakakaha ake i te DNA katahi ano ka whakahiatohia e nga tikanga PCR paerewa, e mohiotia ana ko RT-PCR.

Te tukanga RT-PCR

tangohanga RNA

Hei mahi i tenei tikanga, me tango te RNA viral.Ka taea te whakamahi i nga momo kete purenga RNA mo te wehenga watea, tere me te whai hua.

Ki te tango i te RNA viral ma te whakamahi i te kete arumoni, me tapiri tuatahi te tauira ki te ngongo microcentrifuge ka uru ki te lysis buffer.Ko tenei parepare he tino whakahekehia, a, ko te tikanga he phenol me te guanidine isothiocyanate.I tua atu, kei te nuinga o nga wa kei roto i te papaaru lysis te aukati RNase hei whakarite i te wehenga o te RNA viral mau tonu.

Whai muri i te taapiri i te lysis buffer, whakapourihia te ngongo whakaranu ma te pupuhi me te whakamomi ki te pāmahana rūma.Ka lysedhia te huaketo i raro i nga tikanga tino whakaheke e whakaratohia ana e te buffer lysis.

I muri i te reititanga o te tauira, ka whakamahia he ngongo centrifuge mo te tikanga purenga.Ka utaina te tauira ki roto i te ngongo centrifuge, katahi ka centrifuge.

Ko tenei tikanga he tikanga tangohanga wahanga totoka kei roto i te wahanga noho he matrix gel silica.

I raro i nga tikanga tote me te pH tino pai, ka herea nga ngota ngota RNA ki te kiriuhi silica.

I te wa ano, ka tangohia te pūmua me etahi atu mea poke.

I muri i te centrifugation, kuhua te ngongo centrifuge ki roto i te ngongo kohinga pokekore, makahia te filtrate, katahi ka taapirihia he parepare horoi.

Whakanohia ano te ngongo ki roto i te centrifuge hei tohe i te parepare horoi ki roto i te kiriuhi.Ma tenei ka tango i nga toenga poke katoa mai i te kiriuhi, ka waiho ko te RNA anake e herea ana ki te kirikiri silica.

I muri i te horoinga o te tauira, kuhua te ngongo ki roto i te ngongo microcentrifuge ma, ka tapirihia te parepare elution.

Kātahi ka whakahiatohia ki te whakakaha i te parepare elution i roto i te kiriuhi.Ka tangohia e te kaitao elution te RNA viral mai i te pou hurihuri ka whiwhi i te RNA kua purea kaore he pūmua, aukati, me etahi atu parakino.

KAUPAPA 2

Te whakakotahitanga

Whai muri i te tangohanga o te RNA viral, ko te mahi e whai ake nei ko te whakarite i te ranunga tauhohenga mo te whakakaha PCR.I tenei taahiraa, ka whakamahia te aro.Ko tenei otinga whakakoi he otinga tohanga kua whakauruhia he premix, whakamuri transcriptase, nucleotides, mua whakamua, whakataa whakamuri, TaqMan probe me te DNA polymerase.

Ka mutu, hei whakaoti i tenei ranunga tauhohenga, ka taapirihia te tauira RNA.Ka konatunatuahia nga ngongo ma te pupuhi vortexing, katahi ka utaina te ranunga tauhohenga ki te pereti PCR.I te nuinga o te wa kei roto i te pereti PCR nga puna 96 ka taea te tarai i nga tauira maha i te wa kotahi.

KAUPAPA 3

Whakanui PCR

Muri iho, tuu te pereti ki roto i te miihini PCR, ko te tikanga he kaihuri hiko waiariki.

Ka whakamahia te RT-PCR i te wa-tuuturu ki te kite i te coronavirus hou 2019 ma te whakanui i te raupapa whainga i roto i te ira RdrRP, ira E me te ira N.Ko te kowhiringa o te ira whainga ka whakawhirinaki ki te timatanga me te raupapa rangahau.

Ko te mahi tuatahi o te RT-PCR ko te whakamaoritanga whakamuri.Ka whakahiatotia te whenu tuatahi o te DNA taapiri, i timatahia e te PCR whakamuri tuatahi, e herea ana ki te waahanga taapiri o te ira momo RNA viral.Na ka taapirihia e te transcriptase whakamuri nga nucleotides DNA ki te pito 3′ o te tuatahi ki te whakahiato i te DNA hei taapiri ki te RNA viral.Ko te pāmahana me te roa o tenei taahiraa e whakawhirinaki ana ki nga timatanga, ki te RNA whainga, me te whakamuri transcriptase i whakamahia.

I muri mai, ka tukuna he taahitanga denaturation tuatahi, ka puta ko te denaturation o te ranu RNA-DNA.He mea tika tenei taahiraa hei whakahohe i te DNA polymerase.I te wa ano, ka whakakorehia te transcriptase whakamuri.

Ko te PCR he raupapa o nga huringa waiariki.Kei ia huringa ko te denaturation, annealing me te toronga.

Ko te taahiraa denaturation ko te whakamahana i te ruma tauhohenga ki te 95 nga nekehanga Celsius me te whakamahi mo te denaturation o te tauira DNA whenu-rua.

I te taahiraa e whai ake nei, ka whakahekehia te pāmahana tauhohenga ki te 58 nga nekehanga Celsius, ka taea e te tuatahi o mua te whakauru ki te waahanga taapiri o tana tauira DNA whenu kotahi.Ko te pāmahana whakahiato ka whakawhirinaki ki te roa me te hanganga o te tuatahi.

I te taahiraa toronga, ka whakahiatotia e te polymerase DNA he whenu DNA hou e hono ana ki te whenu tauira DNA.Ma te taapiri i nga karihi kore utu ki te tauira i te ahunga 5′ki te 3′ mai i te ranunga tauhohenga.Ko te pāmahana o tenei taahiraa ka whakawhirinaki ki te DNA polymerase i whakamahia.

I muri i te huringa tuatahi, ka whiwhihia he whaainga DNA paerua.

Na, tomo te huringa tuarua.Ko te DNA whenu-rua ka whakahekehia hei whakaputa i nga ngota ngota DNA e rua.

I te taahiraa e whai ake nei, ka whakahekehia te paanga o te tauhohenga, ka whakahiatohia nga mea tuatahi ki ia tauira DNA whenu-tahi, ka honoa te rangahau Taq-man ki te waahanga taapiri o te DNA whaainga.

Ko te rangahau TaqMan he fluorophore e hono ana ki te pito 5′ o te rangahau oligonucleotide.Ina hikaka ana i te puna marama o te kaihurihuri, ka puta te rewharewha.I tua atu, ko te rangahau he mea tinei i te pito 3′.Ko te tata o te ira kairipoata ki te tineia ka aukati i te kitenga o te rewharewha.

I te taahiraa toronga, ka whakahiato te DNA polymerase i tetahi whenu hou.Ka tae atu te polymerase ki te tirotiro TaqMan, ko tana ngohe 5′nuclease endogenous ka mawehe i te rangahau, ka wehe i te waikano mai i te tiini.

I ia huringa o te PCR, ka nui ake nga ngota ngota waikano ka tukuna, ka piki ake te kaha o te korikori e rite ana ki te maha o nga amplicons kua whakahiatotia.

Ka taea e tenei tikanga te whakatau tata i te maha o te raupapa kua tohua kei roto i te tauira.Ka ruarua te maha o nga whatiwhatinga DNA whenu-rua i ia huringa.Na reira, ka taea te whakamahi PCR ki te tātari i nga tauira iti rawa.

Mo te ine i te tohu rewharewha, rama halogen tungsten, tātari hihiko, whakaataata, arotahi, tātari tuku me te utu honoa te taputapu-whakamahi kamera CCD.

STEP 4 Rapua

Mo te ine i te tohu rewharewha, rama halogen tungsten, tātari hihiko, whakaataata, arotahi, tātari tuku me te utu honoa te taputapu-whakamahi kamera CCD.

Ko te rama kua tohua mai i te rama ka whakaatahia e te whakaata, ka haere i roto i te arotahi condenser, ka aro ki te pokapū o ia kohao.Katahi ka puta mai i te kohao ka puta mai i te whakaata, ka haere i roto i te tātari tuku, ka kitea e te kamera CCD.I ia huringa PCR, ka kitea e te CCD te rama fluorophore hihiko-whaiaro.

Ka huri i te rama kua mau ki te raraunga matihiko.Ko tenei tikanga e kiia ana ko te PCR-a-waa, a ka taea e ia te aroturuki i te wa-a-roa mo te ahunga whakamua o te tauhohenga PCR.