COVID-19 bụ ọrịa na-efe efe nke ọrịa siri ike iku ume Coronavirus Ụdị 2 na-ebute. Mgbe mmadụ butere ọrịa, ihe mgbaàmà ndị a na-ahụkarị gụnyere ahụ ọkụ, ụkwara, na mkpụmkpụ ume.

Enwere ike ịnakọta ihe nlele ndị a na-eji nyocha site na swabs nasopharyngeal ma ọ bụ swabs oropharyngeal.

Kedu ihe bụ PCR?

Usoro ọkọlọtọ nke nchọpụta coronavirus bụ mmeghachi omume polymerase, PCR.Nke a bụ usoro a na-ejikarị eme ihe na bayoloji molekụla.Ọ nwere ike iṅomi nde mmadụ ngwa ngwa gaa na ijeri DNA ndị kpọmkwem.

Coronavirus ọhụrụ ahụ nwere genome RNA nwere ogologo ogologo.Iji chọpụta nje ndị a site na PCR, a ga-atụgharị mkpụrụ ndụ RNA ka ọ bụrụ usoro DNA nkwado ha site na ntụgharị transcriptase, mgbe ahụ enwere ike ịbawanye DNA ahụ emepụtara ọhụrụ site na usoro PCR ọkọlọtọ, nke a na-akpọkarị RT-PCR.

Usoro RT-PCR

mmịpụta RNA

Iji mee usoro a, ekwesịrị ịwepụta RNA viral.Enwere ike iji ngwa nchacha RNA dị iche iche maka nkewa dị mma, ngwa ngwa na dị irè.

Iji wepụta nje RNA site na iji ngwa azụmahịa, buru ụzọ tinye ihe nlele na tube microcentrifuge wee gwakọta ya na ihe nchekwa lysis.Ihe nchekwa a bụ nke ukwuu ma na-enwekarị phenol na guanidine isothiocyanate.Na mgbakwunye, ndị na-egbochi RNase na-anọkarị na ebe nchekwa lysis iji hụ na mwepu nke nje virus RNA adịghị.

Mgbe ị gbakwụnyechara ihe nchekwa lysis, vortex tube agwakọta site na usu wee tinye ya n'ime ụlọ okpomọkụ.A na-ehichakwa nje a n'okpuru ọnọdụ nbipu nke ukwuu nke ihe nchekwa lysis nyere.

Mgbe emechara ihe nlele ahụ, a na-eji tube centrifuge mee ihe maka usoro nchacha.A na-etinye ihe nlele ahụ n'ime tube centrifuge wee kechie ya.

Usoro a bụ usoro mmịpụta siri ike nke na-anọpụ iche nwere matriks silica gel.

N'okpuru ezigbo nnu na ọnọdụ pH, mkpụrụ ndụ RNA na-ejikọta na akpụkpọ anụ silica.

N'otu oge ahụ, a na-ewepụ protein na ihe ndị ọzọ na-emerụ ahụ.

Mgbe centrifugation gasịrị, tinye tube centrifuge n'ime tube nchịkọta dị ọcha, tụfuo nzacha ahụ, wee gbakwunye ebe nchekwa.

Tinye tube na centrifuge ọzọ iji manye ihe nkpuchi na-asa ahụ site na akpụkpọ ahụ.Nke a ga-ewepụ adịghị ọcha niile na akpụkpọ ahụ, na-ahapụ naanị RNA na-ejikọta na gel silica.

Mgbe a sachara ihe nlele ahụ, tinye tube n'ime tube microcentrifuge dị ọcha ma tinye ihe nchekwa elution.

Mgbe ahụ, a na-atụgharị ya ka ọ manye ihe nchekwa elution site na akpụkpọ ahụ.Ihe nchekwa elution na-ewepụ nje virus RNA na kọlụm ogho wee nweta RNA dị ọcha na-enweghị protein, inhibitors na ihe ndị ọzọ na-emerụ emerụ.

Nzọụkwụ 2

Ngwakọta agwakọta

Mgbe iwepuchara nje virus RNA, nzọụkwụ ọzọ bụ ịkwadebe ngwakọta mmeghachi omume maka mmụba PCR.N'ime usoro a, a na-eji itinye uche.Ihe ngwọta a gbanyere mkpọrọgwụ bụ ihe edoziri etinyere nke ọma nke nwere premix, reverse transcriptase, nucleotides, primer forward, reverse primer, TaqMan probe na DNA polymerase.

N'ikpeazụ, iji mechaa ngwakọta mmeghachi omume a, a na-agbakwunye template RNA.A na-agwakọta tubes ndị ahụ site na pulse vortexing, wee tinye ngwakọta mmeghachi omume n'ime efere PCR.Efere PCR na-enwekarị olulu mmiri 96 ma nwee ike nyochaa ọtụtụ ihe nlele n'otu oge.

NKWUKWU 3

PCR nkwalite

Ọzọ, tinye efere ahụ n'ime igwe PCR, nke bụ n'ezie igwe na-ekpo ọkụ.

A na-eji ezigbo oge RT-PCR chọpụta coronavirus akwụkwọ akụkọ 2019 site n'ịkwalite usoro ebumnuche na mkpụrụ ndụ RdrRP, E gene na N gene.Nhọrọ nke mkpụrụ ndụ ihe mgbaru ọsọ na-adabere na mbido na usoro nyocha.

Nzọụkwụ mbụ nke RT-PCR bụ ntụgharị ntụgharị.A na-emepụta eriri mbụ nke DNA nkwado, nke sitere na PCR reverse primer, nke na-ejikọta na akụkụ agbakwunyere nke RNA genome.Mgbe ahụ reverse transcriptase na-agbakwunye DNA nucleotides na 3′ngwụ nke primer iji mejupụta DNA na-akwado RNA viral.Ọnọdụ okpomọkụ na ogologo oge nke usoro a dabere na primers, RNA lekwasịrị anya, na ntụgharị transcriptase eji.

Na-esote, a na-etinye usoro denaturation nke mbụ, nke na-ebute denaturation nke RNA-DNA ngwakọ.Nzọụkwụ a dị mkpa iji mee ka DNA polymerase rụọ ọrụ.N'otu oge ahụ, reverse transcriptase adịghị arụ ọrụ.

PCR nwere usoro okirikiri okpomọkụ.okirikiri nke ọ bụla nwere denaturation, annealing na ndọtị nzọụkwụ.

Nzọụkwụ denaturation na-agụnye ikpo ọkụ ụlọ mmeghachi omume na 95 Celsius ma jiri ya maka denaturation nke DNA nwere eriri abụọ.

Na nzọụkwụ na-esote, a na-ebelata okpomọkụ mmeghachi omume na 58 degrees Celsius, na-ekwe ka primer na-aga n'ihu gbasaa na akụkụ nke mgbakwunye DNA nke nwere otu eriri.Igwe ọkụ na-ekpo ọkụ na-adabere ozugbo na ogologo na nhazi nke primer.

Na nzọụkwụ ndọtị, DNA polymerase na-emepụta eriri DNA ọhụrụ nke dabara na eriri template DNA.Site n'ịgbakwụnye nuclei free nkwado na template na 5′to 3′direction si mmeghachi omume ngwakọta.Okpomọkụ nke nzọụkwụ a dabere na DNA polymerase eji.

Mgbe okirikiri nke mbụ gasịrị, a na-enweta ebumnuche DNA nwere eriri abụọ.

Mgbe ahụ, tinye okirikiri nke abụọ.A napụrụ DNA nke nwere eriri abụọ ka ọ mepụta mkpụrụ ndụ DNA nwere otu eriri abụọ.

N'ọzọ na-esote, a na-ebelata okpomọkụ mmeghachi omume, a na-ewepụ ihe ndị na-emepụta ihe na ụdị DNA nke ọ bụla nwere otu eriri, na nyocha Taq-man na-agbanye n'akụkụ akụkụ nke DNA e lekwasịrị anya.

Ihe nyocha TaqMan nwere fluorophore jikọtara ya na njedebe 5 nke nyocha oligonucleotide.Mgbe ihe na-ekpo ọkụ nke onye na-agbagharị na-enwe obi ụtọ, fluorophore na-ewepụta fluorescence.Na mgbakwunye, nyocha ahụ bụ ihe na-emenyụ ọkụ na 3′nd mejupụtara.Ịdị nso nke mkpụrụ ndụ onye na-ede akụkọ na onye na-eme ihe na-egbochi nchoputa nke fluorescence.

Na nzọụkwụ ndọtị, DNA polymerase na-emepụta eriri ọhụrụ.Mgbe polymerase rutere nyocha TaqMan, ọrụ 5′nuclease ya na-agbajisị nyocha ahụ, na-ekewapụta agba na ihe na-emenyụ ọkụ.

Na okirikiri nke ọ bụla nke PCR, a na-ewepụta mkpụrụ ndụ ihe na-esiji agba karịa, na-ebute mmụba nke ike fluorescence na ọnụ ọgụgụ nke amplicon arụpụtara.

Usoro a na-enye ohere ịkọ ọnụ ọgụgụ nke usoro enyere dị na nlele ahụ.Ọnụ ọgụgụ nke iberibe DNA nwere eriri abụọ na-abawanye okpukpu abụọ na okirikiri nke ọ bụla.Ya mere, enwere ike iji PCR nyochaa obere nlele.

Maka ịlele mgbama fluorescent, tungsten halogen lamp, excitation filter, reflector, lens, emission filter na chaja jikọtara ngwaọrụ-eji igwefoto CCD.

Nzọụkwụ 4 Chọpụta

Maka ịlele mgbama fluorescent, tungsten halogen lamp, excitation filter, reflector, lens, emission filter na chaja jikọtara ngwaọrụ-eji igwefoto CCD.

A na-egosipụta ọkụ a na-ehichapụ site na oriọna ahụ site na onye na-atụgharị uche, na-agafe na oghere condenser, na-elekwasị anya na etiti oghere ọ bụla.Mgbe ahụ, fluorescence na-esi na oghere pụta na-egosipụta site na enyo, na-agafe na nzacha ikuku, igwefoto CCD na-ahụkwa ya.N'ime okirikiri PCR nke ọ bụla, CCD nwere ike ịhụ ọkụ fluorophore nke nwere obi ụtọ.

Ọ na-atụgharị ìhè ewepụtara ka ọ bụrụ data dijitalụ.A na-akpọ usoro a PCR ezigbo oge, ọ na-enye ohere nleba anya n'ezie maka ọganihu nke mmeghachi omume PCR.