A farkon matakin barkewar cutar, saboda saurin ci gaba, saurin gano marasa lafiya da ake zargi shine mabuɗin hana COVID-19.Wasu da aka amince da sake gano abubuwan gano acid nucleic suna da ɗan gajeren lokacin haɓakawa, kuma akwai matsaloli kamar tabbatar da aikin gaggawa, rashin isassun haɓakawa, da manyan bambance-bambance tsakanin batches;Matsalolin dakunan gwaje-gwaje na asibiti daban-daban a fannoni daban-daban na tsarin gano acid nucleic na iya shafar daidaiton sakamakon gano acid nucleic.Wannan labarin zai mayar da hankali kan mahimman hanyoyin haɗin gwiwa da maki a cikin ganowar SARS-CoV-2 na nucleic acid na yanzu, da kuma yin nazarin matsalolin ƙarya mara kyau da tabbataccen sake bincikar binciken binciken nucleic acid da rashin daidaituwa na asibiti.

Ka'idodin gano SARS-CoV-2 nucleic acid

SARS-CoV-2 kwayar cuta ce ta RNA tare da jerin kwayoyin halitta kusan 29kb, tare da kwayoyin halitta 10, wanda zai iya shigar da sunadaran 10 daidai.Kwayoyin cuta sun ƙunshi RNA da furotin, kuma mafi girman Layer shine rufin waje wanda ya ƙunshi lipids da glycoproteins.A ciki, capsid sunadaran suna nannade RNA a cikinsa, don haka yana kare RNA mai saurin lalacewa (P1).

P1 Tsarin SARS-COV-2

Kwayoyin cuta suna mamaye sel ta hanyar takamaiman masu karɓar saman sel don haifar da kamuwa da cuta, kuma suna amfani da ƙwayoyin cuta don yin kwafi.

Ka'idar gano kwayar cutar nucleic acid shine a fallasa kwayar cutar ta RNA ta hanyar kwayar halitta, sannan a yi amfani da ainihin-lokacin fluorescent reverse transcription-polymerase chain reaction (RT-PCR) don ganowa.

Makullin ka'idar ganowa shine a yi amfani da firamare da bincike don cimma "madaidaicin ma'amala" na jerin nucleic acid, wato, nemo jerin nucleic acid na SARS-CoV-2 wanda ya bambanta da sauran ƙwayoyin cuta a cikin kusan sansanonin 30,000 (kwatankwacin nucleic acid zuwa sauran ƙwayoyin cuta) “Low” yanki), masu zanen kaya da masu haɓakawa.

Abubuwan farko da bincike sun dace sosai tare da takamaiman yanki na SARS-CoV-2 nucleic acid, wato, ƙayyadaddun yana da ƙarfi sosai.Da zarar ainihin-lokaci mai kyalli RT-PCR sakamakon haɓaka samfurin da za a gwada ya tabbata, yana tabbatar da cewa SARS-CoV-2 yana cikin samfurin.Duba P2.

Matakan P2 na SARS-CoV-2 ƙaddarar acid nucleic (ainihin mai kyalli RT-PCR)

Yanayi da buƙatun dakin gwaje-gwaje don gano SARS-CoV-2 nucleic acid

Dakunan gwaje-gwaje na gwajin Nucleic acid sune mafi dacewa don yanayin matsa lamba mara kyau, kuma yakamata su mai da hankali kan sanya ido kan matsa lamba, ci gaba da kwarara iska, da kawar da iska.Dole ne ma'aikatan gwajin nucleic acid su sami cancantar dacewa, su sami horon sarkar polymerase mai dacewa kuma su wuce kima.Yakamata a sarrafa dakin gwaje-gwaje sosai, a sanya shi a wuri, kuma an hana ma'aikatan da ba su da mahimmanci shiga.Wurin mai tsabta ya kamata a shaka kuma a shafe shi a wurin.Ana sanya abubuwan da suka dace a cikin yankuna, ana raba tsabta da ƙazanta, ana maye gurbinsu akan lokaci, kuma an lalata su a wuri.Kashewa na yau da kullun: Maganin mai ɗauke da chlorine shine babban mafita ga manyan wurare, kuma ana iya amfani da barasa 75% don ƙananan wurare.Kyakkyawan hanyar magance iska ita ce buɗe tagogi don samun iska, kuma ana iya yin maganin kashe iska ta hanyar hasken ultraviolet, tacewa, da kashe iska.

Maɓallin hanyoyin haɗin kai da sigogi na ƙaddarar SARS-CoV-2 nucleic acid (ainihin mai kyalli RT-PCR)

Kodayake dakunan gwaje-gwaje gabaɗaya suna ba da kulawa sosai ga “ganowa” acid nucleic, a zahiri, “hadin” nucleic acid shima ɗaya ne daga cikin mahimman matakai don samun nasarar ganowa, wanda ke da alaƙa da tattarawa da adana samfuran ƙwayoyin cuta.

A halin yanzu, mafi yawan amfani da samfurori na numfashi, irin su nasopharyngeal swabs, suna amfani da hanya ta biyu, wanda shine maganin rashin kunnawa (tsara) wanda aka shirya bisa ga hakar nucleic acid da kuma maganin lysis.A gefe guda, wannan maganin adana ƙwayoyin cuta na iya cire furotin na ƙwayoyin cuta, ya rasa ayyukansa kuma ya daina kamuwa da cuta, kuma yana inganta amincin hanyoyin sufuri da ganowa;a daya bangaren kuma, tana iya fashe kwayar cutar kai tsaye don sakin sinadarin nucleic acid, ta kawar da sinadarin nucleic acid da ke rubewa, da kuma hana kwayar cutar.RNA ta lalace.

Maganin samfurin ƙwayar cuta wanda aka shirya akan tushen nucleic acid cirewar lysis bayani.Babban abubuwan da aka gyara sune salts ma'auni, ethylenediaminetetraacetic acid chelating agent, guanidine gishiri (guanidine isothiocyanate, guanidine hydrochloride, da dai sauransu), anionic surfactant (dodecane) Sodium sulfate), cationic surfactant (tetradecyl trimethyl ammonium oxalate), phenol, 8-hydrochloride ko protein more.A halin yanzu, akwai nau'ikan kayan cirewar acid nucleic da yawa, kuma ana amfani da su daban-daban na hakar acid da tsarkakewa.Ko da an yi amfani da hakar acid ɗin nucleic iri ɗaya da reagent na tsarkakewa, hanyoyin cirewar kowane kit ɗin sun bambanta.

A halin yanzu, samfuran kayan gano acid nucleic acid da Hukumar Kula da Kayayyakin Kiwon Lafiya ta Ƙasa ta amince da ita an zaɓi su bisa ga ORF1ab, E da N a cikin kwayar halittar SARS-CoV-2.Ka'idodin gano samfuran daban-daban iri ɗaya ne, amma ƙirarsu da ƙirar bincike sun bambanta.Akwai nau'ikan manufa guda ɗaya (ORF1ab), ɓangarori biyu masu manufa (ORF1ab, N ko E), da ɓangarori masu manufa uku (ORF1ab, N da E).Bambanci tsakanin ganowa da fassarar, cirewar acid nucleic da tsarin amsawar RT-PCR na gaske yakamata ya koma ga umarnin kit masu dacewa, kuma ana ba da shawarar cewa masu amfani su bi hanyar fassarar da aka kayyade a cikin umarnin kit don fassarar.Yankunan gama-gari, filaye da jerin bincike waɗanda aka haɓaka ta ainihin mai kyalli RT-PCR ana nunawa a P3.

P3 Wurin da SARS-CoV-2 amplicon ya yi niyya akan kwayoyin halitta da jerin abubuwan farko da bincike.

Fassarar sakamakon SARS-CoV-2 ƙaddarar nucleic acid (Ral-TMai kyalli RT-PCR)

"Shirin Rigakafin Ciwon huhu da Kula da Cutar Cutar SARS-CoV-2 (Bugu na Biyu)" a karon farko ya fayyace ma'auni don tantance sakamakon haɓakar ƙwayoyin cuta guda ɗaya:

1. Babu Ct ko Ct≥40 mara kyau;

2. Ct <37 yana da kyau;

3. Ƙimar Ct na 37-40 shine yanki mai launin toka.Ana ba da shawarar maimaita gwajin.Idan sakamakon sake yin Ct <40 da haɓakar haɓakawa yana da kololuwa a bayyane, ana yin la'akari da samfurin a matsayin tabbatacce, in ba haka ba yana da kyau.

Bugu na uku na jagora da bugu na huɗu na jagora sun ci gaba da ka'idodin da ke sama.Koyaya, saboda maƙasudai daban-daban da aka yi amfani da su a cikin kayan kasuwanci, bugu na 3 da aka ambata na jagorar bai ba da ƙayyadaddun ƙayyadaddun abubuwan haɗin kai ba, yana mai da hankali kan umarnin da masana'anta suka bayar zai yi nasara.An fara daga bugu na biyar na jagororin, an fayyace maƙasudai guda biyu, musamman ma ƙayyadaddun hukunce-hukunce na manufa guda ɗaya da ke da wahalar yin hukunci.Wato, idan dakin gwaje-gwaje yana son tabbatar da cewa shari'ar tana da inganci don gano SARS-CoV-2 nucleic acid, ana buƙatar biyan waɗannan sharuɗɗan 1 cikin 2:

(1) Manufa biyu na SARS-CoV-2 (ORF1ab, N) a cikin samfurin iri ɗaya ana gwada su ta hanyar mai kyalli RT-PCR na ainihi.Idan manufa guda ɗaya ta tabbata, ana buƙatar sake yin samfuri da sake gwadawa.Idan sakamakon gwajin ya kasance Idan makasudin guda ɗaya har yanzu yana da inganci, ana yanke hukunci a matsayin tabbatacce.

(2) Samfuran guda biyu na RT-PCR mai kyalli na ainihi sun nuna maƙasudi guda ɗaya tabbatacce a lokaci guda ko samfuran nau'ikan nau'ikan nau'ikan nau'ikan guda biyu sun nuna sakamakon gwajin tabbatacce guda ɗaya, wanda za'a iya yanke hukunci a matsayin tabbatacce.Koyaya, jagororin sun kuma jaddada cewa mummunan sakamakon gwajin nucleic acid ba zai iya ware kamuwa da cutar SARS-CoV-2 ba.Abubuwan da za su iya haifar da ƙiyayyar ƙarya suna buƙatar cire su, ciki har da ƙarancin samfurin samfurin (samfurin numfashi daga oropharynx da sauran sassa), tarin samfurin da wuri ko kuma latti, Ba a adana samfurori ba, jigilar kaya, da sarrafa su daidai, kuma fasahar kanta tana da matsaloli (bambancin ƙwayar cuta, hana PCR), da dai sauransu.

Abubuwan da ke haifar da mummunan lahani a cikin gano SARS-CoV-2

Ma'anar "ƙarya mara kyau" a cikin gwajin nucleic acid wanda a halin yanzu ya damu, sau da yawa yana nufin "ƙarya mara kyau" wanda sakamakon gwajin nucleic acid bai dace da bayyanar asibiti ba, wato, alamun asibiti da sakamakon hoto ana zargin su da COVID-19, amma gwajin acid nucleic koyaushe yana "mara kyau" sau da yawa.Cibiyar dakin gwaje-gwaje na Clinical na Hukumar Lafiya ta Kasa ta yi bayanin gwajin “karya mara kyau” na SARS-CoV-2.

(1) Akwai takamaiman adadin ƙwayoyin cuta a cikin ƙwayoyin mai cutar.Bayanai na baya-bayan nan sun nuna cewa bayan da kwayar cutar ta kama jiki, kwayar cutar ta kan shiga makogwaro ta hanci da baki, sannan ta shiga cikin iska da buroshi, sannan ta kai ga alveoli.Mutumin da ya kamu da cutar zai fuskanci lokacin kamuwa da cuta, ƙananan alamun bayyanar cututtuka, sa'an nan kuma tsarin cututtuka masu tsanani, da matakai daban-daban na cutar.Kuma adadin kwayar cutar da ke cikin sassa daban-daban na jiki daban.

Dangane da nau'in nau'in kwayar cutar kwayar cutar kwayar cutar kwayar cuta, kwayoyin epithelial na alveolar (ƙananan numfashi na numfashi)> sel epithelial na iska (na sama na numfashi)> fibroblasts, ƙwayoyin endothelial, da macrophages, da dai sauransu;daga nau'in samfurin, ruwan lavage na alveolar (mafi kyawun kyau)>zurfin tari mai zurfi>nasopharyngeal swab>oropharyngeal swab>jini.Bugu da kari, ana iya gano kwayar cutar a cikin najasa.Duk da haka, la'akari da dacewa da aiki da kuma yarda da marasa lafiya, tsarin samfurin asibiti da aka saba amfani dashi shine oropharyngeal swab>nasopharyngeal swab> ruwa mai lavage bronchial (aiki mai rikitarwa) da zurfin sputum (yawanci bushe tari, da wuya a samu) .

Don haka, adadin ƙwayoyin cuta a cikin sel na oropharynx ko nasopharynx na wasu marasa lafiya ƙanƙanta ne ko kaɗan.Idan kawai ana ɗaukar samfuran oropharynx ko nasopharynx don gwaji, ba za a gano kwayar nucleic acid ba.

(2) Ba a tattara ƙwayoyin da ke ɗauke da ƙwayar cuta ba yayin tattara samfurin, ko ƙwayoyin nucleic acid ba a kiyaye su yadda ya kamata ba.

[① Wurin tarawa mara kyau, alal misali, lokacin tattara swabs na oropharyngeal, zurfin tarin bai isa ba, ba a tattara swabs na nasopharyngeal da aka tattara a cikin rami na hanci, da sauransu. Yawancin ƙwayoyin da aka tattara na iya zama ƙwayoyin cuta marasa ƙwayoyin cuta;

②Ana amfani da swabs na samfur ba daidai ba.Misali, ana ba da shawarar zaruruwan roba irin su PE fiber, fiber polyester da fiber polypropylene don kayan swab shugaban.Ana amfani da filaye na halitta irin su auduga a cikin aiki na ainihi (ƙarfin adsorption na furotin kuma ba sauƙin wankewa ba) Da kuma fiber nailan (ƙananan ruwa mara kyau, wanda ke haifar da ƙarancin samfurin samfurin);

③ Yin amfani da bututun ajiyar ƙwayoyin cuta ba daidai ba, kamar rashin amfani da bututun ajiya na polypropylene ko polyethylene filastik waɗanda ke da sauƙin ɗaukar acid nucleic (DNA/RNA), wanda ke haifar da raguwar ƙwayar nucleic acid a cikin maganin ajiya.A aikace, ana ba da shawarar yin amfani da filastik polyethylene-propylene polymer filastik da wasu kwantena filastik polypropylene na musamman don adana ƙwayoyin nucleic acid.]

[① Wurin tarawa mara kyau, alal misali, lokacin tattara swabs na oropharyngeal, zurfin tarin bai isa ba, ba a tattara swabs na nasopharyngeal da aka tattara a cikin rami na hanci, da sauransu. Yawancin ƙwayoyin da aka tattara na iya zama ƙwayoyin cuta marasa ƙwayoyin cuta;

②Ana amfani da swabs na samfur ba daidai ba.Misali, ana ba da shawarar zaruruwan roba irin su PE fiber, fiber polyester da fiber polypropylene don kayan swab shugaban.Ana amfani da filaye na halitta irin su auduga a cikin aiki na ainihi (ƙarfin adsorption na furotin kuma ba sauƙin wankewa ba) Da kuma fiber nailan (ƙananan ruwa mara kyau, wanda ke haifar da ƙarancin samfurin samfurin);

③ Yin amfani da bututun ajiyar ƙwayoyin cuta ba daidai ba, kamar rashin amfani da bututun ajiya na polypropylene ko polyethylene filastik waɗanda ke da sauƙin ɗaukar acid nucleic (DNA/RNA), wanda ke haifar da raguwar ƙwayar nucleic acid a cikin maganin ajiya.A aikace, ana ba da shawarar yin amfani da filastik polyethylene-propylene polymer filastik da wasu kwantena filastik polypropylene na musamman don adana ƙwayoyin nucleic acid.]

(4) Ba a daidaita aikin dakin gwaje-gwaje na asibiti ba.Samfurin sufuri da yanayin ajiya, daidaitaccen aiki na dakunan gwaje-gwaje na asibiti, fassarar sakamako da kula da inganci sune mahimman abubuwan don tabbatar da daidaito da amincin sakamakon gwaji.Dangane da sakamakon kimanta ingancin waje da Cibiyar Nazarin Clinical na Hukumar Lafiya ta Kasa ta gudanar a ranar 16-24 ga Maris, 2020, daga cikin dakunan gwaje-gwaje 844 da suka sami sakamako mai inganci, 701 (83.1%) sun cancanta, kuma 143 (16.9%) ba su samu ba.Cancanta, yanayin gwajin dakin gwaje-gwaje gabaɗaya yana da kyau, amma dakunan gwaje-gwaje daban-daban har yanzu suna da bambance-bambancen iyawar aikin ma'aikata, ikon fassarar maƙasudi guda ɗaya, da sarrafa inganci.

Yadda za a rage mummunan mummunan binciken SARS-CoV-2 nucleic acid?

Ya kamata a inganta rage abubuwan da ba su da kyau a cikin gano nucleic acid daga sassa huɗu na samar da abubuwan da ba su dace ba.

(1)Akwai takamaiman adadin ƙwayoyin cuta a cikin ƙwayoyin mai cutar.Yawan kwayar cutar a sassa daban-daban na jikin wadanda ake zargi da kamuwa da cutar zai bambanta a lokuta daban-daban.Idan babu pharynx, yana iya kasancewa a cikin ruwan lavage na bronchial ko najasa.Idan ana iya tattara nau'ikan samfurori da yawa a lokaci ɗaya ko kuma a matakai daban-daban na ci gaban cuta don gwaji, Zai taimaka wajen guje wa mummunan lahani.

(2) Ya kamata a tattara ƙwayoyin da ke ɗauke da ƙwayoyin cuta yayin tattara samfurin.Ana iya magance wannan matsala mai yawa ta hanyar ƙarfafa horar da masu tattara samfurin.

(3) Amintattun IVD reagents.Ta hanyar gudanar da bincike a kan gano aikin kimantawa na reagents a matakin kasa, da kuma tattauna matsalolin da ake ciki, za a iya inganta aikin ganowa na reagents kuma za a iya inganta ƙwarewar bincike.

(4) Daidaitaccen aiki na dakunan gwaje-gwaje na asibiti.Ta hanyar ƙarfafa horar da ma'aikatan dakin gwaje-gwaje, ci gaba da inganta tsarin kula da ingancin dakin gwaje-gwaje, tabbatar da rarrabuwa mai ma'ana, da inganta iyawar ma'aikata don ganowa, yana yiwuwa a rage rashin kuskuren karya saboda ayyukan da ba daidai ba.

Dalilan sake gwada ingancin gwajin SARS-CoV-2 nucleic acid a cikin marasa lafiya da aka dawo da su.

Shirin "COVID-19 Ganewa da Jiyya (Trial na Bakwai Edition)" ya bayyana a sarari cewa ɗayan ma'auni don warkar da marasa lafiya na COVID-19 kuma a sallame su daga asibiti shine samfuran hanyoyin numfashi guda biyu a jere suna da gwajin nucleic acid mara kyau (aƙalla sa'o'i 24 baya), amma akwai kaɗan kaɗan A SARS-CoV-2 gwajin nucleic acid ya sake faruwa saboda wasu dalilai da aka sake gwadawa.

(1)SARS-CoV-2 sabuwar kwayar cuta ce.Wajibi ne a kara fahimtar tsarinsa na pathogenic, cikakken hoton cutar da aka haifar da kuma halayen yanayin cutar.Sabili da haka, a gefe guda, ya zama dole a ƙarfafa kulawar marasa lafiya da aka sallame su da kuma gudanar da aikin lura da lafiya na kwanaki 14.Gudanar da bin diddigin, kula da lafiya da jagoranci na kiwon lafiya don zurfafa fahimtar dukkanin tsarin abin da ya faru, ci gaba da sakamakon cutar.

(2)Majiyyaci na iya sake kamuwa da cutar.Masanin ilimin kimiyya Zhong Nanshan ya ce: Saboda majinyatan da aka warkar suna da kwayoyin rigakafi, SARS-CoV-2 na iya kawar da su ta hanyar rigakafin idan sun sake mamayewa.Akwai dalilai da yawa, wanda zai iya zama sanadin majinyacin da aka warke, ko kuma yana da alaƙa da maye gurbin kwayar cutar, ko ma dalilin gwajin dakin gwaje-gwaje.Idan kwayar cutar kanta ce, maye gurbi na SARS-CoV-2 na iya haifar da rigakafin da mai haƙuri ya samu ya zama mara tasiri a kan cutar da aka canza.Idan majiyyaci ya sake kamuwa da kwayar cutar da ta canza, gwajin nucleic acid na iya zama tabbatacce kuma.

(3) Dangane da hanyoyin gwajin dakin gwaje-gwaje, kowace hanyar gwaji tana da iyakokinta.Ganewar SARS-CoV-2 nucleic acid ya faru ne saboda zaɓin jerin kwayoyin halitta, abun da ke ciki na reagents, azancin hanyar da sauran dalilai, wanda ke haifar da kayan aikin da ke akwai suna da nasu ƙananan iyakokin ganowa.Bayan an yi wa mara lafiya magani, ƙwayoyin cuta a cikin jiki suna raguwa.Lokacin da kwayar cutar hoto a cikin samfurin da za a gwada yana ƙasa da ƙananan iyaka na ganowa, sakamakon "mara kyau" zai bayyana.Duk da haka, wannan sakamakon ba yana nufin cewa ƙwayoyin cuta a cikin jiki sun ɓace gaba ɗaya ba.Kwayar cutar na iya kasancewa bayan an daina maganin.Tadawa”, ci gaba da kwafa.Saboda haka, ana ba da shawarar yin bita sau ɗaya a mako a cikin makonni 2 zuwa 4 bayan fitarwa.

(4) Nucleic acid shine kwayoyin halittar kwayar cutar.Ana kashe kwayar cutar ne bayan an yi wa majiyyaci maganin rigakafi, amma sauran gutsuttsuran RNA na kwayar cutar har yanzu suna cikin jikin mutum, kuma ba a fitar da su gaba daya daga jiki.Wani lokaci, a ƙarƙashin wasu yanayi, ana iya ƙara riƙe shi.Wani lokaci mai tsawo, kuma a wannan lokacin gwajin nucleic acid zai zama "mai wucewa" tabbatacce.Tare da tsawaita lokacin dawo da majiyyaci, bayan ragowar RNA a cikin jiki sun ƙare a hankali, sakamakon gwajin nucleic acid na iya zama mara kyau.

(5) Sakamakon gwajin nucleic acid na SARS-CoV-2 kawai yana tabbatar da kasancewar ko rashi na kwayar cutar RNA, kuma ba zai iya tabbatar da ayyukan kwayar cutar ba kuma ko kwayar cutar tana iya yaduwa.Wajibi ne a tabbatar ko majinyacin da ya sake gwada gwajin nucleic acid zai sake zama tushen kamuwa da cuta.Wajibi ne don aiwatar da al'adun ƙwayoyin cuta akan samfuran asibiti da haɓaka ƙwayar cutar "rayuwa" don tabbatar da cewa yana da kamuwa da cuta.

Takaitawa

A taƙaice, SARS-CoV-2 nucleic acid na gwajin ƙiyayyar ƙarya, sake gwadawa, da sauran yanayin da ba su dace da bayyanar asibiti gaba ɗaya ba.A cikin gwaje-gwaje na ainihi da gwaji, ana ba da shawarar hada alamun asibiti, gwaje-gwajen hoto (CT) da gwaje-gwajen gwaje-gwajen gwaje-gwajen gwaje-gwajen gwaje-gwajen gwaje-gwaje (gwajin nucleic acid + takamaiman gwajin rigakafin ƙwayoyin cuta) don cikakkiyar ganewar asali don hana ganewar asali da kuskuren da aka rasa.Idan sakamakon gwajin da aka samu a fili saba da na asibiti manifestations, shi bada shawarar don gudanar da wani m bincike na dukan gwajin mahada (samfurin tarin, wurare dabam dabam da kuma aiki links) ware SARS-CoV-2 cutar farkon kamuwa da cuta, m kamuwa da cuta ko a hade tare da sauran numfashi cutar cututtuka, da dai sauransu zai yiwu.Idan sharuɗɗan sun ba da izini, ana ba da shawarar tattara samfuran masu mahimmanci kamar sputum ko ruwan lavage na alveolar don sake gwadawa.

Samfura masu dangantaka:

Sarrafa-CoV-2 Kayan Gane Acid Nucleic (Tsarin Binciken Fluorescent PCR Multiplex)