Kayayyaki
Lifecosm SARS-Cov-2-RT-PCR Gane kit don 2019-nCoV
Amfanin da ake tsammani
Ana amfani da wannan kit ɗin don gano ƙimar sabon coronavirus (2019-nCoV) ta amfani da swabs na makogwaro, swabs na nasopharyngeal, ruwan lavage bronchoalveolar, sputum. Sakamakon gano wannan samfurin kawai don tunani na asibiti ne, kuma bai kamata a yi amfani da shi azaman kawai ba. shaida don ganewar asibiti da magani. Ana ba da shawarar cikakken bincike game da yanayin tare da bayyanar cututtuka na marasa lafiya da sauran gwaje-gwajen dakin gwaje-gwaje.
Ka'idar dubawa
Kit ɗin ya dogara ne akan fasahar RT- PCR mataki ɗaya.A zahiri, sabon coronavirus na 2019 (2019-nCoV) ORF1ab da N genes an zaɓi su azaman yankuna masu haɓakawa.Takamaiman maƙasudi da binciken bincike mai kyalli (N gene probes ana yiwa lakabi da FAM da ORF1ab bincike ana yiwa lakabi da HEX) an tsara su don gano sabon nau'in coronavirus RNA na 2019 a cikin samfuran.Har ila yau, kit ɗin ya haɗa da tsarin gano tsarin sarrafawa na ciki (binciken sarrafa kwayoyin halitta na ciki wanda aka yiwa lakabi da CY5) don saka idanu kan tsarin tarin samfuri, haɓaka RNA da PCR, don haka rage sakamako mara kyau.
Babban abubuwan da aka gyara
| Abubuwan da aka gyara | Ƙarar(48T/Kit) |
| Maganin amsawar RT-PCR | 96ml ku |
| nCOV primer TaqMan probemixture (ORF1ab, N Gene, RnaseP Gene) | 864l ku |
| Sarrafa mara kyau | 1500µl |
| nCOV tabbatacce contro (l ORF1ab N Gene) | 1500µl |
Nasu reagents: hakar RNA ko reagents tsarkakewa.Ikon mara kyau/tabbatacce: Kyakkyawan iko shine RNA mai ɗauke da ɓangarorin manufa, yayin da mummunan iko shine ruwa mara ƙarancin acid nucleic.Lokacin amfani, yakamata su shiga cikin hakar kuma yakamata a yi la'akari da su masu kamuwa da cuta.Ya kamata a sarrafa su kuma a zubar da su daidai da ƙa'idodin da suka dace.
Halin tunani na ciki shine jinsin RnaseP na ɗan adam.
Yanayin ajiya da ranar karewa
-20 ± 5 ℃, kauce wa daskarewa maimaituwa da narke fiye da sau 5, yana aiki na watanni 6.
Kayan aiki mai dacewa
Tare da FAM / HEX / CY5 da sauran kayan aikin PCR masu kyalli da yawa.
Bukatun samfur
1. Abubuwan da ake amfani da su: swabs makogwaro, swabs nasopharyngeal, ruwan lavage bronchoalveolar, sputum.
2. Tarin samfurin (Technical aseptic)
Swab na pharyngeal: Goge tonsils da bangon pharyngeal na baya tare da swabs biyu a lokaci guda, sannan a nutsar da kan swab a cikin bututun gwaji mai ɗauke da maganin samfurin.
Sputum: Bayan majiyyaci ya yi tari mai zurfi, tattara sputum mai tari a cikin bututun gwaji na screw cap wanda ke dauke da maganin samfur;Ruwan lavage bronchoalveolar: Samfurin kwararrun likitoci.3. Storage da sufuri na samfurori
Ya kamata a gwada samfuran keɓewar ƙwayoyin cuta da gwajin RNA da wuri-wuri.Samfuran da za a iya gano su a cikin sa'o'i 24 ana iya adana su a 4 ℃;wadanda ba za a iya gano su a cikin 24 ba
Ya kamata a adana sa'o'i a -70 ℃ ko ƙasa (idan babu yanayin ajiya na -70 ℃, ya kamata su kasance.
an adana shi na ɗan lokaci a -20 ℃ refrigerator).Ya kamata samfurori su guje wa daskarewa maimaituwa da narke yayin sufuri.Ya kamata a aika samfurori zuwa dakin gwaje-gwaje da wuri-wuri bayan tattarawa.Idan ana buƙatar ɗaukar samfurori ta nisa mai nisa, ana ba da shawarar adana busassun kankara.
Hanyoyin Gwaji
1 Samfurin sarrafawa da hakar RNA (yankin sarrafa samfurin)
Ana ba da shawarar ɗaukar 200μl na samfurin ruwa don hakar RNA.Don matakan cirewa masu alaƙa, koma zuwa umarnin kayan aikin hakar RNA na kasuwanci.Duka mara kyau da mara kyau
sarrafawa a cikin wannan kit ɗin sun shiga cikin hakar.
2 PCR reagent shiri (yankin shiri na reagent)
2.1 Cire duk abubuwan da aka gyara daga kit ɗin kuma narke kuma gauraya a zafin jiki.Centrifuge a 8,000 rpm na 'yan seconds kafin amfani;lissafta adadin da ake buƙata na reagents, kuma an shirya tsarin amsa kamar yadda aka nuna a cikin tebur mai zuwa:
| Abubuwan da aka gyara | N hidima (tsarin 25µl) |
| nCOV na farko TaqMan probemixture | 18µl × N |
| Maganin amsawar RT-PCR | 2 µl × N |
| *N = adadin samfuran da aka gwada + 1 (ikon mara kyau) + 1 (nCOVtabbatacce iko) | |
2.2 Bayan an haɗa abubuwan da aka gyara sosai, a saka a cikin ɗan gajeren lokaci don barin duk ruwan da ke bangon bututu ya faɗi ƙasan bututun, sannan a sanya tsarin ƙarawa na 20 µl cikin bututun PCR.
3 Samfura (yankin shirya samfur)
Ƙara 5μl mara kyau da sarrafawa mai kyau bayan hakar.Ana ƙara RNA na samfurin da za a gwada a cikin bututun amsawar PCR.
Rufe bututu da ƙarfi kuma a tsakiya a 8,000 rpm na ƴan daƙiƙa kaɗan kafin a canza shi zuwa wurin gano haɓakawa.
4 PCR haɓakawa (yankin gano haɓaka)
4.1 Sanya bututun amsawa a cikin tantanin halitta na kayan aiki, kuma saita sigogi kamar haka:
| mataki | Zagayowar lamba | Zazzabi(°C) | Lokaci | tarinsite |
| Juya bayarubuce-rubuce | 1 | 42 | 10 min | - |
| Pre-denaturativen | 1 | 95 | 1 min | - |
| Zagayowar | 45 | 95 | 15s | - |
| 60 | 30s | tattara bayanai |
Zaɓin tashar gano kayan aiki: Zaɓi tashar FAM, HEX, CY5 don siginar kyalli.Don tunani BAYA, don Allah kar a zaɓi ROX.
Binciken sakamako 5 (Da fatan za a koma ga umarnin gwaji na kowane kayan aiki don saiti)
Bayan amsawa, ajiye sakamakon.Bayan bincike, daidaita ƙimar farawa, ƙimar ƙarewa, da ƙimar ƙima na asali bisa ga hoton (mai amfani zai iya daidaitawa bisa ga ainihin halin da ake ciki, ana iya saita ƙimar farawa zuwa 3 ~ 15, ƙimar ƙarewa za'a iya saita zuwa ƙimar ƙarshe. 5 ~ 20, daidaitawa) a cikin jadawali na logarithmic A bakin kofa na taga, layin kofa yana cikin lokaci na logarithmic, kuma madaidaicin madaidaicin iko shine madaidaiciyar layi ko ƙasa da layin kofa).
6 Quauty iko (An haɗa tsarin sarrafawa a cikin gwajin) Ikon mara kyau: Babu ƙararrawar haɓakawa ga FAM, HEX, tashoshin gano CY5
COV tabbatacce iko: bayyanannen ƙararrawa lanƙwasa na FAM da tashoshi gano HEX, ƙimar Ct≤32, amma babu ƙaramar tashar CY5;
Abubuwan da ke sama dole ne a cika su lokaci guda a cikin gwaji iri ɗaya;in ba haka ba, gwajin ba shi da inganci kuma yana buƙatar maimaitawa.
7 Ƙaddamar da sakamako.
7.1 Idan babu haɓakar haɓakawa ko ƙimar Ct> 40 a cikin tashoshin FAM da HEX na samfurin gwajin, kuma akwai maɓallin ƙarawa a cikin tashar CY5, ana iya yanke hukunci cewa babu sabon coronavirus na 2019 (2019-nCoV) RNA a cikin samfurin;
.2 Idan samfurin gwajin yana da madaidaicin ƙararrawa a cikin tashoshin FAM da HEX, kuma ƙimar Ct shine ≤40, ana iya yanke hukunci cewa samfurin yana da kyau ga sabon coronavirus na 2019 (2019-nCoV).
7.3 Idan samfurin gwajin yana da madaidaicin ƙararrawa kawai a cikin tashoshi ɗaya na FAM ko HEX, kuma ƙimar Ct shine ≤40, kuma babu maɓallin ƙarawa a cikin ɗayan tashar, sakamakon yana buƙatar sake gwadawa.Idan sakamakon gwajin ya yi daidai, ana iya yanke shawarar samfurin ya zama tabbatacce ga sabon
coronavirus 2019 (2019-nCoV).Idan sakamakon gwajin ya kasance mara kyau, ana iya yanke hukunci cewa samfurin ba shi da kyau ga sabon coronavirus na 2019 (2019-nCoV).
Ƙimar hukunci mai kyau
Ana amfani da hanyar lanƙwasa ROC don tantance ƙimar CT na kit da ƙimar kulawar ciki shine 40.
Fassarar sakamakon gwaji
1.Ya kamata a gwada kowane gwaji don sarrafawa mara kyau da inganci.Za'a iya ƙayyade sakamakon gwaji kawai lokacin da sarrafawa ya cika buƙatun sarrafa inganci
2.Lokacin da tashoshin gano FAM da HEX suna da kyau, sakamakon daga tashar CY5 (tashar kulawa ta ciki) na iya zama mara kyau saboda gasar tsarin.
3.Lokacin da sakamakon sarrafawa na ciki ya kasance mara kyau, idan fam ɗin gwajin gwajin FAM da tashoshin gano HEX shima mara kyau ne, , yana nufin cewa tsarin ya lalace ko aikin ba daidai bane, t gwajin ba daidai bane.Don haka, samfuran suna buƙatar sake gwadawa.
