Huahana Huahana
Ke alakaʻi nei ka World Health Organization (WHO) i hiki i nā antigen-detecting rapid diagnostic tests (Ag-RDRs) ke hāʻawi i kahi ala wikiwiki a ʻoi aku ka liʻiliʻi e ʻike ai i ka maʻi SARS-CoV-2 ikaika ma mua o nā hōʻike hoʻonui ʻana i ka waikawa nucleic acid (NAATs), a ʻo WHO kekahi e paipai. Hiki ke hoʻohana ʻia nā Ag-RDT e hālāwai me nā koi haʻahaʻa haʻahaʻa no ka ʻike ʻana i ka hihia mua, ka huli ʻana i ka pilina, i ka wā o ka hoʻokolokolo maʻi maʻi a me ka nānā ʻana i nā ʻano o ka ulu ʻana o nā maʻi i nā kaiāulu.
Nā hiʻohiʻona
● Hoʻopiha piha:ʻEkolu mau gene i ʻike ʻia i hoʻokahi hoʻokolohua
● kūpono:Hoʻopili i nā mea hana maʻamau me nā ala CY5, FAM, VIC/HEX.
● Hana maikaʻi loa:ʻO ka ʻike kiʻekiʻe a me ka kikoʻī, LOD = 200 kope / ml.
ʻĀpana ʻenehana
| Hōʻike kikoʻī | 50 ho'āʻo/kit, 100 hoʻāʻo/kit |
| Māhele Kūlana | ORF1ab, N, E |
| Laʻana pili | Sputum, Oropharyngeal Swab |
| Ka palena o ka ike | 200 kope/ml |
| Ka huina huikau | 99.55% |
| Waiwai Ct (CV,%) | ≤5.0% |
| Ka helu kuhikahi maikaʻi | 99.12% |
| Ka helu kuhi hewa ʻole | 100% |
| Nā Kūlana mālama a me ka lā pau | Mālama ʻia ma -20±5 ℃, a paʻa ka manawa no 12 mahina. |
| Hoomalu Kuloko | ʻAe |
| Helu Helu | A7793YF-50T, A7793YF-100T |
| Palapala hōʻoia | CE |
| Nā mea hoʻohālike | Nasopharyngeal swab, Oropharyngeal swab, Alveolar lavage wai, Kohu a me ka sputum |
| Mea Hana Pono | ABI 7500, Roche Light Cycler 480Ⅱ , Roche Cobas z 480, SLAN-96P Pūnaehana PCR Manawa maoli |
Kaʻina hana hoʻāʻo
1. Wehe ʻAka Nucleic
Pono e hana ʻia ka hana e like me ka manual of extraction kit.
2. Hoʻomākaukau Pūnaewele:
1) Wehe i ka reagent a hoʻoheheʻe loa i ka reagent.E hoʻohuli koke i ka hui ʻana a me ka centrifuge.N ho'āʻo reactions (N = helu o nā laʻana e ho'āʻo 'ia + maikaʻi mana + ino + 1) ua hoomakaukauia no ka pane nenoaiu, pakahi, e like me keia.
| Vomponents | Volume no ka 1 ʻōnaehana pane | Volume no ka ʻōnaehana pane N |
| ʻO ka hoʻonā ʻana o ka ʻakika nucleic amplification reaction Mix (A7793YF) | 18 µL | 18 µL * N |
| Huihui enzyme | 2 µL | 2 µL * N |
| huina nui | 20 µL | 20 µL * N |
2) Ka hāʻawi ʻana i ka hopena: Ua hui ʻia ka hopena hopena a ua centrifuged, a ua hoʻokaʻawale ʻia kēlā me kēia paipu i ka nui o 20μL i loko o kahi paipu PCR kūpono no kahi mea hana PCR fluorescence.
3. Hoouka ana
Hoʻohui ʻia ka 5μL o ka ʻakika nucleic i unuhi ʻia, ka mana nucleic acid a me ka mana kino nucleic acid i nā ʻōnaehana pane, a ʻo ka nui o ka pane ʻana he 25μL.E hoʻopaʻa i ka uhi paipu a hoʻoneʻe iā ia i kahi hoʻāʻo hoʻonui ma hope o kekahi mau kekona o ka centrifugation.
4. PCR Amplification Assay
1) E hoʻokomo i ka paipu hopena PCR i loko o ka mea hana hoʻonui PCR fluorescent no ka ʻike hoʻonui.
2) Ka hoʻonohonoho ʻana i nā ʻāpana pōʻaiapuni:
| Papahana | Ka helu o nā pōʻaiapuni | Mahana | Manawa pane | |
| 1 | 1 | 50 ℃ | 10 min | |
| 2 | 1 | 95 ℃ | 30 sec | |
| 3 | 45 | 95 ℃ | 5 sec | |
| 60 ℃ | 30 sec | Hōʻiliʻili Fluorescence | ||
3) Nā hoʻonohonoho ʻike:
Hoʻonohonoho ʻia nā ala ʻike i FAM, VIC, ROX a me CY5, e pili ana me ORF1ab, N gene, a me E Gene, RNase P ka mana kūloko.Hoʻonohonoho ʻia ʻo "Quencher Dye" a me "Passive Reference" i "ʻAʻohe" no ka mea kani ABI 7500.E hoʻonoho i ka Mana Positive, Manaʻo ʻino, a me ka Laʻana (ʻAʻole i ʻike ʻia) i mea e kūpono ai nā laʻana, a hoʻonoho i ka inoa hāpana ma ke kolamu "Sample Name".
No X-POCH16, penei ka hana a me ka papahana:
1) Ma hope o ka pau ʻana o ka hoʻāʻo ponoʻī, wehe i ka poʻi a hoʻokomo i nā paipu hopena PCR i nā kūlana i koho ʻia i ka mea kani.
2) E hoʻomaka ma ke koho ʻana i ka "Exper."koho.E koho i ke koho "All" a i ʻole e koho lima i ka wahi pane ma ka ʻaoʻao hema o ka pale.
3) E koho i ke koho "LOAD";koho i ka papahana ho'āʻo;kaomi "DONE" a me ka "RUN".Hoʻopau ka papahana i 30min42s.
Hoʻonohonoho ʻia nā ala ʻike o ka papahana Default i FAM, VIC, ROX a me CY5, e pili ana me ORF1ab, N gene, a me E Gene, RNase P mana kūloko.
ʻO ka palena pōʻaiapuni o ka papahana Default penei:
| Papahana | Helu o | Mahana | Manawa pane |
| 1 | 1 | 50 ℃ | 2min |
| 2 | 1 | 95 ℃ | 30 sec |
| 3 | 41 | 95 ℃ | 2sec |
| 60 ℃ | 13 sec | ʻO ka fluorescence |
5. Hoʻonohonoho paepae
E like me ke kiʻi i kālailai ʻia, e hoʻoponopono i ka waiwai hoʻomaka, ka waiwai hope o Baseline a me ka waiwai paepae (Ua ʻōlelo ʻia ka waiwai hoʻomaka a me ka waiwai hope e hoʻonohonoho ʻia i 3 a me 15 pakahi, a ua hoʻoponopono ʻia ka pihi hoʻonui o ka mana maikaʻi ʻole i palahalaha a i ʻole. haʻahaʻa ma lalo o ka Laina paepae), kaomi i ka Analysis no ka loaʻa ʻana o ka loiloi i ka waiwai Ct hāpana.E nānā i nā hualoaʻa ma ka ʻaoʻao Hōʻike.
6. Kūlana Kūlana Kūlana
Pono kēlā me kēia mana o ka pahu kit e hoʻokō i nā koi e pili ana me ka 'S' curve, inā ʻaʻole pono ka hoʻokolohua.
| Nā ala ʻike | Manaʻo ʻino | Manaʻo maikaʻi |
| FAM(ORF1ab) | ʻAʻole Ct | Ct≤38 |
| VIC(N) | ʻAʻole Ct | Ct≤38 |
| ROX(E) | ʻAʻole Ct | Ct≤38 |
| CY5(RP) | ʻAʻole Ct | Ct≤38 |
【Waiwai oki】
E like me ka hopena o 100 oropharyngeal swab samples a me 100 sputum samples, a me ka ROC curve method, the Cut-off value of the OFR1ab, N genes E Gene of this kit is Ct = 38
FAQ
I loko o kēia pahu, hoʻolālā ʻia nā primers a me nā ʻimi noiʻi o ka ʻenehana PCR fluorescent manawa maoli i nā wahi i mālama ʻia a kikoʻī o ka ORF1ab, N a me E gene o 2019-nCoV.I ka wā o ka hoʻonui ʻana o ka PCR, hoʻopaʻa ka probe i ka template, a ʻo ka hui 5'-end reporter o ka probe i hoʻokaʻawale ʻia e ka Taq enzyme (5'→3' exonuclease activity), a laila e neʻe aku mai ka hui kinai e hana i kahi hōʻailona fluorescent. .Hoʻolālā ʻokoʻa ʻia ka pihi hoʻonui i ka manawa maoli ma muli o ka hōʻailona fluorescence i ʻike ʻia, a helu ʻia ke kumu waiwai Ct.Hoʻopili ʻia nā fluorophores FAM, VIC a me ROX i ka ORF1ab gene, N gene a me E gene probes.Ma ka hoʻohana ʻana i hoʻokahi hoʻāʻo, hiki ke hana ʻia ka ʻike qualitative o nā ʻano ʻekolu o luna o 2019-nCoV i ka manawa like.
Hāʻawi ʻia ka kit me kahi mana kūloko e kuhikuhi ana i ka gen RNase P e nānā i ka hōʻiliʻili ʻana o nā laʻana, ka lawelawe ʻana, ka unuhi ʻana a me ke kaʻina RT-PCR e pale i nā hopena hoʻopunipuni.Hoʻopili ʻia ka mana kūloko me kahi hui fluorescent CY5.
1. E noʻonoʻo a wehewehe i nā hopena hoʻāʻo i ka wā maʻamau ka mea kani, a ʻo ka mana maikaʻi, ka mana ʻino a me ka hopena o ka hoʻāʻo kūloko e kū i ka maʻamau kaohi.
2. Hōʻike ka pihi hoʻonui o ka mana kūloko (CY5) i kahi pihi S maʻamau a me Ct ≤ 38, ʻo ka wehewehe ʻana i nā hopena o nā genes i hoʻopaʻa ʻia ma lalo o nā kūlana.
| Nā ala ʻike | Ka wehewehe ʻana i nā hopena o nā ʻano genes | ||
| FMA | VIC (N gene) | ROX (E gene) | |
| Ct≤38 | Ct≤38 | Ct≤38 | Me ka pihi S amplification maʻamau, ʻo ka waiwai Ct ʻo ≤38, maikaʻi ke ʻano kuʻina e pili ana. |
| 38 ＜ Kt ＜ 40 | 38 ＜ Kt ＜ 40 | 38 ＜ Kt ＜ 40 | Me ka pihi S amplification maʻamau, e hoʻāʻo hou i ke ʻano kumu kūpono o ka hāpana. Inā loaʻa ka waiwai Ct ＜ 40 me ka pihi hoʻonui S maʻamau, ʻoi aku ka maikaʻi o ka gene i koho ʻia;inā ʻo ka Ct value≥40, ʻaʻole i kūpono ka gene target e pili ana |
| Ct≥40 | Ct≥40 | Ct≥40 | ʻAʻole maikaʻi ka gene target e pili ana |
Ka wehewehe ʻana i nā hopena no 2019-nCoV:
E like me nā hopena o ORF1ab, N gene a me E gene, wehewehe penei:
1) Inā maikaʻi ʻelua a ʻEkolu mau genes o nā genes i ʻike ʻia, maikaʻi ka 2019-nCoV;
2) Inā maikaʻi ka 2019-nCoV a i ʻole NONE o nā genes i ʻike ʻia.
'Ōlelo Aʻo: Pono ka pihi hoʻonui o ka hāpana maikaʻi me ka pihi S maʻamau.Eia nō naʻe, inā ʻoi aku ka kiʻekiʻe o ka manaʻo i hoʻopaʻa ʻia, ʻaʻole hiki ke hoʻonui ʻia ka mana maʻamau o loko a hiki ke noʻonoʻo pololei ʻia ka hāpana.Inā loaʻa iā Ct≤38 kekahi mau genes i hoʻopaʻa ʻia, maikaʻi ka 2019-nCoV.Inā loaʻa iā Ct≥40 kekahi mau genes i hoʻopaʻa ʻia, ʻaʻole hewa ka 2019-nCoV.Inā Ct≥40, a i ʻole hōʻike i ka waiwai, ʻaʻole maikaʻi ka wehewehe ʻana i nā hopena o ka gene i manaʻo ʻia.
3. Inā ʻoi aku ka nui o nā koina Ct o FAM, VIC, ROX a me Cy5 ma mua o 38 a i ʻole ʻaʻole ʻike ʻia ka pihi S amplification maʻamau:
1) Aia kekahi mau mea i loko o ka hāpana e kaohi ai i ka hopena PCR.Manaʻo ʻia e dilute i ka hāpana no ka hoʻāʻo hou ʻana.
2) He ʻano ʻē ke kaʻina hana o ka hoʻoneʻe ʻana i ka waikawa nucleic, no laila ua manaʻo ʻia e unuhi hou
nucleic acid no ka ho'āʻo hou.
3) ʻAʻole kēia laʻana he laʻana kūpono i ka manawa o ka laʻana, a i ʻole hoʻohaʻahaʻa ʻia
i ka wā lawe a mālama.
ʻO 2 mau ala e hiki ai iā mākou ke ʻike i kēia mau kūlana: NAAT a me Antigen.
(No ka CDC o Los Angeles)
