Kit e Ncha ea Coronavirus(SARS-Cov-2) Nucleic Acid Detection Kit
New Coronavirus(SARS-Cov-2) Nucleic Acid Detection Kit
(Fluorescent RT-PCR Probe Method) Product Manual
【Product name 】New Coronavirus(SARS-Cov-2) Nucleic Acid Detection Kit(Fluorescent RT-PCR Probe Method)
【Packaging specifications 】25 Liteko / Kit
【Intended uslilemo】
Setsi sena se sebelisetsoa ho bona boleng ba nucleic acid ho tsoa ho coronavirus e ncha ho nasopharyngeal swabs, oropharyngeal (metso) swabs, anterior nasal swabs, mid-turbinate swabs, lihlapa tsa nko le li-nasal aspirates ho tsoa ho batho ba belaelloang ka COVID19 ke mofani oa bona oa tlhokomelo ea bophelo. Ho sibolloa ha liphatsa tsa lefutso tsa ORF1ab le N tsa coronavirus e ncha ho ka sebelisoa bakeng sa tlhahlobo e thusang le ho lekola lefu la tšoaetso ea tšoaetso e ncha ea coronavirus.
【Principles of the procedure 】
Setsi sena se etselitsoe li-probes tse khethehileng tsa TaqMan le li-primers tse khethehileng tse etselitsoeng "coronavirus" e ncha (SARS-Cov-2) ORF1ab le tatelano ea liphatsa tsa lefutso tsa N. Tharollo ea karabelo ea PCR e na le lihlopha tse 3 tsa li-primers le li-fluorescent probes bakeng sa ho lemoha liphofu, 'me sete e eketsehileng ea li-primers le li-fluorescent probes e sebelisoa e le taolo e tloaelehileng ea ka hare ea khiti bakeng sa ho lemoha liphatsa tsa lefutso tsa ho boloka ntlo.
Molao-motheo oa teko ke hore probe e khethehileng ea fluorescent e cheka le ho senyeha ke ts'ebetso ea exonuclease ea enzyme ea Taq karabelo ea PCR, e le hore sehlopha sa motlalehi oa fluorescent le sehlopha sa fluorescent se timang se arohane, e le hore tsamaiso ea fluorescence monitoring e ka fumana fluorescent. sesupo, 'me joale Ka phello ea ntlafatso ea PCR amplification, lets'oao la fluorescence la probe le fihla ho boleng bo behiloeng ba bohlokoa ba Ct (Cycle threshold). Tabeng ea ho se be le amplicon e shebiloeng, sehlopha sa baqolotsi ba litaba se haufi le sehlopha sa quenching. Ka nako ena, ho fetisoa ha matla a fluorescence resonance ho etsahala, 'me fluorescence ea sehlopha sa moqolotsi oa litaba e tingoa ke sehlopha sa ho tima, e le hore letšoao la fluorescent le se ke la fumanoa ke sesebelisoa sa PCR sa fluorescent.
Bakeng sa ho beha leihlo ts'ebeliso ea li-reagents nakong ea tlhahlobo, kit e na le litsamaiso tse ntle le tse mpe: taolo e ntle e na le plasmid e recombinant, mme taolo e mpe ke metsi a hloekisitsoeng, a sebelisetsoang ho lekola tšilafalo ea tikoloho. Ho khothalletsoa ho beha taolo e ntle le taolo e mpe ka nako e le 'ngoe ha ho hlahlojoa.
【Main componets 】
| Cat. No. | BST-SARS-25 | BST-SARS-DR-25 | Kopanyaents | |
| Name | Specification | Quantity | Quantity | |
| Taolo e ntle | 180 μL / pial | 1 | 1 | Li-plasmids tse entsoeng ka maiketsetso, Metsi a hloekisitsoeng |
| Taolo e mpe | 180 μL / pial | 1 | 1 | Metsi a hloekisitsoeng |
| SARS-Cov-2 Mix | 358.5 μL/vial | 1 | / | Li-primer pairs tse khethehileng, lisebelisoa tse khethehileng tsa ho lemoha fluorescent, dNTPs, MgCl2, KCl, Tris-Hcl, metsi a silafetseng, joalo-joalo. |
| Motsoako oa Enzyme | 16.5 μL / pial | 1 | / | Li-enzyme tsa Taq, reverse transcriptase, li-enzyme tsa UNG, jj. |
| SARS-Cov-2 Mix(Lyophilised) | 25 liteko / vial | / | 1 | Li-primer pairs tse khethehileng, li-fluorescent probes tse khethehileng, li-dNTP, li-enzyme tsa Taq, reverse transcriptase, metsi a hloekisitsoeng, joalo-joalo. |
| 2x Buffer | 375 μL / pial | / | 1 | MgCl2, KCl, Tris-Hcl, Metsi a hloekisitsoeng, joalo-joalo. |
Hlokomela:(1) Likarolo tse ka har'a li-batch kits tse fapaneng li ke ke tsa tsoakoa kapa tsa fapanyetsana.
(2) Itokisetse sesebelisoa sa hau: Nucleic acid extract kit.
【Storage conditons le expiration date 】
For BST-SARS-25:Lipalangoang le polokelo ho -20±5℃ nako e telele.
For BST-SARS-DR-25:Lipalangoang ka mocheso oa kamore. Boloka ho -20±5 ℃ nako e telele.
Qoba makhetlo a mangata a ho qhibiliha a leqhoa. Nako ea ho sebetsa e behiloe 12months.
Sheba leibole bakeng sa letsatsi la tlhahiso le tšebeliso.
Kamora ho buloa ka lekhetlo la pele, reagent e ka bolokoa ho -20 ± 5 ° C nako e sa feteng khoeli e le 'ngoe kapa ho fihlela qetellong ea nako ea reagent, ho sa tsotelehe hore na ke letsatsi lefe le tlang pele, ho qoba lipotoloho tse pheta-phetoang tsa leqhoa, le palo ea ho hoama ha reagent. - Lipotoloho tse qhibilihang ha lia lokela ho feta makhetlo a 6.
【Applicable instrument】ABI 7500, SLAN-96P, Roche-LightCycler-480.
【Sample requirements 】
1. Mofuta oa mohlala o sebetsang: Tharollo ea nucleic acid e nkiloeng.
2.Sample polokelo le lipalangoang: Boloka ho-20 ± 5 ℃ bakeng sa likhoeli tse 6. Qhamisa le ho qhibiliha lisampole ho feta linako tse 6.
【TESTing method】
1.Nucleic acid extraction
Khetha khiti e loketseng ea ho ntša nucleic acid ho ntša varal nucleic acid, 'me u latele litaelo tse tsamaellanang le tsona. Ho khothaletsoa ho sebelisa sesebelisoa sa ho ntša le ho hloekisa acid ea Nucleic e hlahisoang ke Yixin Bio-Tech (Guangzhou) Co., Ltd. kapa lisebelisoa tse lekanang tsa ho hloekisa acid ea nucleic.
2. Reaction reagent preparation
2.1 For BST-SARS-25:
( 1) Tlosa Motsoako oa SARS-Cov-2 le Motsoako oa Enzyme, qhibiliha ka ho feletseng mocheso oa kamore o kopane hantle ka sesebelisoa sa Vortex ebe o centrifuge ka nakoana.
(2) 16.5uL Enzyme Mix e kentsoe ho 358.5uL SARS-Cov-2 Mix mme ea tsoakoa hantle ho fumana tharollo e tsoakiloeng.
(3) Lokisetsa 0.2 mL PCR octal tube e hloekileng 'me u e tšoaee ka 15uL ea tharollo e ka holimo e tsoakiloeng ka seliba.
(4) Eketsa 15 μL ea tharollo ea nucleic acid e hloekisitsoeng, taolo e ntle le taolo e mpe, 'me u koahele ka hloko sekoahelo sa octal tube cap.
(5) Kopanya hantle ka ho khelohela holimo, 'me ka potlako centrifuge ho tsepamisa mohopolo mokelikeli botlaaseng ba tube.
1
2.2 For BST-SARS-DR-25:
( 1) Kenya 375ul 2x Buffer ho SARS-Cov-2 Mix((Lyophilised) ho lokisa motsoako oa karabelo Kopanya hantle ka pipetting ebe u centrifuge hakhuts'oanyane. polokelo ea nako e telele.)
(2) Lokisetsa 0.2 mL PCR octal tube e hloekileng 'me u e tšoaee ka 15μL ea motsoako oa karabelo selibeng.
(3) Eketsa 15μL ea tharollo ea nucleic acid e hloekisitsoeng, taolo e ntle le taolo e mpe, 'me u koahele ka hloko sekoahelo sa octal tube cap.
(4) Kopanya hantle ka ho khelohela holimo, 'me ka potlako centrifuge ho tsepamisa mohopolo mokelikeli botlaaseng ba tube.
3. PCR ampbophelocation （Ka kopo sheba bukana ea lisebelisoa bakeng sa litlhophiso tsa ts'ebetso.）
3. 1 Beha PCR 8-tube ka kamoreng ea sampuli ea sesebelisoa sa PCR sa fluorescent, 'me u behe mohlala o lokelang ho lekoa, taolo e ntle le taolo e mpe ho latela taelo ea ho laela.
3.2 Seteishene sa ho lemoha ha Fluorescence:
( 1) ORF1ab gene e khetha mocha oa ho lemoha oa FAM (Motlalehi: FAM, Quencher: Ha ho letho).
(2) N gene e khetha mocha oa ho lemoha oa VIC (Motlalehi: VIC, Quencher: Ha ho letho).
(3) Liphatsa tsa lefutso tsa ka hare li khetha mocha oa ho lemoha oa CY5 (Motlalehi: CY5, Quencher: Ha ho letho).
(4) Passive Reference e behiloe ho ROX.
3.3 PCR tlhophiso ea paramethara ea lenaneo:
| Mohato | Mocheso(℃) | Nako | Palo ea lipotoloho | |
| 1 | Phetoho ea maikutlo a mongolo | 50 | 15 mets | 1 |
| 2 | Ts'ebetso ea enzyme ea Taq | 95 | 2.5 mets | 1 |
| 3 | Ts'ebetso ea enzyme ea Taq | 93 | 10 s | 43 |
| Katoloso ea Annealing le ho fumana fluorescence | 55 | 30 s | ||
Ka mor'a ho beha, boloka faele 'me u tsamaise lenaneo la karabelo.
4.Results analysis
Kamora hore lenaneo le fele, liphetho li bolokoa ka bo eona, 'me sekhahla sa amplification se hlahlojoa. Mokokotlo oa amplification o behiloe moeling oa kamehla oa sesebelisoa.
【Explanation of test results 】
1. Etsa qeto ea bonnete ba teko: Taolo e ntle ea FAM, mocha oa VIC o lokela ho ba le sekhahla se tloaelehileng sa amplification, 'me boleng ba Ct ka kakaretso bo ka tlase ho 34, empa bo ka fetoha ka lebaka la litlhophiso tse fapaneng tsa lisebelisoa tsa lisebelisoa tse fapaneng. Taolo e mpe ea FAM, mocha oa VIC e lokela ho ba Ct e sa ntlafatsoang. Ho lumeloa hore litlhoko tse ka holimo li tlameha ho finyelloa ka nako e le 'ngoe, ho seng joalo tlhahlobo ena ha e sebetse.
2. Sephetho kahlolo
| FAM/VIC mocha | Sephetho sa kahlolo |
| Ct＜37 | Mohlala oa tlhahlobo o nepahetse |
| 37≤Ct＜40 | Mokokotlo oa amplification o bōpehile ka S, 'me lisampole tse belaetsang li hloka ho hlahlojoa bocha; haeba liphetho tsa tlhahlobo e ncha li lumellana, ho nkoa e le tse ntle, ho seng joalo ke tse mpe |
| Ct≥40 Kapa Ha ho Amplification | Teko ea sampole e mpe (kapa e ka tlase ho tekanyo e tlase ea ho fumanoa ha lisebelisoa) |
Ela hloko: ( 1) Haeba kanale ea FAM le kanale ea VIC li le ntle ka nako e le 'ngoe, SARS-Cov-2 e ikemiselitse ho ba ntle.
(2) Haeba mocha oa FAM kapa mocha oa VIC o le ntle 'me mocha o mong o le mpe, tlhahlobo e lokela ho phetoa. Haeba e le ntle ka nako e le 'ngoe, e tla ahloloa e le SARS-Cov-2 positive, ho seng joalo e tla ahloloa e le SARS-Cov-2 negative.
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