Ikhithi Entsha Yokuthola ICoronavirus(SARS-Cov-2) Nucleic Acid
New Coronavirus(SARS-Cov-2) Nucleic Acid Detection Kit
(Fluinsimbiscent RT-PCR Probe Method) Product Manual
【Pi-roduct name 】Ikhithi Yokuthola I-Coronavirus (SARS-Cov-2) entsha ye-Nucleic Acid (Indlela Yokuhlola I-Fluorescent RT-PCR)
【Packaging specifications 】25 Izivivinyo/Kit
【Intended usiminyaka yobudala】
Le kit isetshenziselwa ukutholwa kwekhwalithi ye-nucleic acid evela ku-coronavirus entsha ku-nasopharyngeal swabs, i-oropharyngeal (emphinjeni) swabs, i-anterior nasal swabs, i-mid-turbinate swabs, ukugeza amakhala kanye nama-aspirates aphuma kubantu abasolwa nge-COVID19 ngumhlinzeki wabo wezempilo. Ukutholwa kwezakhi zofuzo ze-ORF1ab kanye no-N ze-coronavirus entsha kungasetshenziswa ekuxilongeni okuyisizayo kanye nokuqapha ubhubhane lokutheleleka okusha kwe-coronavirus.
【Principles of the procedure 】
Le khithi yakhelwe uphenyo oluthile lwe-TaqMan kanye neziqalo ezithile ezidizayinelwe inoveli ye-coronavirus (SARS-Cov-2) ORF1ab kanye nokulandelana kofuzo kwe-N. Isixazululo sokusabela kwe-PCR siqukethe amasethi angu-3 eziqalisi ezithile kanye nama-fluorescent probe ukuze kutholwe okuqondiwe, kanye nesethi eyengeziwe yama-primer athile nama-fluorescent probes asetshenziswa njengokulawula kwangaphakathi okujwayelekile kwekhithi ukuze kutholwe izakhi zofuzo zokugcina indlu.
Umgomo wokuhlola ukuthi i-probe ethize ye-fluorescent iyagaywa futhi yehliswe umsebenzi we-exonuclease we-enzyme ye-Taq ekuphenduleni kwe-PCR, ukuze iqembu lentatheli ye-fluorescent kanye neqembu le-fluorescent elicishiwe lihlukaniswe, ukuze uhlelo lokuqapha lwe-fluorescence lukwazi ukuthola i-fluorescent. isignali, bese Ngomphumela wokunothisa wokukhulisa i-PCR, isignali ye-fluorescence yophenyo ifinyelela inani elimisiwe le-threshold value-Ct (Cycle threshold). Esimeni lapho kungekho i-amplicon eqondiwe, iqembu lezintatheli lophenyo liseduze neqembu lokucisha. Ngalesi sikhathi, ukudluliswa kwamandla e-fluorescence resonance kwenzeka, futhi i-fluorescence yeqembu lentatheli icinywa yiqembu lokucisha, ukuze isignali ye-fluorescent ingabonakali ngensimbi ye-fluorescent PCR.
Ukuze kuqashwe ukusetshenziswa kwama-reagents ngesikhathi sokuhlolwa, ikhithi ifakwe izilawuli ezinhle nezimbi: ukulawulwa okuhle kuqukethe indawo okuhloswe ngayo i-plasmid recombinant, futhi ukulawulwa okungalungile ngamanzi a-Distilled, asetshenziselwa ukuqapha ukungcoliswa kwemvelo. Kunconywa ukusetha ukulawula okuhle kanye nokulawula okungalungile ngesikhathi esisodwa lapho kuhlolwa.
【Main comi-ponents 】
| Cat. No. | BST-SARS-25 | BST-SARS-DR-25 | Inhlanganiselaizi | |
| Name | Ukucaciswaification | Inaniinto | Inaniinto | |
| Ukulawula okuhle | 180 μL/ibhodlela | 1 | 1 | Ama-plasmids akhiwe ngokwenziwa, Amanzi acwecwe |
| Ukulawula okungalungile | 180 μL/ibhodlela | 1 | 1 | Amanzi acwecwe |
| I-SARS-Cov-2 Mix | 358.5 μL/ibhodlela | 1 | / | Amapheya athile okuqala, ama-fluorescent probes athile, ama-dNTP, , MgCl2, KCl, Tris-Hcl, Amanzi acwecwe, njll. |
| I-Enzyme Mix | 16.5 μL/ibhodlela | 1 | / | Ama-enzyme e-Taq, i-reverse transcriptase, ama-enzyme e-UNG, njll. |
| I-SARS-Cov-2 Mix(Lyophilised) | Izivivinyo ezingama-25/ibhodlela | / | 1 | Amapheya athile okuqalisa, ama-fluorescent probes athile, ama-dNTP, ama-enzyme e-Taq, i-reverse transcriptase, Amanzi acwecwe, njll. |
| 2x Ibhafa | 375 μL/ibhodlela | / | 1 | MgCl2, KCl, Tris-Hcl, Amanzi acwecwe, njll. |
Qaphela:(1) Izingxenye kumakhithi eqoqo ahlukene azikwazi ukuxutshwa noma ukushintshaniswa.
(2) Lungiselela eyakho i-reagent: Ikhithi yokukhipha i-Nucleic acid.
【Storage condkuyimions futhi expiration date 】
For BST-SARS-25:Ezokuthutha kanye nesitolo ku -20±5℃ isikhathi eside.
For BST-SARS-DR-25:Ezokuthutha ekamelweni lokushisa. Gcina ku -20±5℃ isikhathi eside.
Gwema imijikelezo yokuqhwaza ephindaphindiwe. Isikhathi sokuqinisekisa simiselwe izinyanga eziyi-12.
Bona ilebula yedethi yokukhiqiza nokusebenzisa.
Ngemuva kokuvulwa kokuqala, i-reagent ingagcinwa ku -20 ± 5 ° C isikhathi esingekho ngaphezu kwenyanga engu-1 noma kuze kube sekupheleni kwesikhathi se-reagent, noma yiluphi usuku olufika kuqala, ukugwema imijikelezo yokuncibilika ephindaphindayo, kanye nenani lokumiswa kwe-reagent. - imijikelezo yokuncibilika akufanele idlule izikhathi eziyisi-6.
【Applicable instrument】I-ABI 7500, SLAN-96P, Roche-LightCycler-480.
【Sample requirements 】
1.Uhlobo lwesampula olusebenzayo: Isixazululo se-nucleic acid esikhishwe.
2.Isitoreji sesampula kanye nezokuthutha: Gcina ku-20±5℃izinyanga ezingu-6. Friza futhi uncibilikise amasampuli izikhathi ezingaphezu kwezingu-6.
【Testing method】
1.Nucleic acid extraction
Khetha ikhithi efanelekile yokukhipha i-nucleic acid ukuze ukhiphe i-viral nucleic acid, bese ulandela imiyalelo yekhithi ehambisanayo. Kunconywa ukusebenzisa ikhithi yokukhipha neyokuhlanza i-Nucleic acid ekhiqizwe yi-Yixin Bio-Tech (Guangzhou) Co., Ltd. noma ikhithi yokuhlanza i-nucleic acid efanayo.
2. Reaction reagent prepisikhathition
2.1 For BST-SARS-25:
( 1) Khipha i-SARS-Cov-2 Mix kanye ne-Enzyme Mix, ncibilika ngokuphelele endaweni yokushisa yegumbi kahle ngedivayisi ye-Vortex bese uyi-centrifuge kafushane.
(2) I-16.5uL Enzyme Mix yengezwe ku-358.5uL SARS-Cov-2 Mix futhi yaxutshwa kahle ukuze kutholwe isisombululo sokusabela esixubile.
(3) Lungiselela ishubhu le-octal elingu-0.2 mL le-PCR elihlanzekile bese ulimaka ngo-15uL wesisombululo esixubile esingenhla emthonjeni ngamunye.
(4) Engeza i-15 μL yesisombululo se-nucleic acid ehlanzekile, isilawuli esihle nokulawula okungalungile, futhi umboze ngokucophelela i-octal tube cap.
(5) Hlanganisa kahle ngokujikisa ubheke phansi, futhi ngokushesha i-centrifuge ukugxilisa uketshezi ngaphansi kweshubhu.
1
2.2 For BST-I-SARS-DR-25:
( 1) Engeza i-375ul 2x Buffer ku-SARS-Cov-2 Mix((Lyophilised) ukuze ulungise ingxube yokusabela. Hlanganisa kahle ngamapayipi bese ubeka i-centrifuge kafushane. （Uma imiksi yokusabela isilungisiwe, kunconywa ukuthi igcinwe ku -20℃ ukugcinwa kwesikhathi eside.)
(2) Lungiselela ishubhu ye-octal engu-0.2 mL ye-PCR ehlanzekile bese uyimaka ngo-15μL wengxube yokusabela emthonjeni ngamunye.
(3) Engeza i-15μL yesisombululo se-nucleic acid ehlanzekile, ukulawula okuhle nokulawula okungalungile, futhi umboze ngokucophelela i-octal tube cap.
(4) Hlanganisa kahle ngokujikisa ubheke phansi, futhi ngokushesha i-centrifuge ukugxilisa uketshezi ngaphansi kweshubhu.
3. I-PCR amplification (Sicela ubheke imanuwali yethuluzi ukuze uthole izilungiselelo zokusebenza.）
3. 1 Beka i-PCR 8-tube ekamelweni lesampula lensimbi ye-fluorescent PCR, bese usetha isampula ukuthi ihlolwe, ukulawula okuhle kanye nokulawula okungalungile ngokohlelo lokulayisha.
3.2 Isiteshi sokubona i-Fluorescence:
( 1) I-ORF1ab gene ikhetha isiteshi sokuthola se-FAM (Intatheli: FAM, Quencher: Ayikho).
(2) I-N gene ikhetha isiteshi sokutholwa se-VIC (Intatheli: VIC, Quencher: Ayikho).
(3) Ufuzo olujwayelekile lwangaphakathi lukhetha isiteshi sokutholwa se-CY5 (Intatheli: CY5, Quencher: Ayikho).
(4) I-Passive Reference isethwe ku-ROX.
3.3 PCR isilungiselelo sepharamitha yohlelo:
| Isinyathelo | Izinga lokushisa(℃) | Isikhathi | Inombolo yemijikelezo | |
| 1 | Hlehlisa ukusabela kokulotshiweyo | 50 | 15 imiz | 1 |
| 2 | Ukusebenza kwe-Taq enzyme | 95 | 2.5 imiz | 1 |
| 3 | Ukusebenza kwe-Taq enzyme | 93 | 10 s | 43 |
| Isandiso se-Annealing kanye nokutholwa kwe-fluorescence | 55 | 30 s | ||
Ngemva kokusetha, gcina ifayela bese usebenzisa uhlelo lokusabela..
4.Results analysis
Ngemuva kokuphela kohlelo, imiphumela igcinwa ngokuzenzakalelayo, futhi ijika lokukhulisa liyahlaziywa. Ijika le-amplification isethwe kumkhawulo omisiwe wensimbi.
【Explanation of test results 】
1. Thola ukufaneleka kokuhlolwa: Ukulawula okuhle kwe-FAM, isiteshi se-VIC kufanele sibe nejika elivamile lokukhulisa, futhi inani le-Ct ngokuvamile lingaphansi kuka-34, kodwa lingashintshashintsha ngenxa yezilungiselelo ezihlukene zomkhawulo wamathuluzi ahlukene. Ukulawula okungekuhle kwe-FAM, isiteshi se-VIC kufanele kube i-Ct engakhulisiwe. Kuvunyelwene ngokuthi izidingo ezingenhla kufanele kuhlangatshezwane nazo ngesikhathi esifanayo, ngaphandle kwalokho lokhu kuhlolwa akuvumelekile.
2. Umphumela wesinqumo
| Isiteshi se-FAM/VIC | Umphumela wokwahlulela |
| Ct＜37 | Uhlolo lwesampula luthi positive |
| 37≤Ct＜40 | Ijika le-amplification linomumo ongu-S, futhi amasampula asolisayo adinga ukuphinde ahlolwe; uma imiphumela yokuhlolwa kabusha ingaguquki, ibhekwa njengephozithivu, ngaphandle kwalokho inegethivu |
| Ct≥40 Noma Akukho Ukukhulisa | Ukuhlolwa kwesampula kunegethivu (noma kungaphansi komkhawulo ophansi wokutholwa kwekhithi) |
Qaphela: ( 1) Uma kokubili isiteshi se-FAM nesiteshi se-VIC kunethemba ngesikhathi esisodwa, i-SARS-Cov-2 inqunywa ukuthi ibe nayo.
(2) Uma isiteshi se-FAM noma isiteshi se-VIC sithi sinalo futhi esinye isiteshi sinegethivu, ukuhlolwa kufanele kuphindwe. Uma i-positive ngesikhathi esifanayo, izobhekwa njenge-positive yakwa-SARS-Cov-2, ngaphandle kwalokho izobhekwa njengenegethivu yakwa-SARS-Cov-2.
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