Ọhụrụ Coronavirus (SARS-Cov-2) Ngwa nchọpụta Nucleic Acid
New Coronavirus(SARS-Cov-2) Nucleic Acid Detection Kit
(Fluorescent RT-PCR Probe Method) Product Manual
【Produkt name 】Ngwa Coronavirus ọhụrụ (SARS-Cov-2) Nchọpụta Nucleic Acid (Ụzọ Nchọpụta Fluorescent RT-PCR)
【Packaging specifications 】25 Nnwale/Kit
【Intended usafọ】
A na-eji ngwa a maka nchọpụta qualitative nke nucleic acid sitere na coronavirus ọhụrụ na swabs nasopharyngeal, swabs oropharyngeal (akpịrị), swabs ihu ihu, swabs etiti turbinate, imi imi na aspirates imi sitere na ndị mmadụ na-enyo enyo na COVID19 site n'aka onye na-ahụ maka ahụike ha. Enwere ike iji nchọpụta ORF1ab na mkpụrụ ndụ N nke coronavirus ọhụrụ mee ihe maka nyocha inyeaka na nlekota ọrịa na-efe efe nke ọrịa coronavirus ọhụrụ.
【Principles of the procedure 】
Emebere ngwa a maka nyocha TaqMan akọwapụtara yana ihe izizi emebere maka coronavirus ọhụrụ (SARS-Cov-2) ORF1ab na usoro mkpụrụ ndụ N. Ngwọta mmeghachi omume PCR nwere usoro 3 nke kpọmkwem primers na fluorescent probes maka nchọpụta kpọmkwem nke ebumnuche, yana mgbakwunye ọzọ nke primers na fluorescent nyocha ka a na-eji dị ka njikwa ọkọlọtọ n'ime nke kit maka ịchọpụta mkpụrụ ndụ ihe nketa ụlọ.
Ụkpụrụ ule bụ na a na-agbaze nyocha nke fluorescent kpọmkwem site na ọrụ exonuclease nke Taq enzyme na mmeghachi omume PCR, nke mere na ndị nta akụkọ fluorescent otu na ndị na-emenyụ ọkụ na-ekewa, ka usoro nlekota fluorescence wee nweta fluorescent. mgbama, wee site na mmetụta nkwalite nke PCR amplification, mgbama fluorescence nke nyocha na-erute uru ọnụ ụzọ setịpụrụ - uru Ct (ọnụ ụzọ okirikiri). N'ọnọdụ enweghị amplicon lekwasịrị anya, otu ndị nta akụkọ nke nyocha ahụ dị nso na ndị na-emenyụ ọkụ. N'oge a, ọkụ ọkụ resonance ike nyefe na-eme, na fluorescence nke ndị na-ede akụkọ otu na-emenyụ site quenching otu, nke mere na fluorescent mgbaàmà enweghị ike ịchọpụta fluorescent PCR ngwá.
Iji nyochaa ojiji nke reagents n'oge ule, ngwa na-onwem na nti na-adịghị mma njikwa: mma akara nwere lekwasịrị saịtị recombinant plasmid, na-adịghị mma akara bụ Distilled mmiri, nke a na-eji nyochaa gburugburu ebe obibi mmetọ. A na-atụ aro ka ịtọ njikwa dị mma na njikwa na-adịghị mma n'otu oge mgbe ịnwale.
【Main components 】
| Cat. No. | BST-SARS-25 | BST-SARS-DR-25 | Components | |
| Ahae | Nkọwantinye | Quantity | Quantity | |
| njikwa dị mma | 180 μl / vial | 1 | 1 | Plasmids arụrụ arụrụ arụ, mmiri a na-agbapụta |
| Njikwa adịghị mma | 180 μl / vial | 1 | 1 | Mmiri distilled |
| Ngwakọta SARS-Cov-2 | 358.5 μl / vial | 1 | / | Ụdị ụzọ abụọ pụrụ iche, nchọpụta fluorescent kpọmkwem, dNTPs, , MgCl2, KCl, Tris-Hcl, mmiri a na-ekpo ọkụ, wdg. |
| Enzyme Mix | 16.5 μl / vial | 1 | / | Taq enzymes, reverse transcriptase, UNG enzymes, wdg. |
| Ngwakọta SARS-Cov-2 (Lyophilized) | 25 ule / vial | / | 1 | Otu ụzọ abụọ pụrụ iche, nchọpụta fluorescent pụrụ iche, dNTPs, Taq enzymes, reverse transcriptase, mmiri distilled, wdg. |
| 2x nchekwa | 375 μl / vial | / | 1 | MgCl2, KCl, Tris-Hcl, Distilled mmiri, wdg. |
Rịba ama:(1) Enweghị ike ịgwakọta ma ọ bụ gbanwee ihe ndị dị na ngwa ngwa dị iche iche.
(2) Kwadebe reagent nke gị: ngwa mmịpụta nucleic acid.
【Storage conditions na expiration date 】
For BST-SARS-25:Ụgbọ njem na ụlọ ahịa na -20 ± 5 ℃ ruo ogologo oge.
For BST-SARS-DR-25:Ụgbọ njem na ụlọ okpomọkụ. Na-echekwa na -20±5 ℃ ruo ogologo oge.
Zenarị okirikiri ifriizi ugboro ugboro. Edebere oge nkwado ahụ maka ọnwa 12.
Hụ akara maka ụbọchị arụpụtara na ojiji.
Mgbe emepechara mbụ, enwere ike ịchekwa reagent na -20 ± 5 Celsius C maka ọnwa 1 ma ọ bụ ruo na njedebe nke oge reagent, ụbọchị ọ bụla na-abịa na mbụ, iji zere usoro ịgbaze ugboro ugboro, yana ọnụọgụ reagent ifriizi. -mgbaze cycles ekwesịghị gafere 6 ugboro.
【Applicable instrument】ABI 7500, SLAN-96P, Roche-LightCycler-480.
【Sample requirements 】
Ụdị ihe atụ nke 1.Adabara: Ihe ngwọta nucleic acid wepụrụ.
2.Sample nchekwa na njem: Chekwaa na-20 ± 5 ℃ maka ọnwa 6. Kwụsị ma mebie ihe nlele ahụ karịa ugboro 6.
【Testing method】
1.Nucleic acid extraction
Họrọ ngwa mmịpụta nucleic acid dabara adaba iji wepụta nje nucleic acid, ma soro ntuziaka ngwa kwekọrọ. A na-atụ aro ka iji ngwa mmịpụta na mkpocha nke Nucleic acid mepụtara nke Yixin Bio-Tech (Guangzhou) Co., Ltd. ma ọ bụ ngwa nrụcha acid nucleic dakọtara.
2. Reaction reagent preparation
2.1 For BST-SARS-25:
( 1) Wepu SARS-Cov-2 Mix na Enzyme Mix , gbazee kpamkpam na ụlọ okpomọkụ ngwakọta nke ọma site na ngwaọrụ Vortex wee centrifuge obere oge.
(2) 16.5uL Enzyme Mix agbakwunyere na 358.5uL SARS-Cov-2 Mix wee gwakọta nke ọma iji nweta azịza mmeghachi omume agwakọta.
(3) Kwadebe 0.2 mL PCR octal tube dị ọcha ma kaa ya na 15uL nke ngwọta mmeghachi omume agwakọta dị n'elu kwa nke ọma.
(4) Tinye 15 μL nke ngwọta nucleic acid dị ọcha, njikwa dị mma na njikwa na-adịghị mma, ma jiri nlezianya kpuchie okpu octal tube.
(5) Gwakọta nke ọma site n'ịtụgharị ihu na ihu, na ngwa ngwa centrifuge ka itinye uche na mmiri mmiri na ala nke tube.
1
2.2 For BST-SARS-DR-25:
(1) Tinye 375ul 2x Buffer na SARS-Cov-2 Mix ((Lyophilised) iji kwadebe ngwakọta mmeghachi omume, gwakọta nke ọma site na pipetting wee centrifuge obere oge. nchekwa ogologo oge.)
(2) Kwadebe 0.2 mL PCR octal tube dị ọcha ma kaa ya na 15μL nke mmeghachi omume ngwakọta kwa nke ọma.
(3) Tinye 15μL nke ngwọta nucleic acid dị ọcha, njikwa dị mma na njikwa na-adịghị mma, ma jiri nlezianya kpuchie okpu octal tube.
(4) Gwakọta nke ọma site n'ịtụgharị ihu na ihu, na ngwa ngwa centrifuge ka itinye uche na mmiri mmiri na ala nke tube.
3. PCR amplification (Biko rụtụ aka na ntuziaka ngwa maka ntọala ọrụ.)
3. 1 Tinye PCR 8-tube n'ime ụlọ nlele nke ngwa PCR fluorescent, ma debe ihe nlele a ga-anwale, njikwa dị mma na njikwa na-adịghị mma dịka usoro nke ntinye.
3.2 Ọwa nchọpụta fluorescence:
(1) ORF1ab gene na-ahọrọ ọwa nchọpụta nke FAM (Onye nta akụkọ: FAM, Quencher: Ọ dịghị).
(2) N gene na-ahọrọ ọwa nchọpụta nke VIC (Onye nta akụkọ: VIC, Quencher: Ọ dịghị).
(3) Mkpụrụ ndụ ihe nketa dị n'ime na-ahọrọ ọwa nchọpụta nke CY5 (Onye nta akụkọ: CY5, Quencher: Ọ dịghị).
(4) Ntụgharị aka na-agafe agafe ka edobere na ROX.
3.3 PCR ntọala oke mmemme:
| Nzọụkwụ | Okpomọkụ (℃) | Oge | Ọnụọgụ okirikiri | |
| 1 | Ntugharị ntụgharị ntụgharị | 50 | 15 min | 1 |
| 2 | Ịrụ ọrụ enzyme Taq | 95 | Nkeji 2.5 | 1 |
| 3 | Ịrụ ọrụ enzyme Taq | 93 | 10 s | 43 |
| Mgbakwunye ndọtị na nnweta fluorescence | 55 | 30 s | ||
Mgbe ịtọlitechara, chekwaa faịlụ ma mee mmemme mmeghachi omume.
4.Results analysis
Mgbe mmemme ahụ gwụchara, a na-echekwa nsonaazụ ya na-akpaghị aka, a na-enyocha usoro nkwalite ahụ. A na-edozi usoro nkwalite ahụ ka ọ bụrụ ebe ndabere ngwaọrụ.
【Explanation of test results 】
1. Kpebisie ike na nnwale ahụ ziri ezi: FAM njikwa dị mma, ọwa VIC kwesịrị inwe usoro mmụba na-ahụkarị, yana uru Ct na-adịkarị obere karịa 34, mana enwere ike ịgbanwe n'ihi ntọala dị iche iche nke ngwa dị iche iche. FAM njikwa na-adịghị mma, ọwa VIC kwesịrị ịbụ Ct. Ekwenyere na a ga-emezurịrị ihe ndị a chọrọ n'otu oge, ma ọ bụghị ya, ule a adịghị mma.
2. Nsonaazụ ikpe
| FAM/VIC ọwa | Nsonaazụ ikpe |
| Ct 37 | Nnwale nlele dị mma |
| 37≤CT40 | Usoro nkwalite ahụ bụ ụdị S, yana ihe nlele ndị enyo enyo kwesịrị ka enyochagharị ọzọ; ọ bụrụ na nsonaazụ nyocha ahụ na-agbanwe agbanwe, a na-ekpe ya ikpe dị ka ihe dị mma, ma ọ bụghị na ọ dị njọ |
| Ct≥40 ma ọ bụ enweghị nkwalite | Nnwale nlele adịghị mma (ma ọ bụ n'okpuru njedebe nke nchọpụta ngwa) |
Mara: ( 1) Ọ bụrụ na ọwa FAM na ọwa VIC dị mma n'otu oge, SARS-Cov-2 kpebisiri ike ịdị mma.
(2) Ọ bụrụ na ọwa FAM ma ọ bụ ọwa VIC dị mma na ọwa nke ọzọ adịghị mma, a ga-emegharị ule ahụ ọzọ. Ọ bụrụ na ọ dị mma n'otu oge, a ga-ekpe ya ikpe dị ka SARS-Cov-2 ziri ezi, ma ọ bụghị ya, a ga-ekpe ya ikpe dị ka SARS-Cov-2 adịghị mma.
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