Nje Virus Monkeypox Real Time PCR Kit
Nkọwa ngwaahịa
Ezubere iji
A na-eji ya maka ịchọpụta Virus Monkeypox na ọbara mmadụ ma ọ bụ ọnya exudate sample site na iji ezigbo PCR sistemu.
Ụkpụrụ ule
Ngwaahịa a bụ sistemụ nyocha PCR ozugbo Taqman® dabere na fluorescent.Emebere ihe izizi na nyocha ndị akọwapụtara maka ịchọpụta mkpụrụ ndụ F3L nke Virus Monkeypox.Njikwa ime na-ezubere iche mkpụrụ ndụ ihe nketa echekwara na-enyocha mkpokọta ihe nlele, njikwa nlele na usoro PCR ozugbo iji zere nsonaazụ na-adịghị mma.Ngwa ahụ bụ sistemu lyophilized premix zuru oke, nke gụnyere ihe ndị achọrọ maka nchọpụta nje Monkeypox: nucleic acid amplification enzyme, UDG enzyme, ihe nchekwa mmeghachi omume, primer kpọmkwem na nyocha.
Isi ọdịnaya
Edepụtara ihe ndị enyere na tebụl.
| Ngwa | ngwugwu | Ngwakọta |
| Nje Virus MonkeypoxLyophilized Premix | 8 eriri PCR tubes× 6 akpa | Primers, nyocha, dNTP/dUTP Mix, Mg2+, Taq DNA polymerase, UDG Enzyme |
| Njikwa mma MPV | 400 μL × 1 tube | Usoro DNA nwere mkpụrụ ndụ Target |
| Njikwa adịghị mma MPV | 400 μL × 1 tube | Usoro DNA nwere akụkụ mkpụrụ ndụ mmadụ |
| Ngwọta Mgbasa | 1 ml × 1 tube | Stabilizer |
| Asambodo Nkwekọrịta | 1 ibe | / |
Usoro ọrụ
1. Ihe atụmkpokọta:Ekwesịrị ịchịkọta ihe atụ n'ime tubes na-adịghị mma dịka
na ọkọlọtọ oru nkọwa.
2. Nkwadebe Reagent (Ebe Nkwadebe Reagent)
Wepụ ihe ndị dị na kit ahụ, dozie ha na ọnụ ụlọ maka ojiji nọrọ na njikere.
3. Nhazi ihe nlere (mpaghara nhazi ụdị)
3.1 Nucleic acid mmịpụta
A na-atụ aro ka ị were ihe nlele mmiri 200μL, njikwa ziri ezi na njikwa adịghị mma maka mmịpụta acid nucleic, dị ka ihe achọrọ na usoro kwekọrọ na ngwa mmịpụta DNA.
3.2 Lyophilized ntụ ntụ dissolving na ndebiri mgbakwunye
Kwadebe Nje Virus Monkeypox Lyophilized premix dabere na ọnụọgụ nlele.Otu ihe nlele chọrọ otu PCR tube nwere ntụ ntụ premix Lyophilized.A ga-emeso njikwa na-adịghị mma & njikwa dị mma dị ka ihe nlele abụọ.
(1) Tinye 15μL Dissolving Solution n'ime PCR ọ bụla nwere Lyophilized premix, wee tinye 5μL amịpụtara sample/Ngative Control/Positive Control n'ime PCR ọ bụla tube n'otu n'otu.
(2) Kpuchie tubes PCR nke ọma, jiri aka tinye ọkpọkọ PCR ruo mgbe agbazere ntụ ntụ lyophilized kpamkpam wee gwakọta ya, nakọta mmiri mmiri na ala nke tubes PCR site na obere obere ọsọ centrifugation ozugbo.
(3) Ọ bụrụ na-eji ngwa PCR oge niile maka nchọpụta, wee bufee PCR ozugbo na mpaghara nkwalite;Ọ bụrụ na-eji BTK-8 maka nchọpụta, mgbe ahụ mkpa ịrụ ọrụ ndị a: nyefee 10 μL mmiri mmiri si PCR tube na mmeghachi omume mgbawa ọma nke BTK-8.Otu tube PCR dabara na otu olulu mmiri dị na mgbawa.N'oge pipetting arụ ọrụ, hụ na pipette bụ 90 degrees vetikal.A ga-etinye ndụmọdụ aerosol mgbochi pipette n'etiti olulu mmiri ahụ na ike dị oke ala ma kwụsị ịkwanye pipette mgbe ọ ruru ihe mbụ (iji zere afụ).Mgbe emejuchara olulu mmiri ahụ, wepụ ihe mgbawa mmeghachi omume iji kpuchie olulu mmiri niile wee bufee mgbawa ahụ na mpaghara nchọpụta mmụba.
4. PCR Amplification (mpaghara nchọpụta)
4.1 Tinye PCR tubes / mmeghachi omume mgbawa n'ime tank mmeghachi omume ma tọọ aha mmeghachi omume ọ bụla nke ọma n'usoro kwekọrọ.
4.2 Ntọala nke nchọta fluorescence: (1) Monkeypox virus (FAM);(2) Njikwa ime (CY5).
4.3 Gbaa usoro ịgba ígwè na-esote
Protocol nke ABI7500, Bio-Rad CFX96, SLAN-96S, QuantStudio:
| Nzọụkwụ | Okpomọkụ | Oge | okirikiri | |
| 1 | Tupu denaturation | 95 ℃ | Nkeji 2 | 1 |
| 2 | Denaturation | 95 ℃ | 10 s | 45 |
| Mwepu, ndọtị, nweta fluorescence | 60 ℃ | 30 s | ||
Protocol nke BTK-8:
| Nzọụkwụ | Okpomọkụ | Oge | okirikiri | |
| 1 | Tupu denaturation | 95 ℃ | Nkeji 2 | 1 |
| 2 | Denaturation | 95 ℃ | 5 s | 45 |
| Mwepu, ndọtị, nweta fluorescence | 60 ℃ | 14 s | ||
5. Nyochaa nsonaazụ (biko rụtụ aka na ntuziaka onye ọrụ akụrụngwa)
Mgbe mmeghachi omume ahụ gasịrị, a ga-echekwa nsonaazụ ya na-akpaghị aka.Pịa "Nyochaa" iji nyochaa, ngwa ahụ ga-akọwa ụkpụrụ Ct nke ihe nlele ọ bụla na kọlụm nsonaazụ.Nsonaazụ njikwa na-adịghị mma na nke dị mma ga-agbaso "6. Njikwa ogo ".
6. Njikwa ogo
6.1 Njikwa adịghị mma: Enweghị Ct ma ọ bụ Ct> 40 na ọwa FAM, Ct≤40 na ọwa CY5 nwere usoro mmụba nkịtị.
6.2 njikwa dị mma: Ct≤35 na ọwa FAM nwere usoro mmụba nkịtị, Ct≤40 na ọwa CY5 nwere usoro mmụba nkịtị.
6.3 Nsonaazụ dị irè ma ọ bụrụ na emezuru njirisi niile dị n'elu.Ma ọ bụghị ya, nsonaazụ ya abaghị uru.
Nkọwapụta nsonaazụ
Nsonaazụ ndị a ga-ekwe omume:
| Ct uru nke ọwa FAM | Ct uru nke ọwa CY5 | Nkọwa | |
| 1# | Enweghị Ct ma ọ bụ Ct> 40 | ≤40 | Ọrịa enwe adịghị njọ |
| 2# | ≤40 | Nsonaazụ ọ bụla | Nje Virus Monkeypox dị mma |
| 3# | 40–45 | ≤40 | Nnwale ọzọ;Ọ bụrụ na ọ ka bụ 40 ~ 45, kwuo dị ka 1 # |
| 4# | Enweghị Ct ma ọ bụ Ct> 40 | Enweghị Ct ma ọ bụ Ct> 40 | Na adịghị mma |
IHE: Ọ bụrụ na nsonaazụ na-ezighi ezi emee, ọ dị mkpa ka achịkọta ihe nlele ahụ wee nwalee ya ọzọ.
Ozi ịtụ
| Aha ngwaahịa | Nwamba.Mba | Nha | Ụdị | Ndụ shelf | Trans.& Sto.Okpomọkụ |
| Nje Virus Monkeypox Real Time PCR Kit | B001P-01 | 48 ule / ngwa | Ọbara / Ọnya Exudate | Ọnwa iri na abụọ | -25 ～-15 ℃ |
