Nje Virus Monkeypox (MPV) Ngwa nchọpụta Nucleic Acid

Ngwa ahụ nwere reagents maka nhazi ule 48 ma ọ bụ njikwa mma, gụnyere ihe ndị a:

Reagent A

ReagentB

Mara: Enweghị ike iji ihe mejupụtara ọnụọgụ ọnụọgụ dị iche iche

【Ọnọdụ Nchekwa na Ndụ Shelf】

1.Reagent A / B nwere ike ịchekwa na 2-30 ° C, na ndụ nchekwa bụ ọnwa 10.

2.Biko mepee mkpuchi tube ule naanị mgbe ị dị njikere maka ule ahụ.

3.Una eji tubes ule gafere ụbọchị njedebe.

4.Adịla eji tube nchọpụta na-agbapụta.

【Ngwa ngwa】

Kwesịrị ekwesị maka usoro nyocha LC480 PCR, Gentier 48E Automatic PCR analysis system, ABI7500 PCR analysis system.

【Ihe atụ chọrọ】

Ụdị ihe atụ 1.Ọdabara: akpịrị swabs sample.

2.Sampling ngwọta:Mgbe nkwenye gasịrị, a na-atụ aro ka iji saline nkịtị ma ọ bụ tube nchekwa nje nke Hangzhou Testsea biology mepụtara maka nchịkọta nlele.

akpịrị akpịrị:hichaa tonsils pharyngeal bilateral na mgbidi pharyngeal nke dị n'azụ na swab nke enwere ike iwepu ya, gbanye swab n'ime tube nke nwere ngwọta nlele 3mL, tụfuo ọdụ, wee mee ka mkpuchi tube sie ike.

3.Sample nchekwa na nnyefe:Ekwesịrị ịnwale ihe nlele a ga-anwale ozugbo enwere ike.The njem okpomọkụ kwesịrị nọ na 2 ~ 8 ℃.The samples na ike nwalere n'ime 24 awa nwere ike ịchekwa na 2 ℃ ~ 8 ℃ na ọ bụrụ na sample-apụghị nwalere n'ime 24 awa, ọ ga-echekwara na-erughị ma ọ bụ hà nhata. ka -70 ℃ (ma ọ bụrụ na e nweghị nchekwa ọnọdụ nke -70 ℃, ọ nwere ike ịchekwa na -20 ℃ nwa oge), zere ugboro ugboro.

kefriza na thawing.

4.Nchịkọta ihe atụ kwesịrị ekwesị, nchekwa, na njem dị oke mkpa maka ịrụ ọrụ nke ngwaahịa a.

【Usoro ule】

1.Sample nhazi na ntinye ihe atụ

1.1 Nhazi nhazi

Mgbe ịgwakọta ihe ngwọta nlele dị n'elu na ihe nlele, were 30μL nke nlele ahụ n'ime tube reagent nke DNA ma gwakọta ya nke ọma.

1.2 na-ebu

Were 20μL nke reconstitution reagent ma tinye ya na MPV nchọpụta reagent, gbakwunye 5μL nke n'elu esichara sample (The mma njikwa na-adịghị mma akara ga-esichara n'otu n'otu na samples), kpuchie tube okpu, centrifuge ya na 2000rpm maka 10. sekọnd.

2. PCR nkwalite

2.1 Ibu Ibu PCR efere / tubes a kwadebere na ngwa PCR fluorescence, njikwa na-adịghị mma na njikwa dị mma ga-edozi maka ule ọ bụla.

2.2 Ntọala ọwa fluorescent:

1) Họrọ ọwa FAM maka nchọpụta MPV;

2) Họrọ ọwa HEX / VIC maka nchọpụta mkpụrụ ndụ n'ime njikwa;

3.Ntụle nsonaazụ

Tọọ ahịrị ntọala n'elu ebe kachasị elu nke mgbanaka fluorescent nke njikwa adịghị mma.

4. Quality njikwa

4.1 njikwa adịghị mma: Enweghị uru Ct achọpụtara na FAM, ọwa HEX / VIC, ma ọ bụ Ct＞40;

4.2 njikwa dị mma: Na FAM, ọwa HEX / VIC, Ct≤40;

4.3 Ihe ndị a chọrọ n'elu kwesịrị inwe afọ ojuju n'otu nnwale ahụ, ma ọ bụghị ya, nsonaazụ ule abaghị uru na nnwale ahụ kwesịrị ka emeghachi ya.

【Bipụ uru】

A na-ahụta nlele anya dị ka ihe dị mma mgbe: Usoro ebumnuche Ct≤40, mkpụrụ ndụ njikwa ime Ct≤40.

【Nkọwapụta nsonaazụ】

Ozugbo agafere njikwa mma, ndị ọrụ kwesịrị ịlele ma ọ bụrụ na ọ nwere ntụgharị mmụba maka nlele ọ bụla na ọwa HEX / VIC, ọ bụrụ na enwere yana yana Ct≤40, ọ na-egosi na mkpụrụ ndụ njikwa nke ime na-abawanye nke ọma na nnwale a dị irè.Ndị ọrụ nwere ike ịga n'ihu na nyocha ndị a:

3.For samples na amplification nke esịtidem akara gene dara (HEX / VIC

ọwa, Ct ＞40, ma ọ bụ enweghị nkwalite nkwalite), obere Viral ibu ma ọ bụ ịdị adị nke PCR inhibitor nwere ike ịbụ ihe kpatara ọdịda, nyocha ahụ kwesịrị ikwughachi ya site na nchịkọta ihe atụ;

4.For nti samples na omenala nje, pụta nke esịtidem akara adịghị emetụta;

Maka ihe nlele nwalere adịghị mma, ọ dị mkpa ka enyocha njikwa ime ahụ nke ọma ma ọ bụghị na nsonaazụ ya abaghị uru ma ọ dị mkpa ka emeghachi nyocha ahụ, malite na usoro nchịkọta ihe atụ.