瑷傝臣(g貌u)淇℃伅锛�
鐢�(ch菐n)鍝佸悕绋� | 銆€璞湏鐠�(hu谩n)鐥呮瘨1鍨�/2鍨�(PCV-1/PCV-2)鏍搁吀妾㈡脯(c猫)瑭﹀姂鐩� |
瑕�(gu墨)鏍� | 48T/鐩� |
椤炲垾 | 鐔掑厜-PCR娉� |
鐢�(ch菐n)鍝佸悕绋憋細璞湏鐠�(hu谩n)鐥呮瘨1鍨�/2鍨�(PCV-1/PCV-2)鏍搁吀妾㈡脯(c猫)瑭﹀姂鐩�
瑕�(gu墨)鏍硷細48T/鐩�
鍒嗛锛氱啋鍏�-PCR娉�
鍎�(ch菙)瀛樻浠讹細-20鈩冮伩鍏変繚瀛�锛岄伩鍏嶅弽寰�(f霉)鍑嶈瀺銆�
閬�(y霉n)杓革細浣庢韩銆侀伩鍏�锛屽揩閬炲厤璨�(f猫i)閫佽波涓婇杸銆�
鐗归粸(di菐n)锛�
1.鍗抽枊鍗崇敤锛岀敤鎴跺彧闇€瑕佹彁渚涙ǎ鍝� DNA 妯℃澘锛屾搷浣滅啊(ji菐n)鍠�锛屽畾閲忔簴(zh菙n)纰哄揩閫熴€�
2. 寮曠墿缍�(j墨ng)閬庡劒(y艒u)鍖�锛岀壒鐣版€у挤(qi谩ng)銆傞爯(y霉)鏈熺殑 PCR 鐢�(ch菐n)鐗╅暦(zh菐ng)搴︾偤 560 bp銆�
3. PCR mix 涓惈涓婃ǎ鏌撴枡锛孭CR 鍚庡彲浠ョ洿鎺ヤ笂妯i浕娉�銆�
4. 鎻愪緵闄�(y谩ng)鎬у皪(du矛)鐓�锛屼究浜庡垎鏋愯│椹�(y脿n)绲�(ji茅)鏋溿€傛敞鎰忎簨闋�(xi脿ng)锛�
1锛庡熀绀�(ch菙)绋嬪簭锛�
2锛�?d膩ng)U澧炴韩搴﹀拰寤朵几婧害锛�
3锛庡弽鎳�(y墨ng)鏅�(sh铆)闁擄紱
4锛庡惊鐠�(hu谩n)娆℃暩(sh霉)锛�
5锛嶱CR 鍙嶆噳(y墨ng)娑茬殑閰嶅埗锛�
6锛嶱CR鎶€琛�(sh霉)鐨勫熀鏈師鐞嗭紱
7锛嶱CR鐨勫弽鎳�(y墨ng)鍕�(d貌ng)鍔涘(xu茅)锛�
8锛嶱CR鎿�(ku貌)澧炵敘(ch菐n)鐗�锛�
9锛嶱CR鍙嶆噳(y墨ng)楂旂郴鑸囧弽鎳�(y墨ng)姊濅欢銆�
浣跨敤鍙婃晥鏋滐細
PCR鍙嶆噳(y墨ng)鐗归粸(di菐n)
(1) 鐗圭暟鎬у挤(qi谩ng)
PCR鍙嶆噳(y墨ng)鐨勭壒鐣版€ф焙瀹氬洜绱犵偤锛�
銆€銆€?銆佸儢類烽敱c妯℃澘DNA鐗圭暟姝g⒑鐨勭祼(ji茅)鍚�锛�
銆€銆€?銆佽冬A鍩洪厤灏�(du矛)鍘熷墖锛�
銆€銆€?銆佽房aq DNA鑱氬悎閰跺悎鎴愬弽鎳�(y墨ng)鐨勫繝瀵�(sh铆)鎬�锛�
銆€銆€?銆侀Θ璋㈩媰铏诲ザ绂烘剫鑰榗淇濆畧鎬�銆�
鍏朵腑寮曠墿鑸囨ā鏉跨殑姝g⒑绲�(ji茅)鍚堟槸闂�(gu膩n)閸�銆傚紩鐗╄垏妯℃澘鐨勭祼(ji茅)鍚堝強寮曠墿閺堢殑寤朵几鏄伒寰牽鍩洪厤灏�(du矛)鍘熷墖鐨勩€傝仛鍚堥叾鍚堟垚鍙嶆噳(y墨ng)鐨勫繝瀵�(sh铆)鎬у強Taq DNA鑱氬悎閰惰€愰珮婧€�锛屼娇鍙嶆噳(y墨ng)涓ā鏉胯垏寮曠墿鐨勭祼(ji茅)鍚�(寰�(f霉)鎬�)鍙互鍦ㄨ純楂樼殑婧害涓嬮€�(j矛n)琛�锛岀祼(ji茅)鍚堢殑鐗圭暟鎬уぇ澶у鍔狅紝琚摯(ku貌)澧炵殑闈跺熀鍥犵墖娈典篃灏辫兘淇濇寔寰堥珮鐨勬纰哄害銆傚啀閫氶亷閬告搰鐗圭暟鎬у拰淇濆畧鎬ч珮鐨勯澏鍩哄洜鍗€(q奴)锛屽叾鐗圭暟鎬х▼搴﹀氨鏇撮珮銆�
(2) 闈堟晱搴﹂珮
PCR鐢�(ch菐n)鐗╃殑鐢熸垚閲忔槸浠ユ寚鏁�(sh霉)鏂瑰紡澧炲姞鐨�锛岃兘灏囩毊鍏�(pg=10-12g)閲忕礆(j铆)鐨勮捣濮嬪緟娓�(c猫)妯℃澘鎿�(ku貌)澧炲埌寰厠(ug=10-6g)姘村钩銆傝兘寰�100钀�(w脿n)鍊�(g猫)绱�(x矛)鑳炰腑妾㈠嚭涓€鍊�(g猫)闈剁窗(x矛)鑳烇紱鍦ㄧ梾姣掔殑妾㈡脯(c猫)涓�锛孭CR鐨勯潏鏁忓害鍙仈(d谩)3鍊�(g猫)RFU(绌烘枒褰㈡垚鍠綅)锛涘湪绱�(x矛)鑿屽(xu茅)涓瓃ui灏忔鍑虹巼鐐�3鍊�(g猫)绱�(x矛)鑿�銆�
(3) 绨�(ji菐n)渚裤€佸揩閫�
PCR鍙嶆噳(y墨ng)鐢ㄨ€愰珮婧殑Taq DNA鑱氬悎閰�锛屼竴娆℃€у湴灏囧弽鎳�(y墨ng)娑插姞濂藉悗锛屽嵆鍦―NA鎿�(ku貌)澧炴恫鍜屾按娴撮崑涓婇€�(j矛n)琛岃畩鎬�-閫€鐏�-寤朵几鍙嶆噳(y墨ng)锛屼竴鑸湪2锝�4 灏忔檪(sh铆)瀹屾垚鎿�(ku貌)澧炲弽鎳�(y墨ng)銆傛摯(ku貌)澧炵敘(ch菐n)鐗╀竴鑸敤闆绘吵鍒嗘瀽锛屼笉涓€瀹氳鐢ㄥ悓浣嶇礌锛岀劇(w煤)鏀惧皠鎬ф薄鏌撱€佹槗鎺ㄥ唬銆�
(4) 灏�(du矛)妯�(bi膩o)鏈殑绱斿害瑕佹眰浣�
涓嶉渶瑕佸垎闆㈢梾姣掓垨绱�(x矛)鑿屽強鍩归(y菐ng)绱�(x矛)鑳烇紝DNA 绮楀埗鍝佸強绺絉NA鍧囧彲浣滅偤鎿�(ku貌)澧炴ā鏉�?锛熻妽鑻綉闂≧搴婃(bi膩o)鏈琛€娑层€侀珨鑵旀恫銆佹礂鍡芥恫銆佹瘺鐧�(f膩)銆佺窗(x矛)鑳�銆佹椿PCR鎶€琛�(sh霉)姒傝珫銆�