{"id":3021,"date":"2024-06-25T23:48:14","date_gmt":"2024-06-25T15:48:14","guid":{"rendered":"https:\/\/opentrons.com.cn\/?post_type=knowledge2&p=3021"},"modified":"2024-09-02T18:57:05","modified_gmt":"2024-09-02T10:57:05","slug":"syrnadvance-viralheot-2pt","status":"publish","type":"post","link":"https:\/\/opentrons.com.cn\/news\/syrnadvance-viralheot-2pt\/","title":{"rendered":"\u4f7f\u7528RNAdvance Viral\u548cOT-2\u5e73\u53f0\uff0c\u8fdb\u884c\u9ad8\u6548\u4e14\u9ad8\u901a\u91cf\u7684SARS-CoV-2\u75c5\u6bd2RNA\u68c0\u6d4b\u53ca\u6d4b\u5e8f\u7814\u7a76"},"content":{"rendered":"\n

\u5e94\u7528\u4ecb\u7ecd
2020\u5e74\u65b0\u51a0\u75c5\u6bd2\u8086\u8650\u5168\u7403\uff0c\u6fc0\u5316\u4e86\u4eba\u7c7b\u5bf9\u4e8e\u4e34\u5e8a\u7814\u7a76\u76ee\u7684\u7684\u9ad8\u901a\u91cf RNA \u68c0\u6d4b\u548c\u57fa\u56e0\u6d4b\u5e8f\u7684\u9700\u6c42\u3002\u4f20\u7edf\u624b\u52a8\u64cd\u4f5c\u65b9\u6848\u96be\u4ee5\u63d0\u5347RNA \u68c0\u6d4b\u7684\u6548\u7387\u3002\u5728\u672c\u9879\u7814\u7a76\u4e2d\uff0c\u6211\u4eec\u5c55\u793a\u4e86\u5728 Opentrons OT-2 \u81ea\u52a8\u5316\u79fb\u6db2\u5de5\u4f5c\u7ad9\u4e0a\u4f7f\u7528 Beckman Coulter Life Sciences RNAd- vance Viral \u8bd5\u5242\u76d2 (C63510) \u7684\u81ea\u52a8\u5316 SARS-CoV-2 RNA \u63d0\u53d6\u5de5\u4f5c\u6d41\u7a0b\u3002 \u6211\u4eec\u63d0\u4f9b\u7684\u5206\u6790\u6570\u636e\u663e\u793a\u4e86\u901a\u8fc7 qRT-PCR \u5b9a\u91cf\u7684SARS-CoV-2 \u68c0\u6d4b\u9650(LoD) \u548c\u5e8f\u5217\u6bd4\u5bf9\u6570\u636e\uff0c\u8fd9\u4e9b\u6570\u636e\u4e3a\u81ea\u52a8\u6d4b\u5e8f\u6837\u54c1\u548c\u6587\u5e93\u5236\u5907\u63d0\u4f9b\u4e86\u4f9d\u636e\uff0c\u4ee5\u4fbf\u5bf9\u7a81\u53d8\u682a\u8fdb\u884c\u9057\u4f20\u9274\u5b9a\u3002<\/p>\n\n\n\n

RNAdvance \u75c5\u6bd2\u8bd5\u5242\u76d2\u548c OT-2 \u5e73\u53f0\u6982\u8ff0
RNAdvance Viral kit \u901a\u8fc7\u5229\u7528 SPRI \u987a\u78c1\u73e0\u6280\u672f\uff0c\u4ece\u6837\u54c1\u4e2d\u9ad8\u6548\u63d0\u53d6\u75c5\u6bd2RNA\u3002 \u6211\u4eec\u5728\u81ea\u52a8\u5316\u7684 Opentrons \u5e73\u53f0\u4e0a\u4f7f\u7528\u4e86\u8fd9\u79cd\u8bd5 \u5242\uff0c\u4ece\u5408\u6210\u9f3b\u57fa\u8d28(110mM NaCl,1%w\/v\u7684\u732a\u80c3I \u578b\u7c98\u6db2 (Sigma M2378-100G) \u548c10\u03bcg\/mL w\/v\u4eba\u7c7b\u57fa\u56e0\u7ec4 DNA(Coriell NA12878), \u4ee590%v\/v \u7684TE\/SNM) \u6837\u54c1\u4e2d\u63d0\u53d6\u4e86\u4e0d\u540c\u6d53\u5ea6\u7684\u70ed\u706d\u6d3b\u7684 SARS-CoV-2(ATCC\u00b0VR-1986HKTM) \u3002 \u6bcf\u4e2a\u6d53\u5ea6\u5305\u62ec20 \u4e2a\u6d4b\u8bd5\u6837\u54c1\u30012\u4e2a\u9633\u6027\u5bf9\u7167\u548c2\u4e2a\u9634\u6027\u5bf9\u7167\u3002<\/p>\n\n\n\n

\u5de5\u4f5c\u6d41\u7a0b\u541e\u5410\u91cf
\u56fe1\u663e\u793a\u4e86\u81ea\u52a8\u5316 RNAdvance \u75c5\u6bd2\u63d0\u53d6\u5de5\u4f5c\u6d41\u7a0b\u7684 OT-2\u7532\u677f\u5e03\u5c40\u3002\u8be5\u5de5\u4f5c\u6d41\u7a0b\u4ece\u5411\u6837\u54c1\u88c2\u89e3\u6db2\u4e2d\u6dfb\u52a0 Bind (BBB)\u5f00\u59cb\uff0c\u6bcf\u4e2a\u6837 \u54c1\u4ec5\u9700\u8981\u6d88\u801710\u4e2a\u5438\u5934\uff0c\u53ef\u7075\u6d3b\u8fd0\u884c8\u4e2a\u6279\u6b21\u5927\u5c0f\u7684\u6700\u591a96\u4e2a\u6837\u672c\u3002\u62a5\u544a\u4e2d\u63cf\u8ff0\u7684\u5de5\u4f5c\u6d41\u7a0b\u6301\u7eed\u65f6\u95f4\u5305\u62ec\u5b9e\u9645\u64cd\u4f5c\u65f6\u95f4\u3001\u6837\u54c1\u88c2\u89e3\u65f6\u95f4\u548c\u4f18\u5316\u540e\u6837\u54c1\u7684\u8fd0\u884c\u65f6\u95f4(\u56fe2)\u3002
\u4f7f\u7528 RNAdvance Viral\u8bd5\u5242\u76d2\u548cOT-2\u5e73\u53f0\uff0c\u5904\u740696\u4e2a\u6837\u672c\u7684\u5de5\u4f5c\u6d41\u7a0b\u603b\u5171\u9700\u89813.5\u5c0f\u65f6\uff0c\u60a8\u53ef\u4ee5\u590d\u5236\u94fe\u63a5\u5230\u6d4f\u89c8\u5668\uff0c\u67e5\u770b\u5177\u4f53\u534f\u8bae\u6d41\u7a0b\uff1a https:\/\/protocols.opentrons.com\/protocol\/bc-rnadvance-viral\u3002\u60a8\u53ef\u4ee5\u6839\u636e\u60a8\u7684\u9700\u6c42\u7075\u6d3b\u5730\u6539\u53d8\u65b9\u6cd5\u53d8\u91cf\uff0c\u4ee5\u83b7\u5f97\u60a8\u6240\u9700\u8981\u7684\u6279\u6b21\u5927\u5c0f\u3001\u6837\u54c1\u8f93\u5165\u548c\u6d17\u8131\u4f53\u79ef\u7684\u6240\u9700\u7ed3\u679c\u3002<\/p>\n\n\n\n

\"OT-2
\u56fe1:\u914d\u5907\u6a21\u5757\u3001\u5b9e\u9a8c\u8017\u6750\u548c<\/strong>\u00a0\u00a0RNAdvance \u00a0\u00a0\u00a0Viral \u00a0\u8bd5\u5242\u7684<\/strong>\u00a0\u00a0Opentrons \u00a0\u00a0\u00a0OT-2 \u00a0\u5b9e\u9a8c\u7532\u677f\u5e03\u5c40<\/strong><\/figcaption><\/figure>\n\n\n\n

\u8be5\u5de5\u4f5c\u6d41\u7a0b\u5728 \u00a0OT-2\u00a0\u4e0a\u662f\u53ef\u4ee5\u5168\u81ea\u52a8\u8fd0\u884c\u00a0\u00a0\u7684\uff0c\u6bcf\u4e2a\u6837\u54c1\u4f7f\u752810\u4e2a\u5438\u5934\uff0c\u5e76\u4ece\u52a0\u5165Bind(BBB) \u00a0\u00a0\u00a0\u5f00\u59cb\u3002\u5b9e\u9a8c\u7532\u677f\u4e0a\u8017\u6750\u5185\u5bb9\u6709\uff1a\u6700\u591a10\u4e2a\u5438\u5934\u67b6\u30011\u4e2a\u00a0NEST\u00a0\u5355\u5b54\u50a8\u6db2\u69fd\u30012\u4e2a\u00a0NEST 12\u00a0\u5b54\u50a8\u6db2\u69fd\u30011\u4e2a NEST\u00a00.1mL\u00a0PCR\u677f\u548c1\u4e2a\u00a0NEST \u00a02mL\u00a0\u6df1\u5b54\u677f\uff0c\u6a21\u5757\u5305\u62ec\u4e00\u4e2a\u78c1\u73e0\u7eaf\u5316\u6a21\u5757\u548c\u4e00\u4e2a\u6e29\u63a7\u6a21\u5757\uff0c\u914d\u5907\u4e00\u4e2a\u00a0P300\u00a0\u591a\u901a\u9053\u79fb\u6db2\u5668\u3002\u6240\u6709\u7684\u00a0NEST \u8017\u6750\u548c\u6a21\u5757\u90fd\u7531\u00a0Opentrons \u63d0\u4f9b\u3002<\/p>\n\n\n\n

\"\u5b9e\u9a8c\u6570\u636e\"<\/figure>\n\n\n\n
\"\u5b9e\u9a8c\u6570\u636e\"<\/figure>\n\n\n\n

\u56fe2:\u5728OT-2\u5e73\u53f0\u4e0a\u4f7f\u7528RNAdvance Viral\u8bd5\u5242\u76d2\u7684\u541e\u5410\u91cf\u60c5\u51b5
\u6574\u4e2a\u5de5\u4f5c\u6d41\u7a0b\u8fd0\u884c\u6240\u9700\u7684\u65f6\u95f4\u5305\u62ec3\u4e2a\u90e8\u5206\uff1a\u64cd\u4f5c\u65f6\u95f4\u3001\u88c2\u89e3\u65f6\u95f4\u548c\u8fd0\u884c\u65f6\u95f4\u3002\u64cd\u4f5c\u65f6\u95f4\u5305\u62ec\u6837\u54c1\u548cOT-2\u7684\u51c6\u5907\u5de5\u4f5c\u3002\u8fd0\u884c\u65f6\u95f4\u6307\u7684\u662f OT-2\u4ece\u5f00\u59cb\u5230\u5b8c\u6210\u6574\u4e2a\u534f\u8bae\u6240\u9700\u7684\u65f6\u95f4\u3002
A)\u5904\u74068\u4e2a\u6837\u54c1\u9700\u898145\u5206\u949f\uff0c\u5904\u740696\u4e2a\u6837\u54c1\u9700\u89812\u5c0f\u65f638\u5206\u3002
B) \u56fe\u5f62\u5316\u5c55\u793a\u4e86\u4e0d\u540c\u6837\u54c1\u6570\u91cf\u4e0b\u7684\u8fd0\u884c\u5904\u7406\u65f6\u95f4\u3002<\/p>\n\n\n\n

RNAdvance \u75c5\u6bd2\u8bd5\u5242\u76d2\u68c0\u6d4b\u9650\u5236
\u68c0\u6d4b\u9650\u5236(LoD)\u662f\u7528\u4e8e\u786e\u5b9aOT-2\u5e73\u53f0\u4e0a\u81ea\u52a8\u5316RNAdvance Viral\u63d0\u53d6\u6548\u7387\u7684\u6307\u6807\uff0c\u5b83\u8868\u793a\u80fd\u591f\u5728\u226595%\u7684\u91cd\u590d\u6d4b\u91cf\u4e2d\u68c0\u6d4b\u5230\u7684\u75c5\u6bd2\u62f7\u8d1d\u6570 \u7684\u6700\u4f4e\u6d53\u5ea6\u3002\u6211\u4eec\u4f7f\u75282019-nCoV CDC N1\u548cRP\u5f15\u7269\u548c\u63a2\u9488(2019-nCoV CDC EUA kit,Integrated DNA Technologies (IDT)) \u8fdb\u884c\u63a2 \u6d4b \uff0cPCR \u6df7\u5408\u6db2\u548c\u9176\u4f7f\u7528Luna\u00b0\u901a\u7528\u63a2\u9488\u4e00\u6b65RT-qPCR \u8bd5\u5242\u76d2(New England BioLabs),PCR\u53cd\u5e94\u5728PCRmax ECO48\u5b9e\u65f6PCR \u7cfb\u7edf\u4e0a \u8fdb\u884c\u3002\u5728SARS-CoV-2 \u6837\u672c (n=20) \u548c\u9633\u6027\u5bf9\u7167\u6837\u672c (n=2) \u4e2d\u68c0\u6d4b\u5230\u75c5\u6bd2RNA (\u62f7\u8d1d\u6570\/\u03bcL), \u4f46\u5728\u9634\u6027\u5bf9\u7167\u6837\u672c (n=2) \u4e2d\u672a\u68c0\u6d4b\u5230\u3002\u6839\u636e\u88681\u7684\u603b\u7ed3\uff0c\u5206\u6790\u8303\u56f4\u5185\u7684\u6240\u6709\u5bf9\u6bd4\u9633\u6027\u6837\u672c\u5747\u88ab\u786e\u5b9a\u4e3a\u9633\u6027\uff0c\u5176\u4e2d\u6700\u4f4e\u6d53\u5ea6\u7684\u75c5\u6bd2RNA \u62f7\u8d1d\u6570\u4e3a1\u62f7\u8d1d\/\u03bcL\u3002PCR \u53c2\u6570\u5982\u4e0b\u3002<\/p>\n\n\n\n

\"\u5b9e\u9a8c\u6570\u636e\"<\/figure>\n\n\n\n

\u88681:\u4f7f\u7528OT-2 \u5e73\u53f0\u548c RNAdvance Viral \u8bd5\u5242\u76d2\u68c0\u6d4b SARS-CoV-2 \u76ee\u6807 N1 \u7684\u75c5\u6bd2\u7075\u654f\u5ea6\u30021 copy\/\u03bcL \u662f\u80fd\u591f\u68c0\u6d4b\u5230\u226595%\u6837\u672c\u7684\u6700\u4f4e\u6d53\u5ea6\uff0c\u88ab\u5b9a\u4e49\u4e3a\u68c0\u6d4b\u9650\u5236 (Limit of Detection,LoD)<\/p>\n\n\n\n

\u4e3a\u4e86\u7814\u7a76 RNAdvance Viral\u5728 OT-2 \u4e0a\u7684\u81ea\u52a8\u5316\u6027\u80fd\uff0c\u6211\u4eec\u5c06850 copy\/\u03bcL \u6d53\u5ea6\u7684\u70ed\u706d\u6d3b SARS-CoV-2 \u75c5\u6bd2\u52a0\u5165\u5408\u6210\u9f3b\u8154\u57fa\u8d28\u4e2d\uff0c\u5e76\u63d0\u53d6RNA\u3002 \u9488\u5bf9N1\u548cRP, \u786e\u5b9a\u4e86\u5747\u503cCt\u503c\u3001\u6807\u51c6\u5dee\u3001\u53d8\u5f02\u7cfb\u6570 (CV%) \u548c\u68c0\u6d4b\u7387\u7b49\u89c4\u683c\u53c2\u6570(\u56fe3A)\u3002
\u5728\u5b9e\u9a8c\u6837\u672c(n=20)\u3001 \u9633\u6027\u5bf9\u7167\u6837\u672c(n=2) \u548c\u9634\u6027\u5bf9\u7167\u6837\u672c(n=2) \u4e2d\uff0c\u5bf9N1 \u7684\u6269\u589e\u66f2\u7ebf\u8fdb\u884c\u5b9a\u91cf\u5206\u6790(\u56fe3B), \u540c\u65f6\u5bf9\u5305\u62ec\u5bf9\u7167\u6837\u672c\u5728\u5185\u7684\u6837\u54c1\u4e2d\u7684RP\u8fdb\u884c\u5b9a\u91cf\u5206\u6790(\u56fe3C)\u3002<\/p>\n\n\n\n

\"\u5b9e\u9a8c\u7ed3\u679c\u8d8b\u52bf\u56fe\"<\/figure>\n\n\n\n

DNA \u6d4b\u5e8f\u548c\u6570\u636e\u5206\u6790
\u4e3a\u4e86\u8fdb\u4e00\u6b65\u4e86\u89e3\u6d4b\u5e8f\u6027\u80fd\uff0c\u6211\u4eec\u4f7f\u7528Swift 2S Turbo DNA\u6587\u5e93\u5236\u5907\u8bd5\u5242\u76d2\u548c Swift Unique Dual Indexing \u5f15\u7269\u677f\u8fdb\u884c N1\u6269\u589e\u3002\u5173\u4e8e850 copy\/\u03bcl \u6837\u672c\u7684\u8be6\u7ec6\u6570\u636e\u548c\u5e8f\u5217\u6bd4\u5bf9\u7ed3\u679c\u53ef\u4ee5\u5728\u56fe4 \u4e2d\u627e\u5230\u3002\u6211\u4eec\u89c2\u5bdf\u5230\u5e73\u5747\u8986\u76d6\u7387\u4e3a 455x, \u5e76\u4e14\u5e73\u5747\u8bfb\u6570\u670991%\u4e0e\u53c2\u8003\u57fa\u56e0\u7ec4 SARS-CoV-2 \u5206\u79bb\u81eaWuhan-Hu-1(NC_045512.2) \u5bf9\u9f50\u3002\u8bfb\u53d6\u957f\u5ea6\u4e0e N1\u6269\u589e\u533a\u57df\u76f8\u5339\u914d\uff0c\u5927\u7ea6\u4e3a72\u4e2a\u78b1\u57fa\u3002\u6839\u636eSwift 2s Turbo DNA \u6587\u5e93\u5236\u5907\u8bd5\u5242\u76d2\u5236\u9020\u5546\u7684\u8bf4\u660e\uff0c\u9884\u671f\u63d2\u5165\u7247\u6bb5\u5927\u5c0f\u7ea6\u4e3a 200bp\u3002 \u6d4b\u5e8f\u662f\u4f7f\u7528 lllumina MiSeq \u8fdb\u884c\u7684\uff0c\u5e76\u5c06\u7b5b\u9009\u6570\u636e\u4e0a\u4f20\u81f3llluminaBaseSpace\u3002\u5728 Galaxy \u4e2d\u4f7f\u7528 Bowtie2 \u786e\u5b9a\u4e86\u8986\u76d6\u5ea6\uff0c\u5e76\u4f7f\u7528 Integrative Genome Viewer(IGV) \u8fdb\u884c\u53ef\u89c6\u5316\u3002NC_045512.2,SAR2-COV-2, \u5206\u79bb\u6e90\u81eaWuhan-Hu-1 \u7684\u57fa\u56e0\u7ec4\u662f\u7528\u4e8e\u6d4b\u5e8f\u7684NCBI\u53c2\u8003\u57fa\u56e0\u7ec4\u3002\u901a\u8fc7\u5728 Galaxy \u4e2d\u4f7f\u7528QualiMap-BAMQC \u5de5\u5177\uff0c\u5206\u6790\u4e86\u8c03\u51c6\u3001\u8bfb\u957f\u3001\u7247\u6bb5\u5927\u5c0f\u548cCG\u542b\u91cf\u7b49\u4fe1\u606f\u3002<\/p>\n\n\n\n

\"\u5b9e\u9a8c\u7ed3\u679c\u5bf9\u6bd4\"<\/figure>\n\n\n\n

A) \u7efc\u5408\u6570\u636e QC \u8868\u63cf\u8ff0\u4e86\u591a\u4e2a\u6570\u636e\u70b9\uff0c\u5305\u62ec455-x \u7684\u5e73\u5747\u8986\u76d6\u7387\u548c\u4e0e\u53c2\u8003\u57fa\u56e0\u7ec4\u5bf9\u9f50\u7684\u5e73\u5747\u8bfb\u6570\uff0cSARS-CoV-2 \u5206\u79bb\u682a Wuhan-Hu-1 (NC_045512.2) \u4e3a91%\u3002\u6839\u636e Swift 2s Turbo DNA Library Prep \u5236\u9020\u5546\u7684\u9884\u671f\u63d2\u5165\u7247\u6bb5\u5927\u5c0f\u7ea6\u4e3a200bp\u3002
B)\u6bcf\u4e2a\u6837\u672c\u4e0e\u53c2\u8003\u57fa\u56e0\u7ec4\u5bf9\u9f50\u7684\u5bf9\u6bd4\u3002<\/p>\n\n\n\n

\u603b\u7ed3
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