【《病原学检测COVID-19的方法概述》1800字】

图1.1）表明ADDINEN.CITEADDINEN.CITE.DATA[4]，SARS-CoV-2属于β属冠状病毒，是一种包膜单链RNA病毒。其基因组包含10个亚基因组：1个非结构蛋白基因开放阅读框1a/b（openreadingframes,ORF1a/b）、4个结构蛋白基因（S,E,M,N）、5个辅助蛋白基因开放阅读框3a,6,7a/b,8,10（ORF3a,6,7a/b,8,10），目前常用方法是对ORF1a/b、N或E基因进行靶向检测以确定样本病毒载量。图STYLEREF1\s1.SEQ图\*ARABIC\s11SchematicPresentationoftheSARS-CoV-2GenomeOrganization,theCanonicalSubgenomicmRNAs,andtheVirionStructureADDINEN.CITEADDINEN.CITE.DATA[4]SARS-CoV-2基因组组织、典型亚基因组mRNA和病毒粒子结构示意图常见的生物标本种类包括上呼吸道拭子（鼻、咽拭子），下呼吸道标本（痰或气道抽取物），其他（血液、粪便、尿液）等，研究表明下呼吸道标本的检测效果更加准确ADDINEN.CITEADDINEN.CITE.DATA[1,5]。根据操作流程及检测原理不同，检测方法可分为三类：变温扩增、恒温扩增和测序方法ADDINEN.CITEADDINEN.CITE.DATA[6]，图1.2总结了当前用于SARS-CoV-2的病原性检测方法，表1.2整理了国家药品监督管理局已批准（截至2021年2月10日）的新型冠状病毒（2019-nCoV）核酸检测试剂盒ADDINEN.CITE<EndNote><Cite><Author>国家药品监督管理局</Author><Year>2021</Year><RecNum>599</RecNum><DisplayText><styleface="superscript">[7]</style></DisplayText><record><rec-number>599</rec-number><foreign-keys><keyapp="EN"db-id="5xrwx9ze32f25qedez6xess8092avpsepvx5"timestamp="1620528365">599</key></foreign-keys><ref-typename="WebPage">12</ref-type><contributors><authors><author><styleface="normal"font="default"charset="134"size="100%">国家药品监督管理局</style></author></authors></contributors><titles><title><styleface="normal"font="default"charset="134"size="100%">国家药品监督管理局</style><styleface="normal"font="default"size="100%">-</style><styleface="normal"font="default"charset="134"size="100%">数据查询</style></title></titles><dates><year>2021</year></dates><urls><related-urls><url>/data_nmpa/face3/dir.html?CbSlDlH0=qGqXcGk_q6Ud0oRuU_p1sXlrf4v4tLnw7G9LlLbNq1GqqiV</url></related-urls></urls><custom1>2021</custom1><custom2>2021.5.9</custom2></record></Cite></EndNote>[7]的类型（按检测原理划分）。图STYLEREF1\s1.SEQ图\*ARABIC\s12AnOverviewoftheClassificationofNucleicAcidTestsADDINEN.CITEADDINEN.CITE.DATA[6]核酸检测方法分类表STYLEREF1\s1.SEQ表\*ARABIC\s11国家药品监督管理局批准新冠核酸检测试剂盒汇总ADDINEN.CITE<EndNote><Cite><Author>国家药品监督管理局</Author><Year>2021</Year><RecNum>599</RecNum><DisplayText><styleface="superscript">[7]</style></DisplayText><record><rec-number>599</rec-number><foreign-keys><keyapp="EN"db-id="5xrwx9ze32f25qedez6xess8092avpsepvx5"timestamp="1620528365">599</key></foreign-keys><ref-typename="WebPage">12</ref-type><contributors><authors><author><styleface="normal"font="default"charset="134"size="100%">国家药品监督管理局</style></author></authors></contributors><titles><title><styleface="normal"font="default"charset="134"size="100%">国家药品监督管理局</style><styleface="normal"font="default"size="100%">-</style><styleface="normal"font="default"charset="134"size="100%">数据查询</style></title></titles><dates><year>2021</year></dates><urls><related-urls><url>/data_nmpa/face3/dir.html?CbSlDlH0=qGqXcGk_q6Ud0oRuU_p1sXlrf4v4tLnw7G9LlLbNq1GqqiV</url></related-urls></urls><custom1>2021</custom1><custom2>2021.5.9</custom2></record></Cite></EndNote>[7]检测原理种类数变温扩增法（Non-isothermalamplification）荧光PCR法18恒温扩增法（Isothermalamplification）恒温扩增芯片法1恒温扩增-实时荧光法1杂交捕获免疫荧光法1RNA捕获探针法1RNA恒温扩增-金探针层析法1双扩增法1测序法（Sequencing）联合探针锚定聚合测序法1总计26其中，较为常用的方法包括逆转录-聚合酶链式反应法（ReverseTranscription-PolymeraseChainReaction,RT-PCR）、CRISPR/Cas(ClusteredRegularlyInterspacedShortPalindromicRepeats/CRISPRAssociation)基因编辑技术、第二代测序法（Next-GenerationSequencing,NGS）等，下文将对不同方法的原理、基本流程及优缺点进行介绍。RT-PCR法（RT-）PCR法被视为诊断病毒感染的“金标准”，在许多国家也是诊断COVID-19最推荐的方法ADDINEN.CITEADDINEN.CITE.DATA[8]。RT-PCR法可分为实时RT-PCR（Real-TimeRT-PCR）、终点RT-PCR（End-PointRT-PCR）、数字RT-PCR（RT-DigitalPCR）等；临床应用中，实时RT-PCR普及范围最广，在全部核酸检测方法中约占77.2%ADDINEN.CITEADDINEN.CITE.DATA[6]。在RT-PCR法中，操作流程包括RNA提取、逆转录、PCR扩增三步：经核酸提取仪获得的病毒RNA首先在逆转录酶的作用下合成单链互补DNA（cDNA）；随后DNA聚合酶将cDNA转化为双链DNA作为下一步PCR扩增的模板；PCR扩增包括三个变温过程，即变性、退火和延伸，为达到足够的DNA浓度，该过程一般需要重复45次左右。为实现病毒RNA的定量分析，采用荧光染料或荧光标记探针对每次PCR循环产物进行标记，同时记录荧光信号达到阈值所需的循环次数（Ct值）ADDINEN.CITE<EndNote><Cite><Author>Heid</Author><Year>1996</Year><RecNum>566</RecNum><DisplayText><styleface="superscript">[9]</style></DisplayText><record><rec-number>566</rec-number><foreign-keys><keyapp="EN"db-id="5xrwx9ze32f25qedez6xess8092avpsepvx5"timestamp="1620351576">566</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Heid,C.A.</author><author>Stevens,J.</author><author>Livak,K.J.</author><author>Williams,P.M.</author></authors></contributors><titles><title>RealtimequantitativePCR</title><secondary-title>GenomeResearch</secondary-title></titles><periodical><full-title>GenomeResearch</full-title></periodical><pages>986-994</pages><volume>6</volume><number>10</number><dates><year>1996</year></dates><publisher>ColdSpringHarborLaboratory</publisher><isbn>1088-9051</isbn><urls><related-urls><url>/10.1101/gr.6.10.986</url></related-urls></urls><electronic-resource-num>10.1101/gr.6.10.986</electronic-resource-num><research-notes>RT-PCR</research-notes></record></Cite></EndNote>[9]，通常认为Ct值<40可判断为病毒核酸阳性。作为对病毒核酸直接进行检测的方法，RT-PCR法在感染早期的检测中具有较大优势，且具有检测需要的样本量小（5μL）、RNA浓度检出限低（100~100.5copies/μLADDINEN.CITE<EndNote><Cite><Author>Prevention</Author><Year>2020</Year><RecNum>567</RecNum><DisplayText><styleface="superscript">[10]</style></DisplayText><record><rec-number>567</rec-number><foreign-keys><keyapp="EN"db-id="5xrwx9ze32f25qedez6xess8092avpsepvx5"timestamp="1620355675">567</key><keyapp="ENWeb"db-id="">0</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>CentersforDiseaseControlandPrevention</author></authors></contributors><titles><title><styleface="normal"font="default"size="100%">CDC2019-NovelCoronavirus(2019-nCoV)Real-TimeRT-PCRDiagnosticPanel</style><styleface="normal"font="default"charset="134"size="100%">（待改）</style></title></titles><dates><year>2020</year></dates><urls></urls><research-notes>COVID-19+PCR</research-notes></record></Cite></EndNote>[10]）、适合进行批量检测等优点ADDINEN.CITEADDINEN.CITE.DATA[8]，因此在临床应用中占比最高。但同时该方法对样本采集、保存、运输、实验室检测等各环节的规范化要求较高，准确率易受到取样位置ADDINEN.CITE<EndNote><Cite><Author>Yang</Author><Year>2020</Year><RecNum>153</RecNum><DisplayText><styleface="superscript">[5]</style></DisplayText><record><rec-number>153</rec-number><foreign-keys><keyapp="EN"db-id="5xrwx9ze32f25qedez6xess8092avpsepvx5"timestamp="1585814796">153</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Yang,Yang</author><author>Yang,Minghui</author><author>Shen,Chenguang</author><author>Wang,Fuxiang</author><author>Yuan,Jing</author><author>Li,Jinxiu</author><author>Zhang,Mingxia</author><author>Wang,Zhaoqin</author><author>Xing,Li</author><author>Wei,Jinli</author><author>Peng,Ling</author><author>Wong,Gary</author><author>Zheng,Haixia</author><author>Wu,Weibo</author><author>Liao,Mingfeng</author><author>Feng,Kai</author><author>Li,Jianming</author><author>Yang,Qianting</author><author>Zhao,Juanjuan</author><author>Zhang,Zheng</author><author>Liu,Lei</author><author>Liu,Yingxia</author></authors></contributors><titles><title>Evaluatingtheaccuracyofdifferentrespiratoryspecimensinthelaboratorydiagnosisandmonitoringtheviralsheddingof2019-nCoVinfections</title><secondary-title>medRxiv</secondary-title></titles><periodical><full-title>medRxiv</full-title></periodical><pages>2020.02.11.20021493</pages><dates><year>2020</year></dates><urls><related-urls><url>/content/early/2020/02/17/2020.02.11.20021493.abstract</url></related-urls></urls><electronic-resource-num>10.1101/2020.02.11.20021493</electronic-resource-num><research-notes><styleface="normal"font="default"charset="134"size="100%">不同取样位置</style></research-notes></record></Cite></EndNote>[5]、人员操作及环境等的影响，因此在应用过程中出现了检出率较低（51.9%ADDINEN.CITE<EndNote><Cite><Author>Xiang</Author><Year>2020</Year><RecNum>259</RecNum><DisplayText><styleface="superscript">[11]</style></DisplayText><record><rec-number>259</rec-number><foreign-keys><keyapp="EN"db-id="5xrwx9ze32f25qedez6xess8092avpsepvx5"timestamp="1588044563">259</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Xiang,Jie</author><author>Yan,Mingzhe</author><author>Li,Hongze</author><author>Liu,Ting</author><author>Lin,Chenyao</author><author>Huang,Shuang</author><author>Shen,Changxin</author></authors></contributors><titles><title>EvaluationofEnzyme-LinkedImmunoassayandColloidalGold-ImmunochromatographicAssayKitforDetectionofNovelCoronavirus(SARS-Cov-2)CausinganOutbreakofPneumonia(COVID-19)</title><secondary-title>medRxiv</secondary-title></titles><periodical><full-title>medRxiv</full-title></periodical><pages>2020.02.27.20028787</pages><dates><year>2020</year></dates><urls><related-urls><url>/content/early/2020/03/01/2020.02.27.20028787.abstract</url></related-urls></urls><electronic-resource-num>10.1101/2020.02.27.20028787</electronic-resource-num><research-notes><styleface="normal"font="default"size="100%">COVID-</style><styleface="normal"font="default"charset="134"size="100%">三种检测法</style></research-notes></record></Cite></EndNote>[11]、64%ADDINEN.CITEADDINEN.CITE.DATA[12]）、假阴性率较高（41%ADDINEN.CITE<EndNote><Cite><Author>Ai</Author><Year>2020</Year><RecNum>149</RecNum><DisplayText><styleface="superscript">[13]</style></DisplayText><record><rec-number>149</rec-number><foreign-keys><keyapp="EN"db-id="5xrwx9ze32f25qedez6xess8092avpsepvx5"timestamp="1585710983">149</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Ai,Tao</author><author>Yang,Zhenlu</author><author>Hou,Hongyan</author><author>Zhan,Chenao</author><author>Chen,Chong</author><author>Lv,Wenzhi</author><author>Tao,Qian</author><author>Sun,Ziyong</author><author>Xia,Liming</author></authors></contributors><titles><title>CorrelationofChestCTandRT-PCRTestinginCoronavirusDisease2019(COVID-19)inChina:AReportof1014Cases</title><secondary-title>Radiology</secondary-title></titles><periodical><full-title>Radiology</full-title></periodical><pages>200642</pages><dates><year>2020</year></dates><publisher>RadiologicalSocietyofNorthAmerica</publisher><isbn>0033-8419</isbn><urls><related-urls><url>/10.1148/radiol.2020200642</url></related-urls></urls><electronic-resource-num>10.1148/radiol.2020200642</electronic-resource-num><research-notes><styleface="normal"font="default"size="100%">CT</style><styleface="normal"font="default"charset="134"size="100%">结果</style><styleface="normal"font="default"size="100%">PCR</style><styleface="normal"font="default"charset="134"size="100%">高假阴性率</style></research-notes><access-date>2020/03/31</access-date></record></Cite></EndNote>[13]）的情况；且假阴性率随病毒感染阶段的变化较为显著，呈现出先下降后上升的趋势，感染后第八天（出现症状后第三天）达到最低值20%（CI,12%~30%）ADDINEN.CITE<EndNote><Cite><Author>Kucirka</Author><Year>2020</Year><RecNum>628</RecNum><DisplayText><styleface="superscript">[14]</style></DisplayText><record><rec-number>628</rec-number><foreign-keys><keyapp="EN"db-id="5xrwx9ze32f25qedez6xess8092avpsepvx5"timestamp="1620811253">628</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Kucirka,LaurenM.</author><author>Lauer,StephenA.</author><author>Laeyendecker,Oliver</author><author>Boon,Denali</author><author>Lessler,Justin</author></authors></contributors><titles><title>VariationinFalse-NegativeRateofReverseTranscriptasePolymeraseChainReaction–BasedSARS-CoV-2TestsbyTimeSinceExposure</title><secondary-title>AnnalsofInternalMedicine</secondary-title></titles><periodical><full-title>AnnalsofInternalMedicine</full-title></periodical><pages>262-267</pages><volume>173</volume><number>4</number><dates><year>2020</year></dates><publisher>AmericanCollegeofPhysicians</publisher><isbn>0003-4819</isbn><urls><related-urls><url>/10.7326/m20-1495</url></related-urls><pdf-urls><url>file://D:\Download\endnote_click\Kucirka-2020-Variation-in-false-negative-rate-of.pdf</url></pdf-urls></urls><electronic-resource-num>10.7326/m20-1495</electronic-resource-num><research-notes>COVID-19+PCR</research-notes></record></Cite></EndNote>[14]，后随着感染天数增加，该方法可靠性持续下降。NGS法第二代测序法（Next-GenerationSequencing,NGS）又称为高通量测序法（High-throughputSequencing），2005年被首次提出。与传统测序方法不同的是，NGS法实现了DNA的合成与测序同时进行，且可对多个DNA并行测序，大大提高了检测通量及检测速度ADDINEN.CITE<EndNote><Cite><Author>Kchouk</Author><Year>2017</Year><RecNum>568</RecNum><DisplayText><styleface="superscript">[15]</style></DisplayText><record><rec-number>568</rec-number><foreign-keys><keyapp="EN"db-id="5xrwx9ze32f25qedez6xess8092avpsepvx5"timestamp="1620356923">568</key><keyapp="ENWeb"db-id="">0</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Kchouk,Mehdi</author><author>Gibrat,JeanFrancois</author><author>Elloumi,Mourad</author></authors></contributors><titles><title>GenerationsofSequencingTechnologies:FromFirsttoNextGeneration</title><secondary-title>BiologyandMedicine</secondary-title></titles><periodical><full-title>BiologyandMedicine</full-title></periodical><volume>09</volume><number>03</number><dates><year>2017</year></dates><isbn>09748369</isbn><urls></urls><electronic-resource-num>10.4172/0974-8369.1000395</electronic-resource-num></record></Cite></EndNote>[15]。与此同时，以NGS法为代表的核酸测序方法可获取病毒更丰富的遗传信息，便于确定感染病毒的种类、源头、变异情况等。但由于该类方法对设备要求高、操作难度大，作为诊断方法使用不如RT-PCR法广泛，目前多用于COVID-19的病理学研究ADDINEN.CITEADDINEN.CITE.DATA[16]。CRISPR/Cas法CRISPR/Cas系统是原核生物的一种免疫防御系统，类似于人体的“获得性免疫”：当受到病毒等外源物质二次入侵时，CRISPR/Cas系统可利用短小的RNA链对病毒核酸进行特异性识别，进而利用Cas蛋白进行切割，从而达到“特异性免疫”的目的ADDINEN.CITE<EndNote><Cite><Author>Qi</Author><Year>2013</Year><RecNum>572</RecNum><DisplayText><styleface="superscript">[17]</style></DisplayText><record><rec-number>572</rec-number><foreign-keys><keyapp="EN"db-id="5xrwx9ze32f25qedez6xess8092avpsepvx5"timestamp="1620360964">572</key><keyapp="ENWeb"db-id="">0</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Qi,L.S.</author><author>Larson,M.H.</author><author>Gilbert,L.A.</author><author>Doudna,J.A.</author><author>Weissman,J.S.</author><author>Arkin,A.P.</author><author>Lim,W.A.</author></authors></contributors><auth-address>UCSFCenterforSystemsandSyntheticBiology,UniversityofCalifornia,SanFrancisco,SanFrancisco,CA94158,USA.stanley.qi@</auth-address><titles><title>RepurposingCRISPRasanRNA-guidedplatformforsequence-specificcontrolofgeneexpression</title><secondary-title>Cell</secondary-title></titles><periodical><full-title>cell</full-title></periodical><pages>1173-83</pages><volume>152</volume><number>5</number><edition>2013/03/05</edition><keywords><keyword>Endodeoxyribonucleases/chemistry/*genetics/metabolism</keyword><keyword>Escherichiacoli/*genetics</keyword><keyword>GeneExpression</keyword><keyword>GeneKnockdownTechniques/*methods</keyword><keyword>*RNAInterference</keyword><keyword>RNA,Guide/*genetics</keyword><keyword>Streptococcuspyogenes/*enzymology/genetics</keyword><keyword>TranscriptionElongation,Genetic</keyword><keyword>TranscriptionInitiation,Genetic</keyword></keywords><dates><year>2013</year><pub-dates><date>Feb28</date></pub-dates></dates><isbn>1097-4172(Electronic) 0092-8674(Linking)</isbn><accession-num>23452860</accession-num><urls><related-urls><url>/pubmed/23452860</url></related-urls></urls><custom2>PMC3664290</custom2><electronic-resource-num>10.1016/j.cell.2013.02.022</electronic-resource-num></record></Cite></EndNote>[17]。利用CRISPR/Cas系统对核酸的特异性识别功能，Fozouni等人开发了基于CRISPR-Cas13a的病毒RNA直接检测方法，因无需PCR扩增大大缩短了检测时间，使实时病毒检测成为了可能ADDINEN.CITE<EndNote><Cite><Author>Fozouni</Author><Year>2020</Year><RecNum>452</RecNum><DisplayText><styleface="superscript">[18]</style></DisplayText><record><rec-number>452</rec-number><foreign-keys><keyapp="EN"db-id="5xrwx9ze32f25qedez6xess8092avpsepvx5"timestamp="1607517339">452</key><keyapp="ENWeb"db-id="">0</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Fozouni,Parinaz</author><author>Son,Sungmin</author><author>DíazdeLeónDerby,María</author><author>Knott,GavinJ.</author><author>Gray,CarleyN.</author><author>D’Ambrosio,MichaelV.</author><author>Zhao,Chunyu</author><author>Switz,NeilA.</author><author>Kumar,G.Renuka</author><author>Stephens,StephanieI.</author><author>Boehm,Daniela</author><author>Tsou,Chia-Lin</author><author>Shu,Jeffrey</author><author>Bhuiya,Abdul</author><author>Armstrong,Max</author><author>Harris,AndrewR.</author><author>Chen,Pei-Yi</author><author>Osterloh,JeannetteM.</author><author>Meyer-Franke,Anke</author><author>Joehnk,Bastian</author><author>Walcott,Keith</author><author>Sil,Anita</author><author>Langelier,Charles</author><author>Pollard,KatherineS.</author><author>Crawford,EmilyD.</author><author>Puschnik,AndreasS.</author><author>Phelps,Maira</author><author>Kistler,Amy</author><author>DeRisi,JosephL.</author><author>Doudna,JenniferA.</author><author>Fletcher,DanielA.</author><author>Ott,Melanie</author></authors></contributors><titles><title>Amplification-freedetectionofSARS-CoV-2withCRISPR-Cas13aandmobilephonemicroscopy</title><secondary-title>Cell</secondary-title></titles><periodical><full-title>cell</full-title></periodical><dates><year>2020</year></dates><isbn>00928674</isbn><urls></urls><electronic-resource-num>10.1016/j.cell.2020.12.001</electronic-resource-num><research-notes>CRISPR-Cas</research-notes></record></Cite></EndNote>[18]。Cas13是原核生物免疫系统中的RNA核酸酶，靶向切割单链RNA，可与CRISPRRNA形成不含核酸酶的核糖核蛋白复合物（ribonucleoproteincomplex,RNP）。当RNP与病毒靶点RNA结合后，Cas13的切割功能被激活，环境中由ssRNA连接的荧光淬灭剂对被切断并发出荧光，检测到的荧光强度可定量反映病毒RNA的含量。该方法与RT-PCR法相比，检测过程中无需提供变温环境，且可对痕量RNA进行检测（10~100copies）因此具有操作简单、用时较短、灵敏度高等优点，有望得到进一步推广ADDINEN.CITEADDINEN.CITE.DATA[19]。图STYLEREF1\s1.SEQ图\*ARABIC\s13Detec