2024, Vol. 64
表1(Table 1)
抗生素抗性基因检测方法发展历程及应用现状
http://dx.doi.org/10.13343/j.cnki.wsxb.20240111
中国科学院微生物研究所，中国微生物学会
中国科学院微生物研究所，中国微生物学会
文章信息
- 孙凌涛, 刘子述, 胡宝兰. 2024
- SUN Lingtao, LIU Zishu, HU Baolan.
- 抗生素抗性基因检测方法发展历程及应用现状
- Development history and current applications of methods for detecting antibiotic resistance genes
- 微生物学报, 64(8): 2591-2609
- Acta Microbiologica Sinica, 64(8): 2591-2609
-
文章历史
- 收稿日期：2024-02-22
- 网络出版日期：2024-03-28
引用本文 |
孙凌涛, 刘子述, 胡宝兰. 抗生素抗性基因检测方法发展历程及应用现状[J]. 微生物学报, 2024, 64(8): 2591-2609.
Sun Lingtao, Liu Zishu, Hu Baolan. Development history and current applications of methods for detecting antibiotic resistance genes[J]. Acta Microbiologica Sinica, 2024, 64(8): 2591-2609.
抗生素抗性基因检测方法发展历程及应用现状
1. 浙江大学环境与资源学院, 浙江 杭州 310058;
2. 污染环境修复与生态健康教育部重点实验室, 浙江 杭州 310058
2. 污染环境修复与生态健康教育部重点实验室, 浙江 杭州 310058
摘要：耐药性的传播已引起全球广泛关注，抗生素抗性基因(antibiotic resistance genes, ARGs)检测技术的开发是研究ARGs在环境-动植物-人群中迁移传播的关键。本文梳理了现有核酸检测技术的发展历程及首次应用于ARGs检测的时间节点，并从检测原理、应用优缺点、开发潜力等方面对各技术进行分类综述。在此基础上提出以等温扩增结合CRISPR/Cas技术为核心的ARGs原位快速检测技术的开发及应用前景展望。本综述旨在回顾各技术发展历程的基础上，为ARGs新检测技术的开发及应用提供参考，为耐药性传播的研究及控制提供技术支撑。
关键词：抗生素抗性基因 聚合酶链式反应 基因测序 等温扩增 原位快速检测
Development history and current applications of methods for detecting antibiotic resistance genes
1. College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China;
2. Key Laboratory of Environment Remediation and Ecological Health, Ministry of Education, Hangzhou 310058, Zhejiang, China
2. Key Laboratory of Environment Remediation and Ecological Health, Ministry of Education, Hangzhou 310058, Zhejiang, China
Abstract: The spread of antibiotic resistance has aroused global concern. The development of technologies for detecting antibiotic resistance genes (ARGs) is essential for curbing the migration and spread of ARGs from the environment to plants/animals and human populations. This paper describes the development timeline of existing nucleic acid detection technologies and their first applications to the detection of ARGs and summarizes their detection principles, advantages and disadvantages, and development potential. Furthermore, this paper prospects that isothermal amplification combined with CRISPR/Cas might be the core technology for the development of in-situ rapid detection methods. By reviewing the development history of each technology, this paper aims to give insights into the development and applications of technologies for detecting ARGs and provide technical support for the research and control of antibiotic resistance transmission.
Keywords:
antibiotic resistance genes polymerase chain reaction gene sequencing isothermal amplification in-situ rapid detection
抗生素广泛应用于人类和动物的疫病预防与治疗[1-2]。过量或未降解的抗生素通过污水、排泄物、废弃物等进入环境，并在养殖场、农田、污水处理设施和制药厂等生境中积累富集[3-5]。在这些生境中，抗生素与其他污染物的选择压力能够诱导产生抗生素抗性基因(antibiotic resistance genes, ARGs)，而ARGs进一步通过垂直基因传递(vertical gene transfer, VGT)、水平基因转移(horizontal gene transfer, HGT)等方式增殖转移，在环境-动植物-人群间迁移传播[6-9]。携带ARGs的宿主细菌会表达对抗生素的耐受性或抗性，放任其发展的结果是出现“超级细菌”无药可医的困境。抗生素耐药性因此成为最严峻的全球性环境问题之一，联合国环境规划署(UN Environment Programme, UNEP, https://www.unep.org/zh-hans)也呼吁给予其与三重行星危机(气候变化、生物多样性丧失、污染)同等的重视程度。
实现快速、准确的ARGs检测为精准防控抗生素耐药性传播的技术支撑。PCR及相关衍生技术和基因测序技术是当前用于ARGs检测的主流技术[10-12]。基于PCR的几种技术往往更具靶向性，其中实时荧光定量PCR (real time quantitative PCR, qPCR)更是凭借其快速、灵敏、可靠等优点成为了检测“金标准”[13-16]；而基因测序能够全面筛查同一样品中的所有已知ARGs，并且可以获得未知的潜在ARGs序列，在ARGs的全面性分析中具有优势。然而，这些主流检测方法依赖专用仪器，仅限于实验室分析测试。一些新兴的分子诊断技术也被应用于ARGs的检测，例如基于等温扩增和基于成簇的规律性间隔短回文重复序列(clustered regularly interspaced short palindromic repeats, CRISPR)及相关蛋白(CRISPR-associated, Cas)系统的快速检测。然而，新兴技术受经济成本、稳定性、检出限等多方面原因限制，对复杂环境样品的检测能力尚未得到广泛认可，因此总体上仍然缺乏可靠的ARGs原位快速检测手段[17]。
本综述对现有核酸检测技术的发明与发展史进行了整理，同时追踪了各技术首次应用于ARGs检测的时间节点。采用统一的方式于Web of Science平台检索相关文献(表 1)，并在检索结果的参考文献中进一步筛查汇总。对各技术进行分类回顾，结合技术开发领域的发展现状，比较各类技术的检测原理和应用优缺点，以期对未来ARGs检测技术的发展及原位快速检测手段的开发提供参考依据。
表 1. 核酸检测技术优缺点与首次应用于ARGs检测的成果检索词条
Table 1. Advantages and disadvantages of nucleic acid detection technologies and retrieval terms for the initial application in ARGs detection
| Type | Technology | Search terms of initial application in ARGs detection | Basic principle | Sensitivity | Specificity | Detection speed | Qualitative/quantitative | Uniformity of amplicons | Amplification efficiency |
| PCR-based | PCR | TS=(PCR OR “Polymerase Chain Reaction”) AND (ARG OR “Antibiotic* Resistance Gene*”) | Enzyme-catalyzed | High | High | Moderate | Qualitative | High | High |
| qPCR | TS=(qPCR OR “quantitative Polymerase Chain Reaction” OR “quantitative PCR” OR real time PCR) AND (ARG OR “Antibiotic* Resistance Gene*”) | Enzyme-catalyzed, fluorescence | High | High | Moderate | Quantitative | High | High | |
| HT-qPCR | TS=(HT qPCR OR high throughput quantitative PCR) AND (ARG OR “Antibiotic* Resistance Gene*”) | Enzyme-catalyzed, fluorescence | High | High | Moderate | Quantitative | High | High | |
| dPCR | TS=(dPCR OR “digital PCR”) AND (ARG OR “Antibiotic* Resistance Gene*”) | Droplet microfluidic, enzyme-catalyzed, fluorescence | Extremely high | High | Moderate | Quantitative | High | High | |
| Sequencing-based | Sanger method | TS=(“Sequencing” OR Sanger method OR dideoxy chain termination method) AND (ARG OR “Antibiotic* Resistance Gene*”) | Short-read, fluorescence | High | Slow | Relatively quantitative | |||
| Next generation | TS=(Next generation sequencing OR High throughput sequencing OR Metagenomics sequencing OR NGS) AND (ARG OR “Antibiotic* Resistance Gene*”) | Short-read, fluorescence/chemical signal | High | Slow | Relatively quantitative | ||||
| Third generation | TS=(Single Molecule Sequencing OR Third Generation Sequencing OR SMS OR TGS OR SMART Sequencing OR Nanopore Sequencing) AND (ARG OR “Antibiotic* Resistance Gene*”) | Long-read, fluorescence/electrical signal | High | Slow | Relatively quantitative | ||||
| Other fluorescence-based | FISH | TS=“Fluorescence in situ hybridization” AND (ARG OR “Antibiotic* Resistance Gene*”) | Fluorescence | Moderate | Moderate | Moderate | Qualitative/quantitative | ||
| DNA microarray | TS=(DNA probe array OR DNA microarray OR gene chip) AND (ARG OR “Antibiotic* Resistance Gene*”) | Fluorescence/isotope | Moderate | Moderate | Moderate | Quantitative | |||
| Molecular beacon | TS=Molecular beacon AND (ARG OR “Antibiotic* Resistance Gene*”) | Fluorescence | High | Moderate | Moderate | Quantitative | |||
| Isothermal amplification and CRISPR/Cas | SDA | TS=Strand Displacement Amplification | Enzyme-catalyzed | Moderate | Moderate | Fast | Quantitative | Low-high | High |
| LAMP | TS=Loop-mediated Isothermal Amplification | Enzyme-catalyzed | Moderate | High | Fast | Quantitative | Low | High | |
| RPA | TS=Recombinase Polymerase Amplification | Enzyme-catalyzed | Moderate | Moderate | Fast | Quantitative | High | High | |
| RCA | TS=Rolling Circle Amplification | Enzyme-catalyzed | Moderate | Moderate | Fast | Quantitative | Low-high | Low-high | |
| HDA | TS=Helicase-dependent Amplification | Enzyme-catalyzed | Moderate | Moderate | Fast | Quantitative | High | High | |
| CRISPR/Cas | TS=CRISPR | Cas protein cleavage | Low | High | Fast | Quantitative |
1 ARGs检测技术的阶段性发展与分类
基于文献检索结果，整理了各类检测技术的发明时间(图 1)。早在20世纪60年代末，以同位素标记的原位杂交技术(in situ hybridization, ISH)的发明为标志，现代核酸分子生物学检测技术兴起[18]。此后的20年间陆续出现了Sanger测序、聚合酶链式反应(polymerase chain reaction, PCR)等奠基性的技术，深远地影响了核酸检测领域的后续发展。1990–2010年间，各类技术大量涌现，这20年的技术研发重点一方面是基于已有技术的优化，例如PCR和测序技术的深入开发；另一方面是延续PCR的核酸体外扩增技术路线，探寻新的引物设计策略和不同的功能酶以实现各式各样的等温扩增。
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| 图 1 核酸检测技术发明发展及首次应用于ARGs检测时间轴 Figure 1 Timeline of the invention, development, and initial application in ARGs detection of nucleic acid detection technologies. |
在ARGs检测方面，各类技术的应用始于20世纪90年代。同样以Sanger法和PCR技术为先导，分别对磺胺类ARGs和氨基糖苷类ARGs进行检测[19-20]。2000–2010年间是ARGs检测技术探索最为活跃的时段，一方面相关技术多集中在1990–2010年被发明，经过几年的优化和改进后才被应用于各个领域；另一方面，20世纪末至21世纪初学术界逐渐认识到了抗生素抗性作为全球性环境问题的严峻性，欧盟细菌耐药性监测系统(European antimicrobial resistance surveillance system, EARSS)的建立、世界卫生组织遏制抗生素耐药全球策略(WHO global strategy for containment of antimicrobial resistance)的提出、多方联合“One Health”理念的形成等一系列事件反映了其受公众关注程度，也促使相关研究成为了科研热点，推动ARGs检测技术的探索。这一时期检测对象也存在一定的偏好性，mecA (一种常见于金黄色葡萄球菌的青霉素结合蛋白编码基因，介导对β-内酰胺的抗性)、rpoB基因(一种RNA聚合酶，为利福平的结合位点，其突变会产生耐药性)、β-内酰胺酶(通过降解失活β-内酰胺而提供耐药性)编码基因是最常用的检测目标，为后续新技术的应用探索提供了参考[21-23]。
通过ARGs检测技术的回顾，根据检测原理与应用普及程度，将涉及到的核酸检测技术分为四大类(图 2)：(1) 检测“金标准”——PCR及衍生技术；(2) 全面性检测技术——基因测序；(3) 新兴检测技术——等温扩增和CRISPR/Cas；(4) 其他基于荧光策略的技术。对每个类别的技术发展进行了更为详细的阐释，并介绍其原理、优缺点及应用现状(表 1)。
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| 图 2 ARGs检测技术分类及原理示意图 Figure 2 Classification of ARGs detection technologies and illustration of principles. |
2 检测金标准——PCR及衍生技术
PCR技术衍生种类众多，在ARGs检测领域应用广泛。对近些年所发表文献的粗略统计显示，PCR及其衍生技术在90%以上的ARGs靶向研究中起到关键作用，在预扩增、检测、验证等流程中被频繁使用。PCR发明于1985年，其发明者Kary Banks Mullis在2年后的一篇文章中正式介绍其原理：通过设定的热循环，在DNA聚合酶的作用下，经过变性、退火、延伸等步骤实现对靶标的指数扩增，理论上经过35个循环可以将靶标浓度扩增至初始浓度的1010倍，然而实际情况下会因为反应抑制而降低扩增效率[24-26]。Webb等[20]首次将该技术引入ARGs检测，他们以琼脂糖凝胶电泳为下游分析手段，定性检测了包含氨基糖苷转移酶ARGs aph3′′的链霉菌(Streptomycete) 16S rRNA基因的扩增子。PCR技术无法单独做到定量检测，通常被用于提高靶标ARGs浓度，联用其他技术从而进一步分析[27]。为了突破这一限制，Heid等[28]于1996年开发了qPCR，该技术以荧光基团或荧光探针为表征手段，直接将扩增产物的浓度可视化。Piatek等[29]首次利用该技术分析结核分枝杆菌(Mycobacterium tuberculosis)中利福平ARGs rpoB的突变。qPCR可在2 h左右实现DNA样品中个位数copies/μL的ARGs精准定量，综合检测能力极佳，是DNA检测的“金标准”[30-33]。
此类技术主要向高通量和高灵敏度2个方向进一步发展。多重qPCR和高通量qPCR (high-throughput qPCR, HT-qPCR)被开发以提高检测通量，然而针对不同基因采用相同的反应条件以及更复杂或更小的反应体系也在一定程度上降低了扩增效率和检测灵敏度[34]。早在2003年HT-qPCR便已问世[35]，然而直到2014年Wang等[36]才首次利用HT-qPCR技术检测ARGs，该研究采用了295对引物，对再生水灌溉的公园土壤中氨基糖苷类、β-内酰胺类等主要ARGs进行了同步定量。HT-qPCR一般只能相对16S rRNA基因定量，或通过16S rRNA基因的测定实现ARGs的间接绝对定量[37]。Pärnänen等采用HT-qPCR对城镇污水处理厂污水中的ARGs进行间接绝对定量，认为20 copies/μL是该方法可以检测到的最低极限浓度[38]。在高灵敏度方面，2011年发明的微滴式数字PCR (droplet digital PCR, ddPCR)是典型代表。其基本原理是在成千上万的油包水小液滴中进行微体系PCR反应，每个体系可以达到纳升甚至皮升级别，由于阳性液滴数量与样本中靶标基因浓度符合泊松分布，因此基于对小液滴的阳性判读可以输出定量结果[39]。该技术2年后便被应用于ARGs检测，Kelley等[40]利用双重ddPCR技术检测了397个临床样本中金黄色葡萄球菌(Staphylococcus aureus)的甲氧西林ARGs mecA，其临床敏感性和特异性都与qPCR相当。Cavé等[41]将qPCR和ddPCR在相同条件下进行比较，结果显示ddPCR的检测限比qPCR低一个数量级。此外，新型荧光探针开发也能够进一步提高基于qPCR技术对低浓度ARGs的检测能力。
PCR类技术的应用主要局限于靶标的定量检测，其他功能仍有待开发。例如，宏基因测序能够同时获得样本中物种、其他基因、可移动遗传元件(mobile genetic elements, MGEs)等信息，而PCR类技术要想获得这些信息则需要花费成倍的时间、经济和人力成本。Xie等[42]开发了一种基于HT-qPCR的毒力因子基因(virulence factor genes, VFGs)高通量检测芯片VFG-Chip，或许可以基于此进一步实现不依赖测序的VFGs和ARGs高通量同步检测。此外，重金属抗性基因芯片与ARGs芯片的结合也可以为相关研究提供便利[43]。然而乳液、配对分离和串联PCR (emulsion, paired isolation and concatenation PCR, epicPCR)则可以将ARGs与未培养宿主关联起来分析，进一步开展ARGs溯源、风险评估等工作[44]。相关手段的研发有助于PCR类技术在ARGs研究领域更多样化地应用。
3 全面性检测技术——基因测序测序技术能够实现对样本中所有碱基的读取，这种特性使其在ARGs筛查方面具有明显的技术优势。最早发明的Sanger测序法使用了双脱氧核苷酸(dideoxy-ribonucleoside triphosphate, ddNTP)，−OH基团的缺失使下一个脱氧核苷酸(deoxy-ribonucleoside triphosphate, dNTP)无法进一步结合，因此链延伸会在结合ddNTP的位置终止。反应在4个体系中进行，每个体系都包含4种dNTP、1种ddNTP以及DNA聚合酶，与引物结合的待测模板单链在其中延伸，在结合ddNTP后终止，得到长短不一的产物。由于每种ddNTP都被不同颜色的荧光标记，根据凝胶电泳分析结果可以推断出对应长度位置上的碱基[45-46]。早在1990年，Sanger测序法就被Martin等[19]成功用于偶发分枝杆菌(Mycobacterium fortuitum)的磺胺类药物ARGs检测，并证实了Tn610转座子中包含整合酶编码基因orf2M和磺胺类ARGs sul3。Sanger法后来也被称作一代测序，得益于其精确性和经济性，目前仍有应用[47-48]。
2005年Roche公司发布了第一台二代测序仪454 GS20，标志着二代测序的诞生[49]。二代测序通过dNTP的可逆修饰，赋予了其可控的终止末端，能够实现边合成边测序，因此大大提高了测序通量。2009年，Allen等[50-51]尝试将二代测序应用于ARGs研究，分析了土壤中的β-内酰胺酶基因和舞毒蛾(Lymantria dispar)幼虫肠道微生物群落的ARGs，证明了二代测序在复杂环境和生物样本中ARGs检测的独特优势。基于二代测序的宏基因组测序可以分析包括环境、动物、反应器等多种样品中的宏基因信息，是混菌体系基因研究最常用的方法之一[52-54]。它不仅可以注释成百上千种ARGs，还能识别上下游遗传信息并筛查MGEs，以评估ARGs的传播风险[55-58]。根据Illumina公司(https://www.illumina.com.cn)提供的参数，旗下的各类平台二代测序耗时4−55 h不等，序列读长在150−300 bp。相较于一代测序，二代测序大幅缩减了获得同等数据量情况下的检测耗时，但一代测序约1 000 bp的序列读长具有更高的精度，因此2种技术并不存在明显的优劣。
2010年，Flusberg等[59]发明了基于荧光信号读取的三代测序技术——PacBio SMRT (single molecule real-time)，这是一种利用甲基化修饰dNTP产生不同特征荧光信号从而识别序列的长读长测序手段。另一种常用的三代测序技术是Oxford Nanopore，该技术的核心原理是基于不同碱基产生不同的电信号实现序列读取。2014年，Wichmann等[60]结合功能宏基因组学和BioPac SMRT分析了奶牛粪便中的ARGs，大大扩展了动物肠道菌群的功能性ARGs清单，同时也为三代测序在该领域的应用提供了借鉴。长读长测序一方面能够大量保存基因的上下游信息，另一方面显著减少了reads拼接过程中出现的错误和遗漏。PacBio SMRT的平均读长为13.5 kb，而Oxford Nanopore最大读长超过4 Mb，相较于前两代测序展现出极大的优势[61-63]。
非靶向筛查的特性赋予测序技术独特的优势。例如远超HT-qPCR的通量，调控基因和遗传元件的同步分析等。与其他靶向检测手段相比，测序也更适合探索新ARGs。功能宏基因组学方法是目前新ARGs发掘最常用的技术之一，其核心思想是首先提取环境样品中的全部DNA并剪切、导入至模式菌构建文库，随后通过扩大培养、药理生化筛选等手段获得具有特定功能的菌株(如耐药菌)，最后对获得的功能菌株进行测序及基因功能分析验证[64-65]。然而，我们仍然不能忽视测序类技术的一些弊端，例如不能像qPCR那样做到绝对定量、单靶检测成本高以及检测耗时长等[66]。
测序技术实现了极高的检测通量和全面的非靶向筛查，但却无法兼顾节省时间和经济成本，弥补这方面的弊端有利于满足更多需求[67]。除了原理导致的反应流程缓慢，庞大的宏基因数据分析也会占用大量的时间。数据库与分析手段的选择极为重要，通过数据库的更新和算法的优化也可以节省该流程的时间，并且可以为研究者提供更有价值的信息[68]。此外，测序类技术不像PCR可以直接实现样品中ARGs的绝对定量检测，推出一种易于使用的、广泛认可的绝对定量方法也是测序技术发展的趋势之一[69-70]。
4 新兴检测技术——等温扩增和CRISPR/Cas常规的PCR和测序等由于对高温、控温仪器的依赖而局限于实验室分析，新兴的等温扩增类技术有望突破该瓶颈。等温扩增与PCR最大的区别在于模板链的解旋、引物的结合与延伸都可以在同一恒定温度下实现，降低了对控温仪器的依赖性[71]。由于反应在蛋白/酶的介导下直接进行，避免了反复的升温与降温程序，从而缩短了检测耗时[72]。此类技术起步相对较晚，1992年Walker等[73]发明的链置换扩增技术(strand displacement amplification, SDA)是最早出现的等温扩增技术之一，其链置换原理在其他等温扩增技术中也有体现。2000–2010年是等温扩增技术发明与应用于ARGs检测的高峰期，环介导等温扩增(loop-mediated isothermal amplification, LAMP)、解旋酶依赖性等温扩增(helicase-dependent isothermal amplification, HDA)、重组酶聚合酶扩增(recombinase polymerase amplification, RPA)等相继出现[74-76]。这3项技术在ARGs的首次应用集中在2006–2008年，而且都选取了耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus, MRSA)作为研究对象[76-78]。
LAMP是最为成熟和广泛运用的等温扩增技术之一，其原理是利用4个不同的引物扩增原始模板。其中有2个特殊的引物，称作前导内引物(forward inner primer, FIP)和后随内引物(backward inner primer, BIP)，它们的5′端片段可以与引物下游的序列互补，从而使初始扩增子形成双端茎环结构的单链，并以此为循环的起点进行扩增，循环产物为长短不一的茎环结构DNA混合物[74]。该技术能够在55−70 ℃的条件下实现个位数copies/μL基因的1 h以内快速检测，相较于PCR具有更强的扩增能力，针对6段序列设计的4个引物虽然增加了设计难度，却也提供了较强的特异性[79]。Suea-Ngam等[80]将LAMP与银纳米板结合，检测MRSA中的mecA并通过智能手机读取信号，在30 min内精准定量了个位数copies/μL的靶标，显示出了LAMP在场地快检的巨大潜力。作为一种新兴的等温扩增技术，RPA在近几年也备受关注。在RPA反应中，重组酶与引物的复合体识别双链DNA上的同源序列并入侵、打开双螺旋结构，单链DNA结合蛋白负责稳定解旋后的D环结构，随后由DNA聚合酶延伸引物完成扩增[81]。Butterworth等[82]利用直接标记的固相RPA检测大肠埃希氏菌(Escherichia coli)中的苯唑西林ARGs，虽然在这项研究中并未优化方法，却也能在1 h内实现个位数copies/μL ARGs的检测。RPA的优势在于快速、低温的反应特性(10−60 min, 37−42 ℃)，然而蛋白/酶的成本和保存等问题在一定程度上限制了该技术的应用[76]。Wang等[83]通过冻干策略使试剂便于保存，但也略微损害了蛋白/酶的活性。
CRISPR/Cas技术同样可以实现低温恒温条件下的ARGs检测。CRISPR/Cas是细菌免疫系统的一部分，具有强大的基因靶向识别和编辑能力。CRISPR是发现于原核生物基因组的特殊序列，通过编码功能性的Cas蛋白可以实现对外源基因的识别、整合及破坏。2010年，Garneau等[84]使用CRISPR/Cas9实现了DNA双链的特异性切割，该技术迅速发展为强大的基因编辑手段。在CRISPR/Cas9体系中，一条人工设计合成的、与靶标DNA互补的单导RNA (single-guide RNA, sgRNA)和Cas9蛋白形成复合体，该复合体将特异性识别并结合至双链DNA靶标，随后在Cas9蛋白的顺式切割(cis-cleavage)活性作用下切断特定位点[85]。2016年，Müller等[86]结合CRISPR/Cas9和DNA光学图谱检测临床菌样品中的超广谱β-内酰胺酶编码基因blaCTX-M-15和碳青霉烯酶编码基因blaKPC、blaNDM，显示出了CRISPR/Cas体系在ARGs检测领域的巨大潜力。随着越来越多的Cas蛋白被发现，CRISPR/Cas的应用领域也不断拓宽。Cas12、Cas13等蛋白还具有额外的反式切割(trans-cleavage)活性。对于Cas12，该活性表现为无差别切割附近所有的游离单链DNA，而对于Cas13则切割单链RNA[87-88]。反式切割活性只有在sgRNA-Cas蛋白复合物结合至靶标后才会激活，因此向反应体系中加入基于荧光共振能量转移(fluorescence resonance energy transfer, FRET)原理设计的单链荧光探针，若探针被切断，荧光信号产生，则说明样品中含有靶标DNA，其浓度在一定范围内与荧光值成正比，由此实现基于CRISPR/Cas的核酸检测方法[89-90]。CRISPR/Cas技术的检测限欠佳但特异性强，使用等温扩增技术预先扩增靶标可以降低整体的检测限[91-92]。Aggarwal等[93]使用HDA结合CRISPR/Cas12a可以实现1 h检测0.6 copies/μL的DNA靶标，在低至0.6 CFU/μL的肺炎克雷伯氏菌(Klebsiella pneumoniae)中检测到碳青霉烯ARGs blaKPC。CRISPR/Cas在ARGs检测领域尚未普及，但针对其他靶标开发的检测技术已有不少报道。例如，Gootenberg等[94]基于LAMP和CRISPR/Cas13a开发的特异性高灵敏度酶报告基因解锁(specific high-sensitivity enzymatic reporter unlocking, SHERLOCK)用于多种病毒和病原菌的检测；Chen[92]基于RPA和CRISPR/Cas12a开发的DNA核酸内切酶靶向CRISPR反式报告基因(DNA endonuclease-targeted CRISPR trans reporter, DETECTR)用于人乳头瘤病毒(human papillomavirus, HPV)的检测；Hu等[95]基于RPA和CRISPR/Cas12a开发的光活化单管法用于多种病毒的检测。
直接在现有等温扩增及CRISPR/Cas等技术的基础上进行二次开发和优化是实现ARGs原位快速检测最高效的方式之一。等温扩增技术众多且发展历史短，在检测速度、稳定性、灵敏度和特异性方面仍有较大优化空间。与探针法qPCR类似，等温扩增可以结合形态功能各异的探针来优化检测效果[96-97]。针对不同的应用选用不同的等温扩增技术可以更好地满足检测需求，例如在靶标序列可以满足复杂引物设计的情况下，选取LAMP获得较强的特异性，或在低温应用场景中采取反应温度更低的RPA。虽然CRISPR/Cas也能在恒温条件下完成检测反应，其检出限(6×105−3×109 copies/μL)却无法满足大多数场景的应用[98]。等温扩增技术与CRISPR/Cas的结合是一种极具潜力的技术组合，靶标的扩增保证了方法的灵敏度，而CRISPR/Cas对靶标的识别则保证了特异性。若将等温扩增与CRISPR/Cas反应同步进行，有利于简化流程并缩短整体反应时间。然而，混合反应体系中组分之间的不良竞争是必须突破的技术瓶颈，引物与sgRNA-Cas复合物会同时竞争靶标导致整体检测效果受到影响，并且Cas12蛋白还会通过反式切割活性切断引物直接降低扩增效率[99]。为了缓解这种不利影响进行了诸多探索，大多以降低CRISPR/Cas反应活性为核心思想，例如采用无切割活性的dCas9 (dead Cas9)蛋白，或基于次优原型间隔子相邻基序(protospacer adjacent motif, PAM)策略或光笼化的sgRNA对CRISPR/Cas反应进行抑制或沉默[85, 95, 98]。等温扩增和CRISPR/Cas在反应条件和反应速度方面有着天然的优势，通过微流控芯片、侧流试纸等商业化产品的研发和推广，这类技术有望实现ARGs的原位快速检测[100-101]。此外也应关注DNA快速提取技术的进步，例如热裂解、纸基材料等；而免去DNA提取直接对样品进行检测同样可以缩短分析周期，但这种方法对样品的纯度和检测技术的可靠性也提出了巨大的挑战[83, 102]。
实现原位快速检测是当下ARGs检测最大的技术需求之一，这意味着在现有技术的基础上同时突破灵敏度和检测分析速度的瓶颈。根据样品特点、检测条件等选取合适的等温扩增方法，结合CRISPR/Cas反应体系构建电化学或荧光表征方法，最有望满足该需求。然而要真正实现在各类复杂环境样品中精准、稳定地检测特定ARGs，不仅需要在反应体系和原理上不断优化与改进，还需要及时开发高效便捷的核酸提取技术和研发配套检测设备，以进一步助推新技术的应用与普及。
5 其他基于荧光策略的技术根据本综述分类筛选方法，还有一些技术与前文所述3种方法的技术原理有较大差别，但都结合了荧光表征手段，因此统一归为其他基于荧光策略的技术，以下作简单介绍。
荧光原位杂交(fluorescence in situ hybridization, FISH)技术发明于1977年，该技术通过荧光标记的探针与互补序列结合，从而将靶标可视化，其前身为1969年发明的同位素标记原位杂交技术[18, 103]。2004年，Zwirglmaier等[104]开发了识别单个基因技术(recognition of individual genes FISH, RING-FISH)，利用这种改进的FISH对大肠埃希氏菌(Escherichia coli)中的ARGs β-内酰胺酶进行了检测，灵敏度可以达到 < 10 copies/cell。FISH适合对靶标基因进行定位、追踪或对微生物进行鉴定，在ARGs定量检测方面应用较少。
1995年，Schena等[105]发明了DNA微阵列(DNA microarray)技术，具有快速、高通量、多靶标等特点。该技术预先使用荧光基团修饰样本中的核酸，并将大量特异性探针固定在微小的芯片上，通过探针捕获互补的靶标实现特定基因的可视化。1999年，2个科研团队先后使用了该技术检测结核分枝杆菌(Mycobacterium tuberculosis)中利福平ARGs rpoB的突变，证明了该技术的ARGs检测应用能力[106-107]。Patterson等[108]利用DNA微阵列从土壤和动物粪便样品中检测出23种四环素类ARGs和10种红霉素ARGs。DNA微阵列在检测灵敏度方面没有优势，只能检测到102 copies/μL的ARGs，此外探针固定、荧光修饰等烦琐处理步骤也限制了该技术的普及[109]。
荧光探针是一种特殊的检测手段，可以与前文所述的许多技术相结合。荧光探针一般由特异性核酸序列、荧光基团、淬灭基团构成，通过靶向识别、结构改变或破坏而产生荧光信号。分子信标(molecular beacon, MB)是一种基于FRET原理设计的茎环结构荧光探针，当它与靶标杂交后，茎环结构打开，荧光基团与淬灭基团空间距离增大从而产生荧光信号[110]。分子信标最常联用的技术是qPCR。1998年，Piatek等[29]利用分子信标与qPCR的联合方法分析结核分枝杆菌(Mycobacterium tuberculosis)中利福平ARGs rpoB的突变，是分子信标与qPCR首次在ARGs检测领域的应用。Singh等[111]同样使用分子信标与qPCR相结合的方法检测地表水中的β-内酰胺ARGs，灵敏度可以达到10 copies/μL。
6 总结与展望综上可见，现有的常用ARGs检测技术尚存在优化与提升空间。检测“金标准” qPCR具有出色的综合检测能力，可以在约2 h定量个位数copies/μL的ARGs，但检测依赖高精密度控温变温设备。测序类技术的非靶标筛查和未知基因探索功能使其在短时间内成为ARGs检测的主流技术之一，不可忽视的是这类技术具有相对较高的时间和经济成本，以及在ARGs绝对定量方面的阻力。等温扩增类技术可以在保证个位数copies/μL灵敏度的基础上将时间缩短至1 h以内，但也一定程度上在特异性、准确性和稳定性方面作出了让步。CRISPR/Cas少于30 min的反应时间使其在检测速度上具有极大的优势，其灵敏度却不够理想。虽然条件的优化可以在一定程度上弥补检测效果上的不足，但技术的突破需要依赖原理层面的改进，因此在未来的技术开发中可以侧重于新型探针的研发、功能蛋白的发掘、信号表征手段的探索等。
现有常用技术尚未同时实现ARGs快速且灵敏的定量检测、难以满足原位快速检测的需求，等温扩增和CRISPR/Cas技术最有望填补该空白。一方面，相近的反应速度和反应温度保证了技术耦合的可行性；另一方面，等温扩增提高了检测的灵敏度，CRISPR/Cas则提供了高特异性的信号表征手段。如何实现单管同步扩增-检测是该方法进一步优化的主要方向，而其关键技术瓶颈是两种体系之间的不良竞争。研究报道通过降低CRISPR/Cas反应活性[85, 95, 98]，以及采用空间/相分离等物理隔离方式[112-114]，可以有效提高整体的检测能力。诸多尝试的初步成功支持了等温扩增和CRISPR/Cas在原位快速检测领域的快速发展，而真正的普及应用则仍需通过实验室的优化论证以及商业化的开发生产来实现，这也是相关科研工作者以后需要重点关注的探索方向。
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