2024, Vol. 51
表1(Table 1)
重组酶聚合酶扩增技术的研究进展
沈方园
,
葛萧
,
张晓宇
,
张郁勃
,
周慧姿
,
李恒
扩展功能
文章信息
- 沈方园, 葛萧, 张晓宇, 张郁勃, 周慧姿, 李恒
- SHEN Fangyuan, GE Xiao, ZHANG Xiaoyu, ZHANG Yubo, ZHOU Huizi, LI Heng
- 重组酶聚合酶扩增技术的研究进展
- Research progress on recombinase polymerase amplification
- 微生物学通报, 2024, 51(5): 1495-1511
- Microbiology China, 2024, 51(5): 1495-1511
- DOI: 10.13344/j.microbiol.china.230558
-
文章历史
- 收稿日期: 2023-07-11
- 接受日期: 2023-10-31
- 网络首发日期: 2023-12-18
沈方园, 葛萧, 张晓宇, 张郁勃, 周慧姿, 李恒. 重组酶聚合酶扩增技术的研究进展[J]. 微生物学通报, 2024, 51(5): 1495-1511.
SHEN Fangyuan, GE Xiao, ZHANG Xiaoyu, ZHANG Yubo, ZHOU Huizi, LI Heng. Research progress on recombinase polymerase amplification[J]. Microbiology China, 2024, 51(5): 1495-1511.
重组酶聚合酶扩增技术的研究进展
潍坊医学院医学检验学院, 山东 潍坊 261053
摘要: 重组酶聚合酶扩增(recombinase polymerase amplification, RPA)技术是一种利用重组酶(T4 UvsX)、单链DNA结合蛋白(T4 Gp32)和链置换DNA聚合酶(Bsu)等蛋白在恒温条件下扩增核酸的技术。由于其可以恒温、高效地扩增靶核酸片段，近几年在细菌、病毒、寄生虫、支原体、衣原体检测方面发挥着独特的作用，逐渐成为床旁检测(point-of-care testing, POCT)的重要技术。本文以RPA技术的原理为起点，对其临床应用、不足之处及改进方法进行阐述，以期为临床疾病的快速检测和诊断方面提供新思路。
关键词:
重组酶聚合酶扩增技术 等温扩增技术 核酸检测
Research progress on recombinase polymerase amplification
School of Medical Laboratory, Weifang Medical University, Weifang 261053, Shandong, China
Abstract: Recombinase polymerase amplification (RPA) is a technology that utilizes proteins such as recombinase (T4 UvsX), single-stranded DNA binding protein (T4 Gp32), and strand replacement DNA polymerase (Bsu) to amplify target nucleic acids at constant temperatures. In recent years, RPA plays a unique role in the detection of bacteria, viruses, parasites, mycoplasma, and chlamydia for it can amplify the target nucleic acid fragment with constant temperature and high efficiency, it gradually becomes a key technology for point-of-care testing (POCT). This review takes the principle of RPA technology as a starting point, it describes the clinical application, shortcomings, and improvement of RPA, and RPA is expected to provide a new idea for the rapid detection and diagnosis of clinical diseases.
Keywords:
recombinase polymerase amplification technique isothermal amplification technique nucleic acid testing
2019年以来，新型冠状病毒感染疫情对人类生命健康造成了巨大的影响。PCR因其灵敏度高、特异性强等优点，作为核酸扩增技术的金标准在新型冠状病毒检测中得到了广泛应用。然而，由于PCR反应过程中需要频繁地加热与制冷，反应全程需在PCR仪中进行，导致以PCR为基础的核酸扩增技术在快速检测和基层应用推广中受到了一定的限制。
重组酶聚合酶扩增(recombinase polymerase amplification, RPA)技术是近年来新兴的核酸扩增技术，其在恒温下就可扩增，受环境限制小[1]。RPA技术操作简便，反应时间短，大约20−30 min即可获得检测结果，对大批量的样本检查和普查有实际意义。其床旁检测(point-of-care testing, POCT)尤其适合资源比较匮乏的偏远地区。除此之外，RPA技术可与多种临床技术结合，使检测灵敏度大大增高，并且实现检测结果的可视化，观察检测结果更便捷。
本文以介绍RPA技术的反应原理为起点，重点介绍了RPA技术在感染性疾病、癌症等方面的临床应用，对其目前存在的不足之处、改进方法进行总结，并对其未来进行展望。
1 RPA的概述RPA技术是在2006年由Twist DX公司提出的等温扩增技术，其反应体系主要由重组酶(T4 UvsX)、单链DNA结合蛋白(T4 Gp32)和链置换DNA聚合酶(Bsu)等多种酶组成。反应时，重组酶与引物配对形成重组酶-引物复合物，其在双链DNA中寻找同源序列，确定同源序列后，在重组酶的作用下，引物与之配对，DNA双链解离为单链，此时，T4 Gp32结合在解离的单链DNA上，防止DNA单链再次复性为双链，随后，重组酶从引物上解离，Bsu聚合酶结合到引物的3′末端启动新的DNA链合成，整个过程反复进行，实现目的DNA序列的指数级扩增(图 1)，反应过程中无须温度变化，在37−42 ℃可进行，另外重组酶等温扩增所需的引物对退火温度无特殊要求，引物设计难度降低，反应无需特殊的仪器，反应时间短，对环境要求低，是具有应用前景的核酸扩增技术[2-3]。
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| 图 1 RPA的作用原理 Figure 1 The action principle of RPA |
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等温扩增技术是一大类技术的总称，它们能在某一恒定的温度条件下扩增特定的DNA或者RNA。该技术包含类型复杂，发展较为迅速，常见的类型包括：重组酶介导等温核酸扩增(recombinase mediated isothermal nucleic acid amplification technique, RAA)技术、环介导等温扩增(loop-mediated isothermal amplification, LAMP)技术、依赖核酸序列的扩增(nucleic acid sequence-based amplification, NASBA)技术、链置换扩增(strand displacement amplification, SDA)技术、转录介导的扩增(transcription-mediated amplification, TMA)技术、酶促重组等温扩增(enzymatic recombinase isothermal amplification, ERA)技术、解旋酶依赖性扩增(helicase-dependent amplification, HDA)和滚环核酸扩增(rolling circle nucleic acid amplification, RCA)技术[4]。下文就几种经典的等温扩增技术进行简要介绍。
重组酶介导扩增(recombinase aided amplification, RAA)技术与RPA有相似的原理，二者都是通过几种酶的互相作用达到等温扩增的目的，不同的是两种等温扩增技术主要酶的来源不同；RPA技术主要依赖T4噬菌体重组酶T4 UvsX、T4 Gp32及链置换Bsu聚合酶发挥作用，而RAA技术所需主要的酶则来自于细菌或真菌，包括UvsX、SSB和DNA聚合酶Klenow三种[5-6]。
LAMP是2000年开发的等温扩增技术，其通过链置换DNA聚合酶如Bst DNA polymerase促进反应进行，最佳反应温度是65 ℃，反应全程可分为复制起始、循环扩增、延伸3个阶段，与PCR的3个反应阶段不同，LAMP反应全程不需要加热、短时间内就可合成新的DNA[7-8]。
NASBA于1991年首次提出，通过AMV逆转录酶、核糖核酸酶H (RNase H)和T7 RNA聚合酶3种酶发挥作用，是由1对带有T7标签的引物引导的等温核酸扩增技术，其反应最佳温度是41 ℃，可在2 h内将模板RNA扩增109倍，比常规PCR的灵敏度高约103倍[9]。
SDA由特定的内切酶和聚合酶组成，目的DNA两端带有被化学修饰的限制性核酸内切酶识别序列，核酸内切酶在其识别位点将DNA打开缺口，DNA聚合酶随后延伸缺口3′端并替换DNA链，被替换下来的DNA单链可与引物结合，被DNA聚合酶延伸成双链[10]。
RCA于1998年提出，主要由Phi29 DNA聚合酶构成，可以模拟自然界微生物环状DNA滚环复制，在具有链置换活性的DNA聚合酶作用下，既实现环状DNA的扩增，又能实现对靶核酸基因的放大，扩增产物一般是连接在固相支持物(如玻片)表面的DNA引物上，因此RCA适合在芯片上进行信号扩增[11-12]。
HDA技术是2004年提出的等温扩增技术，该技术可模拟动物体内DNA复制，利用解旋酶解开DNA双链，随后单链结合蛋白与单链DNA结合，替代PCR过程中通过温度变化来进行变性-退火-延伸的过程，实现靶基因的指数级扩增[13-15]。
随着近年来临床上等温扩增技术的增多，为便于描述而进行分类，主要分为指数扩增、线性扩增和级联扩增三类进行描述(表 1)，对各种等温扩增技术优缺点进行比较(表 2)。
表 1 RPA与其他等温扩增技术的差异
Table 1 Differences between RPA and other isothermal amplification technique
| 反应 Reaction |
反应所需酶 Reactive enzymes |
检测限 Limit of detection |
特异性 Specificity |
所需引物 Primers |
反应最适温度 Reaction optimum temperature (℃) |
反应时间 Reaction time (min) |
反应模板 Template |
产物检测方法 Product detection method |
是否变性 Denatured or not |
是否初始加热 Initial heating or not |
参考文献 References |
| RPA | 重组酶T4 UvsX；单链DNA结合蛋白T4 Gp32；重组酶辅助蛋白T4 UvsY；链置换DNA聚合酶Bsu Recombinase T4 UvsX; single-stranded DNA binding protein T4 Gp32; recombinant enzyme helper protein T4 UvsY; strand displacement DNA polymerase Bsu |
10 copies/μL | High | 1 pair | 37 | 5−20 | DNA | 琼脂糖凝胶电泳；测流试纸条；实时荧光 Agarose gel electrophoresis; lateral flow dipstick; real-time fluorescence |
No | No | [16] |
| NASBA | 逆转录酶AMV；逆转录酶RNase H；T7聚合酶 Reverse transcriptase AMV; reverse transcriptase RNase H; T7 polymerase |
100 CFU/mL | High | 1 pair | 40−55 | 90−120 | DNA and RNA | 琼脂糖凝胶电泳；测流试纸条；酶联免疫吸附；实时荧光 Agarose gel electrophoresis; lateral flow dipstick; enzyme-linked immunosorbent; real-time fluorescence |
Yes | No | [9, 17] |
| LAMP | Bst DNA聚合酶 Bst DNA polymerase |
− | High | 4−6 primers | 65 | 30-60 | DNA and RNA | 浊度仪；实时荧光；琼脂糖凝胶电泳；智能手机；测流试纸条 Turbidity meter; real-time fluorescence; agarose gel electrophoresis; smartphones; lateral flow dipstick |
No | Yes | [2] |
| SDA | 限制性内切酶；DNA聚合酶 Restriction endonuclease enzymes; DNA polymerase |
High | High | 2 pairs | 37 | 15−20 | DNA | 琼脂糖凝胶电泳；实时荧光；测流试纸条；荧光偏振 Agarose gel electrophoresis; real-time fluorescence; lateral flow dipstick; fluorescence polarization |
Yes | Yes | [10, 17] |
| RCA | 特殊聚合酶Phi29 Special polymerase Phi29 |
High | High | 1 primer or 1 pair | 37 | 60 | DNA | 琼脂糖凝胶电泳；实时荧光 Agarose gel electrophoresis; real-time fluorescence |
No | Yes | [11-12] |
| HDA | 解旋酶；单链DNA结合蛋白SSB；DNA聚合酶 Helicase; single-stranded DNA binding protein SSB; DNA polymerase |
High | High | 1 pair | 65 | 75−90 | DNA | 琼脂糖凝胶电泳；测流试纸条；酶联免疫吸附；电化学 Agarose gel electrophoresis; lateral flow dipstick; enzyme-linked immunosorbent; electrochemistry |
No | No | [13-15] |
| TMA | RNA聚合酶；逆转录酶 RNA polymerase; reverse transcriptase |
High | High | 1 pair | 42 | 15−30 | DNA and RNA | 琼脂糖凝胶电泳；化学发光检测 Agarose gel electrophoresis; chemiluminescence detection |
No | No | [18] |
| RAA | 重组酶UvsX；单链结合蛋白SSB；重组酶辅助蛋白UvsY；DNA聚合酶Klenow Recombinase UvsX; single-chain binding protein SSB; recombinant enzyme helper protein T4 UvsY; DNA polymerase Klenow |
10 copies/μL | High | 1 pair | 37 | 15−20 | DNA | 琼脂糖凝胶电泳；测流试纸条；实时荧光 Agarose gel electrophoresis; lateral flow dipstick; real-time fluorescence |
No | No | [19] |
| ERA | 突变的重组酶、外切酶和聚合酶 Mutated recombinase enzymes, mutated exonuclease enzymes and mutated polymerases |
10 copies/μL | High | 1 pair | 35−40 | 20−30 | DNA and RNA | 实时荧光 Real-time fluorescence |
No | No | [20-21] |
| −：文献中未找到相关信息 −: The relevant information was not found in the literature. |
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表 2 各类等温技术的分类及优缺点比较
Table 2 Classification and comparison of advantages and disadvantages of various isothermal technologies
| 扩增类型 Amplification type |
技术名称 Technical name |
优点 Advantages |
缺点 Disadvantages |
参考文献 References |
| 指数扩增 Exponential amplification |
RPA | 此方法检测无须复杂的仪器，常温就可进行检测；可定量；引物设计简单；可进行荧光终点分析；不需要热变性；快速检测；灵敏性高 This method does not require complex instruments to detect, and it can detect at room temperature; it is quantifiable; the primers’ design are simple; it can be detected by fluorescence endpoint analysis; it does not need thermal denaturation; it has a rapid detection and high sensitivity |
此方法琼脂糖凝胶电泳成像前产物需要纯化；核酸提取步骤较烦琐；引物之间可能会结合，出现非特异性扩增 This method’s product needs to be purified before agarose gel electrophoresis imaging; the nucleic acid extraction steps are cumbersome; primers may bind to each other, resulting in nonspecific amplification |
[22] |
| NASBA | 此方法高敏感性、高特异性；反应快，扩增效率高；保真度高 This method has high sensitivity and specificity; it has a fast response and high amplification efficiency; it has a high fidelity |
此方法反应成分复杂；3种酶反应成本高；不适合大批量样本检测；对DNA病毒检测无优势 This method has complex reaction components; the cost of the three enzymes reactions is high; it is not suitable for testing large sample volumes; there is no advantage to DNA virus detection |
[23] | |
| LAMP | 此方法扩增效率高；反应快；特异性强；不需要特殊设备 This method has high amplification efficiency and fast response; it has a strong specificity; it does not need special equipment |
此方法对引物要求高；容易形成气溶胶，造成假阳性；产物不均一，扩增产物不能用于克隆测序 This method has high primers requirements; it is easy to form aerosols and cause false positives; and the product is not exclusive and it cannot be used for clonal sequencing |
[24] | |
| RAA | 此方法操作步骤简单；灵敏度、特异性高；反应快 This method’s operation steps are simple; it has a high sensitivity and specificity; and it has a fast response |
此方法对引物的要求高；前期需提取核酸，比较烦琐 This method has high requirements for primers; nucleic acid needs to be extracted in the early stage, which is more cumbersome |
[17, 22] | |
| SDA | 扩增效率高、反应快；特异性强；不需要特殊设备 This method has a high amplification efficiency and fast response; strong specificity; no special equipment is required |
产物不均一；循环反应中产生单、双链产物，电泳时易出现拖尾；不适合用于基因工程 This method’s products are heterogeneity; single and double-stranded products are produced in the cyclic reaction, and tailing is easy to occur during electrophoresis; it is not suitable for genetic engineering |
[25] | |
| HDA | 此方法反应体系易获得；反应条件等温；操作步骤简单 This method’s reaction systems are readily available; reaction conditions are isothermal conditions; the steps are simple |
此方法反应效率受解旋酶活性的影响；靶序列超过400 bp可显著影响扩增效率；该方法研究少，发展不成熟 This method’s reaction efficiency is affected by helicase activity; target sequences exceeding 400 bp can significantly affect amplification efficiency; there is little research and immature development of this method |
[13-14] | |
| 线性扩增 Linear amplification |
线性RCA Linear RCA |
此方法灵敏度高；特异性强，易操作；高通量；扩增产物磷酸化后可以进行直接测序 This method has high sensitivity and strong specificity, it is easy to operate; it has a high throughput; the amplification products can be phosphorylated and sequenced directly |
此方法信号检测有背景问题，RCA过程中未成环的锁式探针模板DNA或RNA会出现背景信号；仅限于具有环状结构的病毒、质粒及染色体的扩增，探针合成昂贵 This method’s signal detection has background problems, and background signals will appear in unlooped locked probe template DNA or RNA during RCA; it is limited to the amplification of viruses, plasmids and chromosomes with circular structures, the probes are expensive to synthesize |
[12] |
| 线性SDA Linear SDA |
此方法灵敏度高，特异性高，操作简单，反应过程不需要特殊设备 This method has high sensitivity and specificity, it has a simple operation, it does not need special equipment required in the reaction process |
此方法的产物检测需要特殊仪器；靶DNA的含量可能会受到标本中不明抑制物的抑制 This method’s product testing requires special instruments; the content of target DNA may be suppressed by unknown inhibitors in the specimen |
[26-27] | |
| 级联扩增 Cascade amplification |
SDA级联放大扩增 SDA cascade amplification |
此方法可检测多个靶目标，只需要单个引物；特异性高 This method can detect multiple targets; it requires only a single primer; it has high specificity |
此方法操作复杂；反应时间长，产物检测方法需要特殊仪器 This method has a complex operation; it needs more reaction time; the product’s detection requires special instrumentation |
[28] |
| RCA级联放大扩增 RCA cascade amplification |
此方法灵敏度高；特异性高；操作简单；可同时检测多个目标；可在芯片上扩增 This method has high sensitivity and specificity; it has a simple operation; it can detect multiple targets simultaneously; it can amplify on the chip |
此方法芯片上扩增导致操作步骤较多；费用高且需要特殊仪器 This method’s on-chip amplification has more operation steps; it is expensive and requires special instruments |
[29] |
随着近年对RPA技术研究的不断深入，该技术的应用也在不断拓展，其在感染性疾病诊断、癌基因检测等方面的应用进一步成熟，RPA技术已逐渐成为分子诊断领域的重要技术平台和研究工具。
3.1 RPA在感染性疾病诊断中的应用感染性疾病是由致病微生物通过不同方式导致人体发生感染的疾病，RPA技术因其检测快速灵敏的特点而被广泛应用于感染性疾病的检测中。Choi等[30]设计直接离心RPA微装置实时检测牛奶样品中的致病菌，装置的每个反应室中有待检测菌种的特异性探针和引物，上层注入RPA试剂，下层添加检测样品，试剂和待检测样品在离心力作用下等量分配到各个反应室，进行多重RPA反应，在39 ℃时扩增牛奶中待检测细菌的目的基因，无须提取DNA，可检测肠道沙门氏菌、大肠杆菌O157:H7、副溶血弧菌等3种引起食物中毒的病原菌，反应室内目的基因扩增的荧光信号间隔2 min记录1次，RPA技术扩增所需时间短，检测30 min可得到结果。Li等[31]通过对沙门氏菌基因组DNA中的invA基因进行RPA等温扩增，生成DNA双链扩增产物，SYBR Green I与dsDNA结合，通过照射发光二极管进行光敏显色和定量，该方法定量测定沙门氏杆菌的最低检测浓度为5×103 CFU/mL，该方法的灵敏度和特异性分别是90%和95%，在1 h内完成检测。Wang等[32]通过猪圆环3型病毒基因组的保守序列设计特异性RPA引物和探针，在38 ℃扩增20 min，RT-RPA对猪圆环3型病毒具有特异性，与其他检测病原体无交叉反应，以猪圆环3型病毒的DNA为模板，用RT-RPA法进行扩增，RT-RPA符合RT-PCR诊断标准的概率为96.2%。
3.2 RPA在癌基因检测中的应用RPA技术高效、快速、灵敏检测的特点使其应用逐渐广泛，在检测癌症基因中发挥越来越重要的作用。前列腺癌最常见的融合基因是TMPRSS2-ERG，是前列腺癌的高度特异性基因，Koo等[33]用RT-RPA联合辣根过氧化物催化比色反应对TMPRSS2-ERG的mRNA进行检测，通过肉眼可观察结果或分光光度计进行定量检测；检测提取RNA，根据TMPRSS2-ERG设计特异性引物，继而进行RT-RPA扩增，扩增产物用于比色反应，扩增产物-链霉亲和素-HPR结合物多次洗涤后再加入四甲基联苯进行显色反应，在650 nm处记录吸光度值，其检测极限为104 copies/mL，具有单细胞水平的灵敏性，总检测时间约75 min，不同于其他检测，RPA扩增产物检测所需时间短，检测灵敏度高。PIK3CA基因突变是结直肠癌、乳腺癌、胶质母细胞瘤、肺癌和皮肤癌的发病机制之一，Martorell等[34]提出阻断式等温扩增技术检测突变的PIK3CA基因，反应溶液中含有与正常DNA互补的寡核苷酸(阻断剂)，目标核苷酸位于阻断剂的中心位置，此位置可达到更好的阻断效果，上游引物位于封闭寡核苷酸的附近，目的DNA与阻断剂的结合比其与上游引物结合更稳定，通过阻断剂与引物二者的竞争导致RPA扩增的正常DNA大大减少，优先对突变体进行扩增，通过将RPA扩增后产物与芯片上的探针杂交，达到鉴定突变的目的；RPA技术扩增快速的特点与芯片联合检测PIK3CA基因突变在一个微阵列即可完成，对突变体的检测灵敏度可达到95%，可作为辅助手段对个体化治疗提供帮助。随着RPA技术灵敏、快速的特点，其在癌症检测方面的应用会逐渐广泛。
3.3 RPA与其他技术联合应用RPA技术的逐渐成熟促使其与临床上其他检测技术结合，其与成簇的规则间隔短回文重复序列(clustered regularly interspaced short palindromic repeats, CRISPR)、横向流动试纸条技术(lateral flow dipstick, LFS)的联合较为常见，真正做到了高效、快速、准确检测。
3.3.1 RPA与CRISPR技术的结合CRISPR技术是近年来飞速发展的一种分子诊断技术，RPA与CRISPR技术联合可达到快速准确检测的目的。Zhang等[35]开发了RT-RPA结合CRISPR-Cas12a比色法检测新型冠状病毒，针对新型冠状病毒基因组的ORF1ab和N设计特异性引物，使用金纳米颗粒(gold nanoparticles, AuNPs)比色读数，RT-RPA扩增病毒基因组后得到的大量dsDNA识别PAM序列结合特异crRNA，激活Cas12a，在反式裂解过程中，DNA底物会逐渐从AuNPs上水解，通过紫外-可见吸收光谱和肉眼观察可以对其进行监测。Zhang等[1]统计并分析了25篇利用RPA/RAA联合CRISPR技术检测新型冠状病毒的研究，结果表明当等温扩增技术与CRISPR联用检测样本时灵敏性和特异性较高，检测灵敏度和特异性分别可以达到98%和99%，这表明RPA技术与其他检测方法联合使用能提高检测灵敏度和特异性，将来或会成为检测的新方法供人选择。Xiong等[36]将RPA与Cas12a结合检测非洲猪瘟病毒和山羊痘病毒，将RPA试剂和CRISPR/Cas12a试剂分别加到管底和管盖，盖上反应盖进行反应，可减少气溶胶污染；RPA与Cas12a联用不仅实现了crRNA的特异性检测，同时减少假阳性问题发生，还起到了放大检测信号的作用，使RPA技术检测更加精准、可视化。
3.3.2 RPA与侧流试纸条技术的结合RPA与侧流试纸条联合无需特殊仪器，肉眼通过试纸条就可快速、准确观察结果。Srisrattakarn等[37]采用RPA结合侧流试纸(lateral flow dipstick, LFD)方法检测耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus, MRSA)，根据mecA基因设计特异性RPA引物和探针，将RPA扩增产物加入SYBR染料观察反应结果，通过与PCR比较检测结果，发现RPA-LFD法的灵敏度和特异性分别为92.1%和100.0%。Bian等[38]开发RPA-LFD法检测志贺杆菌和肠侵袭性大肠杆菌，根据其共同的毒力基因ipaH设计RPA引物和检测探针；他们根据ipaH基因设计4种不同的引物和探针组合，筛选最适合的引物和探针，减少假阳性发生，该方法的检测极限为1.29×102 copies/μL。Sun等[39]将RPA与LFD联合使用，设计一个试纸条可同时检测A型流感病毒和B型流感病毒，根据病毒保守序列设计引物和探针，探针上带有地高辛、异硫氰酸荧光素标记，在侧流条加入抗地高辛抗体(B型流感)、抗异硫氰酸荧光素抗体(A型流感)与RT-RPA扩增后的dsDNA反应，若带有地高辛的检测线变红说明A型流感存在，带有异硫氰酸荧光素的检测线变红则说明B型流感存在；后期进行临床样本检测，并将检测灵敏度和特异性与PCR进行比较，检测一致性较高，说明RPA技术与试纸条联用可作为高效检测流感病毒的新技术。
3.3.3 RPA技术与临床多种技术的结合RPA技术灵敏、高效的优势使其在临床应用中逐步发展，并且与其他技术联合使用也越来越常见。Asa等[40]将RPA技术联合焦磷酸盐离子探针[pyrophosphateion (PPi)-sensing probe, PK-probe]用于检测新型冠状病毒，在新型冠状病毒的全基因组研究中，PK-probe联合RPA技术对新冠病毒的检测限为1 160 copies/mL，该方法使用RPA扩增目的产物后再和PPi探针结合，根据显色反应可以快速判定检测结果。除此以外，为达到更加灵敏检测的目的，RPA还可与絮凝测定、表面增强拉曼散射[41]、电化学、化学发光、智能手机、基于硅微环谐振器的光子等多种其他检测方法联合使用。
基于RPA技术设计的荧光型RPA试剂盒可在临床上辅助疾病的诊断，RPA与试纸条、CRISPR的联合使用已经成为偏远地区及POCT较常用的方法之一。其能够检测多种样品(表 3)，发挥着越来越重要的作用。
表 3 RPA联合其他技术对较常见样本检测
Table 3 RPA integrated with other techniques for the more common sample detection
| 分类 Classification |
致病菌 Pathogenic bacteria |
检测方法 Detection method |
检测时间 Detection time (min) |
检测限 Limit of detection |
特异性 Specificity |
参考文献 References |
| Bacteria | Staphylococcus aureus | RPA-LF | 25 | 105 CFU/mL | 100% | [42] |
| Vibrio cholerae | RPA-LFS | 35 | 10 copies/μL | High | [43] | |
| Pseudomonas aeruginosa | RPA-LFS | 40 | 3.05 CFU/mL | 98.26% | [44] | |
| Salmonella | RPA-Cas13a | 45 | 102 copies/μL | High | [45] | |
| Listeria monocytogenes | RPA-LFIA | 20 | 9.0 CFU/mL | High | [46] | |
| Mycobacterium tuberculosis | AS-RPA/SYBR | 20 | 4.0 copies/μL | 98% | [47] | |
| Vibrio parahaemolyticus | IMS-RPA-LF | 45 | 104 CFU/mL | 80.3% | [48] | |
| Brucella | RPA-LFD | 10−30 | 4−6 copies/mL | High | [49] | |
| Cryptococcus neoformans | RPA-LFS | 20 | 1 fg/μL | High | [50] | |
| Virus | HBV virus | RPA-LFA | − | 2×105 IU/mL | 88.2% | [51] |
| HPV virus | RPA-Cas13a | 35 | 500 copies/μL | High | [52] | |
| African swine fever virus | RPA-Cas12a | 20 | 2 copies/μL | 100% | [53] | |
| Coronavirus | RPA-CRISPR/Cas12a | − | 10 copies/μL | 100% | [54] | |
| Monkeypox virus | RPA-CRISPR/Cas12a RPA-LFS |
20−30 | 1 copy/μL | 100% | [55] | |
| Norovirus | RPA-CRISPR/Cas12a | 40 | 9.65×102 copies/mL | High | [56] | |
| Lumpy dermatosis virus | RPA-CRISPR/Cas12a | 15 | 5 copies/μL | 96.3% | [57] | |
| H7N9 | LFD-RPA | − | 32 fg | 100% | [58] | |
| Parasite | Schistosoma japonicum | RPA-LFD | 15−20 | 5 fg | 100% | [59] |
| Plasmodium | RPALF | 12 | 10 parasites/μL | 100% | [60] | |
| Trichomonas vaginalis | RPA-CRISPR-Cas12a | 60 | 1 copy/μL | 100% | [61] | |
| Trichomonas vaginalis | RPA-LF | 25 | 100 fg | High | [62] | |
| Others | Mycoplasma pneumoniae | RPA-CRISPR/Cas12a | 60 | 2 copies/μL | 100% | [63] |
| Chlamydia trachomatis | RPA-LFD | 20 | 200 copies | 100% | [64] | |
| Mycoplasma hyopneumoniae | RPA-LFS | 20 | 5.0×102 fg | 100% | [65] | |
| −：文献中未找到相关信息 −: The relevant information was not found in the literature. |
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RPA技术作为一种快速、便捷、高效的核酸扩增技术，与临床上其他核酸扩增技术相比主要具有3个优点：(1) 检测受环境影响小。PCR需要在反应仪器中进行，反应流程包括人工加样、设置程序、等待检测结果等多个步骤，一旦无仪器则反应无法进行；而RPA技术在常温下就可进行反应，反应全程无需复杂的仪器，对环境要求低，操作简单，POCT式的检测适用于不发达地区，可减轻贫困地区因缺少仪器而检测困难的问题，拓宽RPA技术临床的使用范围。(2) 检测时间短。RPA技术反应只需20−30 min，而PCR反应全程需要1−2 h，Ma等[66]用RPA扩增16、18型HPV临床样本DNA，利用SYBR Green I染料监测产物扩增情况，对HPV病毒的检测限达到100 copies/mL，并与其他HPV类型无交叉，准确率达到98.5%，20 min内可通过肉眼观察结果。因此，RPA能够达到快速检测的目的，对于大批量检查或疾病普查有实际意义。(3) 可与其他技术联用，提高检测灵敏度。Su等[67]用RPA与CRISPR-Cas介导多路横向流动法可视化检测新型冠状病毒，双通道能同时检测多个不同基因，提高检测效率，检测新型冠状病毒的灵敏度达到10 copies/μL。除此之外，RPA-LF结合免疫磁分离(immunomagnetic separation, IMS)[68]检测金黄色葡萄球菌，可在15 min内得到检测结果，对金黄色葡萄球菌基因组的检出量可低至每反应600 fg，比PCR的灵敏度高约16.7倍，对提高临床检测灵敏度具有实际意义。
同时RPA也存局限性和挑战，近些年来，科研人员正尝试从4个方面对RPA进行优化：(1) 避免气溶胶污染。在提取核酸和样品加样过程中，反复加样或开盖振荡反应管都易造成气溶胶污染。为减少污染，Li等[69]将RPA和Cas12系统整合到一个试管中，即在一个试管内不开管检测，尽量减少气溶胶污染的操作步骤，在20 min内实现MRSA的可视化检测。(2) 排除抑制剂影响。核酸提取过程中试剂或培养基的选择很重要，Valasevich等[70]发现十六烷基三甲基溴化会严重抑制RPA反应；Liu等[71]发现亚硒酸胱氨酸培养基显著影响RPA反应，会产生大量引物二聚体，出现假阳性结果。因此，研究人员在选择培养基时应注意使用LB培养基，避免选用亚硒酸胱氨酸培养基。(3) 优化引物与探针。Liu等[72]发现同一探针、不同的引物对荧光RPA的效率影响较小，而同一扩增区域不同探针对荧光RPA的效率影响较大。Higgins等[73]提出引物-模板复合物不匹配会影响反应，导致临床诊断易导致假阴性。Higgins等[74]提出Primed RPA软件自动选择RPA引物和探针，通过几对引物、探针序列进行比对来确定保守序列，过滤可能与背景生物发生交叉反应的区域，旨在解决引物与探针不适配的问题。(4) 温度和搅拌。过高、过低的温度都会导致RPA结果不稳定，均匀的搅拌是影响RPA反应外在因素。Wambua等[75]证明当搅拌4 min再进行RPA反应时，阈值荧光值会在5−8 min出现，无搅拌的情况下，荧光值出现的时间是8−14 min，由此可见适当的搅拌可加快反应进程。
4.2 结语RPA技术作为新兴的核酸扩增技术，具有所需设备简单、反应速度快、灵敏度高等优点，克服了荧光定量PCR技术中仪器复杂、成本高昂等缺陷。该技术的应用价值在新型冠状病毒、非洲猪瘟病毒等病原体核酸检验中得到了较好的证明。
综合近几年国内外等温扩增技术的研究情况，RPA检测POCT化和多技术联合应用将是该技术未来几年发展的方向。POCT的最大特点是不需要对标本进行复杂的预处理，在采样现场即可进行检测分析，快速得到检测结果。其对于近几年新型冠状病毒感染的检测有较实际的意义，POCT式的检测可以减少因采集核酸导致的人群大量聚集，降低人群直接接触感染的概率。另外，与临床上其他检测技术结合使检测更灵敏、观察结果更直接便捷。重组酶介导的条形码/扩增诊断方法联合测序高通量方法，可一次筛选10×104个疑似新型冠状病毒感染的病人样本[76]。此外，RPA技术还与光控CRISPR/Cas12a、APHF分析仪等方法联合使用，旨在提高检测灵敏度和特异性[77-78]。
综上所述，RPA是继PCR后有较高实用价值的新技术。由于其还在发展阶段，需要进行不断的探索，相信随着对RPA技术的深入研究，以等温核酸扩增技术为基础的诊断产品会不断被开拓，RPA将发挥自己独特的作用，其产品将更加广泛地应用到日常检测当中，为医疗事业作出贡献。
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