微生物学报

Comparison parameters	PCR	LAMP	References
Temperature conditions	The process requires multiple temperature cycles and imposes high demands on environmental control	Isothermal conditions: maintain at 60-65 °C with lower environmental demands	[12]
Time	2-4 h (including 30-40 cycles)	15-60 min (typically completed within 30 min)	[12]
Equipment requirements	Requires a PCR instrument, which is relatively expensive and has poor portability	The process only requires isothermal equipment such as a water bath or metal heating block, which is low-cost and highly portable	[12]
Sensitivity	Relatively high	High (typically 10-100 times higher than PCR)	[13]
Specificity	Requires 2 primers	Requires 4-6 primers targeting 6-8 distinct regions, ensuring high specificity	[14]
Cost	Instrument and reagent costs are relatively high	Low reagent costs and no expensive instruments required	[14]

Comparison parameters	PCR	LAMP	References
Temperature conditions	The process requires multiple temperature cycles and imposes high demands on environmental control	Isothermal conditions: maintain at 60-65 °C with lower environmental demands	[12]
Time	2-4 h (including 30-40 cycles)	15-60 min (typically completed within 30 min)	[12]
Equipment requirements	Requires a PCR instrument, which is relatively expensive and has poor portability	The process only requires isothermal equipment such as a water bath or metal heating block, which is low-cost and highly portable	[12]
Sensitivity	Relatively high	High (typically 10-100 times higher than PCR)	[13]
Specificity	Requires 2 primers	Requires 4-6 primers targeting 6-8 distinct regions, ensuring high specificity	[14]
Cost	Instrument and reagent costs are relatively high	Low reagent costs and no expensive instruments required	[14]

Indicator	Color change	Principle	Notes	References
SYBR Green I	Orange→ green (UV)	DNA-intercalating dye that binds amplified dsDNA and emits fluorescence. Signal intensity increases with DNA amplification	Non-specific binding; potential toxicity; signal suppression under high dye concentrations	[14]
Calcein	Orange→fluorescent green	Calcein bound to Mn²⁺ is quenched (orange). With target DNA, LAMP releases Mn²⁺ to pyrophosphate, restoring green fluorescence	Calcein and MnCl₂ can inhibit LAMP amplification to some extent	[14]
Hydroxy naphthol blue (HNB)	Purple→ blue	HNB turns purple in the presence of Mg²⁺. During LAMP amplification, abundant magnesium pyrophosphate precipitates, sharply lowering Mg²⁺ and shifting HNB from purple to blue	Color perception varies among observers, introducing subjective error	[14]
Xylenol orange (XO)	Purple→ yellow	The pH range (yellow<pH 6.7<purple). During the LAMP reaction, pyrophosphate and hydrogen ions are released as products, causing the pH to gradually become acidic	The XO concentration significantly affects the coloration	[47]
Phenol red	Pink→ yellow	The pH variation range: 6.8 (pink)-8.4 (yellow). During the LAMP reaction, pyrophosphate and hydrogen ions are released as products, gradually acidifying the pH	Phenol red is susceptible to temperature influences	[48]
Neutral red	Light orange→ pink	The pH transition range: 6.8 (pink)-8.0 (pale orange). During the LAMP reaction, pyrophosphate and hydrogen ions are released as products, gradually acidifying the pH	Buffering conditions have specific requirements	[49]

Indicator	Color change	Principle	Notes	References
SYBR Green I	Orange→ green (UV)	DNA-intercalating dye that binds amplified dsDNA and emits fluorescence. Signal intensity increases with DNA amplification	Non-specific binding; potential toxicity; signal suppression under high dye concentrations	[14]
Calcein	Orange→fluorescent green	Calcein bound to Mn²⁺ is quenched (orange). With target DNA, LAMP releases Mn²⁺ to pyrophosphate, restoring green fluorescence	Calcein and MnCl₂ can inhibit LAMP amplification to some extent	[14]
Hydroxy naphthol blue (HNB)	Purple→ blue	HNB turns purple in the presence of Mg²⁺. During LAMP amplification, abundant magnesium pyrophosphate precipitates, sharply lowering Mg²⁺ and shifting HNB from purple to blue	Color perception varies among observers, introducing subjective error	[14]
Xylenol orange (XO)	Purple→ yellow	The pH range (yellow<pH 6.7<purple). During the LAMP reaction, pyrophosphate and hydrogen ions are released as products, causing the pH to gradually become acidic	The XO concentration significantly affects the coloration	[47]
Phenol red	Pink→ yellow	The pH variation range: 6.8 (pink)-8.4 (yellow). During the LAMP reaction, pyrophosphate and hydrogen ions are released as products, gradually acidifying the pH	Phenol red is susceptible to temperature influences	[48]
Neutral red	Light orange→ pink	The pH transition range: 6.8 (pink)-8.0 (pale orange). During the LAMP reaction, pyrophosphate and hydrogen ions are released as products, gradually acidifying the pH	Buffering conditions have specific requirements	[49]

环介导等温扩增检测特异性与准确性的改善方法

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吴泽勇 ¹ , 欧阳松应 ¹^,² , 王冬梅 ¹

微生物学报 | 综述 2025,65(10): 4326-4339

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微生物学报 | 综述 2025, 65(10): 4326-4339

环介导等温扩增检测特异性与准确性的改善方法

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吴泽勇¹, 欧阳松应¹^,², 王冬梅¹

作者信息

¹ 福建师范大学生命科学学院，福建福州

² 福建师范大学，南方生物医学研究中心，福建福州

Methods to improve the detection specificity and accuracy of loop-mediated isothermal amplification (LAMP)

Zeyong WU¹, Songying OUYANG¹^,², Dongmei WANG¹

Affiliations

¹ College of Life Sciences, Fujian Normal University, Fuzhou, Fujian, China

² Biomedical Research Center of South China, Fujian Normal University, Fuzhou, Fujian, China

出版时间: 2025-09-04 doi: 10.13343/j.cnki.wsxb.20250215

文章导航

摘要

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环介导等温扩增(loop-mediated isothermal amplification, LAMP)是一种快速、灵敏的核酸等温扩增方法，具有良好的特异性和敏感性，在核酸检测领域具有广阔的应用前景。然而，基于LAMP的检测方案开发也面临一些问题，例如在扩增过程中会出现非特异性和非模板扩增等情况，进而影响检测结果的准确性和特异性。本文阐述了近年来LAMP开发过程中在规避假阳性、提升检测效果等方面的研究进展，包括引物设计、优化条件、引入特殊化学物质等策略，并探讨了LAMP在检测领域的应用突破与创新。

关键词

环介导等温扩增 / 引物二聚化 / 假阳性 / 非特异性扩增

Abstract

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Loop-mediated isothermal amplification (LAMP), a rapid and sensitive method of nucleic acid isothermal amplification, is characterized by high specificity and sensitivity and holds broad application prospects in nucleic acid detection. However, the development of detection schemes based on LAMP encounters issues, such as non-specific and non-template amplification during the amplification process, which can affect the accuracy and specificity of the detection results. This article elaborates on the recent research progress in the strategies avoiding false positives and enhancing detection efficacy of LAMP, which include primer design, optimization of conditions, and introduction of special chemical substances. Finally, this article explores the breakthroughs and innovations of LAMP in the field of detection.

Key words

loop-mediated isothermal amplification / primer dimerization / false positive / non-specific amplification

引用本文

吴泽勇, 欧阳松应, 王冬梅. 环介导等温扩增检测特异性与准确性的改善方法. 微生物学报, 2025 , 65 (10) : 4326 -4339 . DOI: 10.13343/j.cnki.wsxb.20250215

Zeyong WU, Songying OUYANG, Dongmei WANG. Methods to improve the detection specificity and accuracy of loop-mediated isothermal amplification (LAMP)[J]. Acta Microbiologica Sinica, 2025 , 65 (10) : 4326 -4339 . DOI: 10.13343/j.cnki.wsxb.20250215

正文

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核酸检测是一种用于检测和分析生物核酸分子的技术，在医学诊断、疾病检测、遗传学研究和环境监测等领域发挥着重要作用^[1]。最常用的核酸检测方法是PCR^[2]。PCR技术需要复杂的设备和特定的实验条件，且操作过程需进行多个温度循环，耗时较长^[3]，这限制了其在现场应用的进一步发展。为克服这些限制，许多新的技术和方法被研发出来。近几年，各类等温扩增技术被提出并应用于核酸检测领域^[4-6]，包括环介导等温扩增(loop-mediated isothermal amplification, LAMP)^[4]技术、重组酶聚合酶扩增(recombinase polymerase amplification, RPA)^[7]技术、核酸序列依赖性扩增(nuclear acid sequence-based amplification, NASBA)^[8]技术、链置换扩增(strand displacement amplification, SDA)^[9]技术、滚环扩增(rolling circle amplification, RCA)^[10]技术和解旋酶依赖扩增(helicase-dependent amplification, HDA)^[11]技术等。其中，LAMP技术具有扩增速度快、扩增温度恒定、操作简便等优点(表1)^[12-14]，在快速检测和高通量分析等领域具有广阔前景。然而，在开发过程中LAMP技术容易导致非特异性扩增和模板污染，这是LAMP检测技术开发过程中需要解决的关键问题^[15]。

1 LAMP的技术原理

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LAMP于2000年由Notomi^[4]提出，是一种利用特殊设计的引物组和DNA聚合酶，在60-65 ℃进行核酸扩增的技术，该技术不需要复杂的温度循环和昂贵设备，具备在简易条件下进行快速核酸检测的潜力^[16]。

LAMP是一种基于链置换DNA聚合酶的恒温核酸扩增方法，其反应体系由脱氧核糖核苷三磷酸(deoxy-ribonucleoside triphosphates, dNTPs)、耐高温Bst DNA聚合酶、荧光染料、4-6条特异性引物及DNA模板组成。引物系统通过靶向目标序列的6-8个特异性区域实现高效扩增：核心引物包括上游内部引物(forward inner primer, FIP)/下游内部引物(backward inner primer, BIP)和上游外部引物(forward outer primer, F3)/下游外部引物(backward outer primer, B3)，其中内引物FIP由5′端F1c反向互补区和3′端F2正向结合区组成，BIP则由5′端B1c反向互补区和3′端B2正向结合区构成，外引物F3/B3分别与模板的F3c/B3c区域完全互补^[12]。扩增原理如图1所示，反应初期BIP通过B2区与模板结合启动链延伸，随后B3引物延伸置换出BIP产物形成单链环，FIP随后结合新生链启动反向延伸，F3引物进一步延伸置换出FIP产物形成哑铃状DNA；哑铃结构通过自引导合成转化为茎环DNA后进入指数扩增阶段，内引物交替结合茎环反向重复区触发链置换反应，环引物上游环状引物(loop primer forward, LF)/下游环状引物(loop primer backward, LB)结合环区加速扩增，最终生成包含不同茎长的茎环DNA、多环花椰菜状结构及嵌套产物的复杂混合物^[12]。

2 LAMP的技术应用难题

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LAMP技术凭借高灵敏度、操作便捷性与低设备依赖性的优势，其应用范围已从病原微生物检测^[17]、转基因检验^[18]拓展至食源性致病菌现场筛查^[19]、环境耐药基因监测^[20]等新兴领域；在临床医学领域，它更成为床旁病原体快速诊断的关键工具^[21]。通过与规律成簇的间隔短回文重复序列系统及相关蛋白(clustered regularly interspaced short palindromic repeats associated, CRISPR-Cas)系统^[22]与微流控芯片技术^[23]相结合，LAMP在提升多重检测精度与自动化程度方面取得了显著突破。尽管LAMP技术在床旁诊断与现场筛查中展现出显著效能，但其临床应用仍面临非特异性扩增与模板污染两大关键挑战，这些问题可能显著降低检测结果的准确性与可靠性，进而引发误诊及治疗错误，最终制约该技术的诊断价值与临床应用安全性。

2.1 非特异性扩增形成原因

2.1.1 引物二聚体

引物二聚体的形成是导致目标DNA产物最终产率降低的主要因素之一。当引物序列中存在互补区域时，引物之间可能会发生非特异性结合，形成引物二聚体。其形成不仅会消耗扩增反应中的核酸聚合酶，降低特异性扩增产物的生成，还会引发非特异性产物的形成^[24]。LAMP技术通常使用4-6条引物靶向目标序列的6-8个特定区域。其中，关键的内引物FIP/BIP是复合引物(典型长度为40-45碱基)，各自与目标序列的2个区域互补，这种结构特性使它们易形成发夹结构^[25]。为确保扩增反应的特异性和效率，体系中需要加入高浓度的FIP/BIP引物。引物组的结构复杂性和高浓度使用增加了引物二聚体形成的潜在风险。LAMP技术的机制决定了引物在反应中的使用浓度，LAMP反应中使用的引物总浓度为3.6-4.4 μmol/L，而PCR反应中使用的引物总浓度仅为0.4-1.0 μmol/L。相较于PCR体系，LAMP扩增引物的总浓度明显更高。高浓度的引物也带来了一些潜在问题：LAMP反应更有可能产生不需要的引物二聚体或其他错配杂交，导致非特异性扩增延伸，在扩增结束时出现明显的背景干扰信号或产生与阳性反应相同的假阳性结果^[26-27]。此外，高浓度的Mg²⁺、dNTPs和链置换DNA聚合酶也可能导致二聚体或非特异性聚合物形成^[28]。

2.1.2 Mg²⁺的增加导致非特异性扩增

在LAMP反应参数中，Bst DNA聚合酶是影响扩增效率的关键因素。二价金属离子是DNA聚合酶扩增体系中需要添加的重要组分之一，用于催化核苷酸转移反应和碱基切除^[29]。DNA聚合酶(如Taq和Bst DNA聚合酶)需要一定水平的二价金属离子作为辅助因子以获得最佳扩增性能。Mg²⁺因存在于细胞中被认为是一种生理激活剂^[30]，在扩增过程中可影响引物退火和DNA聚合酶的活性^[31]。根据其浓度的不同，Mg²⁺可增加或降低聚合酶的保真度^[32-35]。已有研究表明，聚合酶的保真度随着游离Mg²⁺浓度的增加而降低^[32]。LAMP体系中一般使用具有强链置换活性的Bst DNA聚合酶，LAMP反应中终浓度为6-8 mmol/L的MgSO₄被认为是适宜的浓度范围。低于一定浓度的MgSO₄可能会导致酶活性不足，使体系扩增效率不佳；高浓度的MgSO₄会对体系起扩增抑制效果，降低Bst DNA聚合酶的保真度导致体系出现非特异扩增^[16]。因此，合适的MgSO₄浓度至关重要，且针对不同扩增体系环境具有选择差异性。

2.1.3 强链置换DNA聚合酶引发非特异性扩增

一般的DNA扩增反应需要先解开DNA的双链结构，再利用引物与靶标单链部分结合以启动后续的延伸反应。PCR过程利用耐高温的DNA聚合酶，能够耐受95 ℃左右的高温环境，使DNA顺利解开双链^[2]。LAMP与PCR反应不同，LAMP在恒温条件下进行扩增反应，不需要高温解旋的过程，需要具有强链置换活性的聚合酶及特殊设计的引物，在恒温下实现链置换反应，提供有效的双链DNA (double-stranded DNA, dsDNA)变性，进而启动后续扩增延伸^[12-14]。LAMP反应最常用的复制酶是来源于嗜热嗜脂肪地芽孢杆菌(Geobacillus stearothermophilus)的Bst聚合酶，其具有强的链置换活性、中等的热稳定性和高的核酸加工能力，但在特定靶标缺乏的情况下容易引起不正确的扩增反应，例如产生DNA多聚^[36]、非模板^[37]或从头合成^[38]的DNA，导致非特异性扩增产物^[39]。2012年，New England Biolabs (NEB公司)获得了Bst 2.0 DNA聚合酶的专利，这标志着等温核酸扩增技术领域的一项重要进展^[40]。Bst 2.0聚合酶在多个关键性能指标上优于其前身Bst DNA聚合酶，包括聚合速度、温度稳定性、耐盐性、保真度、储存稳定性、对dUTP的耐受性、逆转录酶活性以及修饰核苷酸的掺入能力。Bst 2.0 DNA聚合酶的改进通过在所有结构域中引入数十种突变来实现，同时还融合了多种功能域，包括DNA结合域、来自全长Bst聚合酶的天然或失活的核酸外切酶结构域、His标签、几丁质结合域、内含肽及麦芽糖结合域。市面上最流行的Bst样聚合酶包括Bst 2.0和Bst 3.0。Bst 3.0是在Bst 2.0的基础上通过与额外的DNA结合域融合而开发出的最新版本，这种改进进一步增强了该酶对核酸模板的亲和力^[41]。值得关注的是，Bst 2.0和Bst 3.0 DNA聚合酶通常在等温条件下可能会出现非特异性产物的形成，在无DNA模板或引物的条件下也可能发生从头合成^[42]。Hafner等^[36]研究表明，通过对一个短线性模板的扩增以及应用2个引物，可以促进多聚体的形成，这一过程产生了一组不同长度的DNA产物，这些产物是由模板核苷酸序列的串联重复构建而成的。因此，应用这些聚合酶需要特别注意控制反应条件，并需对扩增产物进行后续的验证和纠错。

2.2 模板污染形成原因

2.2.1 LAMP容易产生更多的DNA片段产物

LAMP技术基于特异性引物组合和Bst DNA聚合酶的链置换活性实现快速、高效的DNA扩增，能在1 h内将DNA数量扩增至 10⁹拷贝，相比之下，PCR技术在同等时间内仅能产生约10⁶ DNA拷贝^[43]。LAMP技术产生大量产物的特性要求格外注意实验操作，避免交叉污染，因为极少量的污染源就可能被快速、大量地扩增，进而影响实验结果的准确性。Tambo等^[44]从纳米比亚北部的卫生设施收集了115个使用过的快速诊断试纸(rapid diagnostic tests, RDTs)，并使用PCR和LAMP来确定寄生虫DNA的存在；以LAMP为金标准，RDTs的敏感性和阳性预测值(positive predictive value, PPV)分别为40.91%和90.00%，PCR的敏感性和PPV分别为72.73%和100%；该研究还显示，在低传播率环境中，LAMP检测应用RDTs试纸检出病原体的阳性率高于PCR，提示LAMP相对PCR具有更高的敏感性和阳性预测值。

2.2.2 LAMP可视化结果判读容易导致模板污染

LAMP技术的扩增速度快且效率高，可在短时间内产生大量目标DNA片段。反应结果通常采用比浊法和比色法进行判断^[12]。比浊法是一种简单且直观的核酸扩增结果判断方法，由于LAMP反应过程中焦磷酸离子与Mg²⁺结合产生白色焦磷酸镁沉淀，通过沉淀物的产生可区分核酸是否被扩增。根据沉淀物产生而增加的反应混合物的浊度与DNA产量相关，可通过实时测量浊度实现对LAMP反应的实时监测^[45]。在早前的研究中很多人通过实时浊度法对LAMP扩增过程进行检测。由于浊度仪成本较高，后期有研究者提出肉眼观察溶液中的白色沉淀进行结果判断^[46]。比色法利用比色指示剂(荧光染料、金属离子指示剂染料、pH指示剂)进行反应结果判断，通过观察颜色变化可判断扩增是否成功以及扩增相对数量。目前应用于LAMP检测的指示剂包括SYBR Green I、钙黄绿素、羟基萘酚蓝(hydroxynaphthol blue, HNB)、二甲酚橙、酚红、中性红等^[16]，各类LAMP比色指示剂的应用特点如表2所示^[14,47-49]。利用比色法可直接通过肉眼观察到结果，但为尽可能降低染料本身或辅助试剂对扩增体系的负面影响，很多方案将染料添加步骤置于扩增程序之后，然而此法需打开反应管盖进行操作^[14]，容易导致气溶胶污染^[50-51]。受污染的核酸可能与扩增目标的靶序列竞争，最终影响结果的准确性和灵敏度^[52]。

3 LAMP的技术应用改进策略

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近年来，研究者对LAMP非特异性扩增和模板问题进行分析，通过引物改进、扩增优化、引入特殊化合物等策略提出多种改进方案，以期提高LAMP检测的准确性、特异性和稳定性。

3.1 引物优化

LAMP反应成功的关键在于引物设计。引物优化需考虑多个因素，如浓度、核苷酸配对位置以及DNA序列区域间的间距^[43]。LAMP过程正常进行依赖于在60-65 ℃温度条件下引物保持单链结构，确保引物能正确与目标DNA序列结合。LAMP扩增涉及复杂的引物体系，引物数量增加可增强引物与靶标之间的相互作用，形成复杂的环状结构或多链结构，使扩增反应更快进行。然而在LAMP检测中增加目标位点性质容易形成引物二聚体，导致非特异性扩增，因此在此过程中需注意避免引物形成稳定的双链结构，以免对体系产生负面影响，降低扩增反应的特异性和效率。选择引物需事先分析目标物种中许多基因组序列的变异^[16,43]。最早的LAMP技术使用4种引物识别目标DNA的6个不同区域，称为“四引物LAMP”^[4]，但其检测速度较慢。为提高扩增效率，研究者通过添加环引物^[53]、茎引物^[54]和群体引物^[55]开发了几种LAMP的变体。在这些变体中，具有2个环状引物的LAMP (称为“六引物LAMP”)已得到广泛应用。同化探针通过标记荧光团和淬灭剂与LAMP引物协同作用，实现对目标DNA的精准检测；其独特的荧光淬灭机制可减少非特异性扩增干扰，提升检测特异性^[16]。此外，同化探针茎引物被提出应用于加速扩增反应、帮助限制引物与非目标序列的结合等，通过促进引物结合DNA以及合成DNA的速度缩短扩增时间。相对于六引物LAMP的引物设计，茎引物在设计上具有挑战性^[54]，茎引物的长度、碱基组成和特定结构等方面都需要研发人员具备丰富经验，以确保其适配目的序列。引入群体引物可减少扩增反应中所需的特异性引物数量，降低检测成本，但仅使用群体引物的LAMP体系扩增效率通常低于使用环或茎引物的LAMP扩增效率^[55]。在不同的技术方案中均需考虑复杂的引物组成对反应体系可能造成的负面影响。Ding等^[56]研究建立了一种“双引物”(“自引物”和“配对引物”)等温扩增(dual-priming isothermal amplification, DAMP)测定法，用于使用超低非特异性信号进行快速核酸检测；该方法利用“双引物”链延伸策略，添加2种配对竞争引物并设计独特的内引物，从而实现高灵敏度和特异性的分子检测。与传统LAMP相比，DAMP具有超低非特异信号、较短的扩增时间和更简单的引物设计等优点。

3.2 反应体系优化

LAMP的体系通常包括DNA模板、6个引物(F3/B3、FIP/BIP、LF/LB)、DNA聚合酶、Mg²⁺、dNTPs和缓冲液。这些成分共同作用形成LAMP反应体系，实现高效、特异的扩增。在实验中可调整这些成分的浓度和比例，增加LAMP反应效率和特异性。此外，一些增强剂或抑制成分对体系优化和改造也具有重要作用。例如，添加甜菜碱可减少碱基堆叠，刺激反应的总体速度，还能增加靶标选择性，显著降低无关序列的扩增^[4]。二甲基亚砜(dimethyl sulfoxide, DMSO)可破坏链间和链内再退火，从而通过改变双螺旋DNA的熔解特性来促进双螺旋DNA的链分离^[57-58]。研究表明适量的DMSO不会抑制非特异性扩增，但过高浓度的DMSO可能会影响Bst 2.0 WarmStart DNA聚合酶的活性^[58]。普鲁兰是一种由麦芽糖单元组成的多糖聚合物，随着普鲁兰浓度的增加，Bst DNA聚合酶的有效浓度降低使非特异性LAMP扩增减少^[59]，此外，普鲁兰在扩增反应速度、稳定性上也有积极影响，但这些都依赖于体系中靶标、引物和普鲁兰的适配扩增能力。此外，Zhang等^[60]发现通过添加盐酸胍能够显著提高逆转录环介导等温扩增(reverse transcription loop-mediated isothermal amplification, RT-LAMP)反应的速度和灵敏度；在结合2-3个LAMP引物组合的体系条件下，盐酸胍在不增加非特异性扩增的情况下进一步增强促进效果；盐酸胍在RT-LAMP中的作用机制尚未阐明，根据其对不同引物组合反应的一致增强效果，推测其增强了引物与靶序列之间的碱基配对，盐酸胍的添加也会显著缩短依赖螺旋酶的扩增反应时间。此外，酰胺通常被用于增加PCR的特异性^[61]，通过与DNA的主要凹槽和次要凹槽结合，阻止引物之间的非特异性相互作用。Tanner和Evans^[62]建议使用特定比例的酰胺混合物来增强等温扩增技术的特异性，在标准检测中添加甲酰胺和N-烷基甲酰胺2种化合物可通过抑制聚合酶的活性来减少LAMP的非特异性扩增，提高检测的特异性。在LAMP技术中选择并优化合适的反应体系对于快速、准确地进行扩增具有重要意义。不同的反应增强试剂或非特异性抑制组分在体系中的选择与使用非常关键^[16]，总的来说，需根据具体情况进行合理选择和优化，以确保反应的准确性、特异性和稳定性。

3.3 酶的改造优化

研究证明，LAMP在扩增经热预变性处理DNA模板时灵敏度和效率能够显著提高^[63]。预热处理的步骤更适配具有高温工作能力的聚合酶，LAMP中一般使用Bst DNA聚合酶及其衍生物^[63]，这些DNA聚合酶表现出强大的链替换活性，但在超过70 ℃的高温下不稳定。PCR一般需要高热稳定性和强大聚合酶活性的酶，如Taq DNA聚合酶，这种酶由于欠缺强链置换活性不适用于等温扩增。具备强链置换能力与热稳定性的DNA酶为LAMP技术的应用提供更多可能。Ignatov等^[64]研究了一种新型的Taq DNA聚合酶突变体SD DNA聚合酶，通过比较Bst DNA聚合酶与SD DNA聚合酶在LAMP反应中的扩增特性，证实两者具有相似的聚合酶和链置换活性，SD聚合酶还表现出比Bst聚合酶更优的热稳定性，其在92 ℃的预变性步骤后仍能高效扩增，而Bst DNA聚合酶则完全失活。此外，SD DNA聚合酶在qPCR和实时荧光定量链置换扩增反应(quantitative polymerase chain displacement reaction, qPCDR)中表现出更高的灵敏度，Cq值分别比Taq DNA聚合酶降低了1个循环和5个循环，灵敏度提升100倍。这些特性使SD DNA聚合酶在需要高灵敏度和热稳定性的扩增体系中具有显著优势^[64]。Li等^[65]通过预测和分析活性位点开发具有增强活性的Bst DNA聚合酶突变体，结果显示单突变K431D和K431E的催化效率相较于野生型Bst酶分别提高了1.93倍和2.03倍；他们通过融合深渊火球菌(Pyrococcus abyssi) DNA连接酶(DBD)的DNA结合域进一步构建嵌合蛋白DBD-K431E，最终得到能够适应高温度条件、增强扩增性能的嵌合酶，其在70 ℃处理8 h后仍保持高活性，且在含50 U/mL肝素、10%乙醇及100 mmol/L NaCl的复杂体系中活性稳定；其对沙门氏菌的检测灵敏度显著优于野生型及商用Bst 2.0酶，可检出低至2×10² ag/μL基因组DNA和1.24 CFU/mL的细菌浓度。LAMP扩增中的链置换酶是非常关键的一个组分，酶的性能直接影响体系的扩增，包括稳定性、灵敏度、特异性等，对酶的改造仍是一个非常重要的研究方向。

3.4 特殊化合物添加

LAMP在扩增过程中会因引物二聚体的形成和杂交错配而产生假阳性结果^[26]。目前，已有多个特殊化合物被应用于LAMP扩增方案中，用于阻止引物二聚体及非特异性扩增产物的形成。Ku等^[26]提出了一种利用聚乙二醇接枝的纳米氧化石墨烯(polyethylene glycol-nano graphene oxide, PEG-nGO)来提高LAMP特异性的方法，PEG-nGO是一种具有高表面积的二维(2D)纳米材料^[66-67]，使用聚乙二醇(PEG)包覆纳米氧化石墨烯(nGO)的表面，可增强nGO在LAMP反应混合物中的溶解性并保持DNA聚合酶的活性，适宜浓度的PEG-nGO可通过吸附多余的DNA引物抑制由引物二聚化引起的非特异性扩增^[27,68-69]。实验数据充分证明了其有效性：添加适宜浓度的PEG-nGO后，目标DNA与空白样本的ΔT _t值从0.15显著提升至33.3，表明特异性大幅增强；在检测丙型肝炎病毒(hepatitis c virus, HCV)样本时，其成功诊断出所有10份阳性样本，而RT-qPCR仅检出7份；同时，与传统LAMP相比，其假阳性判断精度提升了1.75倍；因此PEG-nGO可在背景信号减少的情况下促进目标DNA的特异性扩增，从而显著提高LAMP检测的性能^[26]。传统的等温扩增方法在特异性方面存在局限性，因为引物的非特异性结合可能导致非特异性扩增。Ye等^[70]研究了一种利用金纳米颗粒(gold nanoparticles, AuNP)介导的核酸等温扩增方法，研究人员利用金纳米颗粒作为LAMP反应的介质，通过引物和金纳米颗粒特异性结合提高反应特异性；该方法首先将AuNP表面修饰为亲核酸的分子，然后将引物与AuNP结合，形成引物-AuNP复合物，通过与目标DNA特异性结合来引导扩增反应的进行；实验结果表明，该方法在轮状病毒检测中假阳性率从76%降至0，β-肌动蛋白基因检测中从100%降至0，检测限达1×10³ copies/μL，灵敏度与传统LAMP相当；在对50份临床样本(25份轮状病毒阳性样本和25份阴性样本)的检测中，AuNP介导的LAMP方法成功检测出所有25份阳性样本，且在25份阴性样本中未出现非特异性扩增；同时，其48 ℃热启动效应有效抑制了Bst聚合酶低温活性，显著提升了检测特异性与准确性。Du等^[71]利用纳米金增强的LAMP反应在一体化试管中检测沙门氏菌，将反应时间缩短至30 min内，并表现出优异的特异性。目前，在LAMP中还设计了许多含特殊化合物的反应体系。在进行LAMP实验时，开发工作者可根据具体的实验需求和反应条件选择合适的特殊化合物来优化体系，提高实验的成功率和可靠性。

3.5 UDG-dUTP体系防气溶胶污染

LAMP技术具有快速扩增的特性，极易受到残留污染的影响，前一次LAMP反应中扩增的DNA产物可能会成为下一次反应的模板，导致假阳性结果的出现^[52]。尿嘧啶-DNA-糖基酶补充环介导等温扩增(uracil-dna-glycosylase-supplemented loop-mediated isothermal amplification, UDG-LAMP)是一种防止扩增污染的方法，可有效避免LAMP的残留污染^[52]。通过在LAMP反应中加入dUTP，使扩增产物含有尿嘧啶(U)碱基，扩增DNA产物与原始DNA模板在碱基组成上存在差异：扩增产物含有碱基A、T、C、G、U，原始模板DNA含有A、T、C、G，不含有U。UDG能够识别并切割含U产物，通过UDG反应消化含尿嘧啶的污染DNA分子，从而阻止其再次被扩增，热不稳定的UDG酶在LAMP的扩增温度范围内快速失活。在65 ℃条件下，只有保留完整性的目标DNA存在并在酶参与下进行快速扩增^[52,72-73]。通过扩增反应前在37 ℃温度条件下短暂孵育，UDG可特异性除去含U的DNA分子，而对天然含有胸腺嘧啶的碱基无影响，进而消除残留污染物，避免引起假阳性结果^[72-73]。Hsieh等^[52]进行的实验表明，即使携带污染物含量仅为1 ag时，未添加UDG处理的反应也能观察到扩增现象；当使用UDG处理时，即使携带污染物浓度高出100倍，也能有效消除扩增反应。这表明UDG处理在防止携带污染物对扩增反应产生干扰方面具有显著效果。Zeng等^[74]将UDG掺入实时环介导等温扩增(real-time LAMP, RT-LAMP)中来检测尿液样本中的细菌，并评估集成UDG辅助实时LAMP检测的分析和临床性能，发现添加UDG可有效将残留污染物切割到低至1 fg，并且可通过选择性尿嘧啶切除提高敏感性和特异性。

3.6 可视化LAMP的优化

LAMP产物量大，在结果判断上有更多选择，包括实时检测策略、不同的染料可视化策略等。目前，大部分LAMP是通过染料实现结果可视化。可视化染料对LAMP体系的抑制、染料扩增后添加导致的污染问题等都是值得关注的问题。在染料应用上研究者提出了各种改进方案，包括在反应管中添加封闭剂、开发更优的可视化染料等。Karthik等^[75]建立了一种琼脂染料胶囊封闭管LAMP检测方法，将染料SYBR Green I夹在1.5%琼脂之间制备成琼脂染料胶囊，在反应开始前将琼脂染料胶囊放入已有LAMP反应混合物的PCR管中，保持胶囊不接触PCR管帽以及反应混合物的位置；LAMP反应结束后，在95 ℃下保持5 min，使琼脂高温熔化，离心使染料与产物混合后再进行结果判读；在反应结束后，琼脂胶在室温下变为半固体，降低了LAMP产物溢出或蒸发到空气中的可能性，比较不容易形成气溶胶污染；需要指出的是，琼脂染料胶囊不能在-20 ℃长时间储存，在低温条件下可能会出现琼脂收缩等问题。Tao等^[76]将SYBR Green I嵌入高熔点微晶蜡(熔点为85 ℃)并在反应管中设计染料胶囊，蜡在反应后熔化并释放染料与DNA结合，可实现不开盖的可视化LAMP观察；在扩增过程中蜡染料胶囊未释放干扰物质；在高温加热过程中蜡壳熔化成低密度液体，漂浮在液位顶部，染料沉入反应液中与产物混合；冷却后，蜡凝固形成屏障，将扩增产物局限在管底，降低了污染风险。可视化染料在LAMP体系中可能会受到干扰抑制，导致颜色变化异常。目前，有研究人员开发出更优的可视化染料。Zhang等^[77]利用吡啶偶氮酚金属感应染料对扩增反应进行简单的视觉检测，吡啶偶氮酚金属感应染料与Mn²⁺形成络合物呈红色，但在LAMP反应中生成的焦磷酸二酯(PPi)会取代Mn²⁺，形成不溶性的焦磷酸锰，从而恢复染料的原始黄色。这种检测方法易于通过吸光度定量，肉眼可快速清晰观察，对不同样本条件的适应性强。

3.7 CRISPR-Cas系统增强LAMP特异性

CRISPR-Cas系统与LAMP技术的融合是近年来提升检测特异性的突破性策略。其核心机制在于：LAMP扩增后的产物激活Cas12a或Cas13a的反式切割活性，通过CRISPR RNA (crRNA)引导特异性识别目标序列，进而切割荧光或比色报告分子，实现信号的级联放大与可视化输出^[78]。这种“等温扩增+序列验证”的双重保障机制可有效规避由引物二聚体或非特异性扩增导致的假阳性问题，同时将检测灵敏度提升至单拷贝级别。Habimana等^[79]将LAMP结合最新的CRISPR/Cas12a技术，提出针对高致病性幽门螺杆菌(Helicobacter pylori)菌株的高敏感度检测方法，该方法通过新型优化扩展单链DNA (single-stranded DNA, ssDNA)报告基因，加上新型缓冲体系优化CEXTRAR，显著提高了LbCas12a的反切活性，使检测灵敏度较传统PCR提高了16倍。Yang等^[78]开发了非洲猪瘟病毒(African swine fever virus, ASFV)的“一步法”LAMP-CRISPR检测，其临床样本检测水平与实时荧光定量PCR方法相当，且在一个反应管中一步完成反应，有效避免了污染。Chen等^[80]构建了基于LAMP-CRISPR/Cas12b的犬细小病毒(canine parvovirus, CPV)快速检测平台，通过靶向CPV的VP2基因保守区设计特异性LAMP引物与单链向导RNA (single guide RNA, sgRNA)，开发了“两步法” (LAMP扩增+CRISPR检测分步进行)和“一步法” (磁性纳米颗粒同步富集核酸、扩增与检测) 2种检测模式；该平台在1 h内完成检测，灵敏度达10^-1 copies/μL，较qPCR/LAMP提升100倍，且与犬瘟热病毒、轮状病毒等常见犬腹泻病原体零交叉反应；其中一步法通过闭管操作整合核酸提取至检测全流程，实现“样本进-结果出”的床旁诊断即时检验(point-of-care testing, POCT)，为动物疫病现场筛查提供突破性解决方案。LAMP-CRISPR通过双重级联机制实现检测性能的显著提升，未来需着力攻克crRNA标准化设计及一步法体系兼容性等挑战，以加速该策略成为多场景诊断的可靠工具。

4 总结与展望

收起

近年来，LAMP技术被广泛应用于病原体诊断方案的开发，相较于其他核酸检测技术，LAMP具有扩增速度快、扩增温度恒定、操作简便等优点，但其在反应过程中出现的非特异性扩增和模板污染仍是研究开发工作中不容忽视的问题。本文探讨了LAMP非特异性扩增及阳性污染出现的诸多原因，并对LAMP优化和改进的研究进行总结归纳。未来对LAMP技术的应用研究还可以在以下几个方面寻求突破：(1) 不同的LAMP变体已被提出并应用于疾病检测，但是变体引物设计工作对更多人来说仍是困难的，下一步可通过计算机辅助及机器学习开发引物设计工具，提高设计的准确性和效率；(2) 为解决问题，已搭建或优化了适用于LAMP的不同体系，然而复杂体系需要考虑的成本问题给LAMP的实际应用及大规模生产带来了挑战，后续研究开发低成本、高效率、具有现场检测实用性的LAMP试剂或检测系统仍至关重要，更精密、更方便的配套检测设备或辅助工具在LAMP应用上仍具有开发潜力；(3) 核酸检测的应用场景复杂多样，目前的优化方案或改进手段大多还处于实验研究阶段，能否适应实际环境条件并成功应用还需后续试验。基于以上问题的突破和创新将为LAMP用于病原体检测带来新的可能。

基金

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国家自然科学基金(82172287)

参考文献

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2025年第65卷第10期

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doi: 10.13343/j.cnki.wsxb.20250215

接收时间：2025-03-18
首发时间：2025-11-03
出版时间：2025-09-04

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收稿日期：2025-03-18
录用日期：2025-07-06

基金

the National Natural Science Foundation of China(82172287)

国家自然科学基金(82172287)

作者信息

¹ 福建师范大学生命科学学院，福建福州

² 福建师范大学，南方生物医学研究中心，福建福州

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Comparison parameters	PCR	LAMP	References
Temperature conditions	The process requires multiple temperature cycles and imposes high demands on environmental control	Isothermal conditions: maintain at 60-65 °C with lower environmental demands	[12]
Time	2-4 h (including 30-40 cycles)	15-60 min (typically completed within 30 min)	[12]
Equipment requirements	Requires a PCR instrument, which is relatively expensive and has poor portability	The process only requires isothermal equipment such as a water bath or metal heating block, which is low-cost and highly portable	[12]
Sensitivity	Relatively high	High (typically 10-100 times higher than PCR)	[13]
Specificity	Requires 2 primers	Requires 4-6 primers targeting 6-8 distinct regions, ensuring high specificity	[14]
Cost	Instrument and reagent costs are relatively high	Low reagent costs and no expensive instruments required	[14]

Comparison parameters

PCR

LAMP

References

Temperature conditions

The process requires multiple temperature cycles and imposes high demands on environmental control

Isothermal conditions: maintain at 60-65 °C with lower environmental demands

[12]

Time

2-4 h (including 30-40 cycles)

15-60 min (typically completed within 30 min)

[12]

Equipment requirements

Requires a PCR instrument, which is relatively expensive and has poor portability

The process only requires isothermal equipment such as a water bath or metal heating block, which is low-cost and highly portable

[12]

Sensitivity

Relatively high

High (typically 10-100 times higher than PCR)

[13]

Specificity

Requires 2 primers

Requires 4-6 primers targeting 6-8 distinct regions, ensuring high specificity

[14]

Cost

Instrument and reagent costs are relatively high

Low reagent costs and no expensive instruments required

[14]

Indicator	Color change	Principle	Notes	References
SYBR Green I	Orange→ green (UV)	DNA-intercalating dye that binds amplified dsDNA and emits fluorescence. Signal intensity increases with DNA amplification	Non-specific binding; potential toxicity; signal suppression under high dye concentrations	[14]
Calcein	Orange→fluorescent green	Calcein bound to Mn²⁺ is quenched (orange). With target DNA, LAMP releases Mn²⁺ to pyrophosphate, restoring green fluorescence	Calcein and MnCl₂ can inhibit LAMP amplification to some extent	[14]
Hydroxy naphthol blue (HNB)	Purple→ blue	HNB turns purple in the presence of Mg²⁺. During LAMP amplification, abundant magnesium pyrophosphate precipitates, sharply lowering Mg²⁺ and shifting HNB from purple to blue	Color perception varies among observers, introducing subjective error	[14]
Xylenol orange (XO)	Purple→ yellow	The pH range (yellow<pH 6.7<purple). During the LAMP reaction, pyrophosphate and hydrogen ions are released as products, causing the pH to gradually become acidic	The XO concentration significantly affects the coloration	[47]
Phenol red	Pink→ yellow	The pH variation range: 6.8 (pink)-8.4 (yellow). During the LAMP reaction, pyrophosphate and hydrogen ions are released as products, gradually acidifying the pH	Phenol red is susceptible to temperature influences	[48]
Neutral red	Light orange→ pink	The pH transition range: 6.8 (pink)-8.0 (pale orange). During the LAMP reaction, pyrophosphate and hydrogen ions are released as products, gradually acidifying the pH	Buffering conditions have specific requirements	[49]

Indicator

Color change

Principle

Notes

References

SYBR Green I

Orange→ green (UV)

DNA-intercalating dye that binds amplified dsDNA and emits fluorescence. Signal intensity increases with DNA amplification

Non-specific binding; potential toxicity; signal suppression under high dye concentrations

[14]

Calcein

Orange→fluorescent green

Calcein bound to Mn²⁺ is quenched (orange). With target DNA, LAMP releases Mn²⁺ to pyrophosphate, restoring green fluorescence

Calcein and MnCl₂ can inhibit LAMP amplification to some extent

[14]

Hydroxy naphthol blue (HNB)

Purple→ blue

HNB turns purple in the presence of Mg²⁺. During LAMP amplification, abundant magnesium pyrophosphate precipitates, sharply lowering Mg²⁺ and shifting HNB from purple to blue

Color perception varies among observers, introducing subjective error

[14]

Xylenol orange (XO)

Purple→ yellow

The pH range (yellow<pH 6.7<purple). During the LAMP reaction, pyrophosphate and hydrogen ions are released as products, causing the pH to gradually become acidic

The XO concentration significantly affects the coloration

[47]

Phenol red

Pink→ yellow

The pH variation range: 6.8 (pink)-8.4 (yellow). During the LAMP reaction, pyrophosphate and hydrogen ions are released as products, gradually acidifying the pH

Phenol red is susceptible to temperature influences

[48]

Neutral red

Light orange→ pink

The pH transition range: 6.8 (pink)-8.0 (pale orange). During the LAMP reaction, pyrophosphate and hydrogen ions are released as products, gradually acidifying the pH

Buffering conditions have specific requirements

[49]