QuickConc：基于阳离子辅助捕获的快速高效无动力环境DNA浓缩新方法

本刊推荐一篇环境DNA（eDNA）技术领域的重要研究。该研究创新性地开发了名为QuickConc的eDNA捕获方法，该方法结合了苯扎氯铵（BAC）与分散的玻璃纤维，显著提升了核酸捕获效率。实验表明，与传统玻璃纤维过滤和Sterivex方法相比，QuickConc在河水、海水及池塘水等多种水体中，总eDNA产量提升1.3-3倍，物种特异性qPCR检测灵敏度提高2-10倍。此方法为生物多样性监测与保护策略提供了新的高效工具，尤其适用于现场快速、无动力操作场景。

　　

摘要

环境DNA（eDNA）分析作为一种非侵入性工具，在揭示物种分布与丰度、避免生态系统干扰方面具有重要作用。eDNA分析包含水体保存、DNA捕获、DNA提取和检测四个关键步骤，其中eDNA的捕获因水样的多变性而备受关注。尽管已开发出多种eDNA浓缩方法，如碟式或筒式过滤器过滤以及被动采样器，但由于eDNA分布和水体特性（如浊度）的差异，尚无一种方法能普遍适用。因此，开发替代性的eDNA浓缩方法对推动eDNA研究至关重要。本研究介绍了一种新型核酸捕获方法——QuickConc，该方法将苯扎氯铵（BAC）作为增强剂与分散的硅质玻璃纤维结合，从而改善了核酸与玻璃纤维之间的结合。研究结果表明，这种方法通过增强玻璃纤维与eDNA之间的相互作用，提高了eDNA的捕获和提取效率。研究使用定量聚合酶链式反应（qPCR）和代谢条形码（Metabarcoding）技术在三种环境中对QuickConc进行了测试。在本实验条件下，QuickConc获得的总eDNA量是玻璃纤维过滤法和Sterivex法的1.3-3倍。物种特异性qPCR结果显示，QuickConc检测到的拷贝数比其他两种方法高2-10倍。使用MiFish方法进行的代谢条形码分析表明，在河水样本中，QuickConc检测到的鱼类物种数量多于其他方法；而在海水样本中，检测到的鱼类物种数量与玻璃纤维过滤法和Sterivex法相似。QuickConc为生物多样性监测和保护策略的eDNA分析提供了新的选择。

1 引言

开发微创且破坏性小的生物监测技术对于生物多样性保护（Kerry等，2022；Margules和Pressey，2000）和有效管理政策（Jackson等，2001；Jungmeier和Yenilmez Arpa，2022）至关重要。环境DNA（eDNA）分析已成为生态监测的强大非侵入性工具。它来源于各种生物来源，并以溶解、颗粒结合或封装形式存在（Barnes和Turner，2016；Foote等，2012；Mauvisseau等，2022；Power等，2023；Rees等，2014）。通过使用定量聚合酶链式反应（qPCR）或结合下一代测序（NGS）的代谢条形码技术分析水样，eDNA分析为物种分布和丰度提供了宝贵见解（Civade等，2016；Jackman等，2021；Robson等，2016；Taberlet等，2018）。该技术已广泛应用于水生生态系统调查，并正在进行全球标准化努力（Shu等，2020）。

eDNA的可检测性受到四个关键步骤的显著影响：水体保存、DNA捕获、DNA提取和检测。主要的eDNA捕获方法是过滤（Allison等，2021；Bairoliya等，2022；Muha等，2019）和被动eDNA采样（Kirtane等，2020；Verdier等，2022；Mariani等，2019）。传统的实验室过滤方法可以浓缩数十毫升到数升的水（Tsuji等，2019），但可能受到浊度、耗时或设备要求的限制（Bessey等，2021；Liu等，2024；Ruan等，2022；Sato等，2017）。现场技术，如基于注射器的Sterivex过滤（Miya等，2016）、重力过滤（Gold等，2021；Oka等，2022）或电动采样器（Thomas等，2018）提供了实用的替代方案，但在高浊度水中仍可能面临堵塞或操作限制。被动采样方法（Bessey等，2021；Kirtane等，2020；Rivera等，2021；Verdier等，2022）允许进行简单、长期的监测，但时间分辨率有限，并且使定量解释复杂化。

滤膜孔径的选择影响捕获的eDNA粒径分布（Liu等，2024；Majaneva等，2018；Power等，2023；Rivera等，2021；Zhao等，2021）。Suzuki等（2023）最近开发了一种使用分散玻璃纤维捕获eDNA，然后进行重力过滤的浓缩技术。这种方法利用了DNA对二氧化硅（玻璃的主要成分）的亲和力。该方法的独特之处在于它使用了相对较大的孔径（180 μm）的过滤器，与传统使用的过滤器（玻璃纤维滤膜：0.7–3.0 μm，Sterivex：0.22–0.45 μm）相比（Minamoto等，2021；Takahashi等，2023）。它在过滤前将环境水中的eDNA与玻璃纤维结合。Suzuki等（2023）主要关注现场检测；然而，他们简化的核酸提取方法在eDNA回收效率和抑制剂去除方面仍有改进空间。

硅胶与DNA之间的亲和力众所周知依赖于静电（磷酸盐-硅烷醇）和疏水相互作用（Shi等，2015；Vandeventer等，2012）。促溶剂，如硫氰酸胍，通常用于促进这种结合。苯扎氯铵（BAC）是一种季铵盐化合物和常见的消毒剂，在较高浓度下也已被研究作为eDNA保存剂（Takahara等，2020）；然而，其增强eDNA与分散玻璃纤维特异性结合的能力尚未得到广泛测试。我们假设BAC，凭借其正电荷和疏水尾，可以在带负电荷的DNA和二氧化硅之间形成分子桥，从而促进DNA捕获。

在本研究中，我们开发了QuickConc，一种使用分散玻璃纤维和BAC的新型DNA捕获方法。首先，我们评估了BAC在三种过滤方法（QuickConc、玻璃纤维滤膜和Sterivex滤筒）中是否能提高总eDNA和物种特异性eDNA产量（实验1）。接下来，我们在三种水生环境（河流、海洋、池塘）中比较了QuickConc与玻璃纤维滤膜和Sterivex滤筒在总DNA产量和物种特异性qPCR方面的表现（实验2）。最后，我们使用代谢条形码（MiFish）评估了QuickConc如何影响河水和海水中鱼类群落组成的估计。我们的研究结果表明，QuickConc可以提高eDNA产量，并且在某些情况下，至少在本研究比较的方法中，可以改善物种检测。这为eDNA研究和生物监测提供了一个新工具。

2 材料与方法

我们进行了两个实验：实验1评估了BAC添加对三种过滤方法的影响，而实验2在三种类型的水生环境中比较了QuickConc与传统方法。图1提供了实验设计的概览。

2.1 实验1：评估BAC在三种方法中的添加效果

2.1.1 实验1的样品采集

地表水样（每份400 mL）于2025年3月5日以及2025年6月3-4日从神崎川（34°44′02.8″N 135°27′34.3″E）采集，用于初步评估最佳BAC浓度和提取缓冲液条件。采集了四个重复样本。每个样本立即转移到实验室。非BAC样本未经修改进行处理，而BAC组样本则添加BAC至最终浓度为0.001% w/v（见图1左图）。

2.1.2 实验1的浓缩和DNA提取

我们比较了在存在或不存在BAC（最终浓度0.001% w/v）条件下，使用三种方法处理的样品的eDNA产量：QuickConc、玻璃纤维滤膜（Advantec GA-55）和Sterivex（Millipore SVHV010RS）。基于我们从eDNA产量和操作可行性角度对最佳BAC浓度的研究，比较仅使用0.001%的BAC浓度进行（图S1）。玻璃纤维滤膜和QuickConc的提取条件是在初步研究各种提取缓冲液组成后确定的（图S2）。详细方案如下：

• •
  QuickConc：QuickConc的浓缩过程如图2所示；它是本研究的结果并已商业化。简而言之，将一片玻璃纤维片（1 cm2）加入装有环境水的2升袋中。剧烈搅拌袋子，然后静置1分钟。在此过程中，玻璃纤维片分解成单个纤维，分散到整个水中。然后使用网状过滤器收集这些分散的纤维，并转移到2 mL管中。我们加入400 μL ATL缓冲液和40 μL蛋白酶K，然后在56°C下孵育30分钟。孵育后，加入800 μL 100%乙醇并混合，然后通过离心柱进行纯化。使用DNeasy Blood & Tissue Kit（Qiagen）纯化DNA。
• •
  玻璃纤维滤膜：水样在真空下通过标称孔径为0.6 μm的玻璃纤维滤膜（Advantec GA-55）进行过滤。水过滤和eDNA提取过程遵循环境DNA学会发布的《环境DNA采样与实验手册》（2019）中概述的过程的轻微修改。将滤膜放入Salivette管中，加入400 μL ATL缓冲液和40 μL蛋白酶K，然后在56°C下孵育30分钟，随后以3000 g离心3分钟。加入220 μL无核酸酶水并再次离心后，将400 μL 100%乙醇与溶液混合，然后根据DNeasy Blood & Tissue Kit（Qiagen）方案通过离心柱进行纯化。
• •
  Sterivex：我们使用了先前描述的Sterivex滤筒提取方法（Wu和Minamoto，2023）。简而言之，向装有过滤样品的Sterivex中加入1000 μL ATL缓冲液、495 μL PBS、455 μL AL缓冲液和50 μL蛋白酶K，随后在56°C下孵育30分钟。然后使用2 mL注射器收集提取的DNA，加入1000 μL乙醇，并使用离心柱进行纯化。

所有提取物均用100 μL洗脱，并使用Nanodrop-1000（Thermo Fisher Scientific）进行测量。然后，我们按照下述方案（详见2.3和2.5节）对鲤鱼（Cyprinus carpio）进行了物种特异性qPCR和统计分析。结果总结在图3中。

2.2 实验2：在三种环境中比较QuickConc、玻璃纤维滤膜和Sterivex

2.2.1 实验2的样品采集

水样采集在日本三种水生环境进行，包括2024年4月1日采集的武库川（34°45′32.1″N 135°22′26.8″E）河表水；2024年3月28日采集的神户港（34°38′27.9″N, 135°13′35.8″E）海表水；以及2024年4月11日采集的池塘水（34°44′31.6″N 135°05′48.5″E）。图1展示了实验设计。从河流、海洋和池塘分别采集每份400 mL、1 L和70 mL的样本量。每个地点采集四个重复加一个现场空白样。在添加0.001% w/v BAC后，每个样本立即使用QuickConc、玻璃纤维滤膜或Sterivex进行处理（见图1右图）。

2.2.2 实验2的浓缩和DNA提取

我们使用了与实验1相同的程序，修改之处在于所有方法都包含添加最终浓度为0.001%的BAC，这是基于图3所示的结果。每个提取物最终用100 μL洗脱，并储存在-80°C直至进一步分析。总eDNA浓度通过Nanodrop-1000（Thermo Fisher Scientific）进行定量。

2.3 qPCR分析

为了比较浓缩方法，进行了物种特异性qPCR分析，以定量河流、海水和池塘水样中黑鲷（Acanthopagrus schlegelii）、鲤鱼（C. carpio）和泥鳅（Misgurnus anguillicaudatus）的eDNA。选择这些物种是因为先前的调查确认它们在每种环境中存在且数量充足。引物和TaqMan探针设计与先前研究一致（Jo等，2020；Takahara等，2012；Takahashi等，2020），确保了特异性和可靠性（表S1）。每个qPCR反应总体积为20 μL，包括900 nM引物、125 nM TaqMan探针、1× Environmental Master Mix 2.0和0.1 μL AmpErase Uracil N-Glycosylase（Thermo Fisher Scientific），以及2 μL eDNA提取物。热循环起始于50°C 2分钟和95°C 10分钟聚合酶激活，随后进行55个循环的95°C 15秒和60°C 1分钟。使用标准品的10倍系列稀释液（五个浓度水平：每个反应10–100,000拷贝）建立校准曲线。不同浓缩方法的样品与相应的标准曲线在同一qPCR板上进行分析。所有qPCR实验，包括标准品、eDNA样品、过滤空白和PCR阴性对照，均在单个qPCR板内进行三次重复，确保环境样品DNA浓度计算的准确性。

2.4 扩增子文库制备、MiSeq测序和数据处理

为了评估不同浓缩方法对检测物种数量和群落组成的影响，对鱼类eDNA进行了代谢条形码分析。我们的采样点包括河流和海洋，但值得注意的是，池塘样品被排除在测序过程之外。池塘样品被排除是因为该池塘曾是隔离区域的稻田，不代表广泛的水生生态系统，且已知仅存在两种鱼类。DNA提取后，随后的扩增子文库制备和NGS程序由日本神奈川县的Bioengineering Lab. Co. Ltd.执行。使用DNeasy PowerClean Pro Cleanup Kit（Qiagen）去除PCR抑制物质，DNA最终用50 μL体积洗脱。文库构建开始于使用四个引物组（表S3）的两步尾式PCR方法，这些引物是部分修饰的鱼类通用PCR引物MiFish，由Miya等（2015）开发，用于扩增鱼类物种的eDNA。第一轮PCR反应在10 μL混合物中进行，包含0.375 U ExTaq HS（TaKaRa）、1× ExTaq Buffer、每种dNTP 0.2 mM、每种引物0.5 μM、1 μL eDNA模板和超纯水。热循环程序为94°C 2分钟，随后进行35个循环的94°C 20秒、65°C 15秒和72°C 20秒，最后72°C延伸5分钟。使用VAHTS DNA Clean Beads（Vazyme）纯化第一轮PCR产物后，第二轮PCR总体积为10 μL，包括0.2 U KOD FX Neo（TOYOBO）、1× PCR Buffer、每种dNTP 0.4 mM、每种引物0.25 μM、2 ng第一轮PCR产物和超纯水。第二轮PCR的热循环程序包括94°C 2分钟，12个循环的98°C 10秒、60°C 30秒和68°C 30秒，最后68°C延伸2分钟。使用VAHTS DNA Clean Beads（Vazyme）纯化PCR产物，然后完成配对末端序列文库。使用片段分析仪和dsDNA 915 Reagent Kit（Agilent Technologies）验证构建文库的质量，之后使用MiSeq Reagent Kit v3（Illumina）在2×300 bp条件下在MiSeq系统上进行测序。仅与引物序列开头完全匹配的读段序列使用FASTX Toolkit（ver. 0.0.14）中的fastq barcode splitter提取。使用FASTX-Toolkit的fastx_trimmer从提取的读段中去除引物序列和3'端最后120个碱基。随后，使用Sickle（ver. 1.33）过滤掉质量分数低于20的序列，并丢弃短于40个碱基的序列及其配对读段。此后，使用Qiime2（ver. 2024.2）中的DADA2插件去除嵌合和噪声序列，然后生成代表性序列和ASV表。对于MiFish分析，获得的ASV表使用USEARCH v 10.0.240（Edgar，2010）进行分析，按照Wu等（2021）演示的方法。使用线粒体基因组数据库MitoFish（ver. 3.90）将分类学名称分配给ASV。原始序列数据已上传至Dryad（见数据可用性声明）。

2.5 统计分析

每项分析均使用R ver. 4.3.3（R Development Core Team，2024）进行，使用vegan包版本2.5-6（Oksanen等，2019）和lme4包版本1.1-21（Bates等，2015）。为了比较不同过滤方法在环境水体中的总核酸产量或物种特异性DNA浓度，使用单因素方差分析（one-way ANOVA）进行统计分析，随后使用Tukey's诚实显著差异（HSD）事后检验进行两两比较。对于代谢条形码数据，我们进行了三项分析：物种数量比较、方法内β多样性评估以及非度量多维尺度分析（NMDS）。为了估计浓缩方法是否影响代谢条形码检测到的鱼类物种数量，将读段数据转换为存在/缺失，并使用具有泊松分布的广义线性混合模型。当浓缩方法的影响显著时，我们采用Tukey's多重比较检验。为了评估浓缩方法如何影响β多样性估计，我们计算了方法内β多样性，以下称为“伪β多样性”，代表同一样本独立重复之间的β多样性。伪β多样性通过将检测到的物种总数（γ）除以每个方法中每个样本检测到的物种数（α）来计算。以下分析分别对未稀释（un-rarefied）和稀释（rarefied）数据进行，稀释数据是通过使用vegan R包将总读段数统一到最少读段数而获得的。为了可视化基于存在/缺失的鱼类组成差异，使用Jaccard方法进行了NMDS分析，进行了10,000次置换。在该模型中，缺失赋值为0，存在赋值为1。此外，为了比较三种浓缩方法之间的鱼类组成，我们使用vegan包的“adonis”和“betadisper”函数分别进行了10,000次置换的置换多元方差分析（PERMANOVA）和多元离散度分析（PERMDISP）。我们使用ggplot2包版本3.1.1构建了部分图表。

3 结果

3.1 实验1：BAC对eDNA产量的影响（河水）

为了证明BAC在使用分散玻璃纤维时能提高DNA产量，我们分析了QuickConc、玻璃纤维滤膜和Sterivex在有或无BAC情况下的总eDNA产量和鲤鱼eDNA拷贝数。如图3和表S4所示，添加BAC的QuickConc显示总eDNA比玻璃纤维过滤法和Sterivex法多约1.2-6.6倍（单因素方差分析及Tukey's HSD事后检验，p < 0.001；图3A）。物种特异性qPCR结果显示，添加BAC的QuickConc检测到的拷贝数比其他方法高1.7-8.8倍（单因素方差分析及Tukey's HSD事后检验，p < 0.001；图3B）。鉴于这些结果，我们在后续实验中为QuickConc采用了BAC（0.001%）。

3.2 实验2：QuickConc、玻璃纤维滤膜和Sterivex之间的比较

接下来，我们评估了经BAC增强的核酸产量性能，并与使用玻璃纤维滤膜和Sterivex的其他方法进行了比较（图4）。QuickConc方法在所有水生环境中产生的总eDNA均显著多于其他方法。具体而言，在河水样本中，它回收的总eDNA约为玻璃滤膜和Sterivex法的三倍（方差分析，p < 0.001，Tukey's HSD，p < 0.001，图4A）。在海水样本中，QuickConc产生的总eDNA多约1.3-2.3倍（方差分析，p < 0.001，Tukey's HSD，p < 0.001，图4B），而在池塘水样本中，它回收的总eDNA多约三倍（方差分析，p < 0.001，Tukey's HSD，p < 0.001，图4C）。随后，将特定物种的qPCR分析与这些方法进行比较。标准曲线的总体PCR效率、R2值和拷贝数显示在表S2和S4中。具体而言，R2值均高于0.99，黑鲷、鲤鱼和泥鳅的扩增效率分别为110%、101%和91%。所有qPCR阴性对照和现场空白均显示无扩增，证实无污染。使用QuickConc方法检测到的黑鲷、鲤鱼和泥鳅的拷贝数显著高于其他方法。具体而言，在河水中，使用QuickConc检测鲤鱼的拷贝数比使用Sterivex高约10倍（方差分析，p < 0.001，Tukey's HSD，p < 0.001，图5A）。对于海水中的黑鲷，QuickConc产生的拷贝数比Sterivex高约8倍（p < 0.05，图5B），比玻璃滤膜法高约2.3倍（p < 0.01，图5B）。类似地，对于池塘水中的泥鳅，QuickConc方法导致的拷贝数比使用Sterivex和玻璃纤维滤膜高10倍以上（p < 0.001，图5C）。

最后，我们使用MiFish引物进行了代谢条形码分析。河流和海水分别获得了总共1,781,738和3,272,945条读段，经过滤后分别减少到853,367和981,290条（表S5，图S3）。现场空白和PCR阴性对照中未检测到鱼类DNA。在河流样本中，使用玻璃纤维滤膜、Sterivex和QuickConc分别检测到14、18和21种鱼类（图6）。类似地，在海水样本中，分别检测到13、19和15种鱼类。在河水样本中，使用QuickConc检测到的鱼类物种数量显著高于玻璃纤维滤膜，但与Sterivex相比未观察到显著差异（Tukey's HSD，p < 0.001 和 p = 0.194，图7A）。相比之下，从海水中检测到的鱼类物种数量在不同浓缩方法之间没有显著差异（Tukey's HSD，p = 0.498 和 p = 0.731，图7B）。为了评估这些浓缩方法如何影响样本β多样性的估计，我们计算了每种方法内的伪β多样性。尽管QuickConc方法有下降趋势，但在河流和海水中，任何方法之间均无显著差异（图8A）。接下来，为了评估方法间群落组成的差异，我们进行了带有稀释的NMDS分析以及包括PERMANOVA和PERMDISP的统计分析。这些结果表明，在河水样本中观察到群落组成的显著差异（PERMANOVA，p = 0.026；PERMDISP，p = 0.205，图8B），而在海水样本中未观察到群落组成的显著差异（PERMANOVA，p = 0.638；PERMDISP，p = 0.655，图8B）。相比之下，当使用来自河流和海水样本的非稀释数据时，群落组成的趋势与稀释数据一致（图S4）。

4 讨论

本研究介绍了QuickConc，一种为快速、灵敏分析而设计的新型eDNA捕获方法。我们将QuickConc与传统的玻璃纤维过滤法和Sterivex法进行了比较，定量分析了河流、海水和池塘水样中的总eDNA和物种特异性qPCR目标。QuickConc consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently 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consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently consistently 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