2020, Vol. 12 
2020年2月14日，习近平总书记提出要把生物安全纳入国家安全体系，现阶段全球新冠疫情的暴发更加凸显了生物安全的重要性。水环境是致病菌传播的重要媒介，水环境中致病菌引起的传染病在全球欠发达地区仍频繁发生，严重威胁民众的生命健康安全。由于水环境中致病菌具有丰度低、种类多和风险异质性高等特点，快速准确检测技术的开发与应用是有效控制致病菌传播的关键之一。近年来，水环境中致病菌污染备受关注，水环境中常见的致病菌主要包括铜绿假单胞菌、沙门氏菌、志贺氏菌、梭状芽孢杆菌、军团菌和分枝杆菌等[1-2]，其可通过呼吸道、消化道和皮肤进入人体并引起结核病、伤寒、霍乱和痢疾等多种疾病[3-7]。与此同时，耐药致病菌如金黄色葡萄球菌、肺炎克雷伯菌和结核分枝杆菌等也在水环境中大量检出[8-11]。这些致病菌一般不是水体土著微生物，大部分来自外源污染[12]。携带大量致病菌的污水直接排放或未经彻底处理进入受纳水体，易扩散至水源水，进而进入饮用水处理与管网系统[13-15]。由此可见，水环境已成为致病菌传播的重要媒介。水环境中致病菌通过饮水和直接接触等途径感染人体，存在传播各类疾病的风险。在资源匮乏的国家甚至在发达国家的欠发达农村地区，受致病菌污染的水体导致了多种介水疾病的暴发与流行, 并且介水传染病的发病率和死亡率是巨大的[3-4, 16-17]，严重威胁着公众健康和生态安全。长期以理化污染指标为主的水质监测难以预测人类水源相关致病菌的存在，而传统的水环境致病菌检测主要依靠细菌培养和生化鉴定法，其操作烦琐，检测周期长，且样品中的干扰物会导致目标致病菌检出率下降。目前，水环境中致病菌的监测主要基于对粪便指示菌(FIB)的检测，该方法易于应用且成本较低，但检测结果与致病菌间的相关性较差。因此，水环境致病菌的快速准确检测方法亟须建立，其在水质监测和微生物风险评估中发挥着重要作用，是水安全目标实现的重要基础[18]。例如，2010年海地霍乱暴发的前2年，由于缺少水域中霍乱弧菌的分布和归趋信息，霍乱患者数量高达60多万，其中7 000多人死亡。2年后，Kahler等[19]采用培养法、聚合酶链反应法(PCR)和直接活菌计数法检测水环境中的霍乱弧菌并将其浓度数据与流行病学数据结合，用于后续的风险评估，有效地控制了致病菌的传播，体现了长期以理化污染指标为主的水质监测向环境健康监测与评价体系转变的重要性。
近年来，水环境中致病菌检测的相关研究越来越多，其中分子生物学检测技术因其灵敏度高、特异性强、快速简便、省时省力等优点，在水环境中致病菌的鉴定与检测中扮演着重要角色，但相关研究进展分析总结的报道较少。因此，现总结了水环境中主要致病菌及其导致的疾病，重点介绍了近年来水环境中致病菌的分子生物学检测技术，剖析了不同检测技术的优缺点和应用特点，以期为高效便捷的致病菌检测方法研究与应用提供理论基础和科学依据。
1 水环境中致病菌及相关疾病世界卫生组织报告显示水传播疾病(主要是急性肠胃炎)每年造成220多万人死亡[20]。水中病原体出现的原因包括：水污染、易感人群增加、饮用水处理不达标、商业和旅游全球化等[21]。据统计，目前约有1 400多种病原体会感染人类，其中包括细菌(538种)、病毒(208种)、原生动物(57种)、真菌及寄生虫[22]。其中，能引起人类和动物患病的细菌统称为病原菌或致病菌。致病菌的致病作用与其毒力、侵入机体数量、侵入途径及机体的免疫状态密切相关[7, 23]。水环境中主要致病菌及其导致的疾病见表 1。
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表 1 水环境中主要致病菌及其相关疾病 |
导致介水传染病的典型致病菌包含：(1)霍乱弧菌，其产生的肠毒素是一种剧烈的致泄毒素。该毒素作用于肠壁促使肠黏膜细胞极度分泌从而使水和盐过量排出，导致严重脱水虚脱，进而引起代谢性酸中毒和急性肾功能衰竭[24]；(2)沙门氏菌，菌体通过毒力岛、黏附素、鞭毛等毒力因子入侵肠上皮细胞，可导致全身扩散感染，典型症状包括发热、恶心、呕吐、腹泻及腹部绞痛等[25]；(3)产肠毒素性大肠杆菌，人和多种动物的感染性腹泻的重要病原，会引起胃肠道感染、尿道感染、关节炎、脑膜炎以及败血型感染等[26]。1991—2002年，美国暴发了207起介水传染病，主要致病因子为沙门氏菌、霍乱弧菌、军团菌属、大肠杆菌O157:H7和空肠弯曲杆菌等致病菌和寄生虫，共计433 947人患病[27]。2008—2013年，菲律宾共有42 071例霍乱病例，主要致病因子为霍乱弧菌[28]。中国每年肠道传染病的发病率为97.33例/10万人，主要包括霍乱、伤寒、细菌性痢疾等肠道传染病[29]。由此可见，这些介水传染病具有发病急、传播快、波及范围广和危害严重等特点。
2 水环境中致病菌的检测技术近年来，水环境中致病菌的检测取得了一定的研究进展，已从单一基于培养法的传统致病菌检测方法发展至多种分子生物学检测方法，这些技术的出现和发展为致病菌检测提供了新的思路。水环境中致病菌检测技术的发展历程见图 1。
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图 1 水环境中致病菌检测技术的发展历程 |
目前最普遍的致病菌检测方法是培养法[60]和聚合酶链式反应(Polymerase Chain Reaction, PCR)。这2种方法在选择性和灵敏度方面都很突出，但培养法比PCR法更耗时[61]。一些新兴的方法如生物传感器正在开发中，其未来的发展方向是满足低成本、高选择性要求[62-64]。高通量测序由于通量高和无须特异性引物等优点正逐渐取代传统检测方法，但也存在错误率高等局限性[65-66]。近年来致病菌检测方法的检测限及其应用见表 2。目前，致病菌检测方法还存在相似亚种难以区分、致病性及样品浓缩带来的抑制物等问题，这对新检测方法的开发是重大挑战[31]。
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表 2 致病菌检测方法的检测限及其应用 |
PCR是目前致病菌检测中应用最广泛的分子生物学方法之一，其通过扩增特定的靶基因序列来完成致病菌检测。主要通过变性、退火和延伸(退火和延伸也可以同时进行)3个步骤最终实现目标序列的指数放大。中华人民共和国出入境检验检疫行业标准(SN/T 1896—2007)中采用PCR技术对食品中的多种致病菌(沙门氏菌、志贺氏菌、金黄色葡萄球菌、小肠结肠炎耶尔森氏菌、单核细胞增生李斯特氏菌、空肠弯曲菌、肠出血性大肠埃希氏菌O157:H7、副溶血性弧菌、霍乱弧菌和创伤弧菌)进行快速定性检测。
实时定量PCR(quantitative real-time PCR, qPCR)是在PCR技术基础上利用荧光信号值实时检测目的基因，通过内参或外参法对样品中的特定基因进行定量分析，比常规PCR更为灵敏。已有大量研究采用qPCR方法定量致病菌，如大肠杆菌[71]和沙门氏菌[74]等。Hassard等[61]分别采用qPCR方法与传统培养法检测河口水样中大肠杆菌和肠球菌，发现qPCR方法的检测限能达到1 CFU/100 mL，显著优于传统培养法。但是qPCR也存在DNA回收效率低和对引物特异性要求高的问题[61]。此外，qPCR在检测中还面临其他一些挑战，如反应中存在PCR抑制物[93]。即使少量的PCR抑制物也会延迟复杂样品的阈值循环(Cq)，从而导致模板拷贝数比实际值低。为防止假阴性结果的出现，可在PCR反应中加入对抑制物具有高灵敏度的内部阳性对照(如内参基因)进行检测。此外，提取过程中残留的抑制物如胍盐和苯酚可采用QuickDrop和NanoDrop分光光度计检测，也可以通过凝胶电泳确定是否有RNA、DNA及蛋白污染。如果样品中含有蛋白等其他杂质的污染，可以使用磁珠进行核酸纯化。与此同时，qPCR无法识别活菌或死菌，会产生假阳性结果[71]。目前，一般采用叠氮溴化丙锭(propidium monoazide, PMA)对样品进行前处理，从而避免假阳性结果的产生。PMA是一种高度光敏的DNA结合染料，不能透过完整的活细胞膜，却能选择性地透过不完整的死细胞膜。PMA能与DNA结合形成不可逆共价键，抑制死细胞DNA的扩增以达到区分死、活细胞的目的[76]。
此外，PCR与qPCR对于致病菌的批量检测存在一定的局限性[94]。针对上述问题，多重PCR、微滴式数字PCR等技术相继产生。多重PCR(multiplex PCR, mPCR)，又称多重引物PCR或复合PCR，是在同一PCR反应管中同时加上多种特异性引物进行PCR扩增[95]。微滴式数字PCR(Droplet Digital PCR, ddPCR)是第三代PCR技术，属于单分子分析，可用于绝对定量。Singh等[73]用ddPCR和qPCR计数河流环境中的沙门氏菌，发现水中ddPCR的灵敏度和线性范围与qPCR相当，但沉积物样品中ddPCR的灵敏度和线性范围明显更高。多重PCR和微滴式数字PCR技术都克服了传统PCR方法高成本、通量有限、流程复杂、精确度低等缺点。但是mPCR存在多目标扩增条件不相容的问题[77]，ddPCR检测高浓度样品时的线性度明显下降[71]。
2.2 等温扩增技术近年来新发展起来的等温扩增技术，无论是实际操作还是仪器要求方面都比PCR技术更为简单方便，在临床和现场监测中具有良好的前景，其中，环介导等温扩增(LAMP)已经得到一定的应用。该技术是一种新型的核酸扩增方法[96]，其主要原理是使用4条特异性引物分别识别靶基因的6个特定区域，通过链置换反应实现等温条件下基因的快速扩增[97]。该方法的优点是灵敏度高(检测限比传统的PCR方法低2~5个数量级)、反应时间短(30~60 min)、临床使用不需要特殊仪器和操作简单(反应液、酶和模板的混合液置于63 ℃左右水浴锅或恒温箱中30~60 min)。Lin等[80]使用改性的LAMP定量分析地表水中的大肠杆菌和伤寒沙门氏菌，灵敏度动态范围为0.3~10 000 cells/mL，高浓度抑制物对定量结果影响较小，且1 h内能够完成检测。LAMP技术基于4~6个引物的结合，所以比qPCR扩增效率低[74]，也常常因非特异性扩增出现假阳性结果[97]。目前常用的防止假阳性结果出现的方法主要包括：(1)使用特定结构的PCR管，该管中有一个固定的小隔板将管分成2个区域，分别加入反应液和DNA染料，但这种方法增加了加液次数，不适用于大批量检测；(2)将染料包埋在石蜡中，反应结束后高温熔化石蜡进而释放染料，但该方法增加了前处理时间。
此外，重组酶聚合酶扩增(RPA)[98]、滚环扩增(RCA)[99]、核酸依赖性扩增(NASBA)等技术也在不断发展与完善之中。(1)RPA技术：主要原理是重组酶与引物结合形成的蛋白-DNA复合物，其能在双链DNA中寻找同源序列。一旦引物定位了同源序列，就会发生链交换反应，形成并启动DNA合成，从而对模板上的目标区域进行指数式扩增。该方法属于常温扩增，其灵敏度高、特异性强且检测时间短，可实现致病菌的现场检测[100]。但是RPA反应中的一些物质会干扰试纸上的抗体，不充分稀释样品会出现非特异性结合和假阳性信号[101]。(2)RCA技术：以环状DNA为模板，利用较短的DNA引物(与部分环状模板互补)在酶催化下将三磷酸脱氧核苷酸(dNTPs)转变成包含成百上千个重复的与模板片段互补的单链DNA。其具有很强的拓展性，可以进行原位扩增，但是容易受到复杂溶液体系的干扰[102]。(3)NASBA技术：原理是利用定量mRNA确认目标生物的存在和生存能力。由于NASBA仅扩增RNA，样品中DNA的存在不会导致假阳性结果。与此同时，该方法可以等温进行，减少了对专用设备的需求，是环境样品常规监测工具的理想选择[103]。Fykse等[104]利用NASBA方法检测海水中的霍乱弧菌，检测限为5×103 CFU/100 mL。而Walker等[82]利用NASBA检测地表水和海水中的大肠杆菌，发现其检测限明显高于PCR，可能是抑制性物质如氯化钠和其他菌种RNA影响所致。
2.3 生物传感器目前，致病菌检测不仅对灵敏度、特异性、检测限、检测时间、操作复杂程度和人员培训等关键问题具有较高要求，对经济高效的现场监测设备的需求也越来越高，因此生物传感器逐渐应用于致病菌的快速检测[31]。生物传感器是一种将生物物质浓度转换为电信号进行检测的仪器，有多种分类方式[105-106]：(1)根据分子识别元件可分为酶传感器、微生物传感器、细胞传感器、组织传感器和免疫传感器；(2)根据信号转换器可分为生物电极传感器、半导体生物传感器、光生物传感器、热生物传感器、压电晶体生物传感器和声学生物传感器；(3)根据输出电信号的测量方式可分为电位型生物传感器、电流型生物传感器和伏安型生物传感器；(4)根据被测目标与分子识别元件的相互作用方式可分为生物亲和型生物传感器、代谢型或催化型生物传感器。
在检测复杂水质时，识别元件与待测微生物的不可逆性化学反应会降低识别元件的识别能力，影响传感器的灵敏度[107]。适配体的引入及其在传感技术中的广泛应用可有效增强致病菌检测的敏感性[108]。核酸适配体是一小段经体外筛选得到的寡核苷酸序列或者短的多肽，能与相应的配体进行高亲和力和强特异性的结合。Jin等[83]开发了一种基于纳米粒子与适配体结合的生物传感器来检测大肠杆菌ATCC 8739，该检测器的检测范围为5~1×106 CFU/mL，检测限为3 CFU/mL，检测时间＜20 min。Altintas等[107]开发了基于微流体的全自动电化学生物传感器并对磷酸盐缓冲液(PBS)中的大肠杆菌进行定量检测，发现其检出限低至50 CFU/mL且特异性高。此外，其分析地表水样时的检测限与模拟实验相同，但传感器信号略有下降。生物传感器在致病菌收集和检测之间几乎没有时间延迟[63]，但在检测过程中存在细菌细胞导致薄膜阻抗增加、革兰氏阴性菌检测限高和光流控传感器灵敏度低等问题[109]。
2.4 DNA微阵列技术微流体技术出现于20世纪80年代，该技术精确地控制流体的性能，并将流体限制在一个很小的几何尺度(通常为亚毫米)内，进而产生了芯片实验室或DNA微阵列技术[110]。DNA微阵列技术又称DNA阵列或DNA芯片，一般用于检测不同生长条件下细胞基因的表达、DNA序列的特异性突变或者表征环境样品中微生物的特征[111]。DNA微阵列含有高密度固定化核酸(基因组DNA、cDNA或寡核苷酸)的有序二维矩阵，能够通过核酸杂交同时检测单个样品中的数百个基因[112]，也可以同时快速检测多个生物体的多个基因。该方法能够筛选大量序列[113]，具有很强的自动化能力，并且具有检测时间短、操作简单和检测设备简单易于携带的优势。张子龙等[87]开发了一种能检测12种食源致病菌的芯片，其特异性良好且检测限低至1 CFU/mL。Ramalingam等[114]设计了基于实时PCR的微阵列芯片，该芯片能够同时检测4种水生致病菌(铜绿假单胞菌、嗜水气单胞菌、肺炎克雷伯菌和金黄色葡萄球菌)。但是，目前该方法成本相对较高，不能区分待检样品中的活、死菌且样品量消耗非常大[113]。此外，还可能产生非特异性杂交，导致特异性和敏感性降低[115-116]。而DNA微阵列与靶基因PCR扩增相结合能够提高致病菌检测的灵敏度，即将PCR扩增目标基因产物杂交到一个低密度的DNA芯片上进行检测，信号灵敏度能够增加约1×106倍[115]。
2.5 高通量测序技术近几年，高通量测序技术(High-throughput sequencing, HTS)的开发与应用促进了宏基因组学的发展。HTS可以读取数10亿的环境样品DNA序列(reads)，分析微生物群落组成及功能[11]。目前，第二代测序技术是最主流的测序技术[117]，测序平台主要有Ion Torrent(ABI公司)、Miseq和Hiseq(Illumina公司)[118]、454(Roche公司)[119]。高通量测序流程一般分为检测实验过程(包括样品处理、文库构建、文库质控和上机测序)和生物信息学的数据分析流程(包括数据质控、序列比对、注释和变异识别)[120]，其中文库质控是提高测序准确率的关键。此外，HTS存在一定的系统误差，可通过增加测序深度进行校正，另一方面，测序深度在一定程度上与基因组覆盖度正相关，即测序深度增加，基因组覆盖度也会提高。
目前，HTS主要利用特异性标记基因、毒力因子和16S rRNA基因鉴定致病菌。(1)特异性标记基因：使用特异性标记基因的代表工具是MetaPhlAn2，数据库含有17 000多个参考基因组(包括13 500个细菌和古菌、3 500个病毒和110种真核生物)和多达100万类群特异的标记基因，可以实现快速、精准的分析[121]。目前，运用此方法分析环境致病菌的研究很多[116, 122]。Li等[65]开发了适用于溪流水样的mPCR-HTS方法，该方法利用HTS的高测序深度显著提高了mPCR的多重水平。Wolf-Baca等[118]通过HTS技术分析了供水系统中细菌物种的多样性，发现变形杆菌(40%~97%)是优势菌且检测到梭状芽孢杆菌属和肠杆菌科致病菌。(2)毒力因子：基于毒力因子的鉴别方法广泛应用于致病菌鉴别，应用较多的是利用细菌毒力因子数据库(VFDB)中毒力因子确定致病菌[123]，但是VFDB数据库中致病菌种类较少，且大多数毒力因子在致病菌中不作为特异性基因存在。(3)16S rRNA基因：目前基于16S rRNA基因鉴定致病菌的应用最广泛，16S rRNA是细菌上编码rRNA相对应的DNA序列，包含约50个功能域，存在于所有原核微生物的基因组中，具有高度的保守性和特异性[124]。将细菌16S rRNA序列测序结果与不断更新完善的数据库比对可以获得致病菌在属和种水平的分类信息，从而快速准确地判断致病菌的归趋[116, 125]。但该方法过度依赖引物的质量[126]，导致PCR过程中存在固有的扩增偏差[127]。此外，由于同一属内的16S rRNA基因拷贝数不尽相同，定量结果和实际数量会有差异。由于序列长度原因，尽管使用很严格的筛选条件，HTS也可能存在序列比对上的错误[128]。总体来说，采用基于第二代高通量测序的宏基因组学方法检测致病菌存在精度和准度不高的问题，但是其数据通量高。
随着第二代测序技术的发展和应用，其弊端正在显现，而第三代测序技术在一定程度上可以弥补其在应用中的一些不足。第三代测序技术是指单分子测序技术，不需进行PCR扩增也能实现每一条DNA分子的单独测序。其实现了DNA聚合酶自身的延续性，一个反应就可以测几千个碱基的长序列。技术平台主要有Heliscope/Helicos(Helicos公司)、SMRT(Pacific Biosciences公司)、MinION/GridION/PromethION(Oxford Nanopore Technologies公司)。由于长读长的特点，测序平台在基因组测序中能降低测序后的重叠群(contig)数量，明显减少后续的基因组拼接和注释的工作量，节省大量的时间[66]，因此第三代测序在鉴定新的病原体和细菌基因组测序方面得到了广泛的应用。Hamner等[92]利用MinION设备对河流中存在的水传播疾病病原体进行宏基因组分析，检测到大肠杆菌血清型O104:H4和O1群El Tor型霍乱弧菌等人类致病菌。单分子测序一般存在测序错误率比较高且随机的问题，但可以通过多次测序进行有效纠错[91]。有研究表明[129-130]，MinION测序方法优化后的准确率达到95%以上，可以满足致病菌的检测需要。
3 总结与展望 3.1 研究进展近年来，核酸分子扩增和杂交等分子生物学方法已广泛应用于水环境致病菌检测中，高通量测序技术在致病菌的鉴定及定量领域也处于快速发展阶段，基于分子生物学的快速、灵敏和准确的致病菌检测技术是健康风险评估的关键。综上所述，水环境中致病菌分子生物学检测技术的研究进展主要包括：(1)检测对象从原来的粪源指示菌和少数几种肠道病原菌转变为已知致病菌、不可培养和新出现的致病菌；(2)水环境致病菌分子生物学检测技术的检测通量、灵敏度、特异性和重复性等快速进步，检测技术种类不断丰富；(3)分子生物学检测仪器由原来的手工、半自动发展到标准化检测设备，自动化程度越来越高，检测速度快、通量高且经济成本不断下降。
目前，基于分子生物学的致病菌检测技术可在水环境监测中广泛应用。水环境监测业务化应用过程中可根据不同致病菌检测技术的特点，针对性地选择检测技术：(1) qPCR具有操作简便和灵敏度高的特点，适用于一般地表水和饮用水源的监测，可以定量低丰度的致病菌；(2)LAMP扩增效率较低，但具有操作简便和检测快速的特点，适用于一般地表水的监测；(3)生物传感器具有专一性强、成本低和携带方便等特点，适用于水环境污染应急监测；(4)DNA微阵列技术自动化水平高但灵敏度低，适用于复杂致病菌群落结构的水污染源与污水处理系统的监测；(5)高通量测序技术具有通量高和速度快的特点，适用于水污染源与污水处理系统的监测，可快速识别复杂的致病菌群落结构。
3.2 技术不足目前，分子生物学检测技术仍然存在着以下的一些不足：(1)qPCR检测过程中很容易出现假阳性结果，且对引物的特异性要求很高；(2)LAMP的扩增效率明显低于qPCR，且假阳性/阴性多；(3)生物传感器在真实水环境中存在灵敏度低和检测限高的问题；(4)DNA微阵列技术可能产生非特异性杂交，导致检测方法的特异性和敏感性较低；(5)测序技术正不断向高通量和长读长发展，但是第三代测序技术目前的测序错误率较高。
3.3 未来展望基于水环境中致病菌分子生物学检测技术的优势和存在的不足，未来可在以下几个方向开展研究工作：
(1) 构建致病菌核酸样品制备方法。致病菌核酸样品质量是实现高灵敏度和特异性检测的关键，传统的样品前处理方法具有处理时间长和富集系数低等问题，目前亟须构建致病菌高效预浓缩装备及确定核酸样品快速无损制备方法；
(2) 明确致病菌定量结果与实际数量间的差异。目前大部分研究将qPCR定量的基因拷贝数结果视为细胞数量，忽略其与致病菌实际数量间的差异。可构建致病菌单拷贝基因数据库，以单拷贝基因作为qPCR的标志基因将定量结果转换为致病菌数量；
(3) 构建全面准确的致病菌数据库。采用高通量测序方法检测致病菌需将测序数据与致病菌数据库进行比对，全面准确的致病菌数据库是分析检测的关键；
(4) 开展致病菌活性检测。致病菌活性是其具有感染性的前提条件，也是评估感染风险的重要参数。目前水环境中致病菌分子生物学检测主要关注致病菌浓度和多样性，亟须完善致病菌活性检测方法体系和评价标准；
(5) 开展致病菌耐药性检测。目前抗生素耐药性已成为全球关注的环境问题，致病菌获得耐药性会导致抗生素有效性下降，越来越多的感染难以治愈，威胁人类健康。在完成致病菌检测的同时开展致病菌耐药性分析有助于遏制致病菌耐药发展与蔓延；
(6) 致病菌检测技术适用性分析及耦合使用。在不断发展新技术的同时，将不同的致病菌分子生物学检测方法应用到适合的水环境中，明确更准确灵敏且有效的检测方法。同时，综合评估目前现有检测技术的特点，亟须发挥各类技术优势，建立精准的耦合检测技术。
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