基于PCR技术的产真菌毒素镰刀菌分子诊断研究进展

谢雪钦，刘 舟*

（1.厦门市产品质量监督检验院，福建 厦门 361004；2.厦门医学院药学系，福建 厦门 361008）

基于PCR技术的产真菌毒素镰刀菌分子诊断研究进展

谢雪钦1，刘 舟2,*

（1.厦门市产品质量监督检验院，福建 厦门 361004；2.厦门医学院药学系，福建 厦门 361008）

镰刀菌属于自然界中最频繁产生毒素的真菌类别之一。该属真菌的毒素类次生代谢产物主要包括单端孢霉烯族化合物、伏马菌素和玉米赤霉烯酮3 类，引起人畜各种生理异常甚至癌变。本文综述了3 类镰刀菌毒素的结构和危害、生物合成的分子机理以及聚合酶链式反应（polymerase chain reaction，PCR）技术在其产生菌分子检测中的应用进展，突出了产毒真菌快速分子诊断技术在毒素早期预警、危害前置化干预中的意义。同时，在剖析现有产毒真菌PCR检测体系可能存在问题的基础上，提出克服瓶颈方法和进一步提高体系可靠性的有效策略。

镰刀菌；单端孢霉烯族化合物；伏马菌素；玉米赤霉烯酮；聚合酶链式反应；分子诊断

谢雪钦, 刘舟. 基于PCR技术的产真菌毒素镰刀菌分子诊断研究进展[J]. 食品科学, 2017, 38(11): 291-300. DOI:10.7506/ spkx1002-6630-201711046. http://www.spkx.net.cn

XIE Xueqin, LIU Zhou. Recent advances in PCR-based molecular diagnosis of mycotoxigenic Fusarium[J]. Food Science, 2017, 38(11): 291-300. (in Chinese with English abstract) DOI:10.7506/spkx1002-6630-201711046. http://www.spkx.net.cn

真菌感染普遍发生于作物生长期及贮存期，其代谢过程中产生的生物毒素随谷物基食品及饲料加工过程进入人、畜食物链，严重威胁健康[1-2]。镰刀菌属（Fusarium spp.）包括20余种，与青霉属和曲霉属同为自然界中最主要的3 类真菌毒素产生菌。该属真菌可侵染小麦、玉米、燕麦、马铃薯等农作物，其中禾谷镰刀菌（F. graminearum）、拟枝镰刀菌（F. sporotrichioides）、黄色镰刀菌（F. culmorum）、串珠镰刀菌（F. proliferatum）、轮枝镰刀菌（F. verticillioides）、尖孢镰刀菌（F. oxysporum）和木贼镰刀菌（F. equiseti）等可产生单端孢霉烯族化合物（trichothecenes，TCTs）、玉米赤霉烯酮（zearalenone，ZEA）、串珠镰刀菌素、大镰刀孢菌素、黄色镰刀菌素、丁烯羟酸内酯和伏马菌素（fumonisins，FUMs）等多种生物毒素[3]。不同镰刀菌所产生的生物毒素种类有所差异，其中以TCTs、ZEA和FUMs为最普遍且危害严重的毒素种类。此系列毒素在高温条件下稳定，在染菌食品及饲料贮存、加工及烹饪过程中不易于降解，故正常条件下人畜均有一定的暴露风险[4-6]。

镰刀菌不同种间产毒素种类及能力差异大，对产毒株的快速准确鉴定对预测其产毒潜力并有效预防其可能引发的毒素危害至关重要。目前，产毒镰刀菌的常规鉴定方法多基于形态学及育性分析，其费时、冗繁且专业要求强[7]。为了克服上述缺陷，近年来许多基于聚合酶链式反应（polymerase chain reaction，PCR）及其衍生技术的分子诊断策略被用于产毒真菌的快速检测及鉴定[8-12]，有效地提高了真菌毒素危害预警能力，为保证事前干预、降低危害提供了良好的技术支持。本文就近年来产TCTs、ZEA和FUMs这3 类关键毒素镰刀菌的分子诊断体系发展及其机理进行综述，并在此基础上探讨此技术在毒素危害预警中的意义。

1 玉米赤霉烯酮

ZEA，又称为F-2毒素，是2,4-二羟基苯甲酸酯类化合物，化学式为C18H22O5。ZEA具有雌激素样作用，主要作用于生殖系统，人、畜食用含毒素的食物后，增加流产、死胎和畸胎风险[13]，还可引起恶心、发冷、头痛、神智抑郁等中枢神经系统的中毒症状[14]。ZEA主要由镰刀菌（禾谷镰刀菌、黄色镰刀菌、木贼镰刀菌、轮枝镰刀菌、三线镰刀菌等）产生，其中以禾谷镰刀菌为最常见的产毒菌。

1.1 ZEA的生物合成及其关键基因簇

ZEA首次被Stob等[15]于1962年发现并报道，此后其化学结构获得解析[16]。自1979年起，通过同位素示踪法，经乙酸-丙二酰-辅酶A酶系统以乙酸为前体首尾缩合形成毒素的ZEA生物合成途径逐步被解析[17-19]，但对其合成的分子机制研究仍十分局限。

聚酮合成酶（polyketide synthase，PKS）在ZEA的生物合成中发挥关键催化作用，促成聚酮化合链的形成及毒素的产生。全基因组序列分析表明禾谷镰刀菌中有15 个PKS基因，其中有5 个PKS基因产物已被界定，分别参与ZEA、黄色镰刀菌素、镰菌素C及黑色子囊壳色素的生物合成[20]。Kim[21]、Gaffoor[22]等分别通过基因缺失实验表明，PKS4和PKS13是合成ZEA不可或缺的必需基因，在玉米赤霉菌中删除上述基因导致ZEA毒素缺失。而Lysøe等[23]则认为只有PKS4基因是禾谷镰孢菌中ZEA毒素合成的必需基因。此外，与PKS4和PKS13同在一个基因簇的另外两个基因也是ZEA合成不可或缺的，分别为与异戊醇氧化酶基因高度同源的ZEB1（FG12056）以及转录调控基因ZEB2（FG02398）[21]。

1.2 产ZEA镰刀菌的PCR检测

以ZEA生物合成途径中的关键基因为靶标，近年来多种分子检测技术被开发用于ZEA的间接检测，其结果与酶联免疫吸附测定（enzyme linked immunosorbent assay，ELISA）、高效液相色谱（high performance liquid chromatography，HPLC）法等针对毒素本身的直接分析方法高度一致。如张瑞芳等[24]以PKS4基因为靶标设计一对特异性引物PKS4F/PKS4R，通过PCR法间接检测2 个染菌小麦籽粒及36 株分离自小麦样本的禾谷镰刀菌中的ZEA毒素，均显示阳性，与ELISA直接检测的结果完全吻合。同样以PKS4基因为特异性分子标记基因，Meng Kun等[25]以SYBR实时荧光定量PCR法对食品中的产ZEA镰孢菌进行了定性及定量检测，方法检测限低至10 拷贝PKS4基因/PCR体系或500 个真菌孢子/g食品，其定量线性范围可覆盖102～104共3 个数量级，方法快速、特异、灵敏，可在毒素大量形成前达到预警作用。而Atoui等[26]则选定ZEA生物合成过程中的另一个必需基因PKS13为靶标，通过实时荧光定量（real time）-PCR法建立了玉米样品中ZEA含量与产毒禾谷镰刀菌、黄色镰刀菌PKS13拷贝数间的相关性，经PCR反应Ct值推算产毒菌PKS13拷贝数继而再推测该样品污染ZEA的风险，全程可在8 h内完成，较传统定量方法更简便、快速。Misiewicz等[27]发现禾谷镰刀菌种内PKS4基因的序列也存在多态性，这导致其分离的一株产ZEA的禾谷镰刀菌菌株P7/4以Lysøe等[23]设计的一对引物PKS4F/R经常规PCR无法获得扩增片段。经过充分序列比对分析，一对避开多态性区域而设计的特异性引物PKS_RT_F/R则可在供试的ZEA阳性产毒菌株mRNA上均扩增得91 bp的目标片段，且转录分析表明该基因的转录水平与毒素产量无相关性。此研究提示在后续研究中应需充分考虑PKS4基因的多态性问题，设计兼容性引物以鉴定产ZEA菌株，避免假阴性结果出现。

2 伏马菌素

FUMs是主要由串珠镰刀菌和轮枝镰刀菌产生的致癌性代谢产物。该类毒素为不同多氢醇和丙三羧酸组成的结构类似的双酯化合物，其化学结构中的长碳水化合物链在其毒性中发挥关键作用[28]。FUMs早在1993年已被国际癌症研究机构（International Agency for Research on Cancer，IARC）划定为2B类致癌物。动物实验和流行病学资料已表明，FUMs主要损害肝肾功能，能引起马脑白质软化症和猪肺水肿综合症等[29-30]，并与食道癌的发生有一定的因果关系[31]。

到目前为止，已发现的FUMs分为A、B、C、P共4组，有FA1、FA2、FB1、FB2、FB3、FB4、FC1、FC2、FC3、FC4和FP1共11 种，其中FB1发生频率及毒性最高[32]。FB1为水溶性霉菌毒素，主要污染玉米及其制品，对热稳定，不易被蒸煮破坏，所以控制农作物在生长、收获和储存过程中的霉菌污染至关重要。

2.1 FUMs的生物合成及关键基因

2.1.1 FUMs的生物合成

自1988年FB1被分离[33]且化学结构被解析[34]以来，研究者不断致力于阐明其生物合成途径。生物化学及遗传学研究显示FUMs的碳骨架中的C-3至C-20是聚酮合成的产物[35]，丙氨酸与上述碳链的缩合形成了C2位上的氨基以及C-1和C-2骨架[36]，C12及C16位上的甲基侧链则是由活性腺苷甲硫胺酸转移酶从蛋氨酸上添加[37]，C3位的羟基来自于醋酸盐源羰基，C5、C10、C14和C15位上的羟基则源于游离氧[38]，而丙三羧酸侧链则通过柠檬酸循环代谢生成[39]。对于上述各步骤的先后合成次序，Bojja等[40]通过轮枝镰孢3 个基因缺失突变株Δfum1、Δfum6和Δfum8的共培养，发现与突变株Δfum6共培养可回补基因Δfum1或Δfum8使其恢复产FUMs能力，对共培养代谢物进行LC-质谱（mass spectrometry，MS）分析表明7 d内可形成带1～4 个羟基的侧链结构的FUMs骨架，据此研究者推断FUMs的生物合成始于Fum1p催化的碳链聚合及随后Fum8p催化的丙氨酸缩合，其产物经由Fum6p或其他酶进行后续进一步氧化以形成终产物。

2.1.2 FUMs生物合成基因

借助经典的遗传学手段，参与B系列FUMs合成及调控的相关基因已有一定的研究。野生型轮枝镰孢菌形成4 种B系列FUMs产物（FB1、FB2、FB3、FB4），除线性碳骨架上羟基的数量和位置有所差异之外，其结构完全相同。FB1是其他3 种毒素形式羟基化的产物[32]。上述3 种不同毒素产物的形成与3 个显著毗邻的基因位点（fum1、fum2、fum3）有关，其中fum1（=fum5）编码PKS，是催化C20聚酮骨架形成所必需的，该基因缺陷型菌株不产生FUMs[41-42]；fum2则是毒素骨架C10位羟基化所必需的，若缺失则仅形成FB2，不产生FB1和FB3；fum3（=fum9）缺陷株则丧失C5位羟基化的能力，仅产生FB3

[43-44]。此后，以Ⅰ类PKS的β-酮缩酶区设计兼并引物，Seo等[45]从轮枝镰孢菌cDNA上又克隆得与fum5毗邻的4 个基因fum6、fum7、fum8和fum9。基因阻断分析表明，fum6和fum8是FUMs合成所必需的；fum9基因缺陷株导致FUMs碳骨架中的C5位无法羟基化，功能类似于此前鉴定的fum3，且基因回补实验表明，导入fum9可回补阻断fum3引起的表型缺陷，上述现象充分证明fum9等同于fum3[46]。此外，2003年，Proctor等[47]通过分析上述5 个 FUMs合成相关基因簇发现，其侧翼序列中还有18 个开放阅读框，其中有10 个基因参与毒素合成。敲除编码假定ABC转运蛋白的ORF-19导致FUMs产量的急剧下降，表明外排泵相关基因可能参与毒素转运至胞外。进一步研究发现，上述基因簇中的fum13基因编码的短链脱氢酶参与FUMs碳主链上C3位羰基还原为羟基，且该基因是负责上述转化的主要功能因子，其缺失后轮枝镰孢菌C3位正常羟基化的FUMs产量仅为野生株的10%[48]。在上述对FUMs合成相关基因簇功能解析的基础上，Bojja等[40]通过对缺失突变株共培养代谢产物化学结构进行深入解析，最后绘制了轮枝镰孢菌中各基因调控FUMs逐步合成可能的生物化学过程图。此外，因多数镰刀菌FUMs以B型为主，比对以产C型毒素为主的尖孢镰刀菌O-1890与其他镰刀菌的毒素合成基因簇，并借由回补实验，Proctor等[49]还证实不同结构的fum8决定了FUMs的类型。

2.1.3 FUMs生物合成调控基因

不同于赭曲霉毒素或TCTs等生物毒素，FUMs生物合成相关基因簇中未发现参与毒素合成调控的基因。研究表明，一系列参与镰刀菌pH值调控、糖代谢、氮代谢的基因均参与FUMs的合成调控。如Shim等[50]发现细胞周期素编码基因FCC1调控轮枝镰孢菌中FB1的合成，该基因缺失株在pH 6的基础培养基上生长时，其毒素合成必需基因fum5的表达被阻断，而在pH 3时则可恢复毒素合成。在此基础上，该研究团队进一步研究发现另一参与碱性环境中轮枝镰孢FB1生物合成的负调控因子——PAC1，其缺失导致毒素产量提升[51]。此后，Flaherty等[52]从轮枝镰孢菌FB1生物合成期的表达序列标签（expressed sequence tag，EST）文库中鉴定到一个双核锌簇型基因ZFR1，功能研究表明其参与毒素合成的正向调控，且该基因为FFC1的上游调控基因。后续深入研究推断，ZFR1对FB1合成的控制是通过对FST1等与碳水化合物感应、摄取及转运相关基因的调控实现的[53]。此外，编码异构三聚体（αβγ）G蛋白β亚基的基因GBB1的缺失也导致FUMs合成关键基因fum1及fum8表达下调，致使FB1产量急剧下降，为毒素合成的正调控因子之一[54]。Kim等[55]发现氮代谢调控基因AREA亦参与FB1合成正调控，其缺失导致轮枝镰孢菌丧失产毒能力。除了上述个别调控基因的单独解析外，比较蛋白组学、芯片、EST文库等大数据手段亦被不断用于FUMs生物合成调控基因的全基因组发掘[56-57]。

2.2 产FUMs镰刀菌的PCR检测

当前，对产FUMs菌的分子检测主要靶向于两类目标基因：其一为对直接参与毒素合成的基因（如fum1等）进行扩增；其二是对内转录间隔区（internal transcribed spacer，ITS）等遗传标记核基因进行扩增或多态性分析。2.2.1 基于毒素生物合成相关基因的分子检测

2002年，王晓英等[58]以FUMs生物合成所必需的多酮肽合成酶基因fum5为靶序列，建立了产毒株PCR检测方法。确证某镰刀菌株（ATCC12763）为FUMs生物合成酶基因阴性，判断为非FUMs产毒株，此结果与美国模式培养物集存库（American type culture collection，ATCC）提供的菌株产毒资料相一致。随后，该团队以上述引物鉴别了29 株分离自我国不同省份、不同粮食样本中的串珠镰刀菌分离株，其结果与毒素HPLC分析结果相一致[59]，进一步证实了fum5作为菌株产FUMs能力预测指示基因的可靠性。结合靶向镰刀属ITS的特异性引物，fum5（=fum1）亦被用于多重PCR[60]或TaqMan real time-PCR体系[61]中作为鉴定产FUMs镰孢菌的特异性遗传标记基因。类似的多重检测体系亦被成功用于蔬菜[62]、玉米粒[63]、高粱[64]和稻谷[65]等食物及家禽饲料[66]中产毒镰刀菌的快速鉴定。Gonzalez-Jaen等[67]研究证实fum1（=fum5）、fum6和fum8基因仅存在于F. verticillioides、F. proliferatum、F. fujikuroi和F. nygamai等产FB菌株中。基于fum1基因中的β-酮乙酰还原酶的酮酰还原酶（ketoacyl reductase，KR）功能域设计PCR引物，能高度特异地识别产FUMs的轮枝镰孢菌。据此推断，不产该毒素的菌株缺失了fum1基因或者至少缺失了与该PCR引物配对的部分。镰刀菌能产生多种参与毒素或色素合成的PKS基因，其中KR功能域是产毒相关PKS基因所特有的，在该区设计产毒株鉴定引物更具特异性。研究表明，靶向fum1不同区段序列的引物鉴定产FUMs镰刀菌的效力各不相同，需针对特异性必需片段设计并充分验证其可靠性。如Baird等[68]以fum1为目标基因设计了4 对引物，其中仅B引物对可100%准确地鉴定玉米组织中的产FUMs轮枝镰孢菌，而该引物仅能鉴定出80%的层出镰刀菌产毒阳性株。鉴于此，研究者需结合毒素产物化学测定的结果，引入大量的菌株样本以验证引物甄别真菌产毒与否的能力。此外，随着基因定量技术的发展，以fum1为靶标的TaqMan real time-PCR法近期还被引入用于镰刀菌产FUMs潜能的间接定量预测[69]。

2.2.2 基于非产毒相关基因的分子鉴定

早在1998年，Grimm等[70]就通过分析产毒与非产毒镰刀菌间ITS序列的差异设计了一对特异性引物及生物素标记探针用于扩增产毒株中ITS区的108 bp序列，并通过PCR-ELISA方法测定该序列。同理，基于对产与不产FUMs的轮枝镰孢菌株间核糖体DNA的IGS区序列差异性分析，Patiño等[71]建立了一套高度灵敏的PCR体系用于甄别54 株来自不同地区及宿主的拟枝镰刀菌产毒株。此外，对不同地区来源于香蕉和玉米的29 株镰刀菌分离株ITS扩增产物进行随机引物扩增DNA多态性分型方法（randomly amplif i ed polymorphic DNA analysis，RAPD）及限制性片段长度多态性（restrictionfragment length polymorphism，RFLP）分析，结果显示基于RAPD的序列多态性聚类分析结果可区分产生不同水平FUMs及不同宿主来源的菌株[72]。上述研究所设计的可区分产毒与否的引物对VERTF-1/VERTF-2在后续研究中被用于印度[73]及菲律宾[74]等国玉米中镰孢菌产FUMs情况的预测及分析。Mirete等[75]对分离自多种宿主、地理来源及不同FUMs产量的48 株轮枝镰孢菌的EF-1α基因及基因内间隔区（intergenic spacers，IGS）部分序列进行系统发育分析，聚类结果可将供试菌株分为产及不产FUMs两组，其中产毒株居多，其地理分布广、偏好侵染谷物类、有性繁殖多发且变异大。

3 单端孢霉烯族化合物

根据化学结构中C8位置上是否有酮配基，镰刀菌中TCTs分为A和B两类，其中A类毒性更强[76]。A类包括T-2毒素、HT-2毒素、新茄病镰刀菌烯醇（neosolaniol，NEO）、蛇形霉素（diacetoxyscirpenol，DAS）和单乙酰氧基镰草镰刀菌醇（monoacetoxyscirpenol，MAS）；B类包括脱氧雪腐镰刀菌烯醇（deoxynivalenol，DON）及其衍生物3-AcDON、15-AcDON和雪腐镰刀菌烯醇（nivalenol，NIV）及镰刀菌烯酮X。尽管已有200余种TCTs被鉴定，当前食品及饲料中分离到的污染种类多为T-2、HT-2、DAS、DON和NIV这5 种[77]。此类毒素对胃肠系统、皮肤、免疫功能、血液、基因均有毒性，抑制蛋白质、RNA和DNA的合成，破坏膜功能、抑制免疫反应，引起血液功能异常等[78]。

3.1 TCTs生物合成途经及其相关基因簇

TCTs为倍半萜烯环氧化合物，首次于1948年分离自粉红单端孢，因此而得名，同位素标记代谢前体饲喂实验表明其合成前体为单端孢霉烯（trichodiene，TDN）[79]。以加氧酶抑制剂处理[80-83]或紫外（ultraviolet，UV）辐射[84-85]产毒的镰刀菌，结果发现其体内TDN累积且毒素合成受到抑制，证实TDN也是镰刀菌中该类毒素合成的前体。此外，上述结论通过添加外源同位素标记的人工合成前体实验亦得到进一步的确证[86-87]。以拟枝镰孢菌[88-90]、接骨镰孢菌[91]和黄色镰孢菌[83,92]为对象，以TDN为前体进行氧化、异构化、环化及酯化等反应最后合成各种复杂的TCTs类化合物的过程在后续研究中也被不断解析。Desjardins等[93]总结了上述过程，以图示法解析了镰孢菌中主要TCTs的生物合成途径。

镰刀菌TCTs生物合成遗传机制的解析始于Hohn等[94-95]对拟枝镰孢菌中该类毒素合成第一步的催化酶——TDN合成酶的纯化及其编码基因Tri5的克隆。以Tri5为契机，借由同源片段替换实验，研究者进一步在拟枝镰孢[96]和禾谷镰孢[97]中于该基因侧翼序列发现系列毒素合成相关基因，称为Tri5基因簇，其结构及各基因功能在两个种间保守。随着研究的深入，以禾谷镰孢菌及拟枝镰孢菌为模式种，该属真菌参与TCTs合成及调控的基因簇及其催化毒素生物合成的分子机制也被不断解析。到目前为止，此属真菌至少有3 个基因簇参与该类毒素的合成，分别为Tri5基因簇、Tri1-Tri16双基因簇和Tri101，其中Tri5基因簇包含了7 个合成催化酶编码基因、2 个调控基因和1个转运基因[98]，其中各基因功能详见表1。

2005年，重庆市政府对水稻插秧机的技术特点进行了研究，初步探索出了解决机械化插秧对水稻育秧要求高的技术难题[3]。2006年，全市在36个区县、110个乡镇开展水稻机械育秧技术、水稻插秧机示范推广，在随后的几年里不断加大对水稻插秧机示范推广的力度。 从2006-2010年全市水稻插秧机总量从96台增加到9 000台多，使重庆地区水稻插秧机使用量增长了93倍多，促进了重庆地区的水稻机械化插秧工作。2006-2008年重庆地区机插水稻面积、每公顷产量与增产率如表1所示[4-6]。

表1 参与镰刀菌单端孢霉烯族毒素合成及调控的基因簇及其功能Table 1 Biosynthetic gene clusters for Fusarium trichothecences and their functions

3.2 基于PCR的分子检测技术在产TCTs镰刀菌鉴定中的应用

3.2.1 产TCTs镰刀菌的检测

随着对镰刀菌属中各产毒素代表种参与TCTs合成及调控相关基因功能的解析，多种必需基因被用于此类毒素产毒株的分子鉴定。因Tri5基因负责催化所有产TCTs真菌中此类毒素生物合成的第一步，以该基因为靶标设计特异性产毒株鉴别体系的研究最多见。如Niessen等[115]比对了多种镰刀菌的Tri5序列，发现其中两个高度保守的区域。在此区域设计引物，可在镰刀菌的20多个种中扩增得658 bp的目的片段，上述引物对在后续研究中也被用于多个纯培养菌或染菌样品中Tri5基因的常规PCR定性检测[116-119]；靶向于该基因中一段较短的片段，Schnerr等[120]设计了另一对特异性引物，借由染料法real time-PCR技术定量检测300 个自然染菌小麦样品中TCTs产生菌的Tri5基因拷贝数，发现其与DON毒素产量正相关；基于毒素基因拷贝数与毒素产量存在的相关性，Strausbaugh等[121]采用TaqMan real time-PCR法定量检测了小麦根部及大麦中的产毒黄色镰孢菌。类似地，以real time-PCR技术定量Tri5基因拷贝数继而界定样品中产毒镰刀菌生物量的研究也见诸于后续的研究报道中[69,122-123]。

除Tri5外，其他参与毒素合成或调控的基因及非毒素合成相关基因也被用于产TCTs菌的分子检测。如基于转录因子Tri6设计的引物可特异性地鉴定产TCTs镰刀菌[60]；Niessen等[124]设计了一对可高度特异鉴定禾谷镰刀菌的靶向于半乳糖氧化酶基因gaoA的引物，在后续研究中被证实可特异性检测产TCTs的镰孢菌[125]；以多拷贝rDNA基因间区IGS序列为靶标，Jurado等[126]设计了一对特异性引物以检测产TCTs的禾谷镰刀菌、黄色镰孢、拟枝镰孢、梨孢镰孢及木贼镰孢，其灵敏度高于单拷贝基因。

3.2.2 TCTs不同化学型产毒菌株的分子甄别

除了笼统鉴定TCTs类产毒菌株外，随着产毒生物催化分子机制的阐明，研究者亦建立了多种分子检测体系用于该类毒素中不同化学型毒素产毒菌株的鉴别。如：靶向于Tri5-Tri6基因间序列，Bakan等[127]设计了一对可区分黄色镰孢菌高DON或低DON产毒菌株的引物，测试了17 株高DON产量株和13 株低DON产量株，表明该引物可完全正确地通过扩增片段的大小区分两类产毒菌株。此外，基于Tri13和Tri7基因在DON型和NIV型及其衍生物生物合成过程中的作用[128]，上述两个基因被用于设计特异性引物以区分两种B型TCTs产毒菌株[120,129-132]。随着研究的深入，也有其他毒素合成基因被用于此类毒素的产毒型细分。在Tri3内设计的两对引物Tri303F/Tri303R和Tri315F/ Tri315R被证实可有效区分镰刀菌中产3-AcDON和15-AcDON的菌株，分别产生583 bp和863 bp的片段[133]。基于分别靶向Tri3基因的1对引物及Tri6基因区的3对引物，Suzuki等[134]建立了一套可同时鉴别毒素化学型并区分亚洲镰刀菌和禾谷镰刀菌的多重PCR体系，可用于日本禾谷镰刀菌复合种的鉴别、诊断及流行病学研究。近期，基于Tri11基因的单核苷酸多态性，Wang Jianhua等[135]设计多重PCR体系通过扩增片段长度不同以区分产3-AcDON、15-AcDON和NIV 3 类毒素的禾谷镰刀菌，经对来自不同宿主及地理来源的镰刀菌菌株及染菌小麦的验证，表明该体系具有普适性，可用于预测产毒株或染菌食品及饲料中的B型TCTs化学型。

4 展 望

镰刀菌普遍污染玉米、小麦、水稻等禾谷类作物，不仅造成世界粮食减产，其次生代谢产物——真菌毒素还严重威胁人畜健康[3]。鉴于其危害的严重性，国内外研究者已研发出系列以免疫亲和柱等前处理产品结合色谱-质谱联用的多元化毒素精准检测体系，有效地改善了食品及饲料中真菌毒素的检测能力。

尽管如此，现有仪器法仅靶向于毒素本身，无法在毒素形成前或早期预测样品产毒潜能，有效前置化干预毒素危害。总体而言，若毒素已能从食品或饲料中检测出，其污染已十分严重，受感染的产品仅能毁灭处置，无法挽回经济损失及对受污染产品摄入者的危害。因此，提前监测产毒真菌的存在与否对避免潜在毒素危害意义卓著。就此而言，对产毒菌快速、准确分子检测策略的建立是实现有效防控毒素污染和危害的首要一环，为真菌产毒化学型的诊断新添了一个快速的技术手段，有助于更好地保障人、畜的健康与安全。

因此，加强基于PCR技术的分子诊断手段在我国产毒真菌检测与鉴定中的应用，建立不同产毒真菌的快速、准确分子诊断方法，对早期有效防控真菌毒素污染、保障我国谷物基食品及饲料安全无疑产生深远的意义。

产毒真菌PCR检测的准确性关键取决于目标基因的选择及特异性引物的设计。只有选定该毒素合成的必需基因并针对其特异性片段设计引物进行扩增方能通过产物有无判定其产毒潜能。尽管当前国内外研究者借助已解析的毒素生物合成途径中的关键基因建立了大量针对不同产毒类型真菌的PCR鉴别体系，但不同研究间良莠不齐，体系甄别产毒真菌的准确性需引入大量源自不同宿主及地理位置的菌株结合毒素化学定量结果进行充分验证。

随着微生物基因组学技术的不断发展，在日后研究中可借助全基因组测序对毒素合成代谢途径进行全面分析，进一步发掘新的可用于真菌产毒能力预测的相关基因簇，丰富备选基因库，通过多靶标多重同步验证实现更准确、更科学的检测。在此基础上，通过比较基因组学，筛选共有标记基因，尝试建立关键产毒真菌属通用型检测引物及体系，进一步提高方法的适用范围。此外，仅对基因组水平进行检测还有可能出现假阳性现象，如某些参与毒素合成的关键基因虽存在于基因组，但因个别碱基突变或转录故障而在转录水平上未能获得完整mRNA导致不产毒，故在后续研究还应探索在转录水平上对毒素合成关键基因进行监控的检测方法以更好地保障基于PCR技术的产毒能力预测结果的准确性。

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Recent Advances in PCR-Based Molecular Diagnosis of Mycotoxigenic Fusarium

XIE Xueqin1, LIU Zhou2,*
(1. Xiamen Products Quality Supervision and Inspection Institute, Xiamen 361004, China; 2. Department of Pharmacy, Xiamen Medical College, Xiamen 361008, China)

Fusarium is one of the fungal genera primarily producing mycotoxins. Fusarial toxins, mainly consisting of trichothecene, zearalenone and fumonisin, can cause psychological disorders or even cancers in humans or farmed animals. Their chemical structures and hazards and the molecular mechanisms behind their biosynthesis, together with the application of polymerase chain reaction (PCR) for molecular detection of the mycotoxigenic Fusarium were reviewed in this paper. The significance of rapid molecular diagnosis methodology in early warning and pre-intervention of mycotoxic damage is highlighted. Furthermore, possible problems existing in the present PCR detection systems are presented as well as some strategies to tackle such obstacles and make the systems more reliable.

Fusarium; trichothecenes (TCTs); fumonisins (FUMs); zearalenone (ZEA); polymerase chain reaction (PCR); molecular diagnosis

10.7506/spkx1002-6630-201711046

TS207.4

A

1002-6630（2017）11-0291-10引文格式：

2016-04-27

福建省自然科学基金青年创新项目（2014J01118）；厦门市科技计划项目（3502Z20154086）

谢雪钦（1982—），女，高级工程师，博士，研究方向为食品安全检测及风险评估。E-mail：cherryxie36@163.com

*通信作者：刘舟（1983—），男，副教授，博士，研究方向为食品安全检测及风险评估。E-mail：lau_joe@163.com