基于PCR技術的產(chǎn)真菌毒素鐮刀菌分子診斷研究進展

謝雪欽，劉 舟*

（1.廈門市產(chǎn)品質(zhì)量監(jiān)督檢驗院，福建 廈門 361004；2.廈門醫(yī)學院藥學系，福建 廈門 361008）

基于PCR技術的產(chǎn)真菌毒素鐮刀菌分子診斷研究進展

謝雪欽1，劉 舟2,*

（1.廈門市產(chǎn)品質(zhì)量監(jiān)督檢驗院，福建 廈門 361004；2.廈門醫(yī)學院藥學系，福建 廈門 361008）

鐮刀菌屬于自然界中最頻繁產(chǎn)生毒素的真菌類別之一。該屬真菌的毒素類次生代謝產(chǎn)物主要包括單端孢霉烯族化合物、伏馬菌素和玉米赤霉烯酮3 類，引起人畜各種生理異常甚至癌變。本文綜述了3 類鐮刀菌毒素的結(jié)構(gòu)和危害、生物合成的分子機理以及聚合酶鏈式反應（polymerase chain reaction，PCR）技術在其產(chǎn)生菌分子檢測中的應用進展，突出了產(chǎn)毒真菌快速分子診斷技術在毒素早期預警、危害前置化干預中的意義。同時，在剖析現(xiàn)有產(chǎn)毒真菌PCR檢測體系可能存在問題的基礎上，提出克服瓶頸方法和進一步提高體系可靠性的有效策略。

鐮刀菌；單端孢霉烯族化合物；伏馬菌素；玉米赤霉烯酮；聚合酶鏈式反應；分子診斷

謝雪欽, 劉舟. 基于PCR技術的產(chǎn)真菌毒素鐮刀菌分子診斷研究進展[J]. 食品科學, 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

真菌感染普遍發(fā)生于作物生長期及貯存期，其代謝過程中產(chǎn)生的生物毒素隨谷物基食品及飼料加工過程進入人、畜食物鏈，嚴重威脅健康[1-2]。鐮刀菌屬（Fusarium spp.）包括20余種，與青霉屬和曲霉屬同為自然界中最主要的3 類真菌毒素產(chǎn)生菌。該屬真菌可侵染小麥、玉米、燕麥、馬鈴薯等農(nóng)作物，其中禾谷鐮刀菌（F. graminearum）、擬枝鐮刀菌（F. sporotrichioides）、黃色鐮刀菌（F. culmorum）、串珠鐮刀菌（F. proliferatum）、輪枝鐮刀菌（F. verticillioides）、尖孢鐮刀菌（F. oxysporum）和木賊鐮刀菌（F. equiseti）等可產(chǎn)生單端孢霉烯族化合物（trichothecenes，TCTs）、玉米赤霉烯酮（zearalenone，ZEA）、串珠鐮刀菌素、大鐮刀孢菌素、黃色鐮刀菌素、丁烯羥酸內(nèi)酯和伏馬菌素（fumonisins，F(xiàn)UMs）等多種生物毒素[3]。不同鐮刀菌所產(chǎn)生的生物毒素種類有所差異，其中以TCTs、ZEA和FUMs為最普遍且危害嚴重的毒素種類。此系列毒素在高溫條件下穩(wěn)定，在染菌食品及飼料貯存、加工及烹飪過程中不易于降解，故正常條件下人畜均有一定的暴露風險[4-6]。

鐮刀菌不同種間產(chǎn)毒素種類及能力差異大，對產(chǎn)毒株的快速準確鑒定對預測其產(chǎn)毒潛力并有效預防其可能引發(fā)的毒素危害至關重要。目前，產(chǎn)毒鐮刀菌的常規(guī)鑒定方法多基于形態(tài)學及育性分析，其費時、冗繁且專業(yè)要求強[7]。為了克服上述缺陷，近年來許多基于聚合酶鏈式反應（polymerase chain reaction，PCR）及其衍生技術的分子診斷策略被用于產(chǎn)毒真菌的快速檢測及鑒定[8-12]，有效地提高了真菌毒素危害預警能力，為保證事前干預、降低危害提供了良好的技術支持。本文就近年來產(chǎn)TCTs、ZEA和FUMs這3 類關鍵毒素鐮刀菌的分子診斷體系發(fā)展及其機理進行綜述，并在此基礎上探討此技術在毒素危害預警中的意義。

1 玉米赤霉烯酮

ZEA，又稱為F-2毒素，是2,4-二羥基苯甲酸酯類化合物，化學式為C18H22O5。ZEA具有雌激素樣作用，主要作用于生殖系統(tǒng)，人、畜食用含毒素的食物后，增加流產(chǎn)、死胎和畸胎風險[13]，還可引起惡心、發(fā)冷、頭痛、神智抑郁等中樞神經(jīng)系統(tǒng)的中毒癥狀[14]。ZEA主要由鐮刀菌（禾谷鐮刀菌、黃色鐮刀菌、木賊鐮刀菌、輪枝鐮刀菌、三線鐮刀菌等）產(chǎn)生，其中以禾谷鐮刀菌為最常見的產(chǎn)毒菌。

1.1 ZEA的生物合成及其關鍵基因簇

ZEA首次被Stob等[15]于1962年發(fā)現(xiàn)并報道，此后其化學結(jié)構(gòu)獲得解析[16]。自1979年起，通過同位素示蹤法，經(jīng)乙酸-丙二酰-輔酶A酶系統(tǒng)以乙酸為前體首尾縮合形成毒素的ZEA生物合成途徑逐步被解析[17-19]，但對其合成的分子機制研究仍十分局限。

聚酮合成酶（polyketide synthase，PKS）在ZEA的生物合成中發(fā)揮關鍵催化作用，促成聚酮化合鏈的形成及毒素的產(chǎn)生。全基因組序列分析表明禾谷鐮刀菌中有15 個PKS基因，其中有5 個PKS基因產(chǎn)物已被界定，分別參與ZEA、黃色鐮刀菌素、鐮菌素C及黑色子囊殼色素的生物合成[20]。Kim[21]、Gaffoor[22]等分別通過基因缺失實驗表明，PKS4和PKS13是合成ZEA不可或缺的必需基因，在玉米赤霉菌中刪除上述基因?qū)е耑EA毒素缺失。而Lys?e等[23]則認為只有PKS4基因是禾谷鐮孢菌中ZEA毒素合成的必需基因。此外，與PKS4和PKS13同在一個基因簇的另外兩個基因也是ZEA合成不可或缺的，分別為與異戊醇氧化酶基因高度同源的ZEB1（FG12056）以及轉(zhuǎn)錄調(diào)控基因ZEB2（FG02398）[21]。

1.2 產(chǎn)ZEA鐮刀菌的PCR檢測

以ZEA生物合成途徑中的關鍵基因為靶標，近年來多種分子檢測技術被開發(fā)用于ZEA的間接檢測，其結(jié)果與酶聯(lián)免疫吸附測定（enzyme linked immunosorbent assay，ELISA）、高效液相色譜（high performance liquid chromatography，HPLC）法等針對毒素本身的直接分析方法高度一致。如張瑞芳等[24]以PKS4基因為靶標設計一對特異性引物PKS4F/PKS4R，通過PCR法間接檢測2 個染菌小麥籽粒及36 株分離自小麥樣本的禾谷鐮刀菌中的ZEA毒素，均顯示陽性，與ELISA直接檢測的結(jié)果完全吻合。同樣以PKS4基因為特異性分子標記基因，Meng Kun等[25]以SYBR實時熒光定量PCR法對食品中的產(chǎn)ZEA鐮孢菌進行了定性及定量檢測，方法檢測限低至10 拷貝PKS4基因/PCR體系或500 個真菌孢子/g食品，其定量線性范圍可覆蓋102～104共3 個數(shù)量級，方法快速、特異、靈敏，可在毒素大量形成前達到預警作用。而Atoui等[26]則選定ZEA生物合成過程中的另一個必需基因PKS13為靶標，通過實時熒光定量（real time）-PCR法建立了玉米樣品中ZEA含量與產(chǎn)毒禾谷鐮刀菌、黃色鐮刀菌PKS13拷貝數(shù)間的相關性，經(jīng)PCR反應Ct值推算產(chǎn)毒菌PKS13拷貝數(shù)繼而再推測該樣品污染ZEA的風險，全程可在8 h內(nèi)完成，較傳統(tǒng)定量方法更簡便、快速。Misiewicz等[27]發(fā)現(xiàn)禾谷鐮刀菌種內(nèi)PKS4基因的序列也存在多態(tài)性，這導致其分離的一株產(chǎn)ZEA的禾谷鐮刀菌菌株P7/4以Lys?e等[23]設計的一對引物PKS4F/R經(jīng)常規(guī)PCR無法獲得擴增片段。經(jīng)過充分序列比對分析，一對避開多態(tài)性區(qū)域而設計的特異性引物PKS_RT_F/R則可在供試的ZEA陽性產(chǎn)毒菌株mRNA上均擴增得91 bp的目標片段，且轉(zhuǎn)錄分析表明該基因的轉(zhuǎn)錄水平與毒素產(chǎn)量無相關性。此研究提示在后續(xù)研究中應需充分考慮PKS4基因的多態(tài)性問題，設計兼容性引物以鑒定產(chǎn)ZEA菌株，避免假陰性結(jié)果出現(xiàn)。

2 伏馬菌素

FUMs是主要由串珠鐮刀菌和輪枝鐮刀菌產(chǎn)生的致癌性代謝產(chǎn)物。該類毒素為不同多氫醇和丙三羧酸組成的結(jié)構(gòu)類似的雙酯化合物，其化學結(jié)構(gòu)中的長碳水化合物鏈在其毒性中發(fā)揮關鍵作用[28]。FUMs早在1993年已被國際癌癥研究機構(gòu)（International Agency for Research on Cancer，IARC）劃定為2B類致癌物。動物實驗和流行病學資料已表明，F(xiàn)UMs主要損害肝腎功能，能引起馬腦白質(zhì)軟化癥和豬肺水腫綜合癥等[29-30]，并與食道癌的發(fā)生有一定的因果關系[31]。

到目前為止，已發(fā)現(xiàn)的FUMs分為A、B、C、P共4組，有FA1、FA2、FB1、FB2、FB3、FB4、FC1、FC2、FC3、FC4和FP1共11 種，其中FB1發(fā)生頻率及毒性最高[32]。FB1為水溶性霉菌毒素，主要污染玉米及其制品，對熱穩(wěn)定，不易被蒸煮破壞，所以控制農(nóng)作物在生長、收獲和儲存過程中的霉菌污染至關重要。

2.1 FUMs的生物合成及關鍵基因

2.1.1 FUMs的生物合成

自1988年FB1被分離[33]且化學結(jié)構(gòu)被解析[34]以來，研究者不斷致力于闡明其生物合成途徑。生物化學及遺傳學研究顯示FUMs的碳骨架中的C-3至C-20是聚酮合成的產(chǎn)物[35]，丙氨酸與上述碳鏈的縮合形成了C2位上的氨基以及C-1和C-2骨架[36]，C12及C16位上的甲基側(cè)鏈則是由活性腺苷甲硫胺酸轉(zhuǎn)移酶從蛋氨酸上添加[37]，C3位的羥基來自于醋酸鹽源羰基，C5、C10、C14和C15位上的羥基則源于游離氧[38]，而丙三羧酸側(cè)鏈則通過檸檬酸循環(huán)代謝生成[39]。對于上述各步驟的先后合成次序，Bojja等[40]通過輪枝鐮孢3 個基因缺失突變株Δfum1、Δfum6和Δfum8的共培養(yǎng)，發(fā)現(xiàn)與突變株Δfum6共培養(yǎng)可回補基因Δfum1或Δfum8使其恢復產(chǎn)FUMs能力，對共培養(yǎng)代謝物進行LC-質(zhì)譜（mass spectrometry，MS）分析表明7 d內(nèi)可形成帶1～4 個羥基的側(cè)鏈結(jié)構(gòu)的FUMs骨架，據(jù)此研究者推斷FUMs的生物合成始于Fum1p催化的碳鏈聚合及隨后Fum8p催化的丙氨酸縮合，其產(chǎn)物經(jīng)由Fum6p或其他酶進行后續(xù)進一步氧化以形成終產(chǎn)物。

2.1.2 FUMs生物合成基因

借助經(jīng)典的遺傳學手段，參與B系列FUMs合成及調(diào)控的相關基因已有一定的研究。野生型輪枝鐮孢菌形成4 種B系列FUMs產(chǎn)物（FB1、FB2、FB3、FB4），除線性碳骨架上羥基的數(shù)量和位置有所差異之外，其結(jié)構(gòu)完全相同。FB1是其他3 種毒素形式羥基化的產(chǎn)物[32]。上述3 種不同毒素產(chǎn)物的形成與3 個顯著毗鄰的基因位點（fum1、fum2、fum3）有關，其中fum1（=fum5）編碼PKS，是催化C20聚酮骨架形成所必需的，該基因缺陷型菌株不產(chǎn)生FUMs[41-42]；fum2則是毒素骨架C10位羥基化所必需的，若缺失則僅形成FB2，不產(chǎn)生FB1和FB3；fum3（=fum9）缺陷株則喪失C5位羥基化的能力，僅產(chǎn)生FB3

[43-44]。此后，以Ⅰ類PKS的β-酮縮酶區(qū)設計兼并引物，Seo等[45]從輪枝鐮孢菌cDNA上又克隆得與fum5毗鄰的4 個基因fum6、fum7、fum8和fum9?；蜃钄喾治霰砻?，fum6和fum8是FUMs合成所必需的；fum9基因缺陷株導致FUMs碳骨架中的C5位無法羥基化，功能類似于此前鑒定的fum3，且基因回補實驗表明，導入fum9可回補阻斷fum3引起的表型缺陷，上述現(xiàn)象充分證明fum9等同于fum3[46]。此外，2003年，Proctor等[47]通過分析上述5 個 FUMs合成相關基因簇發(fā)現(xiàn)，其側(cè)翼序列中還有18 個開放閱讀框，其中有10 個基因參與毒素合成。敲除編碼假定ABC轉(zhuǎn)運蛋白的ORF-19導致FUMs產(chǎn)量的急劇下降，表明外排泵相關基因可能參與毒素轉(zhuǎn)運至胞外。進一步研究發(fā)現(xiàn)，上述基因簇中的fum13基因編碼的短鏈脫氫酶參與FUMs碳主鏈上C3位羰基還原為羥基，且該基因是負責上述轉(zhuǎn)化的主要功能因子，其缺失后輪枝鐮孢菌C3位正常羥基化的FUMs產(chǎn)量僅為野生株的10%[48]。在上述對FUMs合成相關基因簇功能解析的基礎上，Bojja等[40]通過對缺失突變株共培養(yǎng)代謝產(chǎn)物化學結(jié)構(gòu)進行深入解析，最后繪制了輪枝鐮孢菌中各基因調(diào)控FUMs逐步合成可能的生物化學過程圖。此外，因多數(shù)鐮刀菌FUMs以B型為主，比對以產(chǎn)C型毒素為主的尖孢鐮刀菌O-1890與其他鐮刀菌的毒素合成基因簇，并借由回補實驗，Proctor等[49]還證實不同結(jié)構(gòu)的fum8決定了FUMs的類型。

2.1.3 FUMs生物合成調(diào)控基因

不同于赭曲霉毒素或TCTs等生物毒素，F(xiàn)UMs生物合成相關基因簇中未發(fā)現(xiàn)參與毒素合成調(diào)控的基因。研究表明，一系列參與鐮刀菌pH值調(diào)控、糖代謝、氮代謝的基因均參與FUMs的合成調(diào)控。如Shim等[50]發(fā)現(xiàn)細胞周期素編碼基因FCC1調(diào)控輪枝鐮孢菌中FB1的合成，該基因缺失株在pH 6的基礎培養(yǎng)基上生長時，其毒素合成必需基因fum5的表達被阻斷，而在pH 3時則可恢復毒素合成。在此基礎上，該研究團隊進一步研究發(fā)現(xiàn)另一參與堿性環(huán)境中輪枝鐮孢FB1生物合成的負調(diào)控因子——PAC1，其缺失導致毒素產(chǎn)量提升[51]。此后，F(xiàn)laherty等[52]從輪枝鐮孢菌FB1生物合成期的表達序列標簽（expressed sequence tag，EST）文庫中鑒定到一個雙核鋅簇型基因ZFR1，功能研究表明其參與毒素合成的正向調(diào)控，且該基因為FFC1的上游調(diào)控基因。后續(xù)深入研究推斷，ZFR1對FB1合成的控制是通過對FST1等與碳水化合物感應、攝取及轉(zhuǎn)運相關基因的調(diào)控實現(xiàn)的[53]。此外，編碼異構(gòu)三聚體（αβγ）G蛋白β亞基的基因GBB1的缺失也導致FUMs合成關鍵基因fum1及fum8表達下調(diào)，致使FB1產(chǎn)量急劇下降，為毒素合成的正調(diào)控因子之一[54]。Kim等[55]發(fā)現(xiàn)氮代謝調(diào)控基因AREA亦參與FB1合成正調(diào)控，其缺失導致輪枝鐮孢菌喪失產(chǎn)毒能力。除了上述個別調(diào)控基因的單獨解析外，比較蛋白組學、芯片、EST文庫等大數(shù)據(jù)手段亦被不斷用于FUMs生物合成調(diào)控基因的全基因組發(fā)掘[56-57]。

2.2 產(chǎn)FUMs鐮刀菌的PCR檢測

當前，對產(chǎn)FUMs菌的分子檢測主要靶向于兩類目標基因：其一為對直接參與毒素合成的基因（如fum1等）進行擴增；其二是對內(nèi)轉(zhuǎn)錄間隔區(qū)（internal transcribed spacer，ITS）等遺傳標記核基因進行擴增或多態(tài)性分析。2.2.1 基于毒素生物合成相關基因的分子檢測

2002年，王曉英等[58]以FUMs生物合成所必需的多酮肽合成酶基因fum5為靶序列，建立了產(chǎn)毒株PCR檢測方法。確證某鐮刀菌株（ATCC12763）為FUMs生物合成酶基因陰性，判斷為非FUMs產(chǎn)毒株，此結(jié)果與美國模式培養(yǎng)物集存庫（American type culture collection，ATCC）提供的菌株產(chǎn)毒資料相一致。隨后，該團隊以上述引物鑒別了29 株分離自我國不同省份、不同糧食樣本中的串珠鐮刀菌分離株，其結(jié)果與毒素HPLC分析結(jié)果相一致[59]，進一步證實了fum5作為菌株產(chǎn)FUMs能力預測指示基因的可靠性。結(jié)合靶向鐮刀屬ITS的特異性引物，fum5（=fum1）亦被用于多重PCR[60]或TaqMan real time-PCR體系[61]中作為鑒定產(chǎn)FUMs鐮孢菌的特異性遺傳標記基因。類似的多重檢測體系亦被成功用于蔬菜[62]、玉米粒[63]、高粱[64]和稻谷[65]等食物及家禽飼料[66]中產(chǎn)毒鐮刀菌的快速鑒定。Gonzalez-Jaen等[67]研究證實fum1（=fum5）、fum6和fum8基因僅存在于F. verticillioides、F. proliferatum、F. fujikuroi和F. nygamai等產(chǎn)FB菌株中。基于fum1基因中的β-酮乙酰還原酶的酮酰還原酶（ketoacyl reductase，KR）功能域設計PCR引物，能高度特異地識別產(chǎn)FUMs的輪枝鐮孢菌。據(jù)此推斷，不產(chǎn)該毒素的菌株缺失了fum1基因或者至少缺失了與該PCR引物配對的部分。鐮刀菌能產(chǎn)生多種參與毒素或色素合成的PKS基因，其中KR功能域是產(chǎn)毒相關PKS基因所特有的，在該區(qū)設計產(chǎn)毒株鑒定引物更具特異性。研究表明，靶向fum1不同區(qū)段序列的引物鑒定產(chǎn)FUMs鐮刀菌的效力各不相同，需針對特異性必需片段設計并充分驗證其可靠性。如Baird等[68]以fum1為目標基因設計了4 對引物，其中僅B引物對可100%準確地鑒定玉米組織中的產(chǎn)FUMs輪枝鐮孢菌，而該引物僅能鑒定出80%的層出鐮刀菌產(chǎn)毒陽性株。鑒于此，研究者需結(jié)合毒素產(chǎn)物化學測定的結(jié)果，引入大量的菌株樣本以驗證引物甄別真菌產(chǎn)毒與否的能力。此外，隨著基因定量技術的發(fā)展，以fum1為靶標的TaqMan real time-PCR法近期還被引入用于鐮刀菌產(chǎn)FUMs潛能的間接定量預測[69]。

2.2.2 基于非產(chǎn)毒相關基因的分子鑒定

早在1998年，Grimm等[70]就通過分析產(chǎn)毒與非產(chǎn)毒鐮刀菌間ITS序列的差異設計了一對特異性引物及生物素標記探針用于擴增產(chǎn)毒株中ITS區(qū)的108 bp序列，并通過PCR-ELISA方法測定該序列。同理，基于對產(chǎn)與不產(chǎn)FUMs的輪枝鐮孢菌株間核糖體DNA的IGS區(qū)序列差異性分析，Pati?o等[71]建立了一套高度靈敏的PCR體系用于甄別54 株來自不同地區(qū)及宿主的擬枝鐮刀菌產(chǎn)毒株。此外，對不同地區(qū)來源于香蕉和玉米的29 株鐮刀菌分離株ITS擴增產(chǎn)物進行隨機引物擴增DNA多態(tài)性分型方法（randomly amplif i ed polymorphic DNA analysis，RAPD）及限制性片段長度多態(tài)性（restrictionfragment length polymorphism，RFLP）分析，結(jié)果顯示基于RAPD的序列多態(tài)性聚類分析結(jié)果可區(qū)分產(chǎn)生不同水平FUMs及不同宿主來源的菌株[72]。上述研究所設計的可區(qū)分產(chǎn)毒與否的引物對VERTF-1/VERTF-2在后續(xù)研究中被用于印度[73]及菲律賓[74]等國玉米中鐮孢菌產(chǎn)FUMs情況的預測及分析。Mirete等[75]對分離自多種宿主、地理來源及不同F(xiàn)UMs產(chǎn)量的48 株輪枝鐮孢菌的EF-1α基因及基因內(nèi)間隔區(qū)（intergenic spacers，IGS）部分序列進行系統(tǒng)發(fā)育分析，聚類結(jié)果可將供試菌株分為產(chǎn)及不產(chǎn)FUMs兩組，其中產(chǎn)毒株居多，其地理分布廣、偏好侵染谷物類、有性繁殖多發(fā)且變異大。

3 單端孢霉烯族化合物

根據(jù)化學結(jié)構(gòu)中C8位置上是否有酮配基，鐮刀菌中TCTs分為A和B兩類，其中A類毒性更強[76]。A類包括T-2毒素、HT-2毒素、新茄病鐮刀菌烯醇（neosolaniol，NEO）、蛇形霉素（diacetoxyscirpenol，DAS）和單乙酰氧基鐮草鐮刀菌醇（monoacetoxyscirpenol，MAS）；B類包括脫氧雪腐鐮刀菌烯醇（deoxynivalenol，DON）及其衍生物3-AcDON、15-AcDON和雪腐鐮刀菌烯醇（nivalenol，NIV）及鐮刀菌烯酮X。盡管已有200余種TCTs被鑒定，當前食品及飼料中分離到的污染種類多為T-2、HT-2、DAS、DON和NIV這5 種[77]。此類毒素對胃腸系統(tǒng)、皮膚、免疫功能、血液、基因均有毒性，抑制蛋白質(zhì)、RNA和DNA的合成，破壞膜功能、抑制免疫反應，引起血液功能異常等[78]。

3.1 TCTs生物合成途經(jīng)及其相關基因簇

TCTs為倍半萜烯環(huán)氧化合物，首次于1948年分離自粉紅單端孢，因此而得名，同位素標記代謝前體飼喂實驗表明其合成前體為單端孢霉烯（trichodiene，TDN）[79]。以加氧酶抑制劑處理[80-83]或紫外（ultraviolet，UV）輻射[84-85]產(chǎn)毒的鐮刀菌，結(jié)果發(fā)現(xiàn)其體內(nèi)TDN累積且毒素合成受到抑制，證實TDN也是鐮刀菌中該類毒素合成的前體。此外，上述結(jié)論通過添加外源同位素標記的人工合成前體實驗亦得到進一步的確證[86-87]。以擬枝鐮孢菌[88-90]、接骨鐮孢菌[91]和黃色鐮孢菌[83,92]為對象，以TDN為前體進行氧化、異構(gòu)化、環(huán)化及酯化等反應最后合成各種復雜的TCTs類化合物的過程在后續(xù)研究中也被不斷解析。Desjardins等[93]總結(jié)了上述過程，以圖示法解析了鐮孢菌中主要TCTs的生物合成途徑。

鐮刀菌TCTs生物合成遺傳機制的解析始于Hohn等[94-95]對擬枝鐮孢菌中該類毒素合成第一步的催化酶——TDN合成酶的純化及其編碼基因Tri5的克隆。以Tri5為契機，借由同源片段替換實驗，研究者進一步在擬枝鐮孢[96]和禾谷鐮孢[97]中于該基因側(cè)翼序列發(fā)現(xiàn)系列毒素合成相關基因，稱為Tri5基因簇，其結(jié)構(gòu)及各基因功能在兩個種間保守。隨著研究的深入，以禾谷鐮孢菌及擬枝鐮孢菌為模式種，該屬真菌參與TCTs合成及調(diào)控的基因簇及其催化毒素生物合成的分子機制也被不斷解析。到目前為止，此屬真菌至少有3 個基因簇參與該類毒素的合成，分別為Tri5基因簇、Tri1-Tri16雙基因簇和Tri101，其中Tri5基因簇包含了7 個合成催化酶編碼基因、2 個調(diào)控基因和1個轉(zhuǎn)運基因[98]，其中各基因功能詳見表1。

2005年，重慶市政府對水稻插秧機的技術特點進行了研究，初步探索出了解決機械化插秧對水稻育秧要求高的技術難題[3]。2006年，全市在36個區(qū)縣、110個鄉(xiāng)鎮(zhèn)開展水稻機械育秧技術、水稻插秧機示范推廣，在隨后的幾年里不斷加大對水稻插秧機示范推廣的力度。 從2006-2010年全市水稻插秧機總量從96臺增加到9 000臺多，使重慶地區(qū)水稻插秧機使用量增長了93倍多，促進了重慶地區(qū)的水稻機械化插秧工作。2006-2008年重慶地區(qū)機插水稻面積、每公頃產(chǎn)量與增產(chǎn)率如表1所示[4-6]。

表1 參與鐮刀菌單端孢霉烯族毒素合成及調(diào)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的分子檢測技術在產(chǎn)TCTs鐮刀菌鑒定中的應用

3.2.1 產(chǎn)TCTs鐮刀菌的檢測

隨著對鐮刀菌屬中各產(chǎn)毒素代表種參與TCTs合成及調(diào)控相關基因功能的解析，多種必需基因被用于此類毒素產(chǎn)毒株的分子鑒定。因Tri5基因負責催化所有產(chǎn)TCTs真菌中此類毒素生物合成的第一步，以該基因為靶標設計特異性產(chǎn)毒株鑒別體系的研究最多見。如Niessen等[115]比對了多種鐮刀菌的Tri5序列，發(fā)現(xiàn)其中兩個高度保守的區(qū)域。在此區(qū)域設計引物，可在鐮刀菌的20多個種中擴增得658 bp的目的片段，上述引物對在后續(xù)研究中也被用于多個純培養(yǎng)菌或染菌樣品中Tri5基因的常規(guī)PCR定性檢測[116-119]；靶向于該基因中一段較短的片段，Schnerr等[120]設計了另一對特異性引物，借由染料法real time-PCR技術定量檢測300 個自然染菌小麥樣品中TCTs產(chǎn)生菌的Tri5基因拷貝數(shù)，發(fā)現(xiàn)其與DON毒素產(chǎn)量正相關；基于毒素基因拷貝數(shù)與毒素產(chǎn)量存在的相關性，Strausbaugh等[121]采用TaqMan real time-PCR法定量檢測了小麥根部及大麥中的產(chǎn)毒黃色鐮孢菌。類似地，以real time-PCR技術定量Tri5基因拷貝數(shù)繼而界定樣品中產(chǎn)毒鐮刀菌生物量的研究也見諸于后續(xù)的研究報道中[69,122-123]。

除Tri5外，其他參與毒素合成或調(diào)控的基因及非毒素合成相關基因也被用于產(chǎn)TCTs菌的分子檢測。如基于轉(zhuǎn)錄因子Tri6設計的引物可特異性地鑒定產(chǎn)TCTs鐮刀菌[60]；Niessen等[124]設計了一對可高度特異鑒定禾谷鐮刀菌的靶向于半乳糖氧化酶基因gaoA的引物，在后續(xù)研究中被證實可特異性檢測產(chǎn)TCTs的鐮孢菌[125]；以多拷貝rDNA基因間區(qū)IGS序列為靶標，Jurado等[126]設計了一對特異性引物以檢測產(chǎn)TCTs的禾谷鐮刀菌、黃色鐮孢、擬枝鐮孢、梨孢鐮孢及木賊鐮孢，其靈敏度高于單拷貝基因。

3.2.2 TCTs不同化學型產(chǎn)毒菌株的分子甄別

除了籠統(tǒng)鑒定TCTs類產(chǎn)毒菌株外，隨著產(chǎn)毒生物催化分子機制的闡明，研究者亦建立了多種分子檢測體系用于該類毒素中不同化學型毒素產(chǎn)毒菌株的鑒別。如：靶向于Tri5-Tri6基因間序列，Bakan等[127]設計了一對可區(qū)分黃色鐮孢菌高DON或低DON產(chǎn)毒菌株的引物，測試了17 株高DON產(chǎn)量株和13 株低DON產(chǎn)量株，表明該引物可完全正確地通過擴增片段的大小區(qū)分兩類產(chǎn)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過程中的作用[128]，上述兩個基因被用于設計特異性引物以區(qū)分兩種B型TCTs產(chǎn)毒菌株[120,129-132]。隨著研究的深入，也有其他毒素合成基因被用于此類毒素的產(chǎn)毒型細分。在Tri3內(nèi)設計的兩對引物Tri303F/Tri303R和Tri315F/ Tri315R被證實可有效區(qū)分鐮刀菌中產(chǎn)3-AcDON和15-AcDON的菌株，分別產(chǎn)生583 bp和863 bp的片段[133]?；诜謩e靶向Tri3基因的1對引物及Tri6基因區(qū)的3對引物，Suzuki等[134]建立了一套可同時鑒別毒素化學型并區(qū)分亞洲鐮刀菌和禾谷鐮刀菌的多重PCR體系，可用于日本禾谷鐮刀菌復合種的鑒別、診斷及流行病學研究。近期，基于Tri11基因的單核苷酸多態(tài)性，Wang Jianhua等[135]設計多重PCR體系通過擴增片段長度不同以區(qū)分產(chǎn)3-AcDON、15-AcDON和NIV 3 類毒素的禾谷鐮刀菌，經(jīng)對來自不同宿主及地理來源的鐮刀菌菌株及染菌小麥的驗證，表明該體系具有普適性，可用于預測產(chǎn)毒株或染菌食品及飼料中的B型TCTs化學型。

4 展 望

鐮刀菌普遍污染玉米、小麥、水稻等禾谷類作物，不僅造成世界糧食減產(chǎn)，其次生代謝產(chǎn)物——真菌毒素還嚴重威脅人畜健康[3]。鑒于其危害的嚴重性，國內(nèi)外研究者已研發(fā)出系列以免疫親和柱等前處理產(chǎn)品結(jié)合色譜-質(zhì)譜聯(lián)用的多元化毒素精準檢測體系，有效地改善了食品及飼料中真菌毒素的檢測能力。

盡管如此，現(xiàn)有儀器法僅靶向于毒素本身，無法在毒素形成前或早期預測樣品產(chǎn)毒潛能，有效前置化干預毒素危害。總體而言，若毒素已能從食品或飼料中檢測出，其污染已十分嚴重，受感染的產(chǎn)品僅能毀滅處置，無法挽回經(jīng)濟損失及對受污染產(chǎn)品攝入者的危害。因此，提前監(jiān)測產(chǎn)毒真菌的存在與否對避免潛在毒素危害意義卓著。就此而言，對產(chǎn)毒菌快速、準確分子檢測策略的建立是實現(xiàn)有效防控毒素污染和危害的首要一環(huán)，為真菌產(chǎn)毒化學型的診斷新添了一個快速的技術手段，有助于更好地保障人、畜的健康與安全。

因此，加強基于PCR技術的分子診斷手段在我國產(chǎn)毒真菌檢測與鑒定中的應用，建立不同產(chǎn)毒真菌的快速、準確分子診斷方法，對早期有效防控真菌毒素污染、保障我國谷物基食品及飼料安全無疑產(chǎn)生深遠的意義。

產(chǎn)毒真菌PCR檢測的準確性關鍵取決于目標基因的選擇及特異性引物的設計。只有選定該毒素合成的必需基因并針對其特異性片段設計引物進行擴增方能通過產(chǎn)物有無判定其產(chǎn)毒潛能。盡管當前國內(nèi)外研究者借助已解析的毒素生物合成途徑中的關鍵基因建立了大量針對不同產(chǎn)毒類型真菌的PCR鑒別體系，但不同研究間良莠不齊，體系甄別產(chǎn)毒真菌的準確性需引入大量源自不同宿主及地理位置的菌株結(jié)合毒素化學定量結(jié)果進行充分驗證。

隨著微生物基因組學技術的不斷發(fā)展，在日后研究中可借助全基因組測序?qū)Χ舅睾铣纱x途徑進行全面分析，進一步發(fā)掘新的可用于真菌產(chǎn)毒能力預測的相關基因簇，豐富備選基因庫，通過多靶標多重同步驗證實現(xiàn)更準確、更科學的檢測。在此基礎上，通過比較基因組學，篩選共有標記基因，嘗試建立關鍵產(chǎn)毒真菌屬通用型檢測引物及體系，進一步提高方法的適用范圍。此外，僅對基因組水平進行檢測還有可能出現(xiàn)假陽性現(xiàn)象，如某些參與毒素合成的關鍵基因雖存在于基因組，但因個別堿基突變或轉(zhuǎn)錄故障而在轉(zhuǎn)錄水平上未能獲得完整mRNA導致不產(chǎn)毒，故在后續(xù)研究還應探索在轉(zhuǎn)錄水平上對毒素合成關鍵基因進行監(jiān)控的檢測方法以更好地保障基于PCR技術的產(chǎn)毒能力預測結(jié)果的準確性。

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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

福建省自然科學基金青年創(chuàng)新項目（2014J01118）；廈門市科技計劃項目（3502Z20154086）

謝雪欽（1982—），女，高級工程師，博士，研究方向為食品安全檢測及風險評估。E-mail：cherryxie36@163.com

*通信作者：劉舟（1983—），男，副教授，博士，研究方向為食品安全檢測及風險評估。E-mail：lau_joe@163.com