Application of LAMP in the rapid detection of aquatic animal pathogens
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摘要:
近年来，一种新的恒温核酸扩增方法LAMP法(loop-mediated isothermal amplification), 即环介导等温扩增法已经被开发利用。它采用能特异识别靶序列上6个位点的4条引物及一种具有链置换活性的DNA聚合酶(Bst DNA polymerase)，在恒温条件(60~65℃)，不到1 h的时间里进行核酸扩增，其扩增效率可达到109~1010个数量级，具有特异性强、等温灵敏、操作简单、产物易检测等优点。LAMP法已经被用来快速检测水产动物病原(细菌、病毒、寄生虫)。文章就LAMP法的反应原理、引物设计及其在水产动物病原快速检测中的应用等做简要的综述。
Abstract:In recent years, a novel isothermal method of nucleic acid amplification known as loop-mediated isothermal amplification (LAMP) has been developed. The LAMP method employs four primers which specifically recognize six distinct sequences on the target DNA and a Bst polymerase that has a strand displacement activity. The target DNA can be amplified from a few original copies to 109~1010 copies in less than an hour under isothermal conditions ranging from 60 to 65℃. The LAMP method has quite a few advantages such as high specificity, isothermal conditions, high sensitivity, simple operation, and simple detection for the LAMP reaction products. The LAMP method has been used for the rapid detection of aquatic animal pathogens (bacteria, virus, parasite). This review briefly summarizes the principle of LAMP, design of primers, and the application for the rapid detection of aquatic animal pathogens.
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Keywords:
- LAMP method /
- principle of LAMP /
- pathogens /
- rapid detection
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合浦珠母贝(P. fucata)(又名马氏珠母贝P. martensii)，分布十分广泛，在太平洋、印度洋和大西洋的主要热带和温带海洋都有分布[1]。合浦珠母贝是我国重要的海水养殖种类，是生产海水珍珠的重要母贝，具有极高的经济价值。也是印度、日本等国珍珠养殖的重要贝类[2-3]。近年来澳大利亚等也开始研究其在珍珠生产中的潜在价值[4-5]。合浦珠母贝在日本叫做P. fucata martensii[2]，在澳大利亚叫P.imbricata[4]，最近的研究表明它们都是同一个种①，为了便于区别，本文仍沿用旧名。日本和澳大利亚群体的地理分布距离大，处于太平洋水域分布区的南北两极[1, 4]。在养殖生产中往往发生不同地理种群的人为引进和混杂，造成当地种群的遗传结构发生改变。因此，不同地理种群的遗传背景分析是科学地引种移植和遗传选育的重要基础。
① Yu D H，Jia X，Chu K H. The common pearl oysters in China, Japan and Australia are conspecific: evidences from ITS sequences and AFLP (submitted).
核糖体DNA(rDNA)是由18 S、5.8 S、28 S和内部转录间隔子(ITS 1、ITS 2)等串联重复单元组成[6]。其中18 S、5.8 S和28 S高度保守，变异小，而ITS变异大，两者结合起来是分析物种遗传变异的重要手段。本文拟对我国、日本和澳大利亚珍珠贝的ITS 2和5.8 S与28 S的部分序列的变异特征进行分析，探讨其遗传亲缘关系，为引种、选育和种群资源管理提供遗传背景资料。
1. 材料和方法
1.1 珍珠贝来源
我国的合浦珠母贝(P. fucata)(简称cn)分别采自广东的大亚湾(db)、广西北部湾(bb)和海南三亚(sb)。日本的合浦珠母贝(P. fucata martensii)(jp)采自日本的三重县(Mie Prefecture)。澳大利亚的样品(P. imbricata)(au)采自Port Stephens(表 1)。每个采样地点各30个个体。采集闭壳肌组织样品，用95%的乙醇保存。
表 1 样品种类、采集地点、基因型和序列号Table 1. Species, collection localities, genotypes and GenBank accession numbers种类及采样地点
species and locality代码
code基因型
genotypeITS 2/bp 总长/bp
total lengthGenBank序列号
accession numberP. imbricata au au1 231 544 AY877606 澳大利亚 Port Stephens au2 237 550 AY877607 au3 237 550 AY877608 au4 231 544 AY877609 au5 237 550 AY877611 P. fucata martensii jp jp1 231 544 AY877612 日本 Mie Prefecture jp2 231 544 AY877613 jp3 233 546 AY877614 jp4 231 544 AY877615 jp5 231 544 AY877616 合浦珠母贝 P. fucata db db1 235 548 AY877581 广东大亚湾 Daya Bay，GD db2 231 544 AY877604 广西北海 Beibu Bay，GX bb bb1 233 546 AY877583 bb2 231 544 AY877605 海南三亚 Sanya, Hainan sb sb1 237 550 AY877592 sb2 230 543 AY877597 1.2 DNA提取、PCR扩增和测序
用QIAamp DNA Mini Kit (QIAGEN)试剂盒提取总DNA，操作步骤按试剂盒提供的程序进行。
ITS 2的引物为自行设计的特异引物，正向引物序列为5.8 SF：5’- GCA GGA CAC ATT GAA CAT CG -3’，反向引物序列为28 SR：5’- CCA AGG ACG TTC TTA GCA GAA G -3’。PCR反应体积为20 μL，含有1×PCR缓冲液(10 mM Tris-HCl (pH 9.0, 25℃), 50 mM KCl, 0.1% Triton X-100)，2.5 mM MgCl2，0.25 mM dNTP，0.15 μM引物，1U Taq DNA聚合酶(Promega)，DNA模板30~50 ng。反应开始为93℃变性4 min，之后30个循环：93℃ 40 s，50℃ 40 s，72℃ 1 min，最后为72℃ 5 min。扩增产物全部上样到1.0%的琼脂糖凝胶进行电泳分离，然后割胶，用凝胶抽提试剂盒(Gel Extraction Kit，QIAGEN)进行PCR产物纯化、回收。纯化产物用于测序分析，测序仪器为ABI3100型。中国、日本和澳大利亚3个地理种群各测序10个样品。测序反应用DNA测序试剂盒(ABI PRISMTM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit，PE Biosystems)，反应体积为20 μL，包括引物3.2 pmol，Terminator Ready Reaction Mix 8.0 μL，PCR产物60~90 ng。反应程序为96℃ 2 min, 30个循环：96℃ 30 s, 50℃ 15 s, 60℃ 4 min。反应产物经乙醇-醋酸钠沉淀纯化后用于测序仪分析。
1.3 数据分析
用Sequence Editor(1.03) (Applied Biosystems) 软件进行序列的校对和编辑。用Clustal X (1.83) 软件[7]进行多重比对 (Multiple alignment) 分析，其参数用缺省 (Default) 设置。之后用MEGA3[8]进行种群内和种群间 Kimura 2-parameter模型的遗传距离计算、碱基组成分析。
2. 结果
2.1 扩增片段的序列特征分析
扩增片段包括部分5.8 S、ITS 2和部分28 S基因。部分个体通过直接测序可获得清晰的测序图谱并用于分析，通过比对分析后获得基因型数据，一个地理群体内序列一致的为同一基因型。日本群体和澳大利亚群体各获得5个基因型，中国群体3个不同采样点各取2个基因型用于分析(表 1)。扩增产物的序列长度在去掉引物后为543~550 bp(表 1)，其中5.8 S基因片段长64 bp，28 S基因片段长249 bp，ITS 2比对长度为237位点。每个地理种群的基因型序列全部在GenBank数据库注册，序列号见表 1。包含引物的序列比对分析结果见图 1。3个群体的5.8 S基因片段的序列全部相同，而28 S基因片段的271 bp(包含引物)序列中只有1个碱基位点(454)发生碱基颠换(transversion)突变(图 1)，在GenBank数据库中同源分析(blastn)和很多物种也具有高度同源性。而ITS 2的长度变异较大，长度范围分布在230~237 bp之间(表 1)。共有20个位点发生突变，其中有12个位点发生缺失/插入突变，4个单突变子(singleton)，4个简约信息位点(parsimony informative site)。
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图 1 基因型序列比对分析结果(图中箭头所示为5.8 S、ITS 2和28 S基因的分界位点)Figure 1. Alignment of genotypic sequences (The arrows show the boundary of ITS 2 gene)从基因型数据来看，3个群体共享的基因型1个，日本和澳大利亚共享1个。从碱基突变位点来看，尚未发现群体特有的突变位点。
2.2 扩增片段各部分基因的碱基组成分析
不同基因的4种碱基组成差异较大。5.8 S的A碱基含量较低，28 S的T碱基含量较低。两者的GC含量远远高于其相应的AT含量，也高于ITS 2的GC含量(表 2)。但各碱基的频率检测未发现显著性的偏差(资料未显示)。3个地理群体之间的碱基组成没有差异。
表 2 rDNA扩增片段各部分基因的碱基组成Table 2. Nucleotide compositions of genes in amplified rDNA fragment% 群体
populationT C A G G+C 5.8 S ITS 2 28 S 5.8 S ITS 2 28 S 5.8 S ITS 2 28 S 5.8 S ITS 2 28 S 5.8 S ITS 2 28 S au 25.0 28.3 18.9 31.3 27.5 28.8 15.6 20.5 23.0 28.1 23.8 29.3 59.4 51.3 58.1 jp 25.0 27.4 18.9 31.3 28.4 28.9 15.6 20.6 22.9 28.1 23.7 29.3 59.4 52.0 58.2 cn 25.0 28.0 18.9 31.3 27.7 28.8 15.6 20.3 23.0 28.1 24.0 29.3 59.4 51.7 58.1 2.3 群体内与群体间的遗传距离分析
中国、日本和澳大利亚3个群体内的遗传距离分别为0.011、0.012和0.010，种群间的遗传距离分别为：中-日：0.013，中-澳：0.010，日-澳：0.011。群体内与群体间的遗传距离基本一致。
3. 讨论
ITS是rDNA上的一段非编码序列，变异较大，多态性较高，适合于亲缘关系较近的种类的遗传多样性分析，已广泛应用于种类鉴定和系统发育研究[9-15]。但用于群体遗传多样性分析的研究报道较少。
Colgan&Ponder基于同工酶的研究发现日本的P.imbricata(即P. fucata或P. martensi)与澳大利亚的P.imbricata遗传距离很近[16]；Atsumi等利用同工酶标记发现日本的P. fucata martensii与中国的P.fucata遗传距离只有0.01[17]。本文首次利用DNA标记对中国、日本和澳大利亚3个群体进行了直接的比较研究，结果与Colgan & Ponder(2002)和Atsumi等(2004)的同工酶研究结果一致[16-17]，3个地理群体具有很近的遗传关系。表明这3个地理群体的遗传关系很近，具有很高的遗传相似度。表明这3个地理群体可能存在基因交流，或分化时间不长，或者兼而有之。但ITS 2序列分析表明，它们的遗传多样性很丰富，基因型较多，变异位点达8.4%。有部分个体直接测序不成功，可能是多拷贝之间存在个体内变异[18]。而我们在白珠母贝3个个体中就发现有序列一致的个体，且都能直接测序，表明它们是纯合子(未发表资料)，说明遗传多样性较低。在长耳珠母贝的克隆测序中也没发现个体内变异[19]，由于该报道仅检测了一个个体，其遗传多样性状况有待进一步研究。因此在珍珠贝类中合浦珠母贝的ITS 2序列变异可能是比较大的。合浦珠母贝分布广泛，适应能力强，是我国、日本、澳大利亚[4]和加勒比海[5]等地区的优势种，这种强大的适应能力可能与其丰富的遗传多样性有关。同时也表明我国的合浦珠母贝具有丰富的可供选育的遗传资源。
从序列的变异特征看，20个突变位点中主要是插入/缺失突变(12个)，4个单突变子有可能是PCR误差造成的，简约信息位点只有4个。因此变异主要表现为长度变异，可能是染色体不等交换(unequal crossover)产生的。也表明合浦珠母贝存在高度的协同进化(concerted evolution)[7]。从基因型数据看，不同地理种群具有较多的独有的基因型，因此不同地理种群的杂交可以增加遗传杂合度。
ITS 2的高度变异性可能与其碱基组成有关。5.8 S和28 S的部分序列碱基组成中GC含量较高，序列变异度低，而ITS 2的GC含量低，序列变异度高。因为GC碱基对之间是由3个共价键连接的，而AC之间只有2个，容易突变。而且ITS 2不具有实质性的生物学功能，受到的选择压力小，有利于DNA序列突变的积累。因此ITS序列适合于种群遗传多样性的研究。
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