基于LM-PCR预扩增法的建立及其在水产品致病菌检测中的应用
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摘要
基于核酸扩增技术的基因检测方法具有高灵敏度与高特异性的优点，但在对实际样品的检测中，核酸扩增检测技术往往受到样品中目标DNA含量极少、破坏程度高、提取得到的模板含有大量背景核酸等因素制约，导致检测效率下降，影响了其在相关领域的应用。如DNA芯片技术可短时间内实现高通量分析但对样品要求较高，此时样品数量及质量往往成为制约检测的首要因素。针对上述问题，对样品DNA进行预扩增（pre-amplification）是有效的解决途径，如全基因组扩增技术（whole genome amplification, WGA）已在单核苷酸多态性分析（SNPs）、基因型（genotyping）和基因组测序（genome sequencing）等大规模分析中发挥了巨大作用。
在现有全基因组扩增方法中，连接介导PCR（ligation-mediated PCR, LM-PCR）具有受样品质量影响小、扩增效率高的优点，但其存在模板片段间可能发生自连的弊端，对此本研究应用mLM-PCR法（modified LM-PCR）解决这一问题，采用IIS型限制性内切酶（BbvI和Alw26I）对模板DNA进行酶切产生具有随机碱基粘性末端的片段以减少片段间自连的概率，并使用一组含有随机碱基的接头混合物与模板片段进行连接，最终使用通用引物实现基因组的全扩增。通过对副溶血弧菌基因组的全扩增结果显示，本方法可实现高灵敏度、均衡的全基因组扩增。通过使用特异性PCR以及荧光定量PCR对全扩增效果进行评价结果显示，与目前广泛应用的MDA法（multiple displacement amplification）相比，本方法具有更高的扩增效率与扩增均衡性，显示出极高的全扩增效率。
WGA法对模板DNA的纯度要求较高，无法应用于大量核酸背景中极微量目标DNA的预扩增，为此本研究建立了基于mLM-PCR法的选择性预扩增方法，可实现大量背景核酸中极微量目标DNA的预扩增：
（1）通过对大量金枪鱼基因组中极微量副溶血弧菌、霍乱弧菌、沙门氏菌、腐败希瓦氏菌的预扩增结果显示，通过本方法与普通PCR法结合使用可检测至数十拷贝目标致病菌（目标致病菌与背景核酸比例约为10-7），与普通PCR方法相比可将检测灵敏度提高2-3个数量级以上；
（2）本方法实现了同时对多个目标的多重预扩增，针对上述四种目标致病菌的四重预扩增结果显示使用多重预扩增法可获得与单重预扩增法相近的检测灵敏度，经过多重预扩增的产物较适合进行特异性多重PCR检测，可显著提高检测效率；
（3）通过使用环介导等温扩增法（LAMP）对预扩增产物进行特异性扩增的结果显示，本预扩增方法可成功实现与LAMP法联用且灵敏度极高，表明预扩增产物可普遍作为PCR类方法或恒温核酸扩增方法等目前常用特异性核酸扩增方法的模板，显示出其灵活性与普遍适用性；
（4）深入探究了接头结构等因素对mLM-PCR预扩增体系的影响，并对预扩增体系设计中关键点进行了总结如下：a）在一定范围内选择较长（大于200bp）的酶切片段作为预扩增目标；b）在一定范围内接头特异性越高，预扩增灵敏度越高；c）使用单条通用引物进行预扩增时茎环结构（loop）会影响PCR反应，但影响程度随模板长度的增长而减小；d）使用较高退火温度、增加引物浓度、增加反应循环数可有效减少茎环结构对预扩增PCR效率的影响；
（5）对人工污染牡蛎样品中副溶血弧菌的检测结果显示，mLM-PCR预扩增法可成功用于实际样品中致病菌的高灵敏度检测，检测灵敏度可达到0.1-1弧菌拷贝/管反应。
综上，本研究建立了一种基于LM-PCR技术的高效预扩增方法，可通过对预扩增体系进行灵活设计而实现对不同状态样品的高效预扩增，可实现大量背景核酸中极微量目标DNA的有效扩增，并对影响预扩增反应的因素进行了总结探讨，通过对水产品中致病菌的检测验证了本方法的可行性与高效性，可普遍应用于食品检测、临床诊断等相关领域。
Gene detection methods based on nucleic acids amplification technique haveadvantages of high sensitivity and specificity and are widely used in various fields,however, under some cases that only little sample DNA could be obtained, sampleDNA was not in good conditions or target DNA existed in massive backgroundnucleic acids thus may hamper its detection efficiency and application. For example,the DNA array technology could perform high-throughput analysis in a short period oftime but need relatively high quality and large quantity of sample. To overcome theseproblems, the pre-amplification of sample DNA is an effective way, one of which isthe whole genome amplification (WGA) technique that already been widely used inanalysis of single nucleotide polymorphisms (SNPs), genotyping and whole genomesequencing.
Among the existing WGA methods, the ligation-mediated polymerase chainreaction (LM-PCR) possessed advantages of high efficiency and less affected by thepoor conditions of template DNA, however, the self-ligation between templatefragments may happen and cause bad results. To solve this problem, a modifiedLM-PCR (mLM-PCR) was developed to carry out WGA: type IIS restriction enzymes(BbvI and Alw26I) were employed to cleave template DNA into numerous fragmentswith random sequences overhangs followed by ligated to a pool of adapters to formthe substrates for PCR, and finally all of the substrates were amplified with auniversal primer. The WGA results showed that V. parahaemolyticus genomic DNAcould be amplified with high sensitivity and uniformity. Compared to multipledisplacement amplification (MDA), the results based on specific PCR and qPCRanalysis showed that the mLM-PCR method obtained higher WGA efficiency thanMDA.
WGA based pre-amplification method require high purity template DNA thus not suitable for pre-amplify little amount of target DNA existed in massive backgroundnucleic acids. For this purpose, this study established a novel selectivepre-amplification method based on mLM-PCR that could selective pre-amplify thelittle amount of target DNA from massive background nucleic acids:
(1) Combined mLM-PCR pre-amplification with specific PCR, the detectionsensitivity of V. parahaemolyticus, S. typhimurium, V. cholera, S. putrefaciensgenomic DNA in massive background nucleic acids achieved about10copies,significantly higher (102to103times) than that with conventional PCR method.
(2) Multiple pre-amplification could be successfully performed with mLM-PCR.According to the multiple pre-amplification results of above four food bornepathogens, the sensitivity of multiple pre-amplification was as same as that of singletarget pre-amplification. The multiple pre-amplified products could be served astemplates for specific multiplex PCR detection.
(3) By using pre-amplified products as templates, loop mediated isothermalamplification (LAMP) could be successfully performed and the results confirmed thehigh sensitivity of the combination of the two methods. The results also indicated theflexibility and general applicability of mLM-PCR pre-amplification method toprovide general templates for various DNA amplification approaches.
(4) The adapter structure was crucial for mLM-PCR pre-amplification and itseffect was discussed as following: a) restriction fragments longer than200bp shouldbe chosen as pre-amplification targets; b) in a certain range, the higher specificity thatthe adapter obsessed, the higher pre-amplification efficiency could be obtained; c)when using single universal primer, the formation of stem-loop structure may affectpre-amplification, and the effect will decrease as the target fragment get longer; d) theeffect of stem-loop structure could be eliminated by using higher annealingtemperature, more PCR cycles and higher primer concentration.
(5) Artificially contaminated oyster samples for V. parahaemolyticus test resultsshowed that mLM-PCR could be successfully applied to the detection of pathogensexisted in actual seafood sample, and the sensitivity could achieve0.1to1pathogencopy per reaction tube.
In conclusion, this study established a novel pre-amplification method that basedon mLM-PCR. Through the variable designation of mLM-PCR, sample DNA underdifferent conditions could be pre-amplified with high efficiency, especially the littleamount of target DNA in massive background nucleic acids could be also effectivelypre-amplified to meet the detection requirements of conventional methods, the highefficiency of mLM-PCR pre-amplification also indicated its wide applications invarious gene detection fields such as food detection and clinical diagnosis.
在现有全基因组扩增方法中，连接介导PCR（ligation-mediated PCR, LM-PCR）具有受样品质量影响小、扩增效率高的优点，但其存在模板片段间可能发生自连的弊端，对此本研究应用mLM-PCR法（modified LM-PCR）解决这一问题，采用IIS型限制性内切酶（BbvI和Alw26I）对模板DNA进行酶切产生具有随机碱基粘性末端的片段以减少片段间自连的概率，并使用一组含有随机碱基的接头混合物与模板片段进行连接，最终使用通用引物实现基因组的全扩增。通过对副溶血弧菌基因组的全扩增结果显示，本方法可实现高灵敏度、均衡的全基因组扩增。通过使用特异性PCR以及荧光定量PCR对全扩增效果进行评价结果显示，与目前广泛应用的MDA法（multiple displacement amplification）相比，本方法具有更高的扩增效率与扩增均衡性，显示出极高的全扩增效率。
WGA法对模板DNA的纯度要求较高，无法应用于大量核酸背景中极微量目标DNA的预扩增，为此本研究建立了基于mLM-PCR法的选择性预扩增方法，可实现大量背景核酸中极微量目标DNA的预扩增：
（1）通过对大量金枪鱼基因组中极微量副溶血弧菌、霍乱弧菌、沙门氏菌、腐败希瓦氏菌的预扩增结果显示，通过本方法与普通PCR法结合使用可检测至数十拷贝目标致病菌（目标致病菌与背景核酸比例约为10-7），与普通PCR方法相比可将检测灵敏度提高2-3个数量级以上；
（2）本方法实现了同时对多个目标的多重预扩增，针对上述四种目标致病菌的四重预扩增结果显示使用多重预扩增法可获得与单重预扩增法相近的检测灵敏度，经过多重预扩增的产物较适合进行特异性多重PCR检测，可显著提高检测效率；
（3）通过使用环介导等温扩增法（LAMP）对预扩增产物进行特异性扩增的结果显示，本预扩增方法可成功实现与LAMP法联用且灵敏度极高，表明预扩增产物可普遍作为PCR类方法或恒温核酸扩增方法等目前常用特异性核酸扩增方法的模板，显示出其灵活性与普遍适用性；
（4）深入探究了接头结构等因素对mLM-PCR预扩增体系的影响，并对预扩增体系设计中关键点进行了总结如下：a）在一定范围内选择较长（大于200bp）的酶切片段作为预扩增目标；b）在一定范围内接头特异性越高，预扩增灵敏度越高；c）使用单条通用引物进行预扩增时茎环结构（loop）会影响PCR反应，但影响程度随模板长度的增长而减小；d）使用较高退火温度、增加引物浓度、增加反应循环数可有效减少茎环结构对预扩增PCR效率的影响；
（5）对人工污染牡蛎样品中副溶血弧菌的检测结果显示，mLM-PCR预扩增法可成功用于实际样品中致病菌的高灵敏度检测，检测灵敏度可达到0.1-1弧菌拷贝/管反应。
综上，本研究建立了一种基于LM-PCR技术的高效预扩增方法，可通过对预扩增体系进行灵活设计而实现对不同状态样品的高效预扩增，可实现大量背景核酸中极微量目标DNA的有效扩增，并对影响预扩增反应的因素进行了总结探讨，通过对水产品中致病菌的检测验证了本方法的可行性与高效性，可普遍应用于食品检测、临床诊断等相关领域。
Gene detection methods based on nucleic acids amplification technique haveadvantages of high sensitivity and specificity and are widely used in various fields,however, under some cases that only little sample DNA could be obtained, sampleDNA was not in good conditions or target DNA existed in massive backgroundnucleic acids thus may hamper its detection efficiency and application. For example,the DNA array technology could perform high-throughput analysis in a short period oftime but need relatively high quality and large quantity of sample. To overcome theseproblems, the pre-amplification of sample DNA is an effective way, one of which isthe whole genome amplification (WGA) technique that already been widely used inanalysis of single nucleotide polymorphisms (SNPs), genotyping and whole genomesequencing.
Among the existing WGA methods, the ligation-mediated polymerase chainreaction (LM-PCR) possessed advantages of high efficiency and less affected by thepoor conditions of template DNA, however, the self-ligation between templatefragments may happen and cause bad results. To solve this problem, a modifiedLM-PCR (mLM-PCR) was developed to carry out WGA: type IIS restriction enzymes(BbvI and Alw26I) were employed to cleave template DNA into numerous fragmentswith random sequences overhangs followed by ligated to a pool of adapters to formthe substrates for PCR, and finally all of the substrates were amplified with auniversal primer. The WGA results showed that V. parahaemolyticus genomic DNAcould be amplified with high sensitivity and uniformity. Compared to multipledisplacement amplification (MDA), the results based on specific PCR and qPCRanalysis showed that the mLM-PCR method obtained higher WGA efficiency thanMDA.
WGA based pre-amplification method require high purity template DNA thus not suitable for pre-amplify little amount of target DNA existed in massive backgroundnucleic acids. For this purpose, this study established a novel selectivepre-amplification method based on mLM-PCR that could selective pre-amplify thelittle amount of target DNA from massive background nucleic acids:
(1) Combined mLM-PCR pre-amplification with specific PCR, the detectionsensitivity of V. parahaemolyticus, S. typhimurium, V. cholera, S. putrefaciensgenomic DNA in massive background nucleic acids achieved about10copies,significantly higher (102to103times) than that with conventional PCR method.
(2) Multiple pre-amplification could be successfully performed with mLM-PCR.According to the multiple pre-amplification results of above four food bornepathogens, the sensitivity of multiple pre-amplification was as same as that of singletarget pre-amplification. The multiple pre-amplified products could be served astemplates for specific multiplex PCR detection.
(3) By using pre-amplified products as templates, loop mediated isothermalamplification (LAMP) could be successfully performed and the results confirmed thehigh sensitivity of the combination of the two methods. The results also indicated theflexibility and general applicability of mLM-PCR pre-amplification method toprovide general templates for various DNA amplification approaches.
(4) The adapter structure was crucial for mLM-PCR pre-amplification and itseffect was discussed as following: a) restriction fragments longer than200bp shouldbe chosen as pre-amplification targets; b) in a certain range, the higher specificity thatthe adapter obsessed, the higher pre-amplification efficiency could be obtained; c)when using single universal primer, the formation of stem-loop structure may affectpre-amplification, and the effect will decrease as the target fragment get longer; d) theeffect of stem-loop structure could be eliminated by using higher annealingtemperature, more PCR cycles and higher primer concentration.
(5) Artificially contaminated oyster samples for V. parahaemolyticus test resultsshowed that mLM-PCR could be successfully applied to the detection of pathogensexisted in actual seafood sample, and the sensitivity could achieve0.1to1pathogencopy per reaction tube.
In conclusion, this study established a novel pre-amplification method that basedon mLM-PCR. Through the variable designation of mLM-PCR, sample DNA underdifferent conditions could be pre-amplified with high efficiency, especially the littleamount of target DNA in massive background nucleic acids could be also effectivelypre-amplified to meet the detection requirements of conventional methods, the highefficiency of mLM-PCR pre-amplification also indicated its wide applications invarious gene detection fields such as food detection and clinical diagnosis.
引文
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