聚合酶链反应中抑制物的检测及去除方法研究
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摘要
目的：恶性淋巴瘤一直是病理诊断的难点。PCR以其快速、灵敏、易于操作等优点成为众多领域的首选技术。近年来，PCR检测基因重排已经成为淋巴瘤诊断不可缺少的手段。然而，假阴性一直是困扰PCR结果的问题之一。尤其是由于PCR抑制物的存在，常常导致PCR出现假阴性结果，影响淋巴瘤的正确诊断。目前，关于抑制物问题，国内尚未见系统研究。因此，如何检出DNA模板中抑制物的存在，通过必要的方法使之去除，同时探究导致PCR假阴性的其他因素，改进固定、DNA提取以及PCR反应中的不适环节，从而提高基因重排的阳性率，成为本课题研究的重点内容。
方法：(1)构建一含有β-globin基因片段的重组质粒，作为检测DNA提取物中是否含有PCR抑制物的内对照，在PCR反应中作为目的基因扩增的模板。选取以淋巴组织为主的新鲜及石蜡组织进行分组，有目的地对新切蜡片和陈旧蜡片设立对照，常规PCR筛查阴性标本，加入内对照体系检测抑制作用的有无。(2)以淋巴瘤细胞系DG75细胞DNA作为PCR反应的模板，选择固定、包埋、HE染色以及消化裂解法、酚／氯仿抽提法提取DNA过程中所用化学试剂10％福尔马林，95％乙醇，二甲苯，苏木素，伊红，Tris.HCl，EDTA，苯酚，氯仿，2％SDS，分别取1μl倍比稀释为1∶10，1∶50，1∶100，1∶1000四个浓度梯度，取原液及上述4个浓度溶液各1μl加入PCR反应体系中，观察不同试剂不同浓度对PCR反应的影响。(3)采用10％福尔马林、95％乙醇、中性缓冲福尔马林、
4%多聚甲醛一O.IM磷酸缓冲液(pH7.3)作为固定剂，0.IM PBS作为对
照组。收集新鲜扁桃体组织16例，每一标本水平剖开，面积2.5 X 1.scm，
及时冰冻切取4阳厚切片共25份，进行4h、12h、24h、72h、120h五
个不同时段的固定，消化法提取DNA，作为PCR的模板，进行看家基因
p一globin的扩增。比较OD26。二/OD:。。二比值、DNA浓度、PCR结果，进行
统计学分析，选出最适固定剂和固定时间。(4)将第一部分实验中p
一globin检测阴性的DNA提取物，分别进行如下处理:加热、稀释、BSA
孵育，Ba顽工、Ec0RI、HindIH内切酶消化，观察各种方法对阴性标本
的处理效果，找出合适的处理方法，为淋巴瘤基因重排检测提供较为可
靠的技术保证。
结果:(1)27例新鲜组织DNA提取物经PCR扩增后，p一globin
基因扩增阳性率88.9%(24/27);65例石蜡组织DNA提取物经PCR扩增
后，p一globin基因扩增阳性率78.5%(51/65)。两者比较，P>0.05，
统计无显著性差异。但新切淋巴瘤组织扩增阳性率89.3ry0(25/28);高
于陈旧淋巴瘤切片扩增阳性率40%(6/15)，尸<0.01，二者存在显著性
差异。17例扩增阴性的标本中，13例抑制了p一globin重组质粒的扩增，
占76.5%。(2)化学试剂10%福尔马林、苏木素、伊红、EDTA、苯酚、
氯仿、2%SDS对PCR存在抑制作用，而95%乙醇、Tri 5.HCI、二甲苯在
上述浓度范围内时对PCR影响不大。(3)对于新鲜淋巴组织基因片段的
扩增，以95%乙醇固定效果最佳，其余三组固定剂随固定时间的延长，
PCR阳性率逐渐降低。尤其是经10%福尔马林固定超过72h，标本中容易
产生PCR抑制成分。(4)经过各种方法的处理，14例阴性标本扩增出了
p一globin目的片段，总有效率为82.4%。对17例阴性标本处理后，PCR
阳性率分别为:加热法23.53%，稀释法64.71%，BSA法82.35%、Ba洲
I法52.94%，EcoRI法35.29%，Hindlll35.29%，各阳性率比较只0.01。
结论:(1)抑制物的存在是导致PCR假阴性的主要原因;(2)某些
试剂10%福尔马林、苏木素、伊红、EDTA、苯酚、氯仿、2%SDS的微量
存在，抑制了目的基因的扩增;(3)经10%福尔马林(A)、95ry0乙醇(B)、
中性缓冲福尔马林(C)、4%多聚甲醛一O.IM磷酸缓冲液(D)4种固定
剂及0.IMPBS液(E)固定4h、12h、24h、72h、120h不同时段，分析
发现，95%乙醇固定效果最好，100k福尔马林固定时间以不超过72h为
宜;(4)对阴性标本的处理，以BSA法效果最好。
Objectives: Malignant lymphoma has been a difficulty in pathological diagnosis. As a general technique in genie diagnosis, polymerase chain reaction (PCR) has been applied in various aspects. In recent years, PCR for rearrangentment of genes has become an essential step in diagnosis of lymphoma. However, false negetive result is a common event in PCR and has been ignored frequently. Especially the presence of inhibitors may be the reasons resulted to false negtive in PCR. At present, the issue of inhibitors has no systematic studies at home. So, how to detect the presence of inhibitors in templates and how to eliminate its function become the aims of this study. In addition, the effects of partial reagents and different fixatives on PCR are the objectives of this study meanwhile.
Methods: (1) The effective measure to demonstrate inhibitors in extractives of DNA is to build an internal control indication systeam. Construct a recombined plasmid containing β -globin gene fraction as a template in PCR (internal control ).Screen negative samples from fresh and embedded tissues and detect if it contains inhibitors. (2) With DNA of lymphoma DG75 as template in PCR.Select regents 10%formalin, 95%ethanol,xylene,Tris.HCl, EDTA,phenol, chloroform, 2%SDS and
-5-
dilute into 1:10, 1:50, 1:100, l:1000,the four different concentration. Place 1 microlitre of every the regents (including the solution not be diluted) into a 20 microlitre systeam of PCR and observe effects resulted from these substances.(3) 16 samples obtained from simple tonsillitis fresh tissue were sliced up in frozen conditions. Each, of them was divided into 25 parts with 2.5cmX 1.5cmX4u m, and fixed for 4h,12h,24h,72h,and 120h with four different fixatives and O.IM PBS as the control at random. Extracted DNA through proteinase-K digestion method, the ratio of OD260nn/OD280nm, the concentration of DNA and the results of PCR was compared.Then select the best fixative and fixation time.(4)In the first part of experiments, after all samples be amplified twice,we select negative extractives as a substrate of some pretreatments such as boiling, dilution, BSA incubation,endonuclease digestion and observe results of these pretreatments on these negative samples.
Results:(1) The positive ratio of 27 fresh tissues is 88.9%(24/27).The positive ratio of 65 paraffin-wax embedded tissues is 78.5%(51/65). However, the positive ratio of lymphoma tissues new-prepared is 89.3%(25/28) and is higher than the sections prepared two years ago 40%(6/15). Within 17 false negative samples , 13 inhibited 3 -globin amplification of a recombined plasmid, account for 76.5%; (2) 10% formalin, hematoxylin, eosin, EDTA, phenol, chloroform, 2%SDS have effects of inhibition on PCR, but 95% ethanol, Tris.HCl, xylene have no obvious effects;(3) With the increases of fix time, the ratio of positive results of samples fixed with 10%formalin has turn down to 66.7%,but the ratio of positive results of samples fixed with 95% ethanol is
-6-
100%;(4)After all means mentioned above, the results of 14 samples turned into positive in 17 negative samples and the whole efficiency is 82.4%.
Conclusions: (l)That the presence of inhibitor(s) is the primary reason for a false positive result; (2) Some reagents such as formalin, hematoxylin, eosin ,EDTA, phenol, chloroform, 2%SDS have inhibition function indeed;(3) After fixed with four different fixatives,we found 95 % ethanol was the best fixative in the amplification of an objective fragment in fresh lymphoid tissue. Especially, the extractives of DNA fixed with 10% formalin perhaps have inhibitors in different concentration and the proper fixation time is less than 72h. (4) BSA pretreatment is the best method for eliminating of inhibition in PCR.
方法：(1)构建一含有β-globin基因片段的重组质粒，作为检测DNA提取物中是否含有PCR抑制物的内对照，在PCR反应中作为目的基因扩增的模板。选取以淋巴组织为主的新鲜及石蜡组织进行分组，有目的地对新切蜡片和陈旧蜡片设立对照，常规PCR筛查阴性标本，加入内对照体系检测抑制作用的有无。(2)以淋巴瘤细胞系DG75细胞DNA作为PCR反应的模板，选择固定、包埋、HE染色以及消化裂解法、酚／氯仿抽提法提取DNA过程中所用化学试剂10％福尔马林，95％乙醇，二甲苯，苏木素，伊红，Tris.HCl，EDTA，苯酚，氯仿，2％SDS，分别取1μl倍比稀释为1∶10，1∶50，1∶100，1∶1000四个浓度梯度，取原液及上述4个浓度溶液各1μl加入PCR反应体系中，观察不同试剂不同浓度对PCR反应的影响。(3)采用10％福尔马林、95％乙醇、中性缓冲福尔马林、
4%多聚甲醛一O.IM磷酸缓冲液(pH7.3)作为固定剂，0.IM PBS作为对
照组。收集新鲜扁桃体组织16例，每一标本水平剖开，面积2.5 X 1.scm，
及时冰冻切取4阳厚切片共25份，进行4h、12h、24h、72h、120h五
个不同时段的固定，消化法提取DNA，作为PCR的模板，进行看家基因
p一globin的扩增。比较OD26。二/OD:。。二比值、DNA浓度、PCR结果，进行
统计学分析，选出最适固定剂和固定时间。(4)将第一部分实验中p
一globin检测阴性的DNA提取物，分别进行如下处理:加热、稀释、BSA
孵育，Ba顽工、Ec0RI、HindIH内切酶消化，观察各种方法对阴性标本
的处理效果，找出合适的处理方法，为淋巴瘤基因重排检测提供较为可
靠的技术保证。
结果:(1)27例新鲜组织DNA提取物经PCR扩增后，p一globin
基因扩增阳性率88.9%(24/27);65例石蜡组织DNA提取物经PCR扩增
后，p一globin基因扩增阳性率78.5%(51/65)。两者比较，P>0.05，
统计无显著性差异。但新切淋巴瘤组织扩增阳性率89.3ry0(25/28);高
于陈旧淋巴瘤切片扩增阳性率40%(6/15)，尸<0.01，二者存在显著性
差异。17例扩增阴性的标本中，13例抑制了p一globin重组质粒的扩增，
占76.5%。(2)化学试剂10%福尔马林、苏木素、伊红、EDTA、苯酚、
氯仿、2%SDS对PCR存在抑制作用，而95%乙醇、Tri 5.HCI、二甲苯在
上述浓度范围内时对PCR影响不大。(3)对于新鲜淋巴组织基因片段的
扩增，以95%乙醇固定效果最佳，其余三组固定剂随固定时间的延长，
PCR阳性率逐渐降低。尤其是经10%福尔马林固定超过72h，标本中容易
产生PCR抑制成分。(4)经过各种方法的处理，14例阴性标本扩增出了
p一globin目的片段，总有效率为82.4%。对17例阴性标本处理后，PCR
阳性率分别为:加热法23.53%，稀释法64.71%，BSA法82.35%、Ba洲
I法52.94%，EcoRI法35.29%，Hindlll35.29%，各阳性率比较只0.01。
结论:(1)抑制物的存在是导致PCR假阴性的主要原因;(2)某些
试剂10%福尔马林、苏木素、伊红、EDTA、苯酚、氯仿、2%SDS的微量
存在，抑制了目的基因的扩增;(3)经10%福尔马林(A)、95ry0乙醇(B)、
中性缓冲福尔马林(C)、4%多聚甲醛一O.IM磷酸缓冲液(D)4种固定
剂及0.IMPBS液(E)固定4h、12h、24h、72h、120h不同时段，分析
发现，95%乙醇固定效果最好，100k福尔马林固定时间以不超过72h为
宜;(4)对阴性标本的处理，以BSA法效果最好。
Objectives: Malignant lymphoma has been a difficulty in pathological diagnosis. As a general technique in genie diagnosis, polymerase chain reaction (PCR) has been applied in various aspects. In recent years, PCR for rearrangentment of genes has become an essential step in diagnosis of lymphoma. However, false negetive result is a common event in PCR and has been ignored frequently. Especially the presence of inhibitors may be the reasons resulted to false negtive in PCR. At present, the issue of inhibitors has no systematic studies at home. So, how to detect the presence of inhibitors in templates and how to eliminate its function become the aims of this study. In addition, the effects of partial reagents and different fixatives on PCR are the objectives of this study meanwhile.
Methods: (1) The effective measure to demonstrate inhibitors in extractives of DNA is to build an internal control indication systeam. Construct a recombined plasmid containing β -globin gene fraction as a template in PCR (internal control ).Screen negative samples from fresh and embedded tissues and detect if it contains inhibitors. (2) With DNA of lymphoma DG75 as template in PCR.Select regents 10%formalin, 95%ethanol,xylene,Tris.HCl, EDTA,phenol, chloroform, 2%SDS and
-5-
dilute into 1:10, 1:50, 1:100, l:1000,the four different concentration. Place 1 microlitre of every the regents (including the solution not be diluted) into a 20 microlitre systeam of PCR and observe effects resulted from these substances.(3) 16 samples obtained from simple tonsillitis fresh tissue were sliced up in frozen conditions. Each, of them was divided into 25 parts with 2.5cmX 1.5cmX4u m, and fixed for 4h,12h,24h,72h,and 120h with four different fixatives and O.IM PBS as the control at random. Extracted DNA through proteinase-K digestion method, the ratio of OD260nn/OD280nm, the concentration of DNA and the results of PCR was compared.Then select the best fixative and fixation time.(4)In the first part of experiments, after all samples be amplified twice,we select negative extractives as a substrate of some pretreatments such as boiling, dilution, BSA incubation,endonuclease digestion and observe results of these pretreatments on these negative samples.
Results:(1) The positive ratio of 27 fresh tissues is 88.9%(24/27).The positive ratio of 65 paraffin-wax embedded tissues is 78.5%(51/65). However, the positive ratio of lymphoma tissues new-prepared is 89.3%(25/28) and is higher than the sections prepared two years ago 40%(6/15). Within 17 false negative samples , 13 inhibited 3 -globin amplification of a recombined plasmid, account for 76.5%; (2) 10% formalin, hematoxylin, eosin, EDTA, phenol, chloroform, 2%SDS have effects of inhibition on PCR, but 95% ethanol, Tris.HCl, xylene have no obvious effects;(3) With the increases of fix time, the ratio of positive results of samples fixed with 10%formalin has turn down to 66.7%,but the ratio of positive results of samples fixed with 95% ethanol is
-6-
100%;(4)After all means mentioned above, the results of 14 samples turned into positive in 17 negative samples and the whole efficiency is 82.4%.
Conclusions: (l)That the presence of inhibitor(s) is the primary reason for a false positive result; (2) Some reagents such as formalin, hematoxylin, eosin ,EDTA, phenol, chloroform, 2%SDS have inhibition function indeed;(3) After fixed with four different fixatives,we found 95 % ethanol was the best fixative in the amplification of an objective fragment in fresh lymphoid tissue. Especially, the extractives of DNA fixed with 10% formalin perhaps have inhibitors in different concentration and the proper fixation time is less than 72h. (4) BSA pretreatment is the best method for eliminating of inhibition in PCR.
引文
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