半巢式 PCR 联合 Southern 杂交检测白血病免疫球蛋白重链(IgH)基因重排

应用ＰＣＲ方法检测免疫球蛋白重链（ＩｇＨ）基因重排可进行白血病微小残留病（ＭＲＤ）研究，应用半巢式ＰＣＲ法检测白血病患者ＩｇＨＣＤＲ－Ⅲ片段，７３．５％（２５／３４）急性淋巴细胞白血病（ＡＬＬ，简称急淋），７５％（３／４）慢性淋巴细胞白血病（ＣＬＬ，简称慢淋），１５．２％（５／３３）急性非淋巴细胞白血病（ＡＮＬＬ，简称急非淋）和０％（０／１９）慢性粒细胞白血病（简称慢粒）存在ＩｇＨ基因重排，所有阳性病例都通过Ｓｏｕｔｈｅｒｎ杂交加以证实，结果显示①半巢式ＰＣＲ较一次ＰＣＲ不仅灵敏度提高，而且在一定程度上能降低假阴性率。②ＩｇＨ重排并不只局限于Ｂ淋巴细胞白血病，还可出现于部分急非淋病例，其机理有待于进一步探讨。     

半巢式PCR联合Southern杂交检测白血病免疫球蛋白重链（IgH）基…

应用ＰＣＲ方法检测免疫球蛋白重链基因重排进行白血病微小残留病研究，应用半巢式ＰＣＲ法检测白血病患者ＩｇＨＣＤＲ－Ⅲ片段，７３．５％（２５／３４）急性淋巴细胞白血产现，７５％（３／４）慢性淋巴细胞白血病，１５．２％（５／３３）急性非淋巴细胞白血病（ＡＮＬＬ，简称急非淋）和０％（０／１９）慢性粒细胞白血病（简称慢粒）丰Ｉdisplay status     

用简并性引物RT—PCR技术检测大鼠舌味蕾细胞谷氨酸受体表达

为快速、有效地用ＰＣＲ检测不同组织，根据大鼠脑中谷氨酸受体家族ＤＮＡ序列，在高同源区设计出对谷氨酸家族有专一性的简并性引物。用此引物通过ＰＣＲ扩增自来大脑的多种谷氨酸受体，而味蕾细胞ＰＣＲ产物克隆后的ＤＮＡ序列，仅有脑内代谢型谷氨酸受体四型相一致；在缺少味蕾的舌上皮组织则无此种ＰＣＲ产物。以含味蕾细胞的ＰＣＲ产物为模板，合成同位素标记的ＲＮＡ探针，用ＲＮＡ酶保护法进一步检测ｍＧｌｕＲ４在不同组织中     

应用YAC探针进行荧光原位杂交检测慢性粒细胞白血病BCR基因重排

为检测慢性粒细胞白血病（ＣＭＬ）中ＢＣＲ基因重排，采用酵母人工染色体（ＹＡＣ）ＤＮＡ的Ｉｎ－ｔｅｒ－Ａｌｕ－ＰＣＲ产物为探针进行荧光原位杂交（ＦＩＳＨ）研究了１０例ＣＭＬ，其中包括初诊患者２例，ＣＭＬ急变并接受化疗２例，α干扰素治疗２例，自体骨髓移植术（ＡＢＭＴ）后３例和Ｐｈ染色体阴性ＣＭＬ１例。同时进行细胞遗传学和ＲＴ－ＰＣＲ检测。结果：９例ＣＭＬ的４６％～１００％的可分析核型显示ｔ（９；２２）易位，其中携带ｔ（９；２２）细胞最少者为１例自体骨髓移植术后８个月的患者，其６４％的分裂相存在ｔ（９；２２），３６％为正常核型；１例Ｐｈ（－）ＣＭＬ未见ＢＣＲ基因易位，而ＲＴ－ＰＣＲ（＋），提示ＡＢＬ基因片段插入２２ｑ１１，造成隐匿性Ｐｈ染色体。结果表明：应用ＹＡＣ探针进行原位杂交的定位明确。ＦＩＳＨ检测微小残留病（ＭＲＤ）比常规细胞遗传学方法更敏感，而且可以完成ＰＣＲ方法不易进行的定量分析。     

冰冻组织特定区域的RNA快速提取及其RT-PCR分析

目的 为减少ＲＮＡ提取过程中不同细胞和组织间的交叉污染，提高逆转录ＰＣＲ（ｒｅｖｅｒｓｅｔｒａｎｓｃｒｉｐｔｉｏｎ ＰＣＲ，ＲＴ－ＰＣＲ）结果的特异性的可靠性。方法 建立冰冻组织特定区域的ＲＮＡ快速提取及ＲＴ－ＰＣＲ检测技术。首先，制备５μｍ和２０μｍ的连续冰冻切片，前者用于常规组织学染色和针对靶基因产物的免疫组织化学染色。依观察结果，在２０μｍ的切片上，分离某一特定区域做快速ＲＮＡ提取和ＲＴ－Ｐ     

应用逆转录PCR技术检测细胞因子基因表达

逆转录ＰＣＲ（ＲＴ－ＰＣＲ）可以快速、灵敏地检测表达很低或只有几个细胞表达折细胞因子ｍＲＮＡ。这项技术又叫细胞因子ｍＲＮＡ扩增测定技术（即ＭＡｐｐｉｎｇ）。可同时测定各种细胞中多种细胞因子ｍＲＮＡ。操作过程包括ＲＮＡ分离、ｍＲＮＡ逆转录为ｃＤＮＡ，ＰＣＲ扩增、产物检测等。根据定量细胞因子ｍＲＮＡ原理和方法的不同，可分为半定量ＰＣＲ法、竞争性定量ＰＣＲ法、内参标定量ＰＣＲ法、ＨＰＬＣ－ＰＣＲ法以及酶     

竞争性定量RT-PCR技术在t(8;21)AML微小残留病变检测中的应用

目的：建立针对ＡＭＬ－ＥＴＯ融合基因转录本的竞争性定量反向－聚合酶链反应（ＲＴ－ＰＣＲ）体系,并探讨其在ｔ（８;２１）急性髓细胞性白血病（ａｃｕｔｅ ｍｙｅｌｏｉｄ ｌｅｕｋｅｍｉａ,ＡＭＬ）微小残留病变跟踪中的应用价值。方法：采用重叠延伸剪接术（ｓｐｌｉｃｉｎｇ ｂｙ ｏｖｅｒｌａｐｐｉｎｇ ｅｘｔｅｎｔｉｏｎ,ＳＯＥ）获得竞争性ＤＮＡ片段,建立竞争性定量ＰＣＲ方法,并应用于ｔ（８;２１）ＡＭＬ患者的检测。结果：成功建立了竞争性定量ＰＣＲ方法〈并获得不同ｔ（８;２１）ＡＭＬ患者的ＡＭＬ１－ＥＴＯ融合基因转录本的竞争性定量ＰＣＲ结果。结论：以ＳＯＥ方法为基础的竞争性定量ＰＣＲ方法简单、适用;不同生存状态下的ｔ（８;２１）ＡＭＬ患者的ＡＭＬ１－ＥＴＯ融合基因转录本表达量具有明显差异。     

肾综合征出血热病毒76－118株的反转录及聚合酶链反应扩增、鉴定的初步研究

用反转录和聚合酶链反应（ＰＣＲ）技术扩增了肾综合征出血热病毒（ＨＦＲＳＶ）７６－１１８株ＭＲＮＡ３＇－端２３７ｂｐ基因片段。制备了长臂光敏氨基已酸生物素标记的２３７ｂｐ探针。经琼脂糖电泳和Ｓｏｕｔｈｅｒｎ印迹，生物素－亲和素系统杂交和显色，对２３７ｂｐ扩增产物进行了特异性和灵敏性鉴定。ＰＣＲ技术与反转录技术相结合可用来对一些ＲＮＡ病毒进行研究和诊断，并能快速制备出质量较好的标记探针。     

一种生物素标记探针DNA—RNA原位杂交方法

一种生物素标记探针ＤＮＡ—ＲＮＡ原位杂交方法王建明，李凌，沈贵华，崔惠云，李申德我们采用国产生物素光标记试剂盒标记探针，用于细胞涂片，冰冻切片及石蜡切片的原位杂交，检测细胞中目的基因的转录产生水平。一、材料和方法１．标本：人大肠肿瘤手术标本，冰冻切片...     

B细胞淋巴瘤肿瘤细胞克隆及微小残留病检测

采用半套式ＰＣＲ对７０例淋巴结活检组织进行了免疫球蛋白重链基因ＣＤＲⅡ片段的体外扩增。Ｂ细胞非何杰金淋巴瘤克隆检出率为８７％，其余病例均为阴性。采用自身特异性生物素探针对３例ＣＤＲⅢ克隆阳性的Ⅳ期病例于缓解期进行了微小残留病检测，２例阳性者皆于１年内复发，１例无杂交信号者仍处缓解期。     

Development of a new strategy for minimal residual disease monitoring in children with B-precursor acute lymphoblastic leukemia

Despite modern regimen of chemotherapy, one-third of children with acute lymphoblastic leukaemia relapse. Recent studies have shown that a high level of minimal residual disease (MRD) at the end of induction is associated with an increased risk of relapse. We have developed and validated a real-time PCR method (RQ-PCR) to quantify MRD. We monitored 57 patients using IgH and TCR (Vdelta2Ddelta3) genes rearrangements as PCR targets. RQ-PCR was performed with a primer and a TaqMan probe designed to consensus sequences in VH segments in combination with one allele specific oligonucleotide primer complementary to the junctionnal region. A sensitivity of 10(-4) was reached for 72% of the IgH alleles (n = 50) and for 54,5% of the Vdelta2Ddelta3 alleles (n = 22). We compared the results with those obtained by competitive PCR in 53 patients: no discordance between the two methods was observed. Seventeen patients were found positive (32%) and 27 negative (51%) with both techniques and 9 children (17%) were positive only with TaqMan technology. RQ-PCR is more sensitive and more specific than competitive PCR. We thus propose that RQ-PCR might be used in first intention for MRD analysis.     

Real-Time polymerase chain reaction of immunoglobulin rearrangements for quantitative evaluation of minimal residual disease in multiple myeloma.

The majority of patients with multiple myeloma (MM) have persistence of minimal residual disease (MRD), as determined by polymerase chain reaction (PCR) detection of clonal immunoglobulin H (IgH) gene rearrangements. As a result, PCR analysis has not provided clinically useful prognostic information in myeloma patients. Instead, quantitative PCR approaches are required to predict patient outcomes and assess response to novel treatment strategies. We adapted real-time PCR technology to quantify myeloma cells using the IgH rearrangement and then assessed the utility of this approach in 29 patients with myeloma who had undergone autologous stem cell transplantation. Because of the high cost of producing a specific reporting probe for each patient, H-chain V-region family-specific consensus probes were used in association with allele-specific oligonucleotides for PCR amplification. Because of the high frequency with which somatic hypermutation at the immunoglobulin locus occurs in MM, a number of mismatches occurred between the patient sequences and the consensus probe. However, construction of a limited number of probes allowed real-time PCR with a sensitivity of 10(-4) to 10(-5). To validate this method, we extensively evaluated assay accuracy and reproducibility. Results indicate that real-time PCR using consensus probes provides a feasible, accurate, and reproducible method for evaluating MRD in M M and possibly in other differentiated B-cell malignancies, and one that is less expensive than the use of patient-specific probes. This technique is being used to assess tumor depletion after immunologic purging and changes in tumor burden in patients undergoing stem cell transplantation and novel treatment approaches.     

以免疫球蛋白和T细胞受体基因重排为标志定量检测MLL基因重排儿童急性淋巴细胞白血病的微小残留病和预后关系

目的探讨MLL基因重排儿童急性淋巴细胞白血病（ALL）微小残留病（MRD）与临床特征的关系及其对预后的指导作用。方法以2003年4月至2009年12月首都医科大学附属北京儿童医院血液肿瘤中心收治的肘址。基因重排的ALL患儿为研究对象。以免疫球蛋白和T细胞受体基因重排、MLL融合转录本为标志,定量PCR方法监测MRD水平。以诱导治疗结束时MRD水平≥10。为MRD阳性组,〈10^-4为MRD阴性组。卡方检验和Kaplan—Meier生存分析分别比较MRD阳性和阴性组临床特征和无事件生存率（EFS）的差异。结果14例ALL患儿在诱导治疗结束时检测了MRD水平,MRD阳性组患儿初诊时外周血WBC计数显著高于MRD阴性组,对泼尼松实验治疗反应显著低于MRD阴性组。MRD阴性组5年EFS显著优于MRD阳性组,100％vs（37．5±17．1）％,P=0．022。结论诱导治疗结束时MRD水平有助于对M比基因重排儿童ALL进行预后分组,指导个体化治疗、改善预后。     

Monitoring HCV RNA viral load by locked nucleic acid molecular beacons real time PCR

Locked nucleic acids (LNA) based real time PCR was used in particular situations where there are difficulties in primer design due to sequence complexity. In this study a new real time RT-PCR assay was developed using LNA modified primers and LNA molecular beacon probes to monitor hepatitis C virus (HCV) viral load in plasma and serum samples. The technique did not suffer from an heterogeneity of the HCV genome and, in addition, an internal RNA control was amplified in the same reaction tube with different short primers and beacon probe. Due to the short consensus LNA primers length, the PCR efficiency was close to 100% with no formation of hairpin loop structures. In summary a new LNA molecular beacon based real time RT-PCR assay was used successfully to measure quantitatively the total level of HCV RNA in both experimental and clinical specimens. The high sensitivity (50 IU/ml), the wide range of genotype detection, increased specificity and robustness obtained with this test are particularly useful for screening large number of specimens and measuring viral loads to monitor the progress of the disease.     

Detection of minimal residual disease (MRD) in canine lymphoma

The objectives of this study were to detect clonal rearrangements of antigen receptor genes by polymerase chain reaction (PCR) assay and minimal residual disease (MRD) in clinical complete remission of chemotherapeutic-treated dogs. For PCR assays to determine clonality for antigen receptor rearrangement genes and MRD from cytologic and peripheral blood samples of 14 dogs with lymphoma either before chemotherapy and during remission, clonality was detected in 13 of the lymphomas before treatment. MRD was demonstrated in seven dogs with lymphoma during remission. Detection of MRD during remission in canine lymphoma using PCR technique is considered as a useful tool for prognosis and monitoring relapsing disease.     

Development of a fluorogenic RT-PCR assay (TaqMan) for the detection of Hendra virus.

A rapid and sensitive one-tube RT-PCR assay using a fluorogenic (TaqMan) probe was developed to improve the diagnosis of Hendra virus (HeV) infection. The TaqMan assay was developed to rapidly and specifically identify Hendra virus. The sensitivity of the new TaqMan-based PCR assay compared favourably with conventional RT-PCR. The major advantage of the TaqMan-based assay was the speed of diagnosis with results available within minutes of completing the PCR, and within 4 h of receiving the specimen. This test greatly reduces the chance of false positives through the elimination of second-round PCR and the requirement for agarose gel. Recombinant primer controls consisting of the Hendra virus primer sequence flanking a rodent GADPH probe sequence and recombinant probe controls consisting of the rodent GADPH primer sequence flanking the Hendra virus probe sequence were designed, cloned and transcribed in vitro to generate RNA. This has alleviated the requirement for viral RNA to be used as positive controls, thus reducing the chance of producing a false positive, at the same time eliminating the biosafety risk associated with handling live virus. This assay will provide a rapid diagnosis of future outbreaks of Hendra virus.