多聚酶链式反应检测FMR—1基因突变

采用多聚酶链式反应结合银染或非同位素杂交的方法，对１５例脆性Ｘ染色体阳性的患者和携带者进行了检查。结果显示，该方法可以检出ＦＭＲ－１正常扩增带、前突变与完全突变，与细胞遗传学检查相比，ＰＣＲ检测更为快速与准确。     

脆性X综合征3个家系的基因诊断

采用快速毛细管ＰＣＲ非同位素检测及Ｓｏｕｔｈｅｒｎ印迹杂交技术，对３个脆性综合征家系１８名成员进行了基因型鉴定，其检出全突变患者４例，前突变女性携者４例及正常男性传递者２例，结果显示直接基因诊断方法较细胞遗传学检查更灵敏，对３个家系进行系谱分析证实其遗传特点符合Ｓｈｅｒｍａｎ推论与Ｓｍｉｔｈｓ的观察，同时提供了一种更加快速筛查脆性Ｘ综合征的非同位素ＰＣＲ诊断方法。     

用Mega-primer PCR方法获得抗HBsAg dsFv抗体重链可变区基因

目前已发展的定点突变方法主要有盒式突变、寡核苷酸引物突变及ＰＣＲ突变等[1]。常用的ＰＣＲ定点突变法,需要四种扩增引物,共进行三轮ＰＣＲ反应,操作步骤繁琐。近来出现的ｍｅｇａｐｒｉｍｅｒＰＣＲ方法[2]克服了这一缺点。我们设计了三条引物,建立了ｍｅｇａｐｒｉｖ...     

一种快速简便的缺失突变方法

为了构建能表达稳定的、不易降解的ＴＭ－ＴＮＦ突变体（ＴＭ－ＴＮＦｍ）的基因，本文用改良的Ｓ－ＰＣＲ和ＯＥ－ＰＣＲ两种定位突变技术，成功地构建了缺失３６个碱基的ｐＢＳＫ－ＴＭ－ＴＮＦ突变重组体。与ＯＥ－ＰＣＲ技术（用两对引物进行两次ＰＣＲ反应）相比较，Ｓ－ＰＣＲ技术（用一对引物做一次ＰＣＲ反应）具有快速、经济、简便等优点，但其突变率较ＯＥ－ＰＣＲ低。     

用PCR-SSCP分析检测睾丸女性化综合征患者雄激素受体基因突变

为进一步探讨睾丸女性化（Ｔｆｍ）综合征患者发病的分子机理，并为研究雄激素受体（ＡＲ）结构与功能关系提供资料，应用银染聚合酶链式反应－单链构象多态性（ＰＣＲ－ＳＳＣＰ）分析方法对４例Ｔｆｍ患者的ＡＲ基因８个外显子中的７个（Ｂ～Ｈ）分别进行了研究。结果表明，所有患者ＡＲ基因的上述外显子经ＰＣＲ扩增后均出现与外显子相应长度一致的条带，表明没有明显的缺失或插入突变。２例患者外显子Ｇ片段在行ＳＳＣＰ分析时分别有泳动变位，经ＤＮＡ双链循环测序证实两外显子各有一点突变（Ｖａｌ８６６Ｍｅｔ，Ｔ２９１９→Δ），这些突变均位于ＡＲ的雄激素结合区内。其中的缺失突变为国际上尚未报道过的新突变。研究表明，ＰＣＲ－ＳＳＣＰ分析是检测ＡＲ基因突变的快速、简便、可靠的方法。     

遗传性非息肉性大肠癌患者中hMLH1和hMSH2基因遗传性突变的分析

目的通过分析遗传性非息肉性大肠癌（ＨＮＰＣＣ）患者错配修复基因的遗传性突变，对患者的家族成员进行遗传咨询和症状前的基因诊断。方法用ＰＣＲ－异源双链体形成、ＰＣＲ－ＳＳＣＰ和ＤＮＡ序列分析技术，检测１４例ＨＮＰＣＣ、１０例有家族史大肠癌患者的外周血细胞ＤＮＡ，分析其错配修复基因ｈＭＬＨ１、ｈＭＳＨ２的所有３５个外显子。结果确认４／１４的ＨＮＰＣＣ、１／１０有家族史的大肠癌患者携带遗传性错配修复基因的突变，其中２例见于ｈＭＬＨ１基因，３例ｈＭＳＨ２基因。突变类型：３例由碱基缺失导致的移码突变，１例无义突变，１例错义突变。结论ＨＮＰＣＣ的发生与错配修复基因的突变密切相关；在大肠癌患者中检测遗传性错配修复基因的突变不宜仅限于严格符合临床诊断标准的ＨＮＰＣＣ患者。     

用非同位素PCR－SSCP法检测平滑肌肉瘤p53基因点突变

用非同位素ＰＣＲ－ＳＳＣＰ法检测平滑肌肉瘤ｐ５３基因点突变赵坡，杨光华，毛新，李甘地我们采用非同位素ＰＣＲ－ＳＳＣＰ分析方法，对５５例平滑肌肉瘤进行检测，旨在探讨ｐ５３基因点突变在其发生机理上的作用。一、材料与方法５５例平滑肌肉瘤均取自１９８７～１９...     

p^53基因与Rb基因在卵巢癌组织中突变的初步分析

为了解卵巢癌组织中抗癌基因Ｐ＾５３与Ｒｂ基因的突变情况，我们把ＰＣＲ单链构象多态（ＰＣＲ－ＳＳＣＰ）银染技术及ＰＣＲ－ＤＮＡ直接测序方法应用于５７例卵巢癌组织细胞的检测，结果发现ｐ＾５３基因可能的突变率为３２％，Ｒｂ基因可能的突变率为２１％，不同分化程度肿瘤的Ｐ５３及Ｒｂ基因突变率未见明显区别，结论：ＰＣＲ－ＳＳＣＰ角染技术是筛查基因突变简便，敏感，无核素污染的最佳方法，而ＰＣＲ－ＤＮＡ直接测序仪     

检测遗传性血色病相关HFE基因突变的复合性ARMS试验诞生

ＰＣＲ限酶消化构型等多种检测方法已证明遗传性血色病相关基因中Ｃ２８２Ｙ和Ｈ６３Ｄ突变，为找寻一种比ＰＣＲ限酶消化法处理标本少、不用放射性核素并能同时检测Ｃ２８２Ｙ及Ｈ６３Ｄ两种突变的方法，作者使用了曾成功地用于筛选ＣＦＴＲ基因突变的放大不应突变系统（...     

唾液口腔粘膜上皮细胞FMR1基因突变的PCR检测

本文采用ＰＣＲ技术对５个已确诊为脆性Ｘ综合征〗Ｆｒａ（Ｘ）【家系的１５名成员唾液中口腔粘上皮细胞ＦＭＲ１基因进行了分析，并与其外周血ＰＣＲ结果进行了比较，发现６例全突变患者有３例两种组织的结果出现了明显关 ，而５全本、４例前突变携带者及另３例全突变患者两种组织的ＰＣＲ结果完全一致，提示唾液中口腔粘膜上皮细胞同样可用于检测ＦＭＲ１基因突变，同时也表明Ｆｒａ（Ｘ）患者体内ＦＭＲ１基因突变存在着组织间的     

微量酶联杂交法定量检测HBV基因 竞争PCR扩增产物

目的 建立一种简便、敏感、精确的微反应板酶联杂交技术,以鉴定HBV基因的竞争PCR扩增产物。方法 设计了两种捕获探针,能分别与竞争PCR扩增产物中的野生片段和突变片段杂交。捕获探针通过3′－端修饰的氨基与微量DNA结合板孔表面的NOS基团化学结合而被“竖直”地包被在反应板上;将热变性后的产物加入两种捕获探针反应孔内,产物中带有生物素的野生或突变片段的一条单链与相应的捕获探针杂交;最后用链亲和素－碱性磷酸酶及底物检测杂交信号。结果 该方法检测PCR产物DNA的灵敏度为80ng/ml,大于琼脂糖凝胶电泳染色鉴定法。获得野生片段和突变片段杂交信号值后,可根据公式计算扩增前野生模板的初始量。结论 本方法操作简单、灵敏度高、结果数据化、特异性强,适用于竞争PCR产物分析。     

Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples.

Two nested PCR assays were developed for the detection of bovine respiratory syncytial virus (BRSV). Primers were selected from the gene encoding the F fusion protein (PCR-F) and the gene encoding the G attachment protein (PCR-G). Biotinylated oligonucleotide probes, termed F and G, were selected for the hybridization of the respective PCR products. The sensitivities of the PCR-F and PCR-G assays were similar, both detecting 0.1 tissue culture infective dose of the virus. The PCR-F assay amplified all bovine strains and one human strain (RS32) tested. No cross-reactions were observed with nine heterologous respiratory viruses. PCR-F products of bovine and human RSV strains were discriminated by using endonuclease restriction enzyme ScaI, which specifically cleaved, products of BRSV. Oligonucleotide probe F was also specific for products of BRSV. The PCR-G assay detected all bovine strains and none of the human strains tested. A faint electrophoretic band was also observed with products of Sendai virus. However, probe G did not hybridize with this product, only with products of BRSV. Nasal swabs collected from cattle with no symptoms and cattle in the acute stage of respiratory disease were analyzed for BRSV by the immunofluorescence (IF) method and by the PCR-F and PCR-G assays. The virus was detected by the PCR assays in 31 of 35 (89%) samples tested. Only 23 samples (66%) were positive by the IF method, and these samples were also positive by both the PCR-F and PCR-G assays. The 31 samples detected as positive by PCR originated from cattle presenting clinical signs of acute respiratory disease; the four PCR-negative samples originated from clinically asymptomatic neighboring cattle. All sampled animals subsequently seroconverted and became reactive to BRSV. Thus, the detection of BRSV by PCR correlated with clinical observations and was considerably more sensitive (66 versus 89%) than IF. These results indicate that both nested PCR assays provide rapid and sensitive means for the detection of BRSV infection in cattle. Considering its higher specificity, the PCR-F assay can be recommended as the method of choice in the analysis of clinical specimens of BRSV.     

Duchenne/becker muscular dystrophy carrier detection using quantitative PCR and fluorescence-based strategies

Dystrophin gene deletions account for up to 68% of all Duchenne (DMD) and Becker (BMD) muscular dystrophy mutations. In affected males, these deletions can be detected easily using multiplex PCR tests which monitor for exon presence. In addition, quantitative dosage screening can discriminate female carriers. We previously analyzed multiplex PCR products by gel electrophoresis and quantitation of fluorescently labeled primers with the Gene Scanner? in order to test carrier status. These multiplex PCR protocols detect DMD gene deletions adequately, but require up to 18 pairs of fluorochrome-labeled primers. We previously described two alternative fluorescent labeling strategies, each with approximately 1,000-fold greater sensitivity than ethidium bromide staining, which can be used to quantify the products of multiplex PCR. The first method uses the DNA intercalating thiazole orange dye TOTO-1 to stain PCR products after 20 cycles. In the second method, fluorescein-12,2′-dUTP is incorporated into products during PCR as a fluorescent tag for subsequent quantitative dosage studies. Both methods label all multiplexed exons including the 506 bp exon 48 fragment that is difficult to detect and quantify by standard ethidium bromide staining. Using this approach, we determined DMD/BMD carrier status in 24 unrelated families using a fluorescent fragment analyzer. Analysis of fluorochrome-labeled PCR products facilitates quantitative multiplex PCR for gene-dosage analysis. © 1993 Wiley-Liss, Inc.     

Detection and diagnosis of parapoxvirus by the polymerase chain reaction

The genus Parapoxvirus includes four members, bovine papular stomatitis virus (BPSV), pseudocowpox virus (PCPV), orf virus (ORFV) and parapoxvirus of red deer in New Zealand (PVNZ). A set of primers for polymerase chain reaction (PCR) was designed to detect viral DNA from cells infected with each of the four parapoxviruses. The set of primers resulted in the amplification of appropriately sized products from cells infected with BPSV, PCPV, ORFV and PVNZ, respectively. The PCR method was applied for the detection of seven field isolates of parapoxvirus from cattle, sheep and free-ranging wild Japanese serows. The expected size of DNA was amplified from cells infected with each of the seven isolates. No specific PCR products were detected from vaccinia virus-, fowlpox virus- and mock-infected cells. Moreover, by a semi-nested PCR with an inner primer and Southern blot analysis, viral DNA was detected from lesions of clinically affected cattle, sheep and Japanese serows. These results suggested that the PCR method used in this study was specific for the detection of parapoxviruses and thus useful for diagnosis of parapoxvirus infections, especially in discrimination from diseases with similar clinical symptoms.     

丁型肝炎病毒在乙肝病毒感染者中分布状况及其分子生物学研究

通过对丁型肝炎病毒（ＨＤＶ）的放免法检测和巢式ＰＣＲ技术检测，在１２０例乙肝病毒感染者中，检测到１３例ＨＤＶ标记阳性（阳性率为１０．８％），５０例乙肝病毒携带者，３例抗ＨＤＶ－ＩｇＭ阳性。经对照所得乙肝患者同乙肝病毒携带者的ＨＤＶ阳性差异结果支持ＨＤＶ的存在常伴随着谷丙转氨酶（ＡＬＴ）活性上升的观点。实验结果还提示了ＨＤＶ和乙型肝炎病毒（ＨＢＶ）重叠感染易发生重症或慢性肝炎。地ＨＤＶ的检测方法进行     

Rapid Detection of Chlamydia pneumoniae by PCR-Enzyme Immunoassay

Chlamydia pneumoniae is an important human respiratory pathogen. Laboratory diagnosis of infection with this organism is difficult. To facilitate the detection of C. pneumoniae by PCR, an enzyme immunoassay (EIA) for analysis of PCR products was developed. Biotin-labeled PCR products generated from the 16S rRNA gene of C. pneumoniae were hybridized to a digoxigenin-labeled probe and then immobilized to streptavidin-coated microtiter plates. Bound PCR product-probe hybrids were detected with antidigoxigenin peroxidase conjugate and a colorimetric substrate. This EIA was as sensitive as Southern blot hybridization for the detection of PCR products and 100 times more sensitive than visualization of PCR products on agarose gels. The diagnostic value of the PCR-EIA in comparison to cell culture was assessed in throat swab specimens from children with respiratory tract infections. C. pneumoniae was isolated from only 1 of 368 specimens tested. In contrast, 15 patient specimens were repeatedly positive for C. pneumoniae by PCR and Southern analysis. All of these 15 specimens were also identified by PCR-EIA. Of the 15 specimens positive by 16S rRNA-based PCR, 13 specimens could be confirmed by omp1-based PCR or direct fluorescent-antibody assay. Results of this study demonstrate that PCR is more sensitive than cell culture for the detection of C. pneumoniae. The EIA described here is a rapid, sensitive, and simple method for detection of amplified C. pneumoniae DNA.     

Detection of Rickettsia tsutsugamushi in Experimentally infected mice by PCR.

We developed a rapid procedure for the detection of Rickettsia tsutsugamushi DNA by the PCR technique. The primer pair used for the PCR was designed from the DNA sequence of the gene encoding a 120-kDa antigen, which was proven to be group specific by immunoblot analysis with mouse hyperimmune sera against various rickettsial strains. This PCR method was able to detect up to 10 ag of plasmid DNA (pKT12). Specific PCR products were obtained with DNAs from R. tsutsugamushi Kato, Karp, Gilliam, TA716, TA1817, and Boryong, but not with DNAs from other rickettsiae, such as R. prowazekii, R. typhi, R. akari, and strain TT118. In a study with experimentally infected mice, the PCR method could detect rickettsial DNA from 2 days after inoculation (DAI), whereas serum antibody against R. tsutsugamushi could be detected from 6 to 8 DAI by an immunofluorescence test. Although clinical manifestations subsided after 14 DAI, rickettsial DNA in blood samples could be detected by PCR for up to 64 DAI. These results suggest that this PCR method can be applied to the early diagnosis of scrub typhus and can also be used to detect the residual rickettsiae after clinical symptoms subside.     

A novel fluorescence polarization based assay for 14 human papillomavirus genotypes in clinical samples

A specific and practical method was developed for high throughput 14 human papillomavirus (HPV) genotypes assay in clinical samples by a single PCR. GP5+/6+ polymerase chain reaction (PCR) system was used to amplify HPV DNA in 1127 samples. The PCR product was assayed by AcycloPrime reaction with fluorescence polarization (FP). Fourteen HPV genotypes specific sequence primers designed within GP5+/GP6+ amplification polymorphism regions of L1 genes for corresponding HPV genotypes were annealed with the type specific PCR products and special fluorescent terminator was added to the end of the primer under direction of the PCR products. AcycloPrime-FP analysis showed specific anneal and incorporation without any cross-reaction. The types detected with FP showed an excellent overall agreement with sequence when the individual monotype results were taken into account. The proposed method could detect more than one type of HPV infection, but the sequence method was limited. AcycloPrime-FP could reach the detection level: 100 ag for representative phylogenetically distant HPV genotypes: HPV6, 18, 31, 39, 42, 51 and 58. The results of AcycloPrime-FP showed excellent reproducibility. The proposed method allowed an economical detection of HPV genotypes without any use of labeled probe. It is expected to be an extremely useful tool for HPV genotypes screening.     

Detection of antigen receptor gene rearrangements in lymphoproliferative malignancies by fluorescent polymerase chain reaction

Abstract: Monoclonal rearrangements of antigen receptor genes in lymphoproliferative diseases are characterized by the specific sequence and the length of their junctional region, which can be used as markers of the proliferating clone. PCR techniques have greatly simplified routine detection of monoclonal rearrangements. But on the one hand, identification of the sequences requires sequencing methods and on the other hand, sizing of rearrangements by conventional analysis of PCR products on agarose or nondenaturing polyacrylamide gels may be uncertain. We have developed an approach based on amplification of rearranged IGH, TCRG and TCRD locus by fluorescent PCR associated to a computerized analysis of generated PCR products allowing their objective sizing. We tested this method on DNA samples from patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia, whose pattern of IGH and TCRG rearrangements had been previously identified by Southern blot techniques. TCRG-PCR assay allowed detection of 100% of rearranged samples. No false-negative results were found but a high rate (60%) of Southern-negative and PCR-positive samples were identified. TCRD PCR-assay detected VD1JD1 or VD2-D2/3 rearrangements in both acute lymphoblastic leukemia and chronic lymphocytic leukemia samples. IGH PCR assay permitted detection of all known rearranged samples. The sensitivity of these three different PCR assays (1% leukemic cells) was equivalent to that of other published PCR protocols. These results show the validity and reliability of the fluorescent PCR method for routine detection of IGH, TCRG and TCRD rearrangements. Sizing of PCR products by computerized analysis was also validated. It provides additional information on rearrangement patterns in lymphoproliferative diseases, as clonal rearrangements can be recognized by their size. This can be of great interest in various circumstances, particularly for detection and follow-up of oligoclonality.     

Rapid epidemiologic analysis of cytomegalovirus by using polymerase chain reaction amplification of the L-S junction region.

A technique based on polymerase chain reaction (PCR) amplification was developed to facilitate the study of the epidemiology of cytomegalovirus (CMV). Consensus oligonucleotide primers from repetitive DNA sequences were designed to amplify interspersed repetitive sequences in an area of heterogeneity within the L-S junction region of the CMV genome, and PCR products were detected by gel electrophoresis. Purified CMV DNAs from 25 CMV isolates, 13 from members of five families in which person-to-person transmission was documented, 9 random clinical isolates of CMV, and 3 laboratory reference strains of CMV (Towne, Davis, and AD169), were analyzed. The gel electrophoretic patterns of DNA bands, or PCR profiles, produced by amplification with the L-S primers were unique for epidemiologically unrelated strains and laboratory reference strains, yet similar patterns were observed for epidemiologically related strains isolated from members of the same family. This method of rapid fingerprinting of CMV DNA within the hypervariable L-S junction region by PCR to produce strain-specific, variably sized PCR products should simplify the molecular epidemiologic analysis of CMV.