A novel buffer system, AnyDirect, can improve polymerase chain reaction from whole blood without DNA isolation

BACKGROUND: Polymerase chain reaction (PCR) using DNA from blood samples is a valuable tool in the field of medical diagnostics. However, DNA isolation from blood is a laborious and sample-consuming step, and hampers the automation of PCR for large-scale studies. Attempts to perform PCR from blood without DNA isolation have been difficult to achieve, since numerous endogenous and exogenous blood constituents may inhibit PCR. METHODS: We used a novel buffer system, 'AnyDirect', that conserves the enzymatic activity of DNA polymerases for effective use in direct PCR from whole blood under various conditions. RESULTS: Using AnyDirect, DNA amplification was achieved from whole blood with a variety of thermostable DNA polymerases. Amplification occurred regardless of target size (up to 1.7 kb), presence of various known PCR inhibitors, and high target GC content. Importantly, low copy number DNA targets were effectively amplified from whole blood. CONCLUSIONS: AnyDirect buffer allows direct PCR from whole blood and may facilitate detection of genetic diseases or infections by eliminating the time and effort for DNA extraction. The use of AnyDirect could facilitate the development of high-throughput PCR for large-scale diagnostic screening or investigation of various medical conditions.     

孕妇外周血胎儿ABO血型基因分型技术的建立

本研究目的是建立孕妇外周血胎儿ABO血型基因分型技术,用于ABO血型不合引起的新生儿溶血病的产前诊断。根据ABO血型基因DNA全序列和mRNA序列设计4对引物,选择20例健康供者血浆,提取血浆中DNA进行扩增,探索最佳的血浆DNA提取及PCR扩增条件,初步建立单人ABO血型基因分型技术。将O型血浆DNA与A型或B型血浆DNA按1：1、2：1、4：1、8：1、10：1、20：1、40：1、100：1进行混合,模拟孕妇外周血胎儿与孕妇自身ABO基因混合状态,建立混合ABO血型基因分型技术。选取14例孕30周以上的孕妇外周血标本,进行胎儿ABO血型基因型鉴定,并对孕妇进行追踪,尽量获取胎儿出生以后的外周血标本进行ABO血型鉴定,以评价孕妇外周血胎儿ABO血型基因分型技术的灵敏度与准确性。结果表明：单人血浆进行准确血型鉴定的最少模板DNA量约为0．625ng,500μl血浆提取的DNA量即可达到PCR扩增要求;当混合血浆中O型DNA所占比例≤10时,可以准确检测出非0基因的存在;14名O型孕妇外周血标本中9例标本扩增出非O型基因,5例未扩增出非0基因;通过血清学方法对5例胎儿出生后外周血进行ABO血型鉴定,其中A型3例,B型1例,O型1例,与其基因分型结果一致,符合率100％。结论：本研究建立的孕妇外周血胎儿ABO血型基因提取、分型技术,可以对妊娠中晚期胎儿ABO血型基因型进行准确鉴定,从而为新生儿溶血病的产前诊断与预防提供指导意见。     

中国汉族人群MN血型相关基因gypa分子多态性的研究

本研究探讨中国汉族人群MN血型相关基因gypa的多态性。随机选取中国汉族人群中无血缘关系的无偿献血者202人份,以血清学方法鉴定样本的MN血型表现型。根据GenBank的NG-007470基因参照序列设计gypa第2外显子引物,对202人份的DNA进行PCR扩增,同时对扩增产物进行直接测序。结果表明:所有样本的gypa第2外显子第1、56位碱基均发生了变异,其中MN表现型杂合子主要以1A>C、22T/C、34A/G、35T/G、56T>C的形式存在;MM表现型纯合子主要以1A>C、22C、34G、35T、56T>C的形式存在;NN表现型纯合子主要以1A>C、22T、34A、35G、56T>C的形式存在。结论:中国汉族人群MN血型相关基因的多态性主要由gypa第2外显子的第1、22、34、35、56共5个位点的多态性所决定,其中第1、56位为非功能性SNP位点。     

T细胞中TCR Vα40与TCR Dδ3,Jδ1和ψJα重排的特点

利用巢式PCR扩增10例正常人外周血单个核细胞、4例分选的外周血CD3^ 细胞和7例心胸外科手术的非免疫性疾病和非肿瘤病人的正常胸腺细胞DNA的TCR Vα40基因与Jδ1，Dδ3和ψJα重排的情况，从对不同含量DNA的PCR分析，了解其重排分布频率。结果显示，TCR Vα40分别与Jδ1，Dδ3和ψJα重排均可见于多6数外周血T细胞和胸腺细胞中，对不同含量DNA的PCR分析显示TCR Vα40重排的分布频率在外周血和胸腺细胞有所不同。研究结果提示，TCR Vα40-ψJα的重排最常见于成熟和未成熟T细胞中，而TCR Vα40-Dδ3则在未成熟T细胞中发生频率更高。     

Detection of human cytomegalovirus in peripheral mononuclear cells and urine samples using PCR.

Samples of peripheral blood lymphocytes from 105 different blood donors were investigated for the presence of human cytomegalovirus (HCMV) DNA using the polymerase chain reaction (PCR) with primers specific for the Pst I w fragment (IE region). Viral DNA sequences were detected in 53 samples, a fifth of which had been previously serotyped as HCMV negative. In the latter cases, Western blot analysis re-determined two out of three individuals that were resampled as seropositive. PCR could therefore be used to extend existing methods employed for the identification of HCMV infected blood samples prior to transfusion to individuals in high risk groups. In addition, the value of PCR as a diagnostic test was evaluated in a small pilot study by comparing the results obtained with urine samples from babies suffering congenital infection and from other high risk patients, with data obtained by isolation of infectious virus or through the detection of immediate early antigens in infected cultures. Data from this study indicated that PCR is at least as sensitive as the other methods used in HCMV diagnosis.     

HBsAg ELISA-/NAT+的HBV血清学模式和病毒变异分析

目的结合乙型肝炎病毒核酸(HBV DNA)的检测结果,分析血清标本血清学模拟及DNA序列,了解HBV DNA以及HBsAg血型模式之间的关系。方法选自该院54例血液标本作为本研究对象;对标本行HBV DNA提取、PCR扩增处理、PCR产物纯化、DNA测序、HBV基因分型和序列分析,并运用生物信息学软件对测序结果进行分析处理,对比分析基因突变情况。结果 PCR扩增结果,54例标本中40例表现为非常显著的PCR扩增不佳,14例标本PCR产物电泳均能够观察到1 400bp特异性条带;22例为HBsAg ELISA阳性,14例为隐匿性HBV阳性,PCR扩增阳性行基因型检测,B型基因在HBsAg ELISA中占81.82%,C型基因在隐匿性HBV阳性中比例为78.57%,差异具有统计学意义(P0.05);14株隐匿性HBV基因中,6例在S区区域内发生基因序列点突变,其中2例在1个碱基点出现突变,另3例在2个位点的碱基点出现突变;3例HBV基因C型感染者出现基因点突变,2例B基因型感染者出现基因点突变;5例标本在9个位点部位的"a"决定族内碱基点出现突变,A-C的突变较多。结论血液筛查中,检测显示为HBsAg的阴性合格血液,仍可能有隐匿性乙肝感染和窗口期漏检,其病毒株主要为C型,且有变异株,B型病毒株相对较少,但突变株相对较高,可能逃避现有筛查试剂。     

Direct method for detecting small quantities of hepatitis B virus DNA in serum and plasma using the polymerase chain reaction.

Serum components inhibit DNA polymerase, thereby obviating direct detection of serum viral DNA sequences by the polymerase chain reaction (PCR). This has necessitated extraction of nucleic acid from sera before performing PCR and has resulted in loss of sensitivity. By adsorbing virus to a solid surface (microcentrifuge tubes or antibody coated microparticles) followed by proteinase K digestion, as little as three viruses per 200 microliters serum may be directly detected by PCR without nucleic acid extraction. The sensitivity is dependent on the surface area of the adsorptive surface and is increased by having antibodies on the adsorptive surface. The nucleic acid sequence of the amplified DNA fragments may be directly determined by the dideoxy method. Of 24 plasma samples from HBsAg+ volunteer blood donors, HBV DNA was detected in 7 by dot blot assay, 7 by liquid hybridization, and 9 by PCR. PCR detected DNA in every sample that was positive by another assay. Analysis of serial samples of two patients with acute self-limited hepatitis B found detectable HBsAg and pre-S2 antigenemia before HBV DNA by the PCR method. These results suggest that surface antigenemia may precede viremia during acute hepatitis.     

Microarray-based method to analyze methylation status of E-cadherin gene in leukemia

BACKGROUND: Aberrant DNA methylation of the CpG sites of the tumor suppressor gene is closely associated with carcinogenesis. Recently, several studies have indicated the aberrant methylation of E-cadherin gene could be a potential marker for leukemic patients. METHOD: We used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated, but not methylated, cytosine into thymine within CpG islands of interest. The amplified product containing a pool of DNA fragments with altered nucleotide sequences was then hybridized with an oligonucleotide-based microarray. Five sets of oligonucleotide probes were designed to detect the methylation patterns of E-cadherin gene CpG islands in leukemia samples. The results were further validated by methylation-specific PCR (MSP). RESULTS: We found that all leukemia samples were methylated at different levels within the target sequences. The specific regions (the CpG sites #16-19 and #20-22) were revealed as hotspots for methylation in leukemic patients. These results showed that the microarray assay could successfully detect methylation changes of E-cadherin gene in leukemia quantitatively. CONCLUSION: The oligonucleotide-based microarray can be a quick and reliable tool to map methylation status in CpG islands. This established microarray could be potentially useful for clinical researches and diagnosis.     

Comparative evaluation of two DNA isolation techniques for PCR-based diagnosis of gastrointestinal nematode infections in sheep

The specific diagnosis of gastrointestinal parasite infections in livestock is central to their control. PCR assays have been developed for routine diagnosis and to overcome limitations of classical methods. Central to the performance of such assays is the effective isolation of the nucleic acids from samples and the elimination of components that are inhibitory to PCR. Here, we directly compared two techniques for the isolation of DNA from strongylid nematode eggs from faecal samples from sheep, and assessed their performance in relation to the sensitivity and specificity of PCR, time required for DNA isolation and ease of use. The results showed differences in the performance of the two isolation techniques, subsequently effecting the PCR results. The main differences related to the time required for DNA isolation, and the elimination of inhibitory substances from the DNA isolated by one technique but not the other.     

Amplification of human genomic DNA sequences with polymerase chain reaction using a single oligonucleotide primer

We present two examples of exponential nucleic acid amplification with the polymerase chain reaction (PCR) in the presence of only one amplification primer. Cloning and sequencing of the PCR products generated by amplification of human genomic DNA revealed that the amplified sequence contained only one primer and its complement, at the two ends of the PCR product. Although these experiments were performed with primers derived from the sequence of the prostate specific antigen (PSA) gene and the normal epithelial cell-specific 1 gene (NES1), the amplified sequences were novel and had no homology with either PSA or NES1 DNA. While both PSA and NES1 genes reside on chromosome 19q13.3-q13.4, the amplified sequences were found by mapping to reside on chromosome 5q12 and 5p15.1-p15.3, respectively. When we examined the mechanism of amplification by PCR using one primer in these two cases, we found that there was a high homology between the PSA primer or the NES1 primer and the two regions flanking the amplified sequence of chromosome 5q12 or 5p15. This indicated that the single PSA or NES1 primer could anneal on both strands of the DNA of that region, and mediate the exponential amplification. Since this phenomenon occurred to us twice with a limited number of different PCR reactions performed in our laboratory (< 20), we believe that it may represent a common artifact of PCR. Moreover, it appears that the palindromic primer binding sites can anneal to each other forming DNA cruciforms.     

多重PCR方法鉴定人巨细胞病毒临床病毒分离株

目的:应用多重PCR技术鉴定体外培养、分离获得的人巨细胞病毒(HCMV)临床病毒株.方法:以先天性感染HCMV的新生儿为研究对象,采集尿液标本进行病毒分离,将分离获得的临床低传代病毒感染HELF细胞,提取病毒DNA,针对较保守基因IE和LA,设计两对引物,同时扩增IE和LA基因,扩增产物经琼脂糖凝胶电泳分析.结果:成功分离获得2株HCMV临床低传代病毒株(D2、D3),经多重PCR同时扩增出209 bp(IE)和401 bp(LA)目的片段.结论:多重PCR可以作为病毒分离后,基础研究中鉴定HCMV临床病毒株的一种方法.     

Analysis of clinically relevant single-nucleotide polymorphisms by use of microelectronic array technology

BACKGROUND: Microelectronic DNA chip devices represent an emerging technology for genotyping. We developed methods for detection of single-nucleotide polymorphisms (SNPs) in clinically relevant genes. METHODS: Primer pairs, with one containing a 5'-biotin group, were used to PCR-amplify the region encompassing the SNP to be interrogated. After denaturation, the biotinylated strand was electronically targeted to discrete sites on streptavidin-coated gel pads surfaces by use of a Nanogen Molecular Workstation. Allele-specific dye-labeled oligonucleotide reporters were used for detection of wild-type and variant sequences. Methods were developed for SNPs in genes, including factor VII, beta-globin, and the RET protooncogene. We genotyped 331 samples for five DNA variations in the factor VII gene, >600 samples from patients with beta-thalassemia, and 15 samples for mutations within the RET protooncogene. All samples were previously typed by various methods, including DNA sequence analysis, allele-specific PCR, and/or restriction enzyme digestion of PCR products. RESULTS: Analysis of amplified DNA required 4-6 h. After mismatched DNA was removed, signal-to-noise ratios were >5. More than 940 samples were typed with the microelectronic array platform, and results were totally concordant with results obtained previously by other genotyping methods. CONCLUSIONS: The described protocols detect SNPs of clinical interest with results comparable to those of other genotyping methods.     

Molecular assessment of post-BMT chimerism using various biologic specimens and automated DNA sizing technology

Highly polymorphic microsatellite markers provide useful genetic markers for detection of complete or mixed chimerism in patients after allogeneic BMT (allo-BMT). We report application of automated DNA sizing technology for detection of post-BMT chimerism using fresh peripheral blood, BM, or archival blood smears and various DNA isolation techniques. Donors' and recipients' DNA was amplified with fluorescent PCR primers specific for short tandem repeat (STR) marker loci: FGA, VWA, TH01, F13A1, D21S11. Chimerism was assessed in 14 recipients after allo-BMT. A complete chimerism was detected in 10 patients, in 3 patients we observed fluctuations of chimerism status, and mixed chimerism was assessed in 1 patient. We show that DNA from different types of biologic specimens (whole peripheral blood, BM suspension, archival blood smears), prepared according to the various isolation techniques (salting-out method, phenol chloroform extraction, Chelex procedure) and amplified with fluorescent PCR primers for microsatellite markers, enable identification of chimerism status following allo-BMT in children.     

Direct genotyping of Cytochrome P450 2A6 whole gene deletion from human blood samples by the SmartAmp method

BackgroundGenetic polymorphisms of the human CYP2A6 gene are considered to be a determinant of smoking behavior and tobacco-related lung cancer risk in male Japanese smokers. We developed a SmartAmp-based genotyping method to detect whole deletion of the CYP2A6 gene directly from blood samples without DNA isolation.MethodsWe validated the new method using CYP2A plasmids, 48 genomic DNA samples and 25 blood samples by utilizing the SmartAmp method, a unique isothermal DNA amplification process.ResultsThis method could discriminate the CYP2A6 gene from highly homologous CYP2A7 and CYP2A13 genes. CYP2A6*1 (wild-type) and CYP2A6*4 (whole gene deletion) were determined by the new method in perfect accordance with sequence analysis data.ConclusionsA SmartAmp assay for genotyping the CYP2A6 gene was developed, and the reliability of the method was validated using the conventional PCR method.     

Absence of human T-lymphotropic virus types I and II infection in an Ontario hemophilia population

Two hundred ninety-three serum samples from Ontario hemophiliacs and 200 samples from human immunodeficiency virus-positive blood donors were screened for the presence of antibodies to human T-lymphotropic virus type I (HTLV-I) by enzyme-linked immunosorbent assay, radioimmunoassay, and Western blot techniques. None of the serum samples provided unequivocal positive results, but several samples gave inconclusive results. Of the hemophiliacs with inconclusive serologic results from whom peripheral blood lymphocyte DNA could be obtained, all were negative for HTLV-I and HTLV type II (HTLV-II) sequences as determined by polymerase chain reaction (PCR). PCR was also performed on a lymph node biopsy sample taken from a hemophiliac who developed a rare T-cell lymphoma; the sample was negative for HTLV-I and -II sequences. These results indicate that Ontario hemophiliacs have not been exposed to HTLV-I or HTLV-II.     

Development of novel PCR assays to detect azole resistance-mediating mutations of the Aspergillus fumigatus cyp51A gene in primary clinical samples from neutropenic patients

The increasing incidence of azole resistance in Aspergillus fumigatus causing invasive aspergillosis (IA) in immunocompromised/hematological patients emphasizes the need to improve the detection of resistance-mediating cyp51A gene mutations from primary clinical samples, particularly as the diagnosis of invasive aspergillosis is rarely based on a positive culture yield in this group of patients. We generated primers from the unique sequence of the Aspergillus fumigatus cyp51A gene to establish PCR assays with consecutive DNA sequence analysis to detect and identify the A. fumigatus cyp51A tandem repeat (TR) mutation in the promoter region and the L98H and M220 alterations directly in clinical samples. After testing of the sensitivity and specificity of the assays using serially diluted A. fumigatus and human DNA, A. fumigatus cyp51A gene fragments of about 150 bp potentially carrying the mutations were amplified directly from primary clinical samples and subsequently DNA sequenced. The determined sensitivities of the PCR assays were 600 fg, 6 pg, and 4 pg of A. fumigatus DNA for the TR, L98H, and M220 mutations, respectively. There was no cross-reactivity with human genomic DNA detectable. Sequencing of the PCR amplicons for A. fumigatus wild-type DNA confirmed the cyp51A wild-type sequence, and PCR products from one azole-resistant A. fumigatus isolate showed the L98H and TR mutations. The second azole-resistant isolate revealed an M220T alteration. We consider our assay to be of high epidemiological and clinical relevance to detect azole resistance and to optimize antifungal therapy in patients with IA.     

Polymerase chain reaction: amplification of DNA from fixed tissue

The polymerase chain reaction (PCR) allows the analysis of DNA from biologic samples containing only nanogram quantities of DNA. We used DNA purified from fresh or frozen peripheral blood (PB) leukocytes and formalin, or B-5 fixed bone marrow aspirate clots (BM). A sequence of the beta-globin gene was amplified via the PCR then hybridized with allele specific oligonucleotide probes for hemoglobin A, S, and C. All DNA preparations, including formalin and B-5 fixed BMs, were successfully amplified; the hybridization of the amplified products resulted in patterns consistent with the hemoglobin phenotype for all patients. PCR can be used on DNA from many sources; retrospective studies using paraffin embedded fixed tissue are possible because extremely small amounts of DNA present in fixed tissue can be successfully amplified.     

套式聚合酶链反应在母婴传播巨细胞病毒研究中的应用

本文建立了套式ＰＣＲ技术加限制酶分析方法，对１０５份母脐血配对进行套式ＰＣＲ，病毒分离，特异性ＩｇＭ及ＩｇＡ测定。结果母血ＨＣＭＶ ＤＮＡ阳性６份，脐血３份，母－脐传播率为３／６。三对被证实为母婴传播ＨＣＭＶ套式的标本中，二对套式ＰＣＲ、病毒分离、特异性ＩｇＭ及ＩｇＡ均阳性。一对套式ＰＣＲ病毒分离、特异性ＩｇＡ阳性，但特异性ＩｇＭ阴性，提示：套式ＰＣＲ是一种敏感、特异、简便快速，能早期诊断ＨＣＭＶ     

多重PCR在瘢痕疙瘩Fas基因突变分析中的应用

目的:建立瘢痕疙瘩Fas基因常见突变外显子的多重PCR扩增反应体系,以利进行批量瘢痕疙瘩基因突变的筛选和检测。方法:取正常皮肤、增生瘢痕和瘢痕疙瘩组织DNA样本,根据瘢痕疙瘩Fas基因易发突变的外显子6,8,9之序列,按照多重PCR引物设计原则,建立3对相应引物,先分别找出各外显子的最佳扩增条件,再进行综合分析,最终找到一个理想的多重PCR扩增条件。结果:各外显子单一扩增片段和多重PCR扩增条带均清晰,产量较高,无非特异性扩增,产物长度与理论值一致;DNA测序证实,各扩增产物即各外显子基因片段。结论:成功建立了瘢痕疙瘩Fas基因常发突变外显子的多重PCR扩增体系,与以往单基因分次扩增相比,实现了一次同时扩增3个基因片段,为瘢痕疙瘩基因诊断和进一步相关的分子生物学研究提供了一个经济、快捷的方法,对于瘢痕疙瘩Fas基因的突变研究具有较高的应用价值。     

杀菌蛋白rBPI23与haFGF融合基因及真核表达质粒的构建

目的利用克隆人杀菌／通透性增加蛋白（rBPI23）和人酸性成纤维细胞生长因子（haFGF）cDNA,构建BF融合基因和真核表达载体,为能制备既能杀菌又能促进创伤愈合的双功能多肽创造条件。方法从人中性粒细胞和人胎脑组织中提取mRNA,经逆转录聚合酶链式反应（RT—PCR）分别获得rBPI23和haFGF编码序列的eDNA,运用重组PCR技术将两段基因通过一疏水性多肽接头（Gly4Ser）3DNA序列进行体外基因重组,合成融合基因,并将其梅建于真核表达载体peDNA3．1（＋）中。结果所扩增的rBPI23和haFGFCDNA序列与Genebank中报道的rBPI23、haFGFCD-NA序列一致。构建的BF融合基因及真核表达质粒DNA序列,经测序分析,与设计序列符合率为100％。结论本研究结果为进一步探讨BF融合蛋白的表达、生物学活性和特性奠定了基础。