多重聚合酶链式反应技术及其应用

标准的聚合酶链式反应是使用一对引物扩增一个特定的核酸序列,即单一引物对的PCR。但是在具体工作中有时需要同时对多个核酸序列进行扩增分析。显然,单引物对PCR要完成这类工作,需要耗费较多的时间和金钱。1988     

肺炎支原体快速聚合酶链式反应（PCR）法检测

本文根据Ｂｅｒｎｅｉ设计的肺炎支原体ＰＣＲ引物，采用国产的耐热ＤＮＡ聚合酶，建立了双温循环肺炎支原体ＰＣＲ快速检测法。４０例儿童肺炎咽拭子标本中，８例呈阳性结果。     

聚合酶链式反应生物芯片/微装置技术在疾病诊断中的应用

聚合酶链式反应（Polymerase chaim reaction，PCR）技术已经在分子生物学、疾病诊断等领域得到广泛应用。PCR生物芯片／微装置由于具有所需样品和反应混合物体积小、反应时间短、轻便等优点倍受人们关注。本文综述了PCR生物芯片／微装置在疾病诊断领域中的应用，并对其应用和发展作出了展望。     

聚合酶链式反应法检测血HBsAg阳性产妇乳汁中的HBV-DNA

我们应用PCR技术检测了24例血HBsAg阳性产妇初乳中的HBVDNA,结果发现,24例中19例乳汁HBV_-DNA为阳性(阳性率高达79.2％),表明该19例乳汁是排毒的。HBV可通过体液排出体外,除乳汁外,还有唾液、泪液、月经、精液、尿以及汗等。过去较为重视的是唾液和月经。本研究表明血HBsAg阳性产妇乳汁HBV排出率颇高,为此,以往认为HBV携带者产妇只要乳头不破溃可以哺乳的观点值得再商讨。本文作者建议乳汁HBV-DNA阳性,而其婴儿血HBV标志物检测阴性者行人工喂养。     

PCR二小时法性别鉴定

本文报告采集受检者之微量外周血、血斑、漱口液、毛发根等4种样品,经5～15分钟的简单预处理后,应用先进的聚合酶链式反应技术作二小时法性别鉴定。     

聚合酶链式反应（PCR）及其应用

聚合酶链式反应(简称PCR),又称体外基因放大技术,是用DNA聚合酶在体外系统中诱发一对引物间的DNA双链的合成过程。经过20～30次循环后,可使目的基因片段成百万倍地扩增,使得对该基因片段的分析研究更为便利。PCR技术自1985年创立以来,经过不断改进,目前已成为分子生物学研究的重要手段。PCR技术已经应用于遗传病的基因诊断和产前诊断,病原体的鉴定,癌基因的分析研究等。最突出的是经PCR放大后的DNA片段可以直接进行顺序分析。     

聚合酶链反应技术在病理研究中的应用

本文综述了ＰＣＲ应用于病理的有关问题，主要包括石蜡切片ＰＣＲ技术，固定剂的选择，原位ＰＣＲ和定点ＰＣＲ等。     

孕妇血中胎儿细胞在产前诊断中的初步应用──胎儿SRY基因的鉴定

以Ｎｙｃｏｄｅｎｚ和Ｐｒｅｃｏｌｌ作为分离介质，对２３名孕龄为６～４１周，年龄为２１～２８岁的孕妇外周血中胎儿细胞进行了密度梯度离心富集。其中１５名怀男胎孕妇外周血富集前后各类细胞ＳＲＹ基因的ＰＣＲ扩增检查表明，大部分孕妇外周血多核细胞及低密度单个核细胞扩增结果为阳性，与胎儿实际性别符合率分别为８０．０％和９３．３％．这一结果提示：（１）ＰＣＲ分析母血中胎儿细胞单拷贝基因应采用低密度细胞多核细胞，且以前者为优；（２）密度梯度离心法已在一定程度上从母血中富集胎儿细胞，且所获得的细胞已基本上满足了ＰＣＲ扩增单拷贝基因所需的模板量；（３）富集实验证实母血中胎儿细胞大致由多核细胞及低密度单个核细胞两部分组成，而未经富集的孕妇血单个核细胞及高密度细胞中仅有少数几例呈ＳＲＹ阳性，符合率分别为１３．３％和６．７％；提示从未富集的孕妇外周血中按常规方法难以扩增出含量极微的胎儿单拷贝基因。在对３名怀女胎的孕妇外周血富集前后各类细胞共３２份标本进行ＳＲＹ基因的检测时，出现３例假阳性结果，其原因不明，不能排除外源男性ＤＮＡ的污染或前次妊娠的残留胎儿细胞的干扰所致。     

聚合酶链式反应法检测婴儿肝炎综合征患者尿人巨细胞病毒特异脱氧核糖核酸

人巨细胞病毒(HCMV)是广泛感染人类的重要病原之一。胎儿、新生儿以及免疫力低下者(器官移植者、免疫缺陷者)感染HCMV可致严重疾患,甚至是死亡的直接原因。聚合酶链式反应法(PCR)检测病毒核糖核酸是当今最灵敏的方法。本文应用PCR法检测了20例婴儿肝炎综合征患者尿HCMV脱氧核糖核酸,结果阳性者为17例。阳性率达85％。     

Specific identification of Bordetella pertussis by the polymerase chain reaction

Oligonucleotide primers were used to amplify specific DNA regions of the Bordetella pertussis genome by the polymerase chain reaction. One pair of primers, PTp1/PTp2, identified a 191-bp DNA fragment located in the regulatory region of the pertussis toxin operon; a second pair of primers led to amplification of a 121-bp DNA piece located in an insertion-like element specific to B. pertussis. Both sets of primers were able to discriminate between the pathogen and related Bordetella species; they detected down to 6 bacteria and appeared suitable for routine detection of B. pertussis in clinical specimens.     

Sex identification by polymerase chain reaction using X-Y homologous primer

A method of sex identification using the polymerase chain reaction technique is described. Using a pair of nucleotide primers from an X-Y homologous region, both the X and the Y sequences can be amplified simultaneously, and more importantly, they result in fragments of different lengths. The success of the procedure is therefore monitored by the presence of a X-specific band while sex is identified by the presence or absence of a Y-specific band.     

Detection and identification of human influenza viruses by the polymerase chain reaction

A series of oligonucleotide primers are described which hybridize to conserved regions of influenza virus cDNA and prime DNA synthesis in Taq polymerase catalyzed amplification reactions (PCR). Primers were designed to hybridize as nested pairs and, following a two-step amplification, produce uniquely sized DNA fragments diagnostic for viral type and subtype. Influenza A and B matrix-protein genes and the influenza C haemagglutinin gene were targets for the type-specific primers. Subtype-specific primers targeted conserved sequences within the three haemagglutinin or two neuraminidase subtypes of different human influenza isolates. The utility of this method was demonstrated using computer search methods and by accurately amplifying DNA from a variety of influenza A, B, and C strains. Type-specific primer sets showed a broad type specificity and amplified DNA from viral strains of unknown sequence. Restriction mapping and DNA sequencing showed that fragments amplified in this manner derived from the input template, confirming the accuracy of the method and demonstrating how PCR can be used to quickly derive sufficient sequence information for analysis of viral relatedness. Subtyping primers were able to distinguish accurately between the three haemagglutinin (H1, H2, H3) and two neuraminidase (N1, N2) alleles of human influenza A isolates. Again DNA was amplified from viruses of unknown sequence confirming that most of these primer sets may prove useful as broad range subtyping reagents. In order to simplify the work associated with analysis of many samples, we have also devised a rapid method for the isolation of viral RNA and synthesis of cDNA. Using this 'mini-prep' technique, it is possible to detect, amplify, and identify picogram quantities of influenza virus in a single day, confirming that PCR provides a useful alternative to existing methods of influenza detection.     

Detection and identification of vaccine-related polioviruses by the polymerase chain reaction.

We have used the polymerase chain reaction (PCR) to obtain sensitive detection and identification of poliovirus RNA genomes. Primer pairs were designed to permit identification of each Sabin poliovaccine strain by the electrophoretic mobilities of the amplified DNA products (Sabin 1: 97 bp; Sabin 2: 71 bp; Sabin 3: 44 bp). The compositions of samples containing mixtures of vaccine strains could be readily determined by PCR. When the amplified products were visualized by ethidium bromide fluorescence, as few as 250 genomic copies in the original sample could be detected. When PCR was used in combination with strain-specific 32P-labeled oligonucleotide probes, the limit of detection was less than or equal to 2.5 poliovirus genomes, exceeding the sensitivity of poliovirus isolation in cell culture by at least 100-fold. PCR amplifications may be performed on virion RNAs extracted directly from clinical specimens, potentially eliminating the requirement for virus isolation in routine identifications while yielding reliable results within 8 h.     

Serotype identification of Actinobacillus pleuropneumoniae by arbitrarily primed polymerase chain reaction.

Rapid and accurate determination of the Actinobacillus pleuropneumoniae serotype involved in a disease outbreak is important both in limiting the severity of an outbreak and for tracing the source of the infecting organism. This study describes the use of arbitrarily primed polymerase chain reaction (AP-PCR) as a rapid, precise, and genetically based procedure to identify A. pleuropneumoniae. AP-PCR amplification of bacterial genomic DNA results in specific DNA profiles, which can be used to differentiate currently recognized serotypes. This technique is especially useful for identifying previously nontypeable and serologically cross-reactive A. pleuropneumoniae field isolates. Consecutive passages of isolates on different media, freezing, and subsequent infection of pigs did not alter the AP-PCR genomic profile. We propose the use of M13 and T3-T7 oligodeoxynucleotide primers for diagnostic and epidemiological identification of A. pleuropneumoniae by AP-PCR techniques.     

Detection and identification of Helicobacter pylori by the polymerase chain reaction.

A polymerase chain reaction for the specific detection of Helicobacter pylori was developed using a primer pair derived from the nucleotide sequence of the urease A gene of H pylori. Specific amplification of a 411 base pair DNA fragment from all strains of H pylori tested was achieved. Ten organisms were detected using the PCR and the technique permitted direct detection of H pylori in clinical biopsy samples. PCR will be useful for both prospective and retrospective investigation of the aetiology and epidemiology of H pylori associated disease.     

Sex identification by polymerase chain reaction using a Y-autosome homologous primer set

Summary We have developed a one-step polymerase chain reaction (PCR) technique which detects a sequence on the human Y chromosome and an autosomal sequence in one reaction. The method is very reliable for the sex determination, as the detection of the autosome-specific signal ensures the presence of DNA in the specimen even in the absence of the Y-specific signal.     

Rapid identification of Debaryomyces hansenii/Candida famata by polymerase chain reaction.

Primers designed in this study were used in a polymerase chain reaction test to amplify a species-specific fragment of approximately 340 bp of the large subunit ribosomal DNA of Debaryomyces hansenii/Candida famata. None of the other medically relevant yeasts including C. guilliermondii, and also the related species, D. nepalensis and C. saitoana, were amplified by this primer pair.     

Detection of cytomegalovirus in human placental cells by polymerase chain reaction

Human cytomegalovirus (HCMV) is the most common cause of viral intrauterine infection. Progress in rapid, specific, and dependable detection of HCMV has recently been achieved by the use of DNA hybridization techniques and other molecular methods. We examined 21 placentas after delivery for the presence of HCMV DNA by polymerase chain reaction (PCR). To test the reliability of the PCR for the detection of HCMV DNA in clinical specimens, two simple PCR assays and a real-time quantitative PCR were used. PCR analysis of villous and decidual cells showed that HCMV DNA was present in 16 placentas (76.2%). Transmission of HCMV infection to chorionic villi was confirmed in 11 organs (52.4%), and congenital infections in newborns were detected in 9 cases (42.8%). These results suggest that HCMV genome detection in placentas at later gestational ages is common. Our results demonstrated that detection of HCMV DNA in placental tissues by DNA amplification provides a specific and sensitive method for diagnosis of intrauterine HCMV infection.