套式聚合酶链反应检测巨细胞病毒的初步研究

目的建立套式聚合酶链反应（ＰＣＲ）加限制性酶切分析方法检测人巨细胞病毒（ＨＣＭＶ）。方法在ＨＣＭＶ直接早期蛋白ＥｃｏＲＩＪＤＮＡ片段内，自行设计两对引物，建立套式ＰＣＲ检测ＨＣＭＶＤＮＡ，同时结合病毒分离检测临床标本。结果２３例新生儿肝炎综合征患儿中，１０例病毒分离及套式ＰＣＲ均阳性；１例病毒分离阴性，但套式ＰＣＲ阳性。对５８例妊娠早期孕妇血标本进行检测，套式ＰＣＲ阳性率为９％，病毒分离阳性率则为７％。结论套式ＰＣＲ加限制性酶切分析是一种临床检测ＨＣＭＶ的快速有效手段，值得临床推广。     

Development of a quadriplex polymerase chain reaction for human cytomegalovirus detection

The development of a quadriplex PCR method with amplification of HCMV in a single‐step procedure using primers taken from four different regions of the viral genome is described. Different concentrations of dNTPs and MgCl₂ were assayed in order to optimize the constitution of the buffer for the multiplex PCR. The specificity of the PCR was tested with 100ng, 10ng, and 1ng of genomic MRC‐5 cell DNA infected with CMV in the presence of 10μg of uninfected MRC‐5 cell DNA. The sensitivity of the PCR was evaluated by the amplification of various amounts (100ng, 10ng, 1ng, and 0.1ng) of genomic MRC‐5 cell DNA infected with CMV. The specificity and sensitivity assays were performed for each pair of primers and for the combined four primer pairs in the multiplex PCR. CMV was consistently detected from 10ng of genomic MRC‐5 cell DNA with each primer pair. When all four sets of primers were combined in a single reaction tube, the sensitivity of the assay was equivalent to 10ng of genomic MRC‐5 cell DNA, whereas amplification from 1ng genomic MRC‐5 cell DNA produced only a subset of the amplimers. By amplifying four target‐sequences of HCMV simultaneously with minimum incubation time at each temperature, a quadriplex, highly sensitive PCR assay was performed. The use of four primer sets designed in different genomic regions of HCMV allowed the detection of variants and achieved maximal sensitivity and specificity which are essential for a diagnostic utilization. J. Clin. Lab. Anal. 13:99–105, 1999. © 1999 Wiley‐Liss, Inc.     

Detection of cytomegalovirus in blood donors by the polymerase chain reaction

A polymerase chain reaction (PCR) assay was developed and optimized to detect cytomegalovirus (CMV) DNA in the blood of 86 normal donors who had originally tested seropositive for CMV. Evidence of previous or current infection with CMV was determined by rescreening of the blood for CMV antibodies and by detecting the presence of infectious virus in the white cells by cell culture. DNA was extracted from the blood of donors by a manual or an automated method and amplified by PCR using primers from the major immediate early gene of CMV DNA. The amplified product was detected by visualization of a fluorescent 435-base pair DNA band in an electrophoretic agarose gel after ethidium bromide staining and confirmed by slot-blot DNA hybridization using an oligonucleotide probe with complementarity for the major immediate early gene. Seven (8%) of the 86 donors were positive for CMV DNA in both fluorescence and hybridization studies. These donors were also antibody positive. While 74 (86%) of the 86 donors were positive for the presence of CMV antibodies in enzyme-linked immunosorbent assay, none was positive for virus in cell culture. PCR has the potential to be an effective and reliable procedure for the detection of CMV DNA in donor blood, but further study is required for this technique to be used for diagnostic or routine screening purposes.     

Comparison of quantitative competitive polymerase chain reaction-enzyme-linked immunosorbent assay with LightCycler-based polymerase chain reaction for measuring cytomegalovirus DNA in patients after hematopoietic stem cell transplantation

Development of highly sensitive quantitative assays for cytomegalovirus (CMV) DNA detection is crucial for identification of immunodeficient patients at high risk of CMV disease. We designed 2 internally controlled competitive quantitative assays, enzyme-linked immunosorbent assay (ELISA)-based and real-time polymerase chain reaction (PCR) tests, using amplification of the same segment of the CMV genome. The aim of this study was to compare sensitivity, specificity, and laboratory performance characteristics of these assays. In both assays, a 159-bp segment of UL83 gene was amplified. External and internal controls were constructed by cloning the amplification product and heterogenous DNA segment flanked by target sequences for CMV-derived primers into bacterial plasmids, respectively. Real-time PCR was performed on LightCycler (Roche Diagnostics, Mannheim, Germany), and amplicons were detected using fluorescence resonance energy transfer probes. Alternatively, PCR products were labeled by digoxigenin, hybridized to immobilized probes, and detected by ELISA. The assays were tested on genomic DNA isolated from laboratory strains of CMV, QCMD control panel, and CMV DNA-positive peripheral blood DNA samples from hematopoietic stem cell transplant recipients, previously characterized by pp65 antigenemia and qualitative nested PCR. Real-time and ELISA-based PCR assays showed a linear course of 1-10(8) and 10-10(5) copies of CMV DNA per reaction, respectively. When compared with ELISA-based PCR, real-time PCR showed superiority in inter- and intra-assay reproducibility. Both assays were highly specific in detecting CMV DNA. No difference in amplification efficiency of internal or external standards and wild-type CMV DNA was found. The assays exhibited 83% concordance in CMV DNA detection from clinical samples, all discrepant samples having low CMV DNA copy numbers. There was a good correlation between viral DNA loads measured by the 2 assays. Statistically significant correlation was observed between the numbers of CMV DNA copies and pp65-positive leukocytes in the samples tested. Both variants of competitive PCR are adequately sensitive to be used for CMV DNA quantitation in clinical samples. LightCycler PCR, having superior performance characteristics and being less time-consuming, seems to be more suitable for routine diagnosis.     

Early virus isolation, early structural antigen detection and DNA amplification by the polymerase chain reaction in polymorphonuclear leukocytes from AIDS patients with human cytomegalovirus viraemia.

Fifty AIDS patients were investigated for human cytomegalovirus (HCMV) viraemia when potentially HCMV-related clinical symptoms or syndromes were observed. Nine patients underwent prolonged virologic follow-up, while 41 additional patients were examined only once or sporadically. Concentrated preparations of polymorphonuclear leukocytes (PMNL) from 153 blood samples were obtained for monitoring: (1) early virus isolation in cell cultures 24 h p.i. (viraemia); (2) early structural antigen detection in cytospin preparations (antigenemia); and (3) HCMV DNA in blood (DNAemia) through DNA amplification by the polymerase chain reaction (PCR). Viraemia and antigenemia were quantitated, whereas evaluation of DNAemia was only qualitative. A good correlation between levels of viraemia and antigenemia was consistently found except during ganciclovir treatment. HCMV-related clinical symptoms were observed when the number of infected PMNL was greater than 100 per 2 x 10(5) cells examined. All 56 blood samples positive for viraemia and antigenemia were also PCR-positive, whereas 44 samples (39 of which taken from patients with ascertained HCMV infection in blood) were positive by PCR only. Viraemia and antigenemia were often unrelated to HCMV organ syndromes, such as retinitis, in which only DNAemia was often detected. Prolonged ganciclovir treatment kept viraemia, antigenemia and even DNAemia at a low or negative level, yet drug discontinuation led to rapid progression of HCMV infection in blood. In addition, prolonged antiviral treatment could induce appearance of ganciclovir-resistant HCMV strains, requiring alternative foscarnet therapy. In conclusion, determination of viraemia and antigenemia appears essential for correct clinical management and antiviral treatment of disseminated HCMV infections in AIDS patients. However, PCR is the most sensitive method for diagnosis and monitoring of HCMV infections in blood at a pre-clinical stage.     

Evaluation by polymerase chain reaction of cytomegalovirus reactivation in intensive care patients under mechanical ventilation

Objective The study was undertaken to determine if critically ill patients under mechanical ventilation could reactivate latent cytomegalovirus (CMV) in either lung or blood.Design Prospective study in critically ill patients.Setting The study was performed in a multidisciplinary intensive care unit in a university hospital.Patients 23 non-immunocompromised, mechanically ventilated patients who were anti-CMV immunoglobulin G-positive. Ten immunocompromised patients with active CMV infection and 16 asymptomatic CMV seropositive non-immunocompromised patients constituted the positive and negative control groups.Measurements and results The presence of CMV in blood and bronchoalveolar lavage (BAL) was evaluated by both viral cultures and polymerase chain reaction (PCR). Thirty-seven blood and 22 BAL samples were investigated. Sequential samples were evaluated in 8 patients. For PCR, a 290 bp fragment in the first exon of the immediate early 1 gene was amplified. In order to exclude inhibitors of PCR amplification, a 268 bp fragment of the -globin gene was concurrently amplified in all samples. Viral cultures of blood and BAL were negative in all 23 non-immunocompromised, mechanically ventilated patients. Moreover, no CMV DNA could be amplified in blood or BAL samples, whereas a -globin amplification was observed in all samples.Conclusion In a series of 23 critically ill patients under mechanical ventilation who were seropositive for CMV, no reactivation of CMV in blood or lung was demonstrated.     

Rapid detection of Salmonella by polymerase chain reaction

Salmonella enterotoxin gene (stn) was sequenced from Salmonella enterica serotypes: Typhimurium, Typhi, Paratyphi A and B. The sequences from all the four serotypes showed complete homology with the already reported stn gene sequence of the serotype Typhimurium. As a tool for detection of this organism, four pairs of oligonucleotide primers were designed to amplify different fragments of this important pathological marker. The protocols were standardized with serotype Typhimurium in such a way so as to complete the PCR reaction in 75-90 min. These primers were found to generate specific amplicons with all the serotypes of Salmonella tested. The PCR protocols were found to be highly specific as no amplifications, specific or non-specific, were found when reactions were run using non-Salmonella DNA as template. The employment of a nested PCR markedly increased the sensitivity of the assay system in natural water samples. The protocol described herein is highly sensitive as it detects less than 10 cells of Salmonella in 250 microl of blood and approx. 1 cell in 1 ml of water without any enrichment. For the validation of this protocol, 72 coded samples of 11% skimmed milk spiked with different pathogens were received from NICED, Kolkata and analyzed for the presence of Salmonella. Our procedures detected correctly the presence of Salmonella in nine samples. 50 samples of raw milk were subjected to this PCR after enrichment for 8 h and 6 samples were found positive for Salmonella. The study indicates that Salmonella enterotoxin (stn) gene is highly conserved and the protocol devised in this study can be used as rapid and reliable method for detection of Salmonella spp. in water, milk and blood samples.     

聚合酶链反应检测SEN病毒的方法学探讨

目的探讨聚合酶链反应(PCR)扩增SEN病毒(SENV)核酸的优化条件。方法将标本分别以Acupure DNA／RNA提取法(方法1)、SDS-蛋白酶K方法(方法2)及裂解液提取’兀V核酸法(方法3)抽提SENV DNA模板，并用3组针对SENV DNA不同区的引物进行扩增，比较不同模板提取方法、不同扩增引物及不同体积标本对SENV DNA检出率的影响。结果在191份血清中，方法1、方法2和方法3提取核酸后可分别检测出15份、9份和7份。采用方法1提取SENV DNA的基础上，采用2组套式引物分别扩增出15份和11份，一次PCR引物仅检出2份SENV DNA阳性标本；应用引物2扩增至少需要50μl血清。结论不同引物扩增方法和不同模板提取方法可能是影响SENV DNA检出率重要因素。     

The detection of Bordetella pertussis in swab samples by polymerase chain reaction

Polymerase chain reaction (PCR) is revolutionizing the diagnosis of many infectious diseases, particularly those caused by organisms that are difficult to cultivate. Rapid and sensitive detection of Bordetella pertussis in clinical samples is important for the early diagnosis of pertussis, the prevention of transmission of the organisms and vaccine efficacy trials. In this study dacron and calcium-alginate nasopharyngeal swabs (NPSs) taken from patients with suspected pertussis were analysed by PCR using primers derived from the DNA adjacent to the B. pertussis porin gene. NPSs in Regan-Lowe medium were transported to the laboratory for bacterial culture and PCR. Chelex 100 resin was used to extract bacterial DNA from NPSs. Three hundred and five NPSs including 127 dacron and 178 calcium-alginate swabs from 165 children with suspected pertussis were tested. Fourteen (11%) dacron swabs and 10 (6%) calcium-alginate swabs were culture positive. However, 23 (18%) dacron swabs and nine (5%) calcium-alginate swabs were positive by PCR using the ethidium bromide staining method. When the digoxigenin immunoblot system was used to increase the sensitivity of PCR, 79 (62%) dacron NPSs including 14 culture-positive samples were PCR positive; however, only 28 (16%) calcium-alginate NPSs including one culture-positive sample were positive by PCR. This data indicates that PCR is more sensitive than culture and the sensitivity of PCR can be increased using the digoxigenin immunoblot system. In addition, many fewer PCR positives were found in calcium-alginate swabs compared to dacron NPSs, which may be due to the quality and the Taq polymerase inhibitory effect of calcium-alginate fibre.     

The development and testing of a molecular biological test system for DNA detection of anthrax pathogen by real-time polymerase chain reaction assay

The paper presents the results of the development and testing of a molecular biological test system for DNA detection of anthrax pathogen (Bacillus anthracis) by real-time polymerase chain reaction assay. The test system has shown high sensitivity, specificity, and reproducibility of results of analysis, as exemplified by aqueous suspensions of daily agar cultures of Bacillus anthracis strains, related and heterologous species of microorganisms, and clinical materials of experimental animals. There is evidence for the persistence of the basic characteristics of the test system when stored at 22 +/- 2 degrees C for 12 months.     

Detection of Mycoplasma hyopneumoniae DNA by the polymerase chain reaction

DNA amplification by the polymerase chain reaction (PCR) was examined to detect DNA of Mycoplasma hyopneumoniae, an etiological agent of porcine pneumonia. A pair of synthetic primers was selected that specify the amplification of a 520-basepair DNA fragment in a reiterative sequence of M. hyopneumoniae genome. The PCR product was detected by direct gel electrophoresis or by blot hybridization to a synthetic oligonucleotide probe. The specificity of PCR for M. hyopneumoniae was confirmed by lack of cross-reactivity to DNA from other porcine mycoplasmas.     

Detection of Borrelia burgdorferi DNA by the polymerase chain reaction

DNA amplification by the polymerase chain reaction (PCR) was used to detect DNA of the Lyme disease spirochaete Borrelia burgdorferi. Primers that specify the amplification of a 145 basepair DNA fragment of the OspA gene of B. burgdorferi were used. The amplification product was detected by gel electrophoresis and ethidium bromide staining or by hybridization to a radiolabelled oligonucleotide probe. The hybridization method was found to be more sensitive. As little as 50 fg of purified B. burgdorferi DNA could be detected by PCR. This corresponds to fewer than 50 spirochaetes. The specificity of PCR for B. burgdorferi was tested by using DNA from other organisms as templates for amplification. No cross-reactivity was found. The data shown provide useful information for the development of a PCR-based diagnostic test for Lyme disease.     

Failure to detect human cytomegalovirus DNA in peripheral blood leukocytes of healthy blood donors by the polymerase chain reaction

The transmission of cytomegalovirus (CMV) by blood transfusion may have a major effect on certain immunocompromised patients. To protect susceptible blood recipients from infection, it is advisable to use blood components from CMV-seronegative donors. However, serologic tests are not capable of indicating which blood component actually harbors infectious virus and can transfer it to the recipient. Therefore, a sensitive method is needed for the detection of the virus itself. There have been three reports on the detection of CMV in healthy volunteer blood donors by the polymerase chain reaction (PCR). CMV DNA was found in all seropositive and most seronegative blood donors. However, many other authors have failed to confirm these data. A highly sensitive and specific PCR assay was developed for the detection of CMV DNA in peripheral blood leukocytes. With this protocol, blood samples from 116 volunteer blood donors were investigated. None of these samples proved to be positive for CMV DNA. In contrast, CMV DNA was detected in 10 of 10 renal transplant patients early in the course of active CMV infection. It can be concluded that the CMV genome copy number in the peripheral blood leukocytes of healthy individuals is beyond the detection limit of current PCR technology.     

磁捕法对传染性非典型肺炎冠状病毒RNA富集体系的初步研究

目的 初步研究建立磁捕法对传染性非典型肺炎又称严重急性呼吸综合征(SARS)冠状病毒RNA的富集、纯化体系,以提高逆转录聚合酶链反应(RT-PCR)检测SARS冠状病毒RNA的敏感性。方法 根据国际基因库公布的SARS冠状病毒基因序列,自行设计引物、探针,体外克隆目的RNA片段作为标准物质;利用标准RNA以及模拟SARS血清标本的RNA,评价自行设计的SARS冠状病毒巢式RT-PCR检测体系和磁捕法SARS冠状病毒RNA富集检测体系的检测效果。结果SARS冠状病毒巢式RT-PCR检测体系,对于模拟血清SARS标本RNA的最低检测样本浓度为20拷贝/μl,而磁捕法SARS冠状病毒RNA富集检测体系的最低检测样本浓度为1拷贝/μl。结论 初步建立的磁捕法SARS冠状病毒RNA富集检测体系,可有效地对低浓度的RNA样本进行浓缩、纯化,有效提高SARS RT-PCR检测方法的敏感性。     

乙型肝炎病毒实时荧光定量PCR检测方法的建立

目的建立一种采用Lightcycler系统进行HBVDNA实时荧光定量PCR的方法,并探讨其临床应用价值。方法针对HBV基因组S区设计一对扩增引物,并通过预实验严格优化反应体系的组成和条件;将T载体与HBVRT区扩增后纯化的产物进行连接反应,然后转染大肠杆菌（DH5a）,经蓝、白斑筛选后挑取阳性菌落,提取质粒,制备外标准品。结果用于制成外标准品的质粒经1：10的缓冲液倍比稀释,制作标准曲线,线性方程为：Y=-3．344X＋37（r2=0．9999）;通过检测已知浓度并经倍比稀释的HBVDNA,表明其最低检测限为5×10^2 IU／mL;拷贝数介于5×10^2～5×10^8 IU／mI。之间的HBVDNA浓度与ct值具有良好的线性关系。结论外标法实时荧光定量PCR是一种定量相对准确、灵敏度高、特异性强、操作相对简便的方法;该方法可用于乙型肝炎患者病情监测,有效指导临床用药;联合该法与血清标志物检测,可更准确评价HBV感染者病情。     

Sensitive detection of polyalanine expansions in PHOX2B by polymerase chain reaction using bisulfite-converted DNA

Congenital central hypoventilation syndrome, also known as Ondine’s curse, is characterized by idiopathic abnormal control of respiration during sleep. Recent studies indicate that a polyalanine expansion of PHOX2B is relevant to the pathogenesis of this disorder. However, it is difficult to detect the repeated tract because its high GC content inhibits conventional polymerase chain reaction (PCR) amplification. Here, we describe a bisulfite treatment for DNA in which uracil is obtained by deamination of unmethylated cytosine residues. Deamination of DNA permitted direct PCR amplification that yielded a product of 123 bp for the common 20-residue repetitive tract with replacement of C with T by sequencing. It settled allele dropouts accompanied by insufficient amplification of expanded alleles. The defined procedure dramatically improved detection of expansions to 9 of 10 congenital central hypoventilation syndrome patients examined in a previous study. The chemical conversion of DNA before PCR amplification facilitates effective detection of GC-rich polyalanine tracts.     

Single-run, parallel detection of DNA from three pneumonia-producing bacteria by real-time polymerase chain reaction

A molecular assay for parallel detection of three bacteria, Chlamydia (C.) pneumoniae, Legionella (L.) spp., and Mycoplasma (M.) pneumoniae, in clinical specimens by a set of real-time polymerase chain reactions (PCRs) in a single run was evaluated. Bacterial DNAs were extracted by an automated DNA extraction protocol on the MagNA Pure LC System. Amplification and detection were done by real-time PCR on the LightCycler (LC) instrument. For amplification, specific oligonucleotides derived from the 16s rRNA genes of C. pneumoniae, L. spp., and M. pneumoniae were used. The three assays were complemented with an internal control (IC), a specially designed DNA fragment which contains the specific primer binding sites for the three PCRs. The IC was added to the samples, co-extracted, and co-amplified. Primers and hybridization probes were designed to suit one LC PCR program. LC PCRs were established, detection limits were determined, and clinical samples were tested. The detection limits were found between 5.0 and 0.5 IFU/CFU per PCR reaction for each of the bacteria. A total number of 100 clinical specimens were tested for validation of the molecular assay. Tested samples included 63 bronchoalveolar lavages (BALs) and 37 induced sputa specimens. The internal control was detected in all negative and low-positive samples; no inhibition was found throughout the whole study. Additionally, samples underwent testing by culture for L. spp., and M. pneumoniae; for C. pneumoniae, the serological microimmunofluorescence (MIF) test was used. In conclusion, the developed set of LC PCR assays permits parallel detection of C. pneumoniae, L. spp., and M. pneumoniae in a single LC run. This molecular assay may lead to accurate and early diagnosis of pneumonia produced by these three types of bacteria. The assay proved to be suitable for the high-throughput routine diagnostic laboratory.     

聚合酶链反应方法检测造血干细胞移植患者巨细胞病毒感染状况

目的 评价PCR检测血浆和尿巨细胞病毒(CMV)对CMV病发生的预告意义。方法 对1999年8月～2001年7月进行异基因造血干细胞移植的131例患，自预处理开始，每周留取血及尿标本，经PCR法检测血浆和尿沉渣中的CMV-DNA。结果 血浆病毒血症阳性89例，尿CMV-DNA阳性99例；发生CMV病37例，累计发生率为28．2％。CMV病的发生率在血CMV-DNA阴性组为15．7％，在1次血CMV-DNA阳性组为31．3％，2次以上血CMV-DNA阳性的患组为47．3％，三组间CMV病发生率差异有显性(P=0．0126)，2次以上血CMV-DNA阳性组比阴性组患CMV-DNA病发生率明显增高。CMV病的发生率在尿CMV-DNA阴性组为24．8％，尿CMV-DNA1次阳性组为43．5％，尿CMV-DNA2次以上阳性组为33．0％，三组间CMV病发生率无统计学差异(P=0．845)。血浆CMV-DNA对预测CMV病的阳性预告值为40．5％，阴性预告值为84.4％，灵敏度75．0％，特异度69.2％。结论 血浆CMV PCR检测结果对CMV病的发生有一定预测意义，单独尿CMV PCR检测结果不能预测CMV病的发毕．     

多重聚合酶链反应检测呼吸机相关性气管-支气管炎和肺炎患者常见致病菌的研究

目的 探讨多重聚合酶链反应(PCR)技术在快速检测呼吸机相关性气管-支气管炎(VAT)和呼吸机相关性肺炎(VAP)常见致病菌中的临床作用和价值.方法 留取75例外科重症监护病房(ICU)机械通气并发VAT或VAP患者的痰标本,进行细菌培养、普通PCR、多重PCR检测,比较3种方法对致病菌检出率的差异.结果 细菌培养法对金黄色葡萄球菌、鲍曼不动杆菌、大肠埃希杆菌、铜绿假单胞菌和肺炎克雷伯杆菌分离阳性检出率分别为50.7％、45.3％、30.7％、41.3％和58.7％,普通PCR阳性检出率分别为88.0％、89.3％、78.7％、85.3％和93.3％,多重PCR阳性检出率分别为92.1％、90.7％、82.7％、89.3％和96.0％.普通PCR和多重PCR对5种致病菌的阳性检出率均高于细菌培养(均P＜0.05);且多重PCR较普通PCR具有快速检测的优点.结论 与细菌培养比较,普通PCR和多重PCR对VAT和VAP5种常见致病菌的阳性检出率高,且多重PCR可有效节省人力、财力.