应用循环逆转录PCR技术检测丙型肝炎病毒RNA

循环逆转录（ｃｉｒｃｕｌａｔｏｒｙ ｒｅｖｅｒｓｅ ｔｒａｎｓｃｒｉｐｔｉｏｎ,ＣＲＴ）是线性增长逆转录ｃＤＮＡ产量的一种新技术。为了将该技术用于检测ＨＣＶ ＲＮＡ,通过改变ＣＲＴ的循环次数,结合竞争ＰＣＲ,作出标准曲线。采用１６次ＣＲＴ加３４次循环ＰＣＲ检测了１３６例ＨＣＶ ＥＬＩＳＡ阳性、５４例ＨＣＶ ＥＬＩＳＡ阴性和１０８例临床可疑病人全血标本,并与逆转录ＰＣＲ（ＲＴ－ＰＣＲ）和巢式ＰＣＲ（     

Detection of DNA of borrelia circulating in Novosibirsk region

Detection of DNA of Borrelia burgdorferi sensu lato was performed by PCR in taiga ticks Ixodes persulcatus, in blood samples and skin bioptates of small forest mammals, and in blood and urine samples of humans after attaching of ticks events. In Novosibirsk region both in natural reservoir and in patients with Ixodes ticks-borne borreliosis DNA of Borrelia garinii and Borrelia afzelii are detected. DNA of these borrelia were detected in 8 from 72 of taiga ticks, in 36 from 298 of blood and skin samples of small forest mammals, and in 32 from 102 of human blood and urine samples. In all studied samples DNA of B. garinii from NT29 subgroup was predominated. Borrelia DNA in which sequence of intergenic spacer region was homologous to sequence Chy13p first detected in China has been detected in one blood sample from red-backed vole (Clethrionomys rutilus).     

半定量RT-PCR检测运动发酵单胞菌中外源基因转录水平的研究

本研究运用半定量RT-PCR法检测运动发酵单胞菌重组菌中外源基因xylB的转录水平。提取野生型运动发酵单胞菌CP4及其2个重组菌的总RNA, 检测无DNA污染后定量至同一浓度、并反转录为cDNA。观测目的基因xylB和内标基因16S rRNA的PCR扩增曲线、并确定合适的循环数, 选用相同量的cDNA为模板, PCR检测各样本中xylB相对16S rRNA的转录水平。结果表明野生型菌株CP4中xylB基因没有转录, 而两株重组菌中皆有xylB的转录本, 且转录丰度基本一致, 酶活测定也进一步证实该基因在重组菌中有效表达。该方法可用于鉴定运动发酵单胞菌中特定基因的转录水平, 是一种快速有效的检测方法。     

Highly sensitive revealing of PCR products with capillary electrophoresis based on single photon detection

Post-PCR fragment analysis was conducted using our single photon detection-based DNA sequencing instrument in order to substantially enhance the detection of nucleic biomarkers. Telomerase Repeat Amplification Protocol assay was used as a model for real-time PCR-based amplification and detection of DNA. Using TRAPeze XL kit, telomerase-extended DNA fragments were obtained in extracts of serial 10-fold dilutions of telomerase-positive cells, then amplified and detected during 40-cycle real-time PCR. Subsequently, characteristic 6-base DNA ladder patterns were revealed in the post-PCR samples with capillary electrophoresis (CE). In our CE instrument, fluorescently labeled DNA fragments separate in a single-capillary module and are illuminated by a fiberized Ar-ion laser. The laser-induced fluorescence (LIF) is filtered and detected by the fiberized single photon detector (SPD). To assess the sensitivity of our instrument, we performed PCR at fewer cycles (29 and 25), so that the PCR machine could detect amplification only in the most concentrated samples, and then examined samples with CE. Indeed, PCR has detected amplification in samples with minimum 10(4) cells at 29 cycles and over 10(5) cells at 25 cycles. In contrast, the SPD-based CE-LIF has revealed 6-base repeats in samples with as low as 10(2) cells after 29 cycles and 10(3) cells after 25 cycles. Thus, we have demonstrated 100- to 1000-fold increase in the sensitivity of biomarker detection over real-time PCR, making our approach especially suitable for analysis of clinical samples where abundant PCR inhibitors often cause false-negative results.     

The diagnosis of malaria and identification of plasmodium species by polymerase chain reaction in Turkey

More than half of the world's population is exposed to malaria in approximately 100 countries. Rapid diagnosis and correct treatment of cases are the main objectives of control programs in malaria endemic areas. We have developed a PCR method to determine the presence of plasmodium DNA in blood. The method can also identify the species of the plasmodium by restriction enzyme analysis of the amplified product. We evaluated the performance of this method in the diagnosis of malaria suspected cases in Turkey by comparing to microscopy of the blood smears: blood samples were obtained from 114 patients with malaria symptoms, including fever and/or chills lasting for several days, before starting treatment. Thin and thick blood smears were prepared immediately in the region of specimen collection. After isolation of DNA from blood samples, DNA was amplified by PCR and digested by restriction enzyme AluI. The obtained fragments were analyzed by agarose gel electrophoresis. The number of parasites in the thick and thin smears of the blood samples was evaluated microscopically after staining by Giemsa and results were compared by PCR results. Among 114 plasmodium positive cases detected by microscopy, 100 were also detected by PCR. There were 14 false negatives and no false positive by PCR. Compared to microscopy, the sensitivity, specificity and Positive Predictive Value (PPV) of PCR were determined as 76%, 100% and 100%, respectively.     

Detection and typing of Helicobacter pylori cagA/vacA genes by radioactive,one-step polymerase chain reaction in stool samples from children

The detection and molecular typing of Helicobacter pylori virulence genes in human stool specimens by polymerase chain reaction (PCR) require an adequate amount of bacterial DNA and an appropriately adjusted PCR protocol. DNA was isolated from stool samples of 39 H. pylori-infected and nine uninfected Colombian children using the QIAamp Kit following the manufacturer's instructions but with modifications. DNA templates were amplified for the vacA s and m regions and for the cagA gene by PCR using radioactively labeled (32P) primers. The modifications in the standard Qiagen protocol of stool DNA extraction increased the final concentration of eluted total stool DNA 4.7 times (117 +/- 17 versus 22 +/- 3 ng/microl; P < 0.0001). Nevertheless, its amplification by regular PCR programs (30-40 cycles) did not generate visible signals because of the very low ratio of H. pylori DNA to other DNA. PCR for 80 cycles successfully amplified vacA in 36/39 samples (sensitivity, 92.3%) and cagA fragments in 21/39 (53.8%) fecal DNA samples. Both s and m vacA regions were amplified in 33/36 (91.7%) DNA samples. The s1m1 genotype was the most commonly isolated variant, accounting for 17/36 or 47.2% of positive samples. The s2m2 genotype was ascertained to be frequent also (14/36 or 38.9%). Almost all (94.1%) s1m1 genotypes were cagA positive. The majority of s2m2 genotypes (78.6%) were not associated with the cagA gene. Neither cagA nor vacA fragments were amplified from DNA isolates of H. pylori-uninfected children nor from DNA isolated from six gastrointestinal bacterial strains (specificity, 100%). The data suggest that the proposed modified technique of DNA extraction and PCR assay of stool samples may be an effective and reliable noninvasive tool for the detection and typing of H. pylori cagA/vacA virulence genes in infected individuals.     

Development and evaluation of a PCR method for diagnosis of Salmonella enteric fever, based on DNA sequences of the hilA gene

Typically, diagnosis of enteric fever due to Salmonella spp. is by bacterial isolation from blood culture; however, the blood culture method is slow, not always available, and not informative in patients with antibiotic treatment. Salmonella spp. uses the hilA gene (component of the pathogenicity island I) to invade epithelial cells and produce infection. Using the hilA gene sequence a PCR test was designed to detect Salmonella in blood samples. The sensitivity (S), specificity (SP), positive predictive value (PPV) and negative predictive value (NPV) of the PCR method were obtained by testing the blood samples from 34 patients with suspected of enteric fever. Presence of S. typhi was confirmed by blood culture. Blood samples were also tested from 35 patients with infections due to other non-Salmonella pathogens, again corroborated by blood culture (Klebsiella pneumoniae, 9; Serratia marcescens, 5; Escherichia coli, 4; Pseudomonas aeruginosa, 9; Providencia alcalifaciens, 4; Enterobacter cloacae, 4). Control samples were obtained from 150 healthy volunteers. The S, SP, PPV and NPV for the PCR method were all 100%. The lowest number of colony forming units/ml detected by PCR in blood samples was 10.     

猕猴巨细胞病毒(RhCMV)nested PCR检测方法的建立与初步应用

目的建立猴巨细胞(RhCMV)病毒的nested PCR检测方法,并初步应用。方法根据GenBank中报道的RhCMV全基因序列,针对其中的保守区域Rh85设计两对引物进行nested PCR反应,利用此方法对20份猕猴全血标本进行检测,将检测到的猕猴阳性标本扩增片段进行克隆测序。结果利用保守区域Rh85设计的引物可对人HCMV阳性对照进行扩增。用此方法检测的20份猕猴全血标本,出现2例阳性。其中一例扩增片段经纯化、回收克隆测序后用BLAST软件进行同源性对比,与GenBank中报道的RhCMV序列基本相同。结论建立了从猴全血中直接检测猴RhCMV病毒DNA的敏感、特异的nested PCR方法。     

Identification of Phytoplasmas Associated with Grapevine Yellows in Serbia

The molecular identification and characterization of phytoplasmas from infected grapevines in four locations in Serbia are reported. Phytoplasmas were detected and identified by restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR) amplified 16S rDNA. Grapevine yellows were associated with three molecularly distinguishable phytoplasmas: Flavescence dorée phytoplasmas (elm yellows group: 16SrV‐C subgroup) were present only in the Župa Aleksandrovac region; Bois noir phytoplasmas (stolbur group: 16SrXII‐A subgroup) were detected in the other surveyed regions; a mixed infection of European stone fruit yellows (apple proliferation group: 16SrX‐B subgroup) and Bois noir phytoplasmas was identified in one sample. A finer molecular characterization by RFLP analysis of rpS3 and SecY genes of Flavescence dorée phytoplasmas from Župa Aleksandrovac confirmed that the Serbian genotype is indistinguishable from a strain from the Veneto region, Italy. Characterization of the tuf gene of Bois noir phytoplasmas showed lack of amplification of samples from Erdevik. HpaII profiles of tuf gene PCR products of samples from Pali and Radmilovac were identical, and were indistinguishable from one of the two profiles produced by samples from Italian grapevines used as reference strains.     

A novel clinical syndrome and detection of Anaplasma ovis in Mongolian reindeer (Rangifer tarandus)

The Tsaatan (or Dhuka) peoples of northern-western Mongolia are one of the few remaining reindeer-herding cultural groups in the world. Recently a disease condition that involves sudden death of reindeer and cases involving fever, lethargy, and pale mucous membranes has been reported. Examination of blood smears collected in the 2005 field season resulted in the identification of intra-erythrocytic inclusions resembling Anaplasma spp. in smears from clinically sick animals. Using universal polymerase chain reaction (PCR) primers for the amplification of the 60 kDa chaperonin gene (cpn60, also known as hsp60 or groEL), we detected sequences corresponding to Anaplasma ovis in reindeer blood samples. Species-specific PCR primers for A. ovis were designed and validated and used to screen blood samples from Mongolian reindeer. Screening of 66 blood samples collected in the 2006 field season resulted in the detection of A. ovis in 80% of the samples. Our results indicate a high prevalence of A. ovis in the Tsaatan reindeer herds and an association with clinical disease that is likely to be anaplasmosis. To our knowledge this is the first report of natural A. ovis infection in reindeer.     

Development of a PCR-based method for diagnosing Mycoplasma pneumoniae infections

The polymerase chain reaction was used to detect clinical samples of Mycoplasma pneumoniae. A 245-bp region of the cytoadhesin P1 gene was shown to be specifically amplified in Myc. pneumoniae , but not in other species of Mollicutes. Picogram amounts of Myc. pneumoniae DNA could be detected per ml blood serum by use of a simple and reliable protocol for sample preparation and a PCR reaction involving two rounds of amplification. Application of the PCR-based method for the detection of Myc. pneumoniae in serum samples and throat swabs from patients with atypical pneumonia showed that it could be used in clinical diagnosis.     

Detection of Banana streak virus in field samples of bananas from Uganda

Banana streak virus (BSV) is one of the major constraints to banana production in Uganda. To develop a diagnostic technique, 59 samples were taken from 30 farms at 14 locations across Uganda; a further three samples were taken from infector plants for BSV epidemiology experiments. BSV was found in 51 of the field samples and in the three infector plants. The possible variation of the virus was assessed by serology (ISEM and ELISA) using a broad‐spectrum antiserum and by PCR. Virus was poorly detected in many of the samples by serological tests even though other techniques showed its presence. Virus was detected in most samples by PCR with a degenerate primer set on extracted viral DNA and on immune‐captured (1C) or directly bound (DB) virus particles. The epidemiology experiment samples did not give a product with these degenerate primers but did with other primer sets. A diagnostic procedure was developed involving concentrating the virus in sap by polyethylene glycol precipitation followed by 1C‐ or DB‐PCR using a degenerate primer set which detected virus in most samples.     

猕猴结核分枝杆菌PCR检测方法的建立与初步应用

目的建立猕猴结核分枝杆菌PCR快速检测方法。方法根据GenBank中报道的人型结核分枝杆菌标准株H37RV全基因序列,针对其中致病性结核分枝杆菌所特有的保守区域ESAT-6设计一对引物进行TouchdownPCR反应,利用此方法对100份野生来源猕猴全血标本进行检测,并与传统的PPD实验和咽拭子抗酸染色实验结果作比对。结果抽取33份野生来源猕猴的外周血,提取DNA进行扩增,扩增片段经纯化、回收克隆测序后用BLAST软件进行同源性对比,与GenBank中报道的序列基本相同,检测标本中有4份（4／33）为阳性,其中3份（3／33）标本与结核菌素试验和咽拭子抗酸染色试验结果相符合。结论建立了从猴全血中直接检测猴结核分枝杆菌DNA的TouchdownPCR方法,具有敏感性和特异性高,且简便的优点。     

Relative Sensitivity of Conventional and Real-Time PCR Assays for Detection of SFG Rickettsia in Blood and Tissue Samples from Laboratory Animals

Studies on the natural transmission cycles of zoonotic pathogens and the reservoir competence of vertebrate hosts require methods for reliable diagnosis of infection in wild and laboratory animals. Several PCR-based applications have been developed for detection of infections caused by Spotted Fever group Rickettsia spp. in a variety of animal tissues. These assays are being widely used by researchers, but they differ in their sensitivity and reliability. We compared the sensitivity of five previously published conventional PCR assays and one SYBR green-based real-time PCR assay for the detection of rickettsial DNA in blood and tissue samples from Rickettsia- infected laboratory animals (n = 87). The real-time PCR, which detected rickettsial DNA in 37.9% of samples, was the most sensitive. The next best were the semi-nested ompA assay and rpoB conventional PCR, which detected as positive 18.4% and 14.9% samples respectively. Conventional assays targeting ompB, gltA and hrtA genes have been the least sensitive. Therefore, we recommend the SYBR green-based real-time PCR as a tool for the detection of rickettsial DNA in animal samples due to its higher sensitivity when compared to more traditional assays.     

肺癌循环肿瘤细胞的单细胞EGFR基因突变检测

循环肿瘤细胞（Circulating tumor cells,CTCs）是从肿瘤原发病灶脱落并侵入外周血循环的肿瘤细胞。由于CTCs存在较大的异质性,其与癌症发展转移密切相关,但目前尚缺乏有效的CTCs单细胞异质性检测方法。鉴于此,本文发展了在单细胞层面对CTCs进行基因突变的检测方法并用于单个肺癌CTC的EGFR（Epidermal growth factor receptor）基因突变检测。首先用集成式微流控系统完成血液中稀有CTCs的捕获,接着将CTCs释放入含有多个微孔的微阵列芯片中,得到含有单个CTC的微孔,通过显微操作将单个CTC转入PCR管内完成单细胞基因组的放大,并进行单细胞的EGFR基因突变检测。以非小细胞肺癌细胞系A549、NCI-H1650和NCI-H1975为样本,通过芯片与毛细管修饰、引物扩增条件（复性温度、循环次数）的优化,结果显示在复性温度59℃、30个循环次数的条件下,引物扩增效果最优。利用该方法成功地对非小细胞肺癌（Non-small cell lung cancer, NSCLC）患者的血液样本进行了测试。从患者2 mL血液中获取5个CTCs,分别对其EGFR基因的第18、19、20、21外显子进行测序,发现该患者CTCs均为EGFR野生型。研究结果证明此检测方法可以灵敏地用于单个CTC基因突变的检测,在临床研究上具有重要的指导意义。     

Long-term dynamics of Sin Nombre viral RNA and antibody in deer mice in Montana

Infections with hantaviruses in the natural host rodent may result in persistent, asymptomatic infections involving shedding of virus into the environment. Laboratory studies have partially characterized the acute and persistent infection by Sin Nombre virus (SNV) in its natural host, the deer mouse (Peromyscus maniculatus). However, these studies have posed questions that may best be addressed using longitudinal studies involving sequential sampling of individual wild-caught, naturally infected mice. Using enzyme immunoassay and polymerase chain reaction (PCR) analysis of monthly blood samples, we followed the infection status of deer mice in a mark-recapture study in Montana for 2 yr. Only six of 907 samples without IgG antibody to SNV contained detectable SNV RNA, suggesting that there is a very brief period of viremia before the host develops detectable antibody. The simultaneous presence of both antibody and viral RNA in blood was detected in consecutive monthly samples for as long as 3 mo. However, chronic infection was typified by alternating characteristics of PCR positivity and PCR negativity. Two possible interpretations of these results are that 1) viral RNA may be consistently present in the blood of chronically infected deer mouse, but that viral RNA is near the limits of PCR detectability or 2) SNV RNA sporadically appears in blood as a consequence of unknown physiological events. The occurrence of seasonal patterns in the proportion of samples that contains antibody and that also contained SNV RNA demonstrated a temporal association between recent infection (antibody acquisition) and presence of viral RNA in blood.     

Molecular investigation of the distribution, abundance and diversity of the genus Pseudoalteromonas in marine samples

The genus Pseudoalteromonas has attracted interest because it has frequently been found in association with eukaryotic hosts, and because many Pseudoalteromonas species produce biologically active compounds. One distinct group of Pseudoalteromonas species is the antifouling subgroup containing Pseudoalteromonas tunicata and Ps. ulvae, which both produce extracellular compounds that inhibit growth and colonization by different marine organisms. PCR primers targeting the 16S rRNA gene of the genus Pseudoalteromonas and the antifouling subgroup were developed and applied in this study. Real-time quantitative PCR (qPCR) was applied to determine the relative bacterial abundance of the genus and the antifouling subgroup, and denaturing gradient gel electrophoresis (DGGE) was applied to study the diversity of the genus in 11 different types of marine samples from Danish coastal waters. The detection of Ps. tunicata that contain the antifouling subgroup was achieved through specific PCR amplification of the antibacterial protein gene (alpP). The Pseudoalteromonas species accounted for 1.6% of the total bacterial abundance across all samples. The Pseudoalteromonas diversity on the three unfouled marine organisms Ciona intestinalis, Ulva lactuca and Ulvaria fusca was found to be low, and Ps. tunicata was only detected on these three hosts, which all contain accessible cellulose polymers in their cell walls.     

A polymerase chain reaction for the detection of Brucella canis in semen of naturally infected dogs

The objective was to evaluate a PCR assay for the detection of Brucella canis in canine semen, comparing its performance with that of bacterial isolation, serological tests and PCR assay of blood. Fifty-two male dogs were examined clinically to detect reproductive abnormalities and their serum was tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR assays were performed on blood and semen samples. The findings of the semen PCR were compared (Kappa coefficient and McNemar test) to those of blood PCR, culture of blood and semen, RSAT, and 2ME-RSAT. Nucleic acid extracts from semen collected from dogs not infected with B. canis were spiked with decreasing amounts of B. canis RM6/66 DNA and the resulting samples subjected to PCR. In addition, semen samples of non-infected dogs were spiked with decreasing amounts of B. canis CFU and the resulting suspensions were used for DNA extraction and amplification. Of the 52 dogs that were examined, the following tests were positive: RSAT, 16 (30.7%); 2ME-RSAT, 5 (9.6%); blood culture, 14 (26.9%); semen culture, 11 (21.1%); blood PCR, 18 (34.6%); semen PCR, 18 (34.6%). The PCR assay detected as few as 3.8 fg of B. canis DNA experimentally diluted in 444.9 ng of canine DNA (extracted from semen samples of non-infected dogs). In addition, the PCR assay amplified B. canis genetic sequences from semen samples containing as little as 1.0 x 10(0) cfu/mL. We concluded that PCR assay of semen was a good candidate as a confirmatory test for the diagnosis of brucellosis in dogs; its diagnostic performance was similar to blood culture or blood PCR. Furthermore, the PCR assay of semen was more sensitive than the 2ME-RSAT or semen culture. Examination of semen by PCR should be included for diagnosis of brucellosis prior to natural mating or AI; in that regard, some dogs that were negative on serological and microbiological examinations as well as blood PCR were positive on PCR of semen.     

Identification of pathogenic bacteria in blood cultures: Comparison between conventional and PCR methods

Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Acinetobacter baumanii, and Klebsiella pneumoniae were found to be the most prevalent bacteremia-causing bacteria in a survey in a medical center. A PCR method for identification of these five most common pathogens in blood cultures was developed. A unique sequence was chosen for each pathogen and used for primer design. Sixty-one blood samples (from hospitalized patients) in which bacterial growth was detected were processed in parallel by conventional microbiological methods and by the PCR method. The results obtained by PCR were identical to those obtained by conventional methods in 93.4% of the cases. PCR failed to identify bacteria which were found conventionally in only 6.6% of the cases (mostly bacteria not included in the PCR cassette). Another group of eighty-eight blood samples from patients were processed immediately upon their arrival at the laboratory by taking aliquots for the PCR method. The blood sample bottles were processed in parallel by conventional methods. In 78.4% of the cases the results of both methods were identical. In 12.5% of the cases, PCR afforded identification of bacteria but conventional methods showed no bacteria in the sample. On the other hand, PCR afforded 9.1% negative results while conventional methods identified bacteria not included in the PCR cassette. It is concluded that the molecular method appears to be a specific and precise method for identifying pathogenic bacteria in blood samples.     

The effect of hyperbaric oxygenation on thein vitro growth ofEscherichia coli in environments with and without blood cells

The aim of this study was to investigate whether thein vitro presence of blood cells influences the anti-microbial activity of hyperbaric oxygen (HBO) againstEscherichia coli. FiftyE. coli isolates from clinical samples were used in the study. A small number of colonies belonging to each isolate from the nutrient media were transferred into two K₃EDTA tubes (the blood group) and two Mueller-Hinton broth tubes (the broth group). Then, both groups were divided into subgroups according to whether HBO was administered (HBO subgroup) or not (non-HBO subgroup). HBO treatment was applied for one hour at 2.5 absolute atmospheres. The tubes in the non-HBO subgroup were left at room temperature during this period. Subsequently, all the tubes were cultured on Mueller-Hinton and Eosin Methylene Blue agar using the quantitative counting technique. After 18 to 24 h incubation at 37 °C, the colonies formed in the plates were counted. In the blood group, compared with non-HBO subgroup samples, the number of colonies decreased in 56% of samples, increased in 32% of samples and did not change in 12% of samples in the HBO subgroup. Whereas, in the broth group the number of colonies decreased in only 32% of samples increased in 38% of samples and did not change in 30% of samples in the HBO subgroup compared with the non-HBO subgroup. The difference between the blood and the broth groups revealed a statistical significance using Pearson’s Chi-square test (P=0.025). We concluded that the antibacterial effect of HBO onE. coli increases in the cellular environment belonging to the host organism.