Usefulness and efficiency of formalin-fixed paraffin-embedded specimens from laryngeal squamous cell carcinoma in HPV detection by IHC and PCR/DEIA

The use of formalin-fixed paraffin-embedded (FFPE) tissues for HPV DNA detection by PCR from biopsy materials is not entirely clear in retrospective studies. The aim of our study was to evaluate the usefulness and efficiency of FFPE tissues from laryngeal cancer (LSCC) in HPV detection by immunohistochemistry reaction (IHC) and PCR-DNA enzyme immunoassay method (PCR/DEIA) and to compare with HPV detection from DFT. HPV-DNA was amplified from 54 FFPE tissues from LSCC specimens by the short PCR fragment (SPF10) primer set using PCR/DNA method and monoclonal anti Human Papillomavirus antibodies in IHC. In the same patients 54 specimens were collected and immediately deep-frozen and stored at (-70°C) to (-80°C). All the FFPE and deep-frozen tissue (DFT) specimens were positive for β-globin amplification. HPV was detected by two methods (SPF10 PCR/DEIA and IHC) in 14 (25.92%) out of 54 specimens from FFPE. Significant differences were found between the HPV detection using PCR/DEIA method and IHC method in FFPE tissues. The comparative analysis of the 54 samples after assuming PCR method in FFPE tissues showed accuracy of 92.6%, sensitivity of 90.5% and specificity of 93.9%. The FFPE tissues method has high sensitivity, specificity and accuracy when used to detect HPV DNA by PCR reaction and it is comparable to DFT results. DNA quality of FFPE samples is adequate and it can be used in HPV-DNA detection and in retrospective studies on LSCC.     

'Real-time' PCR-based detection of Coxiella burnetii using conventional techniques

The diagnosis of Q fever (Coxiella burnetii infection) relies primarily on the serological detection of specific antibodies. Recently, PCR-based methods have been introduced in diagnostic laboratories. Unfortunately, the fastest and most reliable 'real-time' detection method, which employs the 'online' detection of target nucleotide sequences while the amplification process is still in progress, requires expensive devices and consumables. In this study, we present a simple method that combines the simplicity of conventional PCR with new technical and methodical enhancements, resulting in a fast, specific and easy method for the molecular detection of C. burnetii. A collection of C. burnetii reference strains was tested with the modified conventional gel-based PCR approach applying a particluar PCR buffer (QIAGEN(?) Fast Cycling PCR kit) and using a closed ready-to-use gel-cassette-system (FlashGel(?)) for the visualization of specific PCR products. The modified conventional PCR method reached nearly the speed of the LightCycler(?) HybProbe real-time PCR assay (120 vs. 90 min) and showed equal sensitivity and specificity. The general cost per PCR run was 25% less than that for the LightCycler method. These improvements make this method suitable for small laboratories with limited resources and for deployable PCR diagnostics in field laboratories.     

PCR MP (PCR melting profile) technique--possibilities of application in epidemiological studies

The performance and convenience of a PCR melting profile (PCR MP) technique based on using low denaturation temperatures during ligation mediated PCR (LM PCR) of bacterial DNA is shown. We found that PCR MP technique is a rapid method that offers good discriminatory power, excellent reproducibility and may be applied for epidemiological studies. Results from strain genotyping illustrate that PCR MP is useful for the study of intraspecific genetic relatedness of strains and is as effective in discriminating closely related strains as the PFGE method, which is currently considered to be the gold standard for epidemiological studies. The usefulness of the PCR MP for molecular typing was shown for clinical strains of Escherichia coli, Enterococcus faecium VRE and Stapylococcus aureus.     

Nested double PCR法对献血者血浆中HCV-RNA的检出——兼评作为献血筛选试验的Anti-HCV抗体检查

本文报道了在位于5’端非编码区的引物的诱导下用Nested double PCR技术检测献血者血浆中HCV-RNA的方法。我们发现Nested double PCR敏感性及特异性均优于单次PCR。anti-HCV(C100-3)阳性血浆样品中只有35.2％(19/54)能同时被证实为PCR阳性。由于anti-HCV(C100-3)假阳性率太高,作为献血筛选试验检测方法不能令人满意,而本文报导的Nestel double PCR方法可以弥补anti-HCV试验的不足。     

Bacterial etiology of otitis media with effusion; focusing on the high positivity of Alloiococcus otitidis

The etiology of otitis media with effusion (OME) is unclear. The bacterial analyses of middle ear effusion (MEE) in OME may reveal important information regarding its etiology. Alloiococcus otitidis, Heamophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis were investigated by using microbiologic culture and a multiplex PCR method in the middle ear fluid of 32 children (54 samples) with chronic OME. PCR yielded positive results in 18 (33.3%) middle ear effusions while culture resulted positive for 3 (5.6%). The PCR method detected A. otitidis in 10 (18.5%) specimens, H. influenzae in 7 (13%), M. catarrhalis in 4 (7.4%) and S. pneumoniae in 2 (3.7%) specimens. The multiplex PCR method enhances the detection rate significantly compared to that of the conventional culture method. A. otitidis is the most common detected pathogen in the MEE of the OME.     

PCR技术对人IL-3 cDNA进行定点突变的研究

本文利用PCR技术对人IL-3cDNA体外进行定点突变,将人IL-3cDNA第3位Met,第116位Lys密码子突变为Val密码子GTT。PCR扩增片段核苷酸序列与引物设计相应的cDNA突变体序列完全一致。结果证实此方法比经典寡聚核苷酸方法简单、迅速、成本低、效率高,也为基因的修饰,蛋白质工程研究提供了简便、稳定的方法。     

Evaluation of mass spectrometric techniques for charaterization of engineered proteins

A simple and versatile method of in vitro site-specific mutagenesis based on polymerase chain reaction (PCR) is described. The complete method required the use of three oligonucleotide primers and two PCRs. The product of the first PCR was used as one of the primers (megaprimer) in the second PCR. Essentially 100% of the final product incorporated the desired mutation. The various aspects of the procedure and its application is described in detail.     

Rapid detection and quantification of five periodontopathic bacteria by real-time PCR

The quantity of periodontopathic bacteria in plaque samples is an important determinant for understanding the etiologic role of bacteria. The real-time PCR method was used to detect and quantify periodontopathic bacteria, such as Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Porphyromonas gingivalis, Treponema denticola, and Treponema socranskii, in saliva and subgingival plaque samples. There was good agreement between the results of conventional PCR and real-time PCR methods. Using the LightCycler system we were able to determine the amount of periodontopathic bacteria within an hour. The real-time PCR method was linear for samples containing from 10(3) to more than 10(8) cells (r2 = 0.999). The application of the real-time PCR method should be useful in the rapid detection and quantification of periodontopathic bacteria in clinical samples.     

Comparison of disk-diffusion method and PCR for detection of methicillin resistance in Staphylococcus aureus strains

The aim of the study was to compare the disk-diffusion (oxacillin 1 microg, cefoxitin 30 microg) method and PCR for detection of methicillin-resistance in S. aureus. The investigation were carried out on 120 S. aureus strains isolated from clinical materials of patients hospitalized in the University Hospital at the L. Rydygier Collegium Medicum in Bydgoszcz, University of Nicolaus Copernicus in Toruń. Of the 120 S. aureus strains tested, 60 (50%) were mecA-positive by PCR. Consistency of results between oxacillin disk-difussion method and PCR amounted 92.5% and cefoxitin disk-diffusion method and PCR--98.3%. The oxacillin disk-difussion method falsely identified 3 (2.5%) strains as MSSA (sensitivity 95.0%) and 4 strains as MRSA (specificity 93.3%) in comparison with PCR. The cefoxitin disk-diffusion method falsely identified 2 (1.6%) strains as MSSA (sensitivity 96.7%) and there were no false resistant results (specificity 100%). Our results showed that in disk-diffusion tests, cefoxitin is a better than oxacillin for the identification of MRSA.     

Multiplex SNP-SCALE: a cost-effective medium-throughput single nucleotide polymorphism genotyping method

We describe a convenient, cost-effective and flexible medium-throughput single nucleotide polymorphism (SNP) genotyping method, Multiplex SNP-SCALE, which enables the simultaneous amplification by polymerase chain reaction (PCR) of up to 25 (or potentially more) loci followed by electrophoresis in an automated DNA sequencer. We extended the original SNP-SCALE method to include (i) use of a commercial multiplex PCR kit, (ii) a four-dye system, (iii) much-reduced (2-μL) reaction volumes, (iv) drying down of template DNA before PCR, (v) use of pig-tailed primers, (vi) a PCR product weighting system, (vii) a standard optimized touchdown PCR thermocycling programme, and (viii) software (SNP-SCALE Primer Designer) that automatically designs suitable SNP-SCALE primers for a batch of loci. This new protocol was validated for different types of SNPs. The method is cost- and time-effective for medium-scale evolutionary and ecological projects involving 10s to 100s of loci.     

快捷的定点突变效率近100%的大引物PCR方法

报道了一种新的PCR突变方法,它不需要纯化大引物或设计特别的旁侧引物．利用一个诱变引物和两个测序引物(T_m≤58℃)作为旁侧引物．第一轮PCR产物12.5 μl直接加入到50 μl的第二轮PCR反应体系作为模板和大引物,在开始第二轮PCR反应时,增加在68℃退火温度下进行10个循环的不对称PCR,这一步骤大大提高了通过600 bp或800 bp大引物所导致的突变效率．结果表明,该方法的产物能够达到高保真、97%～98%的突变效率和高产率．     

转基因棉花MON88701品系特异性实时荧光PCR检测方法的建立

【目的】建立转基因棉花MON88701品系特异性实时荧光聚合酶链反应(polymerase chain reaction,PCR)检测方法。【方法】利用实时荧光PCR技术,根据转基因棉花MON88701品系特异性序列设计引物和探针,建立转基因棉花MON88701实时荧光PCR检测方法,并测定本方法的灵敏度、特异性及可重复性。【结果】建立的检测方法特异于转基因棉花MON88701成分的检测,灵敏度测试表明其定量下限为34拷贝;重复性试验显示其相对标准偏差在可接受范围内。【结论】本研究建立的转基因棉花MON88701品系特异性实时荧光PCR检测方法具有良好的特异性和高灵敏度,适合对转基因棉花MON88701品系进行检测。     

Random PCR-based screening for soluble domains using green fluorescent protein

A novel approach to screen soluble protein domains is presented, by combining tagged random primer polymerase chain reaction (PCR) method and protein-folding assay using green fluorescent protein. Tagged random primer PCR method was used to amplify random DNA fragments from a template cDNA. The PCR products were fused to the green fluorescent protein (GFP) gene. Then solubilities of their translation products were rapidly monitored by the fluorescence of transformed Escherichia coli colonies on plates. We succeeded in cloning four soluble domains from Vav protein using this method. The present method is applicable to all proteins when cDNAs are available.     

PCR技术在猴免疫缺陷病毒(SIV)感染模型中的应用

目的(1)建立RT PCR方法,定性测定SIV感染猴血浆中病毒RNA,比较其与传统血浆病毒分离方法的敏感性;(2)建立DNA PCR方法,检测SIV感染猴外周血淋巴细胞(PBMCs)中的前病毒DNA。(3)检验DNA PCR和RNA PCR方法在猴SAIDS模型应用中的实用性和可操作性。方法用SIVmac251静脉感染恒河猴,定期采血,从血浆中提取病毒RNA,以RNA为模板通过RT PCR法扩增,凝胶电泳定性;从感染猴PBMC中提取带有整合的SIV前病毒DNA的细胞基因组DNA,巢式PCR扩增,凝胶电泳定性。结果DNA PCR和RNA PCR经两轮扩增后均得到一长度为477bp的特异条带,测序鉴定确为目的片段。9只实验猴感染SIV后7d,RNA PCR结果为79阳性,DNA PCR结果为100%阳性,而血浆病毒分离只有59阳性;此后一直到感染后的42d,RNA PCR和DNA PCR的结果一直为100%阳性,而血浆病毒分离阳性率在感染后35d下降到49,到42d时下降为零。结论PCR方法比病毒分离方法的敏感性高。尤其是DNA PCR,既可检测具有活跃病毒复制的受感染细胞,又可检测那些携带病毒处于转录休眠期的细胞,所以在感染的早期和中后期———血浆病毒水平较低的情况下或病毒处于潜伏感染的阶段,它作为猴艾滋病(SAIDS)模型病毒学指标之一有其必要性和重要性。这个指标的检测方法应该是较血浆病毒RNA检测更为敏感。     

应用real-timePCR法快速定量人类粪便中双歧杆菌的研究

目的建立快速、准确从粪便标本中定量双歧杆菌的RT—PCR技术。方法传统培养定量法,普通PCR定量法,real—timePCR比较测量。结果（I）粪便标本前处理采取简单的离心和清洗、稀释步骤能去除粪便标本中的抑制物,实现不提取DNA直接进行PCR、real—time定量粪便中双歧杆菌。（2）本实验建立的PCR方法直接半定量粪便双歧杆菌技术在双歧杆菌值介于10^3～10^7CFU／ml时具有较好的分辨率,粪便标本普通PCR得理论菌数与培养得菌数值之间差异无显著性（P〉0．05）;real-timePCR直接定量双歧杆菌技术在双歧杆菌值介于10^1-10^7CFU／ml时具有较好的分辨率,粪便标本RT—PCR得理论菌数与培养得菌数值之间差异无显著性（P〉0．05）。结论利用PCR、real—timePCR直接半定量和定量粪便中的双歧杆菌可行。     

GA21转基因玉米实时荧光PCR检测方法的建立

成功建立了实时荧光PCR鉴定检测转基因玉米GA21品系的方法。该方法通过GA21玉米品系的OTPmEPSPS边界的270bp和133bp靶序列,设计品系特异性检测引物和探针,同时针对Pactin1mEPSPS边界的430bp靶序列设计品系特异性检测引物,应用实时荧光PCR和PCR技术,特异性检测GA21玉米品系。结果表明,应用实时荧光PCR的TaqMan探针技术检测转基因作物边界序列,不仅可以达到品系鉴定的目的,而且该方法和常规PCR比特异性强,简便快速,同时实验采用完全闭管检测,又降低了污染机会,为转基因作物的品系鉴定检测提供了新方法。     

Increased amplification efficiency of microchip-based PCR by dynamic surface passivation

Surface passivation is critical for effective PCR using silicon-glass chips. We tested a dynamic polymer-based surface passivation method. Polyethylene glycol 8000 (PEG 8000) or polyvinylpyrrolidone 40 (PVP-40) applied at 0.75% (w/v) in the reaction mixture produced significant surface passivation effects using either native or SiO2-precoated silicon-glass chips. PCR amplification was achieved from human genomic DNA as a template as well as from human lymphocytes. The dynamic surface passivation effect of PEG 8000 remained similar under both conditions. Dynamic surface passivation offers a simple and cost-effective method to make microfabricated silicon-glass chips PCR friendly. It can also be used in combination with static passivation (silicon oxide surface layer) to further improve PCR performance using silicon-glass PCR chips.     

A two-step quantitative pathogen detection system based on capillary electrophoresis

Rapid identification of bacterial pathogens is important for patient management and initiation of appropriate antibiotic therapy in the early stages of infection. Among the several techniques, capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis combined with small subunit rRNA gene-specific polymerase chain reaction (PCR) has come into the spotlight owing to its sensitivity, resolution, and reproducibility. Despite the advantages of the method, the design of PCR primers and optimization of multiplex PCR conditions remain to be studied so that as many pathogens as possible can be analyzed in a single run. Here we describe a novel two-step technique involving multiplex PCR pathogen detection by CE-SSCP analysis followed by singleplex PCR pathogen quantification by CE-SSCP. Specific PCR primers were designed for optimal separation of their products by CE-SSCP based on molecular weight. PCR conditions were then optimized for multiplex analysis of the targets. Subsequently, detected pathogens were quantified by PCR with specific primers. Eight clinically important strains were simultaneously identified under the optimized conditions. Each individual pathogen was then quantified at a level of sensitivity of tens of cells per milliliter. In conclusion, the two-step pathogen detection method based on CE-SSCP described here allows for sensitive detection of pathogens by multiplex PCR (first step) and quantification by specific PCR (second step). The results illustrate the potential of the method in clinical applications.     

The use of melting curves as a novel approach for validation of real-time PCR instruments

Validation of PCR thermal cycler performance is crucial in order to obtain reliable results. In this study, high resolution melting curve (HRM) analysis is presented as a novel validation method for real-time PCR instruments. By applying HRM analysis using a defined PCR amplicon and EvaGreen dye, information about the temperature accuracy and thermal homogeneity of the heating block was obtained. This pilot study shows the potential of our technique for temperature validation of real-time quantitative PCR thermal cyclers. Our data correlated well with the temperature accuracy data obtained from the Mobile Temperature Acquisition System (MTAS; r2 = 0.93), which conforms to the National Institute of Standards and Technology criteria, and our method was reproducible in independent runs (r2 = 0.95). The advantages of this HRM-based method include: (i) temperature measurement under real world conditions in the reaction liquid in closed reaction tubes; (ii) temperature measurement of all wells; and (iii) applicability to all real-time PCR instruments capable of HRM analysis.