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。     

A PCR assay for the detection of Campylobacter jejuni and Campylobacter coli in water

A PCR assay has been developed for the detection of Campylobacter jejuni and Camp. coli in water samples. The sample is filtered through a membrane which is subjected to sonication to release the impacted cells. After removal of the filter from the cell suspension and a freeze/thaw cell lysis step, a semi-nested PCR is carried out on the filtrate using the primers CF02, CF03 and CF04 ( Camp. jejuni fla and flaB gene sequences). Incorporation of a sonication stage allows removal of the filter membrane since they have been shown to inhibit the PCR. In experiments with spiked water samples (20 ml) a theoretical sensitivity of 10–20 Campylobacter cells ml^-1 was achieved. Using a sample volume of 100 ml this sensitivity can be increased to approximately 2 Campylobacter cells ml^-1.     

双标准曲线相对定量PCR试验原理与方法

实时荧光定量PCR(FQ-PCR)是一种准确有效的核酸定量分析技术,具有易操作、高通量、高敏感性、高特异性、高度自动化和低污染等优点,并随新定量PCR仪及新操作方法的发展而得到广泛应用,但是,定量PCR的高敏感性特点使得实验操作严格而繁琐。阐述了一种改进的相对定量方法——双标准曲线法的试验原理和特点,描述了定量PCR体系的优化方式,探讨了试验误差分析方法及试验操作技巧,并就试验数据的处理方法进行讨论。试验证明,双标准曲线法是一种经济、简单而准确的定量方法。     

Clathrin senses membrane curvature

The ability of proteins to assemble at sites of high membrane curvature is essential to diverse membrane remodeling processes, including clathrin-mediated endocytosis. Multiple adaptor proteins within the clathrin pathway have been shown to sense regions of high membrane curvature, leading to local recruitment of the clathrin coat. Because clathrin triskelia do not bind to the membrane directly, it has remained unclear whether the clathrin coat plays an active role in sensing membrane curvature or is passively recruited by adaptor proteins. Using a synthetic tag to assemble clathrin directly on membrane surfaces, here we show that clathrin is a strong sensor of membrane curvature, comparable with previously studied adaptor proteins. Interestingly, this sensitivity arises from clathrin assembly rather than from the properties of unassembled triskelia, suggesting that triskelia have preferred angles of interaction, as predicted by earlier structural data. Furthermore, when clathrin is recruited by adaptors, its curvature sensitivity is amplified by 2- to 10-fold, such that the resulting protein complex is up to 100 times more likely to assemble on a highly curved surface compared with a flatter one. This exquisite sensitivity points to a synergistic relationship between the coat and its adaptor proteins, which enables clathrin to pinpoint sites of high membrane curvature, an essential step in ensuring robust membrane traffic. More broadly, these findings suggest that protein networks, rather than individual protein domains, are likely the most potent drivers of membrane curvature sensing.     

Rapid high resolution western blotting: from gel to image in a single day.

A streamlined protocol is described that allows high sensitivity antigen detection by Western blotting in a single day. The choice of membrane blotting matrix, as well as blocking reagents, has been optimized in order to allow rapid development of the blot with chemiluminescent reagents. The entire process, from gel to blot to a permanent, hard copy image on x-ray film, can be accomplished within six hours.     

Development and evaluation of real competitive PCR for high-throughput quantitative applications

Real competitive PCR (rcPCR) has been shown to have high sensitivity, reproducibility, and high-throughput potential. We describe further development and evaluation of this methodology as a tool for measuring nucleic acid abundance within a cell. Modifications to the original protocol allow analysis of gene expression levels using standard conditions regardless of mRNA abundance and assay type, thereby increasing throughput and ease of reaction setup while decreasing optimization time. In addition, we have developed a software package, TITAN, to automatically analyze the results. The details are relevant to researchers performing competitive PCR using any detection technique. The effectiveness of the described developments is demonstrated using 12 genes known to have differential expression in cell lines grown under normal and hypoxic conditions. Quantitative and qualitative comparisons to real-time PCR are presented. It is also demonstrated that the technique is capable of detecting submicroscopic chromosomal DNA deletions.     

基孔肯雅病毒和辛德毕斯病毒可视化基因芯片与荧光基因芯片的建立

根据GenBank中收录的基孔肯雅病毒和辛德毕斯病毒E蛋白基因序列,设计及筛选针对2种病毒的寡核苷酸探针及引物,制备基孔肯雅病毒与辛德毕斯病毒可视化基因芯片与荧光基因芯片,对芯片的灵敏性、特异性进行了验证,并将可视化基因芯片、荧光基因芯片进行灵敏性比较.结果显示,制备的两种基因芯片都能检测到基孔肯雅病毒和辛德毕斯病毒特异性杂交信号.可视化基因芯片、荧光基因芯片检测两种病毒质粒的灵敏度达到9.1×103 copies/mL, 6.8×101 copies/mL和9.1×104 copies/mL, 6.8×103 copies/mL,与普通PCR比较差异显著. 荧光基因芯片灵敏度是PCR方法的10倍,可视化基因芯片是荧光基因芯片灵敏度的100倍. 模拟病毒检测过程特异性检验证明,可视化基因芯片都具有良好的特异性.本试验建立了基孔肯雅病毒与辛德毕斯病毒两种特异的可视化和荧光基因芯片检测方法,两种方法灵敏度高、特异性强,适用于基孔肯雅病毒与辛德毕斯病毒的流行病学调查和种特异性鉴定.     

Improving Precision of Proximity Ligation Assay by Amplified Single Molecule Detection

Proximity ligation assay (PLA) has been proven to be a robust protein detection method. The technique is characterized by high sensitivity and specificity, but the assay precision is probably limited by the PCR readout. To investigate this potential limitation and to improve precision, we developed a digital proximity ligation assay for protein measurement in fluids based on amplified single molecule detection. The assay showed significant improvements in precision, and thereby also detection sensitivity, over the conventional real-time PCR readout.     

Molecular diagnosis of medical viruses

The diagnosis of infectious diseases has been revolutionized by the development of molecular techniques, foremost with the applications of the polymerase chain reaction (PCR). The achievable high sensitivity and ease with which the method can be used to detect any known genetic sequence have led to its wide application in the life sciences. More recently, real-time PCR assays have provided additional major contributions, with the inclusion of an additional fluorescent probe detection system resulting in an increase in sensitivity over conventional PCR, the ability to confirm the amplification product and to quantitate the target concentration. Further, nucleotide sequence analysis of the amplification products has facilitated epidemiological studies of infectious disease outbreaks, and the monitoring of treatment outcomes for infections, in particular with viruses which mutate at high frequency. This review discusses the applications of qualitative and quantitative real-time PCR, nested PCR, multiplex PCR, nucleotide sequence analysis of amplified products and quality assurance with nucleic acid testing (NAT) in diagnostic laboratories.     

Polymerase chain reaction-ELISA to detect Bacillus anthracis from soil samples—limitations of present published primers

A polymerase chain reaction (PCR)-ELISA technique to detect Bacillus anthracis from soil samples has been developed. The application of streptavidine-coated microtitre plates as well as plates covered with covalently linked oligonucleotides as catching probes led to a test sensitivity of about 100 fg pure genomic DNA or of less than 10 spores seeded into 100 g soil material. Some non-suspicious soil samples collected from different locations yielded positive results with presently published primers or probes targeting the B or C gene of pX02 and with primers targeting the chromosomal sequence B813. The former PCR products were sequenced. The number and mode of base exchanges led to the assumption that there will exist at least one unknown soil organism with high similarity within highly conserved capsule-encoding genes.     

产肠毒素大肠杆菌快速检测方法的建立和评价

目的利用环介导等温扩增(LAMP)技术,建立产肠毒素大肠杆菌(ETEC)的快速、便捷、敏感、特异的检测方法,并对该方法的特异性和敏感性进行评价,为实验动物检测和细菌性腹泻的诊断提供技术支持。方法根据GenBank公布的产肠毒素大肠杆菌的LT毒素基因序列(S60731.1)设计外引物和内引物进行LAMP扩增,对LAMP特异性和敏感性与PCR方法做比较。结果建立的LAMP方法检测最低浓度为100 pg/μL,灵敏度是PCR的10倍以上并具有较高的特异性,利用该方法对27份猴腹泻样品进行LAMP和PCR方法检测,发现PCR检出率为33.3%,LAMP(60 min内)结果与PCR相同,而LAMP(90 min内)检出率为92.6%,约是PCR检出率的3倍。结论建立了一种用于检测肠毒性大肠杆菌(ETEC)的LAMP检测方法,该方法特异性强,灵敏度高,方便快捷,适合于ETEC临床快速检测。     

Automated polymerase chain reaction product sample preparation for capillary electrophoresis analysis

The analysis of crude polymerase chain reaction (PCR) products by capillary electrophoresis (CE) is often compromised due to the presence of a high concentration of salt. Salt interferes with the electrokinetic injection and induces localized heating within the column; hence, PCR products must be desalted or cleaned-up prior to CE analysis. A variety of commercial clean-up systems are available that have been traditionally used to prepare PCR products for cloning, sequencing and digestion with restriction enzymes. These systems were tested for their effectiveness in preparing PCR products for CE analysis and were evaluated based on CE resolution, salt removal, DNA recovery, processing time and cost. One particularly effective clean-up system, membrane dialysis, was automated using a robotic workstation.     

A PCR-based semiquantitative assay of DNA impurities in recombinant protein preparations

A semiquantitative assay of DNA impurities in preparations of human recombinant insulin is described. The assay is based on the detection of a fragment of the ampicillin-resistant gene within the producer strain DNA by PCR. The analysis of PCR products of the studied preparations and PCR products containing known amounts of E. coli total DNA enabled a quantitative determination of the producer strain DNA content in the preparations under study. The sensitivity of the method is 7 pg of E. coli DNA per 10 microg of human recombinant insulin. The high sensitivity of the method allows us to recommend it for the quantitative determination of DNA content in recombinant preparations that do not inhibit PCR.     

Single-base mutational analysis of cancer and genetic diseases using membrane bound modified oligonucleotides.

A convenient format for the detection of PCR amplified sequences is the hybridization of the PCR products to oligonucleotide probes which are immobilized on a solid phase. We describe a new method for site-specific attachment of such probe oligonucleotides to nylon membranes. The method is based on the formation of an amide bond between carboxyl groups present on the membranes and amino-linkers situated on the 5' end of the oligonucleotides. The covalent attachment is via a carbodiimide mediated condensation. The single, 5' end attachment of the oligonucleotides to the membrane surface leaves the probe free to interact with complementary sequences, thus increasing the hybridization efficiency relative to methods where heat or ultraviolet light is used for non-specific fixation. Using biotinylated PCR products in hybridization reactions along with a non-radioactive chemiluminescent detection system, high efficiency hybridization is obtained as well as a very good signal to noise ratio. The method has been applied successfully to the detection of RAS point mutations, cystic fibrosis deletion and point mutations and others. The sensitivity, simplicity and reproducibility of this method make it an ideal tool for the diagnosis of infectious and genetic diseases, as well as analysis of mutations in neoplasias, HLA typing and other areas.     

Conversion-specific detection of DNA methylation using real-time polymerase chain reaction (ConLight-MSP) to avoid false positives

Methylated cytosines appear as sequence variations following bisulfite treatment and polymerase chain reaction (PCR) amplification. By using methylation-specific PCR (MSP), it is possible to detect methylated sequences in a background of unmethylated DNA with a high level of sensitivity. MSP is frequently used to identify methylated alleles in carcinogenesis, and may be combined with the TaqMan real-time PCR system, which uses fluorescence-based detection of amplification products during the amplification phase of the PCR and increases the sensitivity of detection (MethyLight). Sequences that have been incompletely converted during the bisulfite treatment are frequently coamplified during MSP, resulting in an overestimation of DNA methylation. The presence of amplified sequences originating from partially unconverted material may be determined by sequencing or by restriction digests or Southern blots of MSPs. Alternately, we have developed a method where the PCR and conversion assay are combined within a single TaqMan reaction by using an additional fluorescent probe directed against unconverted DNA (ConLight-MSP). We recommend that MSP detection always should include a step to detect unconverted DNA to avoid overestimation of the frequency or level of methylated DNA in the sample.     

Filipin-sterol complexes in molluscan gill ciliated epithelial cell membranes: intercalation into ciliary necklaces and induction of gap junctional particle arrays

Summary Freeze-fracture electron microscopy has been used in conjunction with the antibiotic filipin to investigate possible differences in the distribution of sterols in ciliary and somatic cell membranes of scallop and mussel gill epithelial cells. Contrary to previous reports, we find that filipin-sterol lesions can occur among the strands of the ciliary necklace but they are partially excluded from the smooth neck region above the necklace where the membrane is tightly apposed to the axonemal microtubules. No obvious differences in filipin-sterol lesions occur in the membranes of mussel gill cilia of varying mechanical sensitivity. Although abundant in the apical plasma membrane, filipin-sterol complexes are rare within the membranes of microvilli. Filipin-sterol lesions form outside the loosely parallel particle strands of septate junctions, sometimes increasing their relative orderliness. At sufficiently high density, filipin-sterol protrusions within the plasma membrane result in mass aggregation of gap junctions, possibly through recruitment of unorganized connexons.     

Optimized adaptor polymerase chain reaction method for efficient genomic walking

Genomic walking is one of the most useful approaches in genome-related research. Three kinds of PCR-based methods are available for this purpose. However, none of them has been generally applied because they are either insensitive or inefficient. Here we present an efficient PCR protocol, an optimized adaptor PCR method for genomic walking. Using a combination of a touchdown PCR program and a special adaptor, the optimized adaptor PCR protocol achieves high sensitivity with low background noise. By applying this protocol, the insertion sites of a gene trap mouse line and two gene promoters from the incompletely sequenced Xenopus laevis genome were successfully identified with high efficiency. The general application of this protocol in genomic walking was promising.     

High-sensitivity quantitative PCR platform

Real-time PCR methods have become widely used within the past few years. However, real-time PCR is rarely used to study chronic diseases with low pathogen loads, presumably because of insufficient sensitivity. In this report, we developed an integrated nucleic acid isolation and real-time PCR platform that vastly improved the sensitivity of the quantitative detection of the intracellular bacterium, Chlamydia spp., by fluorescence resonance energy transfer real-time PCR. Determinants of the overall detection sensitivity were analyzed by extracting nucleic acids from bovine milk specimens spiked with low amounts of chlamydial organisms. Nucleic acids were optimally preserved and recovered by collection in guanidinium stabilization buffer, binding to a matrix of glass fiber fleece, and elution in low volume. Step-down thermal cycling and an excess of hot-start Taq polymerase vastly improved the robustness and sensitivity of the real-time PCR while essentially maintaining 100% specificity. The amplification of Chlamydia 23S rRNA allowed for the differentiation of chlamydial species and was more robust at low target numbers than amplification of the omp1 gene. The best combined method detected single targets per a 100-microL specimen equivalent in a 5-microL real-time PCR input. In an initial application, this high-sensitivity real-time PCR platform demonstrated a high prevalence of chlamydial infection in cattle.     

A new fluorescent pH probe for imaging lysosomes in living cells

A new rhodamine B-based pH fluorescent probe has been synthesized and characterized. The probe responds to acidic pH with short response time, high selectivity and sensitivity, and exhibits a more than 20-fold increase in fluorescence intensity within the pH range of 7.5–4.1 with the pK_a value of 5.72, which is valuable to study acidic organelles in living cells. Also, it has been successfully applied to HeLa cells, for its low cytotoxicity, brilliant photostability, good membrane permeability and no ‘alkalizing effect’ on lysosomes. The results demonstrate that this probe is a lysosome-specific probe, which can selectively stain lysosomes and monitor lysosomal pH changes in living cells.     

A real-time PCR assay for quantifying Plasmodium falciparum infections in the mosquito vector

Transmission-blocking vaccines prevent the development of Plasmodium parasite within the mosquito vector, thereby thwarting the spread of malaria through a community. The gold standard for determining the efficacy of a transmission-blocking vaccine is the standard membrane feeding assay. This assay requires the dissection of mosquitoes and microscopic counting of oocysts present on the mosquito mid-gut, typically at 7-10 days p.i. Here we describe a real-time quantitative PCR assay that is rapid, target-specific and robust, with a sensitive detection threshold and which may be employed earlier p.i. than the standard membrane feeding assay and is applicable to preserved material. The real-time PCR assay utilises the LightCycler platform and SYBR Green I detection system to amplify 180 bp of the asexual form of the Plasmodium falciparum rRNA gene. It has a quantitative range of greater than four orders of magnitude and a detection threshold of 10 parasites. Validation experiments using a monoclonal antibody of known blocking activity revealed the real-time PCR assay to give equivalent results to the standard membrane feeding assay. In addition, the PCR assay can establish the effect of such a monoclonal antibody on the parasites' development within the oocyst and on the sporozoite (the transmissible stage) yield, providing a more pertinent assessment of transmission blocking activity than is possible by the standard membrane feeding assay. This assay may also be employed to monitor the sporogonic development of P. falciparum parasites within the mosquito vector.