Use of degenerate primers and touchdown PCR for construction of cDNA libraries

Optimized construction of low-redundancy cDNA mini-libraries using low-stringency RT-PCR is described cDNAs are generated using arbitrary consensus-degenerate hybrid oligonucleotide primers and nanogram amounts of Schistosoma mansoni mRNA. A number of conditions such as temperature and salt concentration are combined to create balanced, low-stringency conditions that permit a normalized amplification of a diversified, random set of sequences. On average, 350 different sequences are obtained per mini-library, which represents a significantly higher diversity of messages per library when compared to previously published conditions (ie., 20-40 sequences/ mini-library). The optimized high-throughput approach described here is likely to help in the discovery of expressed genes in any complex organism.     

Early detection of Schistosoma mansoni infection by touchdown PCR in a mouse model

A detection assay for Schistosoma mansoni DNA in mouse serum samples based on touchdown PCR was developed and evaluated. The serum samples could be assayed directly without the need to extract DNA. No cross reactions between S. mansoni and related species inducing human schistosomiasis were observed. After the infection, mouse sera and feces were collected for 8 weeks. Anti-worm antigen IgG and anti-soluble egg antigen IgG were detected in the sera at 6 weeks post-infection by ELISA. The parasite's eggs were detected in the feces at 8 weeks. In contrast, S. mansoni DNA was detected in the sera at 2 weeks post-infection. These data suggest that touchdown PCR is a potential tool for the early diagnosis of S. mansoni infection.     

Mapping transposon insertion sites by touchdown PCR and hybrid degenerate primers

A novel mapping method based on touchdown PCR was developed for identifying a transposon insertion site in genomic DNA using a hybrid consensus-degenerate primer in combination with a specific primer that anneals to the transposon. The method was tested using Xanthomonas citri transposon mutants. PCR products contained adjacent DNA regions that belonged to both X. citri genomic DNA and the transposon. Products were directly sequenced from PCRs using only the specific primer. Different PCR conditions were tested, and the optimized reaction parameters that increased product yields and specificity are described. Best results were obtained with the HIB17 hybrid primer, which is a 25-mer oligonucleotide having degenerate bases at 6 different positions within the last 12 bases at the 3' end. An X. citri mutants library was produced by random transposition using the EZ::TN transposon, and we identified the insertion sites within the genome of 90 mutants. Insertions were found within both the chromosomal and the plasmid DNA in these X. citri mutants. Restriction mapping and Southern blot analysis confirmed the insertion sites for eight randomly chosen mutants. This method is a very useful tool for large-scale characterization of mutants in functional genomics studies.     

Detection of mycoplasma contamination in cell cultures by a mycoplasma group-specific PCR.

The suitability of a 16S rRNA-based mycoplasma group-specific PCR for the detection of mycoplasma contamination in cell cultures was investigated. A total of 104 cell cultures were tested by using microbiological culture, DNA fluorochrome staining, DNA-rRNA hybridization, and PCR techniques. A comparison of the results obtained with these techniques revealed agreement for 95 cell cultures. Discrepant results, which were interpreted as false negative or false positive on the basis of a comparison with the results obtained with other methods, were observed with nine cell cultures. The microbiological culture technique produced false-negative results for four cell cultures. The hybridization technique produced false-negative results for two cell cultures, and for one of these cell cultures the DNA staining technique also produced a false-negative result. The PCR may have produced false-positive results for one cell culture. Ambiguous results were obtained with the remaining two cell cultures. Furthermore, the presence of contaminating bacteria interfered with the interpretation of the DNA staining results for 16 cell cultures. For the same reason the hybridization signals of nine cell cultures could not be interpreted. Our results demonstrate the drawbacks of each of the detection methods and the suitability of the PCR for the detection of mycoplasmas in cell cultures.     

A method for amplification of unknown flanking sequences based on touchdown PCR and suppression-PCR

Thermal asymmetric staggered PCR is the most widely used technique to obtain the flanking sequences. However, it has some limitations, including a low rate of positivity, and complex operation. In this study, a improved method of it was made based on suppression-PCR and touchdown PCR. The PCR fragment obtained by the amplification was used directly for sequencing after gel purification. Using this improved method, the positive rate of amplified flanking sequences of the ATMT mutants reached 99%. In addition, the time from DNA extraction to flanking sequence analysis was shortened to 2 days with about 6 dollars each sample.     

Competitive touchdown PCR for estimation of Escherichia coli DNA recovery in soil DNA extraction

Competitive approaches have shown promise for overcoming some of the difficulties in the use of PCR for assessment of specific bacterial species in soil. A competitive touchdown PCR (cTD-PCR) protocol specific for the rrsB gene of Escherichia coli was developed for tracking the organism in environments impacted by human wastes. Regression of product ratios from co-amplification of varying amounts of analyte and competitor DNA templates was linear. To test the robustness of the method, reactions were titrated with an extract of sterilized soil; no significant effect was detected. The cTD-PCR was used to assay recovery of E. coli DNA from soil. Stock DNA was spiked onto two sterilized soils during extraction, and the purified extracts were analyzed by cTD-PCR. Recovery of DNA spiked at a rate of 180 ng g(-1) was 34+/-7% (mean+/-S.D.) for an agricultural silt loam. DNA spiked at 1.8 pg g(-1) was recovered at a mean rate of 6.1+/-1.3%. DNA in these extracts was not directly quantifiable by image analysis. The cTD-PCR method provides a useful means of quantifying small amounts of E. coli DNA, and could be modified for other specific targets in a mixture of DNA from a variety of organisms.     

直接培养法检测支原体方法的建立

采用牛心消化液,加入支原体营养成份及琼脂,制备成固体培养基。以此培养基检测支原体,并根据其灵敏度及适用性确定培养基的配方。结果表明,用此法可以检测牛血清、胰酶、细胞及疫苗中的支原体生长,并且检测时间短,重复性好,灵敏度高,可以用于生物制品的质量控制。     

Validation of a real‐time PCR for Haemophilus parasuis

Aims: To validate a real‐time PCR test for the diagnosis of Glässer’s disease, a major pig disease caused by Haemophilus parasuis. Methods and Results: The specificity of a real‐time PCR amplifying the inf B gene was validated with 68 H. parasuis isolates and 36 strains of closely related species. As well, 239 samples of DNA from tissues and fluids of 16 experimentally challenged animals were tested with the real‐time PCR, and the results were compared with culture and a conventional PCR. The real‐time PCR produced significantly more positive results than the conventional PCR (165 vs 86). Conclusions: The sensitivity of the real‐time PCR combined with high specificity makes it a very valuable tool for the diagnosis of Glässer’s disease. Significance and Impact of Study: This new method will improve the ability of laboratories to diagnose Glässer’s disease, especially in laboratories where the culture method for H. parasuis is not optimal.     

The emerging international regulatory framework for biotechnology

Debate about the potential risks of genetically modified organisms (GMOs) to the environment or human health spurred attention to biosafety. Biosafety is associated with the safe use of GMOs and, more generally, with the introduction of non-indigenous species into natural or managed ecosystems. Biosafety regulation--the policies and procedures adopted to ensure the environmentally safe application of modern biotechnology--has been extensively discussed at various national and international forums. Much of the discussion has focused on developing guidelines, appropriate legal frameworks and, at the international level, a legally binding international biosafety protocol--the Cartagena Protocol on Biosafety. The Protocol is one among various international instruments and treaties that regulate specific aspects relevant to agricultural biotechnology. The present article presents the main international instruments relevant to biosafety regulation, and their key provisions. While international agreements and standards provide important guidance, they leave significant room for interpretation, and flexibility for countries implementing them. Implementation of biosafety at the national level has proven to be a major challenge, particularly in developing countries, and consequently the actual functioning of the international regulatory framework for biotechnology is still in a state of flux.     

Development of a novel PCR assay specific for Riemerella anatipestifer

AIMS: Riemerella anatipestifer is a significant pathogen of waterfowl and turkeys. Due to their similar ecology and morphological and cultural characteristics it is important to differentiate R. anatipestifer infections from those caused by Pasteurella multocida. Present study describes a novel PCR assay that is capable of rapid and species-specific identification of R. anatipestifer from bacterial cultures. METHODS AND RESULTS: An ERIC (enterobacterial repetitive intergenic consensus)-PCR fragment common to all tested isolates was used as a target for primer design. After optimization, the assay was tested on 72 R. anatipestifer strains isolated from clinical samples and identified using biochemical tests. All of these gave positive results, while heterologous pathogens, including different serotypes of P. multocida, proved to be negative. The assay was also capable of demonstrating R. anatipestifer directly from five clinical samples. CONCLUSIONS: The presented PCR is suitable for proper identification of R. anatipestifer from culture. Preliminary investigation showed that the test could be suitable for detection of the pathogen from clinical samples as well. SIGNIFICANCE AND IMPACT OF THE STUDY: The described PCR assay will improve the fast and proper identification of R. anatipestifer.     

Development and Validation of a HPV-32 Specific PCR Assay

Background

The present work aims at determining HCV genotypes in patients with chronic HCV infection, in Gaza strip, Palestine. The most common risk factors for HCV transmission were also evaluated in conjunction with the genotyping data.

Results

The study shows that there are only two major genotypes of HCV in Gaza Strip: Genotype 1 (subtypes 1a and 1b) collectively contribute to 28.3% of the cases, and genotype 4 (subtypes 4a and 4c/d) collectively contribute to 64.1% of the cases. Mixed infection with the two genotypes was also present among 7.6% of the cases. In this study a statistically significant relationship was established between the distribution of these genotypes and the patients' living place, traveling history, history of blood transfusion and history of surgical operations.

Conclusion

The present study is the first to link HCV genotyping in Gaza strip with its possible roots of transmission. Traveling to endemic countries, especially Egypt; blood transfusion and surgical operations are major roots of HCV infection in Gaza strip. The results indicate that iatrogenic and nosocomial procedures may be responsible for the majority of HCV infections in Gaza strip.     

Development and evaluation of a real-time quantitative PCR assay for Aspergillus flavus

Aspergillus flavus is a ubiquitous mold and the most common mold contaminating foodstuffs. Many strains of A. flavus produce aflatoxins. In addition it is an allergen and an opportunistic pathogen of animals and plants. A. flavus often is underestimated in traditional culture analyses due to the expertise required and the cost associated with speciating members of the genus Aspergillus. The goal of this study was to develop and validate a primer and probe set for the rapid detection and quantitation of A. flavus in pure culture using real-time quantitative polymerase chain reaction (QPCR) amplification. Unique DNA regions were located in the genome of the target organism by sequence comparison with the GenBank database, and several candidate oligonucleotides were identified from the scientific literature for potential use with the TaqMan QPCR technology. Three primer and probe sets were designed and validated for specificity and sensitivity in laboratory experiments. Initial screening to test for sensitivity was performed with seven A. flavus isolates and selected nontarget fungi. Specificity testing was conducted with the selected primer and probe set, which amplified all nine A. flavus isolates tested, including an aflatoxin producing strain. The primers did not amplify DNA extracted from 39 other fungal species (comprising 16 genera), including 18 other Aspergillus species and six Penicillium species. No amplification of human or bacterial DNA was observed; however cross-reactivity was observed with Aspergillus oryzae. PCR analysis of DNA dilutions and the use of an internal positive control demonstrated that 67% of the fungal DNA samples assayed contained PCR inhibitors. The assay validated for the target organism is capable of producing PCR results in less than 1 h after DNA extraction. The results of this research demonstrate the capabilities of QPCR for the enhanced detection and enumeration of fungi of significance to human health.     

Development and field testing of a real-time PCR assay for cylindrospermopsin-producing cyanobacteria

Aims: To develop and test a real-time PCR assay to detect and quantify genes specific to Cylindrospermopsis sp. and cylindrospermopsin-producing cyanobacteria. Method and Results: A duplex real-time PCR assay was developed that targets a cylindrospermopsin-specific and Cylindrospermopsis raciborskii-specific DNA sequence. The C. raciborskii-specific sequence was based on the rpoC1 DNA-dependent RNA polymerase gene, whilst the cylindrospermopsin-specific sequence was selected by surveying an extensive number of potential cylindrospermopsin-producing cyanobacterial strains for genes implicated in toxin production, aoaA, aoaB and aoaC. In toxic strains, sequences of each of these three genes were always present; whilst in nontoxic strains the distribution of these sequences was patchy, resulting in what are likely to be natural deletion mutants. The real-time assay was optimized on a fixed and portable device, with results indicating that the reliable limit of detection for the assay was 100 copies per reaction or 1000 cells ml⁻¹ for both target sequences on both devices. In routine environmental samples enumerated by microscopy, the assay results were positive for all samples where C. raciborskii cells were observed at >1000 cells ml⁻¹ and negative in 15 samples where no C. raciborskii cells were observed. In field samples, the number of copies of the rpoC1 sequence more closely approximated the number of cells enumerated by microscopy, the number of copies of the pks sequence and detection of the toxin-specific sequence matched the results of toxin testing. Conclusions: The duplex real-time PCR assay was a sensitive and rapid method for detecting potential cylindrospermopsin-producing cyanobacteria in the laboratory or in the field. The observation of probable natural deletion mutants provides further evidence that the aoaA, aoaB and aoaC genes are involved in toxin production. Significance and Impact of the Study: This assay provides a new monitoring capability for tracking cylindrospermopsin-producing cyanobacteria that are an emerging threat to water quality.     

Validation of a multiplexed and targeted lipidomics assay for accurate quantification of lipidomes

A major challenge of lipidomics is to determine and quantify the precise content of complex lipidomes to the exact lipid molecular species. Often, multiple methods are needed to achieve sufficient lipidomic coverage to make these determinations. Multiplexed targeted assays offer a practical alternative to enable quantitative lipidomics amenable to quality control standards within a scalable platform. Herein, we developed a multiplexed normal phase liquid chromatography-hydrophilic interaction chromatography multiple reaction monitoring method that quantifies lipid molecular species across over 20 lipid classes spanning wide polarities in a single 20-min run. Analytical challenges such as in-source fragmentation, isomer separations, and concentration dynamics were addressed to ensure confidence in selectivity, quantification, and reproducibility. Utilizing multiple MS/MS product ions per lipid species not only improved the confidence of lipid identification but also enabled the determination of relative abundances of positional isomers in samples. Lipid class-based calibration curves were applied to interpolate lipid concentrations and guide sample dilution. Analytical validation was performed following FDA Bioanalytical Method Validation Guidance for Industry. We report repeatable and robust quantitation of 900 lipid species measured in NIST-SRM-1950 plasma, with over 700 lipids achieving inter-assay variability below 25%. To demonstrate proof of concept for biomarker discovery, we analyzed plasma from mice treated with a glucosylceramide synthase inhibitor, benzoxazole 1. We observed expected reductions in glucosylceramide levels in treated animals but, more notably, identified novel lipid biomarker candidates from the plasma lipidome. These data highlight the utility of this qualified lipidomic platform for enabling biological discovery.     

Real-time PCR assay for quantitative mismatch detection

We describe here a quantitative real-time PCR assay for the detection of single-base-pair differences that does not require fluorescently labeled gene-specific probes or complicated primer combinations. Following PCR or RT-PCR of a gene segment that may contain allele-specific differences, 100 pg amplified product are used for a real-time PCR with allele-specific primers and SYBR Green. The use of HEPES buffer at a pH of 6.95 together with AmpliTaq DNA polymerase results in a threshold difference between the correct template and the mismatched template of as many as 20 cycles, depending on the mismatch. Correct matches can be detected in an excess of mismatched template at least at the 0.01 level for the six primer-template matches versus mismatches tested: GC vs. A.C, AT vs. G.T, GC vs. C.C, GC vs. G.G, AT vs. C.T, and GC vs. G.A. Because the initial amplification is separate from real-time detection, conditions can be independently optimized for each step, making the assay particularly suitable for the detection of allele-specific expression in single cells.