Capillary Electrophoresis-based Single-strand Conformation Polymorphism Research Articles

Multiplex quantitative analysis of microRNA expression via exponential isothermal amplification and conformation-sensitive DNA separation

Expression profiling of multiple microRNAs (miRNAs) generally provides valuable information for understanding various biological processes. Thus, it is necessary to develop a sensitive and accurate miRNA assay suitable for multiplexing. Isothermal exponential amplification reaction (EXPAR) has received significant interest as an miRNA analysis method because of high amplification efficiency. However, EXPAR cannot be used for a broader range of applications owing to limitations such as complexity of probe design and lack of proper detection method for multiplex analysis. Here, we developed a sensitive and accurate multiplex miRNA profiling method using modified isothermal EXPAR combined with high-resolution capillary electrophoresis-based single-strand conformation polymorphism (CE-SSCP). To increase target miRNA specificity, a stem-loop probe was introduced instead of a linear probe in isothermal EXPAR to allow specific amplification of multiple miRNAs with minimal background signals. CE-SSCP, a conformation-dependent separation method, was used for detection. Since CE-SSCP eliminates the need for probes to have different lengths, easier designing of probes with uniform amplification efficiency was possible. Eight small RNAs comprising six miRNAs involved in Caenorhabditis elegans development and two controls were analyzed. The expression patterns obtained using our method were concordant with those reported in previous studies, thereby supporting the proposed method’s robustness and utility.

Accurate and effective multidrug-resistant Mycobacterium tuberculosis detection method using gap-filling ligation coupled with high-resolution capillary electrophoresis-based single strand conformation polymorphism

Tuberculosis (TB) has severely threatened public health via emerging multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (MTB) strains. For effective TB treatment, rapid, accurate, and multiplex detection of drug resistance is extremely important. However, conventional methods for TB diagnosis are time consuming and have a limited effect on treatment. Nucleic acid-based molecular detection methods have been developed as an effective MDR/XDR-TB diagnosis technology. Among the nucleic acid-based methods, ligation-dependent methods are attractive as MDR/XDR-MTB detection technologies, but multiplex analysis is limited by the detection method. Although an electrophoresis-based method is considered for multiple target detection because it is free from the errors pertaining to hybridization-based systems, the procedure of multiplex analysis is quite complicated owing to the DNA size-based separation system. In this study, we report an accurate, rapid, and simple multiple MDR/XDR-MTB detection technology using gap-filling ligation reaction coupled with high-resolution capillary electrophoresis-based single-strand conformation polymorphism. Using this system, rapid and accurate MDR/XDR-MTB detection is feasible via similar length probes without the complicated step of probe design. We found that this method could accurately and effectively detect highly polymorphic regions in specific codons associated with drug resistance.

Simultaneous Detection of 10 Foodborne Pathogens using Capillary Electrophoresis-Based Single Strand Conformation Polymorphism

This report outlines the development of a rapid, simple, and sensitive detection system for pathogenic bacteria using a capillary electrophoresis-based, single strand conformation polymorphism (CE-SSCP) combined with PCR. We demonstrate that this method, used with primers targeting the V4 region of the16S rRNA gene, is capable of the simultaneous detection of 10 microbes that could be associated with foodborne illness, caused by animal-derived foods: Salmonella enterica, Listeria monocytogenes, Escherichia coli O157:H7, Campylobacter jejuni, Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, Yersinia enterocolitica, Vibrio parahaemolyticus, and Enterobacter sakazakii. The traditional detection techniques are time-consuming and labor-intensive, due to the necessary task of separate cultivation of each target species. As such, the CE-SSCP-PCR method, that we have developed, has the potential to diagnose pathogens rapidly, unlike the traditional technique, in order to prevent foodborne illness in a much more efficient manner.

Parallel analysis of 7 food-borne pathogens using capillary electrophoresis-based single-strand conformation polymorphism

Capillary electrophoresis-based single-strand conformation polymorphism (CE-SSCP) coupled with multiplex polymerase chain reaction (PCR) method was used for the detection of 7 pathogens associated with foodborne illness, including Salmonella enterica, Clostridium perfringens, Bacillus cereus, Listeria monocytogenes, Yersinia enterocolitica, Vibrio parahaemolyticus, and Escherichia coli O157:H7. The method was applied to model food systems, both of culture medium and cooked rice. The detection limit of individual microbes was in the range of 101–103 CFU/g, and that of the mixture of 7 microbes was 103 CFU/g in the cooked rice sample. This method allowed the detection and identification of all 7 food-borne pathogens within 5 hr without the requirement for enrichment steps.

Synergic degradation of phenanthrene by consortia of newly isolated bacterial strains

Three different bacteria capable of degrading phenanthrene were isolated from sludge of a pulp wastewater treatment plant and identified as Acinetobacter baumannii, Klebsiella oxytoca, and Stenotrophomonas maltophilia. Phenanthrene degradation efficiencies by different combinations (consortia) of these bacteria were investigated and their population dynamics during phenanthrene degradation were monitored using capillary electrophoresis-based single-strand conformation polymorphism (CE-SSCP). When a single microorganism was used, phenanthrene degradation efficiency was very low (48.0, 11.0, and 9.0% for A. baumannii, K. oxytoca, and S. maltophilia respectively, after 360 h cultivation). All consortia that included S. maltophilia degraded ∼80.0% of phenanthrene and reduced lag time to 48 h compared to the 168 h of pure A. baumannii culture. CE-SSCP analysis showed that S. maltophilia was the predominant species during phenanthrene degradation in the mixed culture. The results indicate that mixed cultures of microorganisms may effectively degrade target chemicals, even if the microorganisms show low degradation activity in pure culture.

Simultaneous identification of seven foodborne pathogens and Escherichia coli (pathogenic and nonpathogenic) using capillary electrophoresis-based single-strand conformation polymorphism coupled with multiplex PCR.

The objective of this study was to develop a novel technique for parallel analysis of eight important foodborne microbes using capillary electrophoresis-based single-strand conformation polymorphism (CE-SSCP) coupled with multiplex PCR. Specific primers for multiplex PCR amplification of the 16S rRNA gene were designed, corresponding to eight species of bacteria, including Escherichia coli, Clostridium perfringens, Campylobacter jejuni, Salmonella enterica, Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, and Bacillus cereus, for the species-specific identification and optimal separation of their PCR products in subsequent analysis by CE-SSCP. Multiplex PCR conditions including annealing temperature, extension time, the number of PCR cycles, and primer concentrations were then optimized for simultaneous detection of all target foodborne bacteria. The diagnostic system using CE-SSCP combined with multiplex PCR developed here can be used for rapid investigation of causative agents of foodborne illness. The simplicity and high sensitivity of the method may lead to improved management of safety and illness related to food.

A rapid and sensitive method for detecting foodborne pathogens by capillary electrophoresis-based single-strand conformation polymorphism

A parallel analysis method using capillary electrophoresis-based single-strand conformation polymorphism (CE-SSCP) combined with 16S rRNA gene-specific PCR was developed for rapid and effective detection of five pathogens associated with food poisoning. Conventional detection and identification methods for these pathogens are time consuming and labor intensive due to the necessity of separate cultivation of each target strain. Using the developed CE-SSCP method, Escherichia coli, Clostridium perfringens, Campylobacter jejuni, Salmonella enterica, and Bacillus cereus were simultaneously identified with individual corresponding peaks from a mixture of their genomic DNA. This method can be used for rapid investigation of causative agents of food poisoning. Its simplicity and high sensitivity may lead to improved management of food safety.

Prevalence, Spectrum, and Functional Characterization of Melanocortin-4 Receptor Gene Mutations in a Representative Population-Based Sample and Obese Adults from Germany

Autosomal dominant inheritance of mutations in the melanocortin-4 receptor gene (MC4R) is currently regarded as the most relevant genetic cause for extreme obesity and affects 2-4% of extremely obese individuals. Our objective was to assess the relevance of MC4R mutations in a German population-based sample. We conducted a mutation screen of the MC4R gene by capillary electrophoresis-based single-strand conformation polymorphism analysis and denaturing HPLC. Subjects included 4068 individuals of a German population-based study group [Kooperative Gesundheitsforschung im Raum Augsburg, Survey 4 (KORA-S4); i.e. Cooperative Health Research in the Region of Augsburg] and 1003 German obese adults (body mass index >or= 30 kg/m(2)). Samples with aberrant capillary electrophoresis-based single-strand conformation polymorphism analysis/denaturing HPLC patterns were resequenced. Functional studies including agonistic receptor stimulation (Nle-D-Phe-alpha-, alpha-, and beta-MSH) and cell surface expression assays were performed. Sixteen (six novel) coding nonsynonymous mutations were detected in 27 heterozygous individuals of KORA-S4. Four of the mutation alleles led to impaired receptor function in vitro; however, none of these six heterozygous mutation carriers was obese (body mass index >or= 30 kg/m(2)). In the obese adults, six coding nonsynonymous and a nonsense mutation were detected in 13 individuals. Only the nonsense mutation allele entailed impaired receptor function. Our study depicts prevalence, spectrum, and functional characterization of MC4R mutations in the German population-based sample KORA-S4. In this epidemiological study group, individuals heterozygous for nonsynonymous MC4R mutation alleles entailing impaired function were not obese. Furthermore, nonsynonymous MC4R mutations causing impaired receptor function were rare in German obese adults (two in 1003 = 0.2%).

Capillary electrophoresis-based single strand DNA conformation analysis in high-throughput mutation screening.

The generation of the draft human genome sequence has created new possibilities for diagnosis, prevention, and treatment of human disease. One consequence of these new possibilities is an increasing need for methods and technology that can be used for high-throughput screening for mutations in large DNA sample materials. In recent years, a number of mutation screening methods have emerged that are based on the analysis of sequence-dependent changes in the conformation of single- and double-stranded DNA using capillary electrophoresis. Common features of these methods are high sensitivity and reproducibility as well as the possibility for automation and massive parallelization. Thus, at present they are among the most attractive technologies for high-throughput mutation screening. This review describes the recent advances in capillary electrophoresis-based single strand conformation polymorphism (CE-SSCP) for detection of unknown mutations, and assesses its practical usability for high-throughput mutation screening based on the available literature. In addition, future prospects are outlined in light of the recent advances in microchip-based capillary electrophoresis.