An accurate multiplex antibiotic susceptibility test using a high‐resolution <scp>CE</scp>‐<scp>SSCP</scp>‐based stuffer‐free multiplex ligation‐dependent probe amplification system

Chung, Boram; Shin, Gi Won; Choi, Woong; Hwang, Hee Sung; Oh, Mi‐Hwa; Jung, Gyoo Yeol

doi:10.1002/elps.201200372

Cited by 4 publications

(5 citation statements)

References 24 publications

(28 reference statements)

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“…Particularly for applications in multiplex analyses, the low resolution frequently results in unexpected peak overlaps when separating multiple DNA fragments having similar sizes. [28][29][30] However, baseline-level separation based on a sequence difference is now feasible using our high-resolution CE-SSCP, and consequently constraints in the analysis of stuffer-free products can be minimized.…”

Section: Discussionmentioning

confidence: 99%

A robust and simple-to-design multiplex DNA methylation assay based on MS-MLPA-CE-SSCP

Shin

Jung³

et al. 2013

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Aberrant DNA methylation is a potential diagnostic marker for complex diseases, such as cancer. With the increase in the number of genes known to exhibit disease-associated aberrant methylation, the need for accurate multiplex assays for quantifying DNA methylation has increased. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) is one method that has been highlighted in this context. However, two limitations make the custom design of MS-MLPA assays impractical: the need for long probes containing stuffer sequences and a reliance on only one restriction enzyme. Here, we developed a variation of MS-MLPA that employs a simpler probe-design process. To overcome the above-mentioned limitations, we used stuffer-free MS-MLPA probes that are subsequently analyzed using high-resolution capillary electrophoresis-based single-strand conformational polymorphism (CE-SSCP) instead of conventional length-dependent CE. Moreover, multiple methylation-sensitive restriction enzymes (HhaI, HpaII, and AciI) were used simultaneously; thus, probes satisfying desired criteria were available for all targets. Using this assay concept, we analyzed 17 genes associated with hepatocellular carcinoma. Our results showed that the custom-designed assay based on MS-MLPA-CE-SSCP provided robust multiplex quantification of DNA methylation levels.

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Section: Discussionmentioning

confidence: 99%

A robust and simple-to-design multiplex DNA methylation assay based on MS-MLPA-CE-SSCP

Shin

Jung³

et al. 2013

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“…Seven of these bacterial species ( Yersinia enterocolitica, Vibrio parahaemolyticus, Escherichia coli O157:H7, Staphylococcus aureus, Shigella sonnei, Listeria monocytogenes, S. enteritidis ) are major causes of foodborne illness or respiratory tract infection, whereas two ( E. coli, Klebsiella pneumonia ) are responsible for lower respiratory tract infections. The stuffer‐free MLPA probe sets were designed as reported in our previous study . Detailed information about target genes and probe sets is presented in Table .…”

Section: Methodsmentioning

confidence: 99%

“…CE‐SSCP analyses were performed using an ABI 3130xl Genetic Analyzer (Applied Biosystems) with a 50 cm capillary array. The capillary was fully filled with a 15% solution of Pluronic F108 polymer (Sigma‐Aldrich, St. Louis, MO, USA), prepared as described in our previous study .…”

Section: Methodsmentioning

confidence: 99%

Precise characterization method of antibody‐conjugated magnetic nanoparticles for pathogen detection using stuffer‐free multiplex ligation‐dependent probe amplification

et al. 2014

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Antibody-conjugated magnetic nanoparticles (Ab-MNPs) have potential in pathogen detection because they allow target cells to be easily separated from complex sample matrices. However, the sensitivity and specificity of pathogen capture by Ab-MNPs generally vary according to the types of MNPs, antibodies, and sample matrices, as well as preparation methods, including immobilization. Therefore, achieving a reproducible analysis utilizing Ab-MNPs as a pathogen detection method requires accurate characterization of Ab-MNP capture ability and standardization of all handling processes. In this study, we used high-resolution CE-single strand conformational polymorphism coupled with a stuffer-free multiplex ligation-dependent probe amplification system to characterize Ab-MNPs. The capture ability of Ab-MNPs targeting Salmonella enteritidis and nine pathogens, including S. enteritidis, was analyzed in phosphate buffer and milk. The effect of storage conditions on the stability of Ab-MNPs was also assessed. The results showed that the stuffer-free multiplex ligation-dependent probe amplification system has the potential to serve as a standard characterization method for Ab-MNPs. Moreover, the precise characterization of Ab-MNPs facilitated robust pathogen detection in various applications.

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“…However, traditional antimicrobic susceptibility testing is troublesome and time consuming. Therefore, Chung et al [70] employed stuffer-free MLPA-CE-SSCP to develop a novel multiplex antibiotic susceptibility test. The values obtained following parallel multiple analyses are consistent with those estimated using a traditional broth dilution approach, but the handling time was reduced to < 40% of that of traditional method and avoided false results from subjective observation, indicating that the stuffer-free MLPA-CE-SSCP method is a feasible alternative to the traditional approach.…”

Section: Detecting and Diagnosing Bacteriamentioning

confidence: 99%

Capillary electrophoresis based on nucleic acid detection for diagnosing human infectious disease

Lian

Zhao

2016

Clinical Chemistry and Laboratory Medicine (CCLM)

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Rapid transmission, high morbidity, and mortality are the features of human infectious diseases caused by microorganisms, such as bacteria, fungi, and viruses. These diseases may lead within a short period of time to great personal and property losses, especially in regions where sanitation is poor. Thus, rapid diagnoses are vital for the prevention and therapeutic intervention of human infectious diseases. Several conventional methods are often used to diagnose infectious diseases, e.g. methods based on cultures or morphology, or biochemical tests based on metabonomics. Although traditional methods are considered gold standards and are used most frequently, they are laborious, time consuming, and tedious and cannot meet the demand for rapid diagnoses. Disease diagnosis using capillary electrophoresis methods has the advantages of high efficiency, high throughput, and high speed, and coupled with the different nucleic acid detection strategies overcomes the drawbacks of traditional identification methods, precluding many types of false positive and negative results. Therefore, this review focuses on the application of capillary electrophoresis based on nucleic detection to the diagnosis of human infectious diseases, and offers an introduction to the limitations, advantages, and future developments of this approach.

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An accurate multiplex antibiotic susceptibility test using a high‐resolution CE‐SSCP‐based stuffer‐free multiplex ligation‐dependent probe amplification system

Cited by 4 publications

References 24 publications

A robust and simple-to-design multiplex DNA methylation assay based on MS-MLPA-CE-SSCP

A robust and simple-to-design multiplex DNA methylation assay based on MS-MLPA-CE-SSCP

Precise characterization method of antibody‐conjugated magnetic nanoparticles for pathogen detection using stuffer‐free multiplex ligation‐dependent probe amplification

Capillary electrophoresis based on nucleic acid detection for diagnosing human infectious disease

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