Forty-five of 50 Data consistent with PCR-ribotype were generated in place of PCR-ribotype by centre B indicating likely transcription of the test isolate. The profile for PCR-ribotype was obscured with additional peaks consistent with sample contamination at centre C. PCR-ribotypes and were associated with amplification failure at centre C and likely to represent attempted testing of an organism other than C.
An error in the construction of the challenge set resulted in the circulation of PCR-ribotype in place of PCR-ribotype All test failures highlighted errors associated with the manipulation of test isolates as opposed to inconsistencies with the prescribed method, or data quality. A dataset of sized fragments for 60 blinded ribotypes was distributed electronically from the UK laboratory. All isolates were correctly identified by a minimum of three centres.
Failure to identify the correct ribotype was only associated with Centre B for the remaining three isolates. Analysis of the discrepancies revealed that an incomplete set of PCR-ribotype profiles generated from the training set had been used to formally identify PCR-ribotypes at this centre, resulting in three unidentified profiles.
This process highlighted an error during database identification at a single centre as opposed to inconsistencies with the prescribed method, or data quality. Increased awareness of CDI is accompanied by the need to develop a consensus typing and analysis method that will allow the establishment of a standardised nomenclature and greater understanding of C. This should be robust, reliable and generate high resolution, portable data, and function at local, national and international levels.
Capillary Electrophoresis - Methods and Protocols | Philippe Schmitt-Kopplin | Springer
Existing reports involving CE-ribotyping have represented only single-centre studies and inter-laboratory variability has not been formally assessed [ 18 , 20 ]. We report a high level of reproducibility and accuracy associated with a consensus CE-ribotyping protocol. Our results demonstrate that this consensus protocol generates sufficiently low level inter-laboratory variation to support accurate PCR-ribotyping for international C. More recently, Xiao et al. Several epidemiological studies have been conducted using subtly different CE-ribotyping protocols, making future inter-laboratory comparison of results difficult [ 18 , 20 , 24 , 25 ].
We hope that the provision of a consensus CE-ribotyping protocol will encourage laboratories to use the technique in the knowledge that data will be directly comparable across centres. We have shown that a basic crude DNA extraction method is sufficient to demonstrate good reproducibility of the system. However, many laboratories have more sophisticated higher-throughput DNA extraction platforms, which invariably yield good quality DNA, and these could be expected to be viable alternatives.
The primers originally described by Stubbs et al. It is generally accepted that these primer sets offer equivalent levels of performance and discrimination, but generate different relative fragment lengths. Bidet et al. The loss of accuracy associated with sizing larger DNA fragments when using electrophoresis should ideally be minimised, and the selection of primers that generate smaller fragments would therefore be preferable. In order to avoid potential primer:template mismatches and minimise inter-laboratory variation, the primers designed by Bidet et al.
It is important that standardized protocols for multi-centre surveillance can be easily adopted by participating laboratories. PCR consumables can be acquired or changed relatively easily, but access to specific DNA sequencing instrumentation can present difficulties. It is therefore essential that a standardised protocol can operate on more than one instrument type without significant variation in ribotype profiles. Our study has incorporated the use of two different sequencing instruments with little effect on PCR-ribotype recognition or fragment-size variation. These results suggest that laboratories can adopt the consensus protocol for use on different automated sequencers; further validation is required to confirm this issue.
Previous reports using CE-ribotyping have called for validation of the technique using internationally accepted reference strains [ 18 , 20 ]. The standardised consensus method used here was tested on a well characterised collection of 70 different PCR-ribotypes [ 22 , 23 ] to evaluate the performance of the protocol against prevalent and clinically relevant PCR-ribotypes. Several examples of profiles that shared a very high level of similarity were incorporated in this study eg.
CE ribotyping was able to consistently discriminate between all these isolates. It is generally accepted that such discrimination is difficult with conventional agarose gel-based PCR-ribotyping unless gel quality is particularly high. The number of unique PCR-ribotypes has expanded rapidly in recent years and now exceeds Fawley, Wilcox, unpublished.
As this library expands, the number of ribotypes with similar profile is likely to increase. In turn, the interpretation of PCR-ribotype profiles will become more complex.
Capillary Electrophoresis of Proteins
CE-ribotyping offers a solution for future ribotyping strategies by offering accurate fragment sizing and increased discrimination between ribotypes, including those with similar profiles that can be confused using conventional agarose gel-based PCR-ribotyping. CE-ribotyping has already confirmed reproducible differences in DNA profile of isolates previously assigned to the same ribotype using agarose gel electrophoresis, most notably those associated with ribotypes , and [ 18 , 20 , 27 ]. Multi-locus sequence typing studies have further shown that individual ribotypes tend to be associated with a single ST-type, but that a small number are associated with multiple ST-types [ 22 , 27 , 28 ].
CE-ribotyping represents a cost-effective technique to further discriminate between ribotypes of very similar DNA profile in order to further understand phylogenetic relationships.
Current opinion would indicate that the largest barrier to widespread adoption of CE-ribotyping remains the acquisition of an automated sequencing instrument. However, such instruments are now in widespread use; most medical and academic institutions use automated sequencing technology and many offer access to their instruments on or off site on a service provision basis.
In this way CE-ribotyping could reasonably be performed in any laboratory and need not be reserved for larger reference centres. Our results suggest that data generated using the consensus method are fully portable; Hitherto, surveillance programs have been impeded due to the inability of laboratories to exchange typing data efficiently. America and Europe was affected by inefficient inter-laboratory exchange of PCR-ribotyping data, involving the laborious exchange of bacterial isolates to establish significant epidemiological links [ 2 , 6 ].
There have been calls for improvements to PCR-ribotype nomenclature [ 19 , 29 ]. Stubbs et al.
clublavoute.ca/map63.php These have only local immediate applicability. The UK library was developed using agarose gel-based ribotyping, and further development is hindered by interpretation issues associated with this technique. Standardised and fully portable ribotyping data would be a significant step forwards to consolidating a single library of C.
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Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract PCR-ribotyping has been adopted in many laboratories as the method of choice for C. The work is made available under the Creative Commons CC0 public domain dedication Data Availability: All relevant data are within the paper. Introduction Clostridium difficile infection CDI is a major nosocomial disease, placing a considerable burden on healthcare resources.
The optimum pH value for the separation was set at 9. The influence of pH values of the background electrolyte on the effective electrophoretic mobilities of the studied penicillins is presented in Figure 1. In order to optimize the electrophoretic conditions, we studied the influence of the applied voltage and temperature on the separation. The increase of the voltage and temperature, respectively, results in the decrease of the migration times, but with little effect on the resolution. The migration order can be explained as AMO is the more polar molecule in the mixture because of the - OH substituent , it consequently has the lowest affinity towards micelles and migrates fastest; while OXA is the less polar molecule in the mixture because of the large izoxazolyl substituent , it has the best affinity towards micelles and migrates last.
As it is usual, the precision for migration times was better than of peak areas.
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The LD and LQ were calculated as the sample concentration that produces a peak signal-to-noise ratio of and , respectively. The individual linear regression equations were calculated according to six concentrations in a specific range and three replicates per concentration.
Linearity was evaluated taking into account the correlation coefficients; as correlation coefficients higher than 0. Another method to verify linearity was the application of a lack of fit ANOVA test, a statistical test, which has an F-distribution under the null hypothesis.
The calculated F values for the four penicillins were below the F critical value 3. Penicillins dissolved in water undergo a rapid hydrolysis, being gradually converted to different degradation products Block, Beale, ; Kummerer, After dissolution of the penicillins in water, the sample solutions were reinjected several times over the duration of two weeks, using ciprofloxacin hydrochloride as internal standard.
Ciprofloxacin is a fluoroquinolone derivative, a zwitterionic compound, which can ionize in both acid and alkaline environment. Ciprofloxacin exhibits a smaller electrophoretic mobility than penicillin derivatives; consequently, it will migrate after the last studied penicillin. D, in order to obtain shorter analysis times. The degradation of the compounds depends highly on the temperature of the medium, with an increase of the degradation rate at higher temperatures.
The degradation depends also on the pH, but further investigations are necessary.