Rapid identification and classification of bacteria by 16S rDNA restriction fragment melting curve analyses (RFMCA)

Rudi, Knut; Kleiberg, G.H.; Heiberg, Ragnhild; Rosnes, Jan Thomas

doi:10.1016/j.fm.2006.09.006

Cited by 9 publications

(5 citation statements)

References 12 publications

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“…The software included in the PCR instrument produces several distinct graphs, each representing one sub‐type of target pathogen. This is all done without any additional laboratory work (Rudi et al., 2007a).…”

Section: Current Developmentsmentioning

confidence: 99%

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Rapid detection, characterization, and enumeration of foodborne pathogens

Hoorfar

2011

APMIS

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As food safety management further develops, microbiological testing will continue to play an important role in assessing whether Food Safety Objectives are achieved. However, traditional microbiological culture-based methods are limited, particularly in their ability to provide timely data. The present review discusses the reasons for the increasing interest in rapid methods, current developments in the field, the research needs, and the future trends. The advent of biotechnology has introduced new technologies that led to the emergence of rapid diagnostic methods and altered food testing practices. Rapid methods are comprised of many different detection technologies, including specialized enzyme substrates, antibodies and DNA, ranging from simple differential plating media to the use of sophisticated instruments. The use of non-invasive sampling techniques for live animals especially came into focus with the 1990s outbreak of bovine spongiform encephalopathy that was linked to the human outbreak of Creutzfeldt Jakob's Disease. Serology is still an important tool in preventing foodborne pathogens to enter the human food supply through meat and milk from animals. One of the primary uses of rapid methods is for fast screening of large number of samples, where most of them are expected to be test-negative, leading to faster product release for sale. This has been the main strength of rapid methods such as real-time Polymerase Chain Reaction (PCR). Enrichment PCR, where a primary culture broth is tested in PCR, is the most common approach in rapid testing. Recent reports show that it is possible both to enrich a sample and enumerate by pathogen-specific real-time PCR, if the enrichment time is short. This can be especially useful in situations where food producers ask for the level of pathogen in a contaminated product. Another key issue is automation, where the key drivers are miniaturization and multiple testing, which mean that not only one instrument is flexible enough to test for many pathogens but also many pathogens can be detected with one test. The review is mainly based on the author's scientific work that has contributed with the following new developments to this field: (i) serologic tests for large-scale screening, surveillance, or eradication programs, (ii) same-day detection of Salmonella that otherwise was considered as difficult to achieve, (iii) pathogen enumeration following a short log-phase enrichment, (iv) detection of foodborne pathogens in air samples, and finally (v) biotracing of pathogens based on mathematical modeling, even in the absence of isolate. Rapid methods are discussed in a broad global health perspective, international food supply, and for improvement of quantitative microbial risk assessments. The need for quantitative sample preparation techniques, culture-independent, metagenomic-based detection, online monitoring, a global validation infrastructure has been emphasized. The cost and ease of use of rapid assays remain challenging obstacles to surmount.

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Section: Current Developmentsmentioning

confidence: 99%

“…One of the most promising developments is taking place in the areas of intestinal microbiology and geomicrobiology using metagenomics techniques, i.e. detection and identification of specific genes directly from the sample without any cultivation steps (Rudi et al., 2007a, 2007b; Morgan et al., 2010).…”

Section: Future Trendsmentioning

confidence: 99%

Rapid detection, characterization, and enumeration of foodborne pathogens

Hoorfar

2011

APMIS

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“…Resolution can be further enhanced or normalized by several methods. 63,64 This has enabled identification of bacterial and viral species. 65 Our application of this tool to species of Candida shows that the separation between species is great enough to call species without any postamplification handling.…”

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confidence: 99%

High-Throughput Identification and Quantification of Candida Species Using High Resolution Derivative Melt Analysis of Panfungal Amplicons

Mandviwala

Shinde

Kalra

et al. 2010

The Journal of Molecular Diagnostics

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Fungal infections pose unique challenges to molecular diagnostics; fungal molecular diagnostics consequently lags behind bacterial and viral counterparts. Nevertheless , fungal infections are often life-threatening , and early detection and identification of species is crucial to successful intervention. A high throughput PCR-based method is needed that is independent of culture , is sensitive to the level of one fungal cell per milliliter of blood or other tissue types , and is capable of detecting species and resistance mutations. We introduce the use of high resolution melt analysis , in combination with more sensitive , inclusive , and appropriately positioned panfungal primers , to address these needs. PCRbased amplification of the variable internal transcribed regions of the rDNA genes generates an amplicon whose sequence melts with a shape that is characteristic and therefore diagnostic of the species. Simple analysis of the differences between test and reference melt curves generates a single number that calls the species. Early indications suggest that high resolution melt analysis can distinguish all eight major species of Candida of clinical significance without interference from excess human DNA. Candida species , including mixed and novel species , can be identified directly in vaginal samples. This tool can potentially detect , count , and identify fungi in hundreds of samples per day without further manipulation , costs, or delays , offering a major step forward in fungal molecular diagnostics. Rapid and economical detection, identification, and quantification of fungal species directly from clinical samples is a long-sought goal of clinicians that has still not been fulfilled.

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“…2007; Patel 2009). The melt analysis has previously been applied for identification and classification of bacterial communities by 16S rRNA restriction fragment melting curve analyses (Rudi et al. 2005, 2007).…”

Section: Introductionmentioning

confidence: 99%

“…DNA from samples or pure strains can thus be characterized according to their melting profile and specific melting temperature (T m ), T m being defined as the temperature where 50% of the DNA duplexes are melted (Reed et al 2007;Patel 2009). The melt analysis has previously been applied for identification and classification of bacterial communities by 16S rRNA restriction fragment melting curve analyses (Rudi et al 2005(Rudi et al , 2007. Since then, improvement of instrumentation has increased the resolution and specificity of HRM, making it even more attractive as a molecular microbiology tool (Vossen et al 2009).…”

Section: Introductionmentioning

confidence: 99%

Rapid lactic acid bacteria identification in dairy products by high-resolution melt analysis of DGGE bands

Porcellato

Grønnevik

Rudi

et al. 2012

Letters in Applied Microbiology

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Aim: To investigate the application of high‐resolution melt (HRM) analysis for rapid species‐level identification of lactic acid bacteria (LAB) communities in dairy products, as well as for bacterial community profiling and monitoring. Methods and Results: First, comparisons of HRM profiles of known reference strains of LAB and their denaturing gradient gel electrophoresis (DGGE) bands showed very good agreement, allowing species recognition and identification from DGGE bands by HRM. Second, samples of cheese, kefir grains and kefir were characterized by PCR‐DGGE, and melting profiles of DGGE bands were compared with known reference strains. Of the 13 DGGE bands, ten were identified by HRM by comparison with the reference strains and only three required sequencing for identification. Use of HRM profiling for comparison and monitoring of total LAB communities from dairy products or starter cultures was also evaluated, and good agreement was found when comparing clustering of DGGE band profiles with clustering of HRM melting profiles. Conclusion: Identification of DGGE bands is possible by comparison of HRM melting profiles with known reference strains. Significance and Impact of the Study: HRM profiling is suggested as an additional approach for identification of DGGE bands.

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Rapid identification and classification of bacteria by 16S rDNA restriction fragment melting curve analyses (RFMCA)

Cited by 9 publications

References 12 publications

Rapid detection, characterization, and enumeration of foodborne pathogens

Rapid detection, characterization, and enumeration of foodborne pathogens

High-Throughput Identification and Quantification of Candida Species Using High Resolution Derivative Melt Analysis of Panfungal Amplicons

Rapid lactic acid bacteria identification in dairy products by high-resolution melt analysis of DGGE bands

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