Total bacterial and species-specific 16S rDNA micro-array quantification of complex samples

Treimo, Janneke; Vegarud, Gerd Elisabeth; Langsrud, Thor; Marki, S.; Rudi, Knut

doi:10.1111/j.1365-2672.2006.02805.x

Cited by 11 publications

(11 citation statements)

References 43 publications

(93 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The supernatant was discharged, and the pellet was resuspended in 1 ml Tris-EDTA (TE) buffer (pH 7.8). The cells were disrupted mechanically in a Fast-Prep bead beater (Bio 101, La Jolla, California) (44). Free DNA was obtained by centrifugation of 5-ml samples for 5 min at 10,000 rpm (Beckman J2-MC centrifuge, JA-17 rotor) and at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…Separate detection of the two targets was done using real-time PCR (ABI 7900 HT; Applied Biosystems) with the Fast Start SYBR green Master (Rox) reagents (Roche). We used a primer pair equal to the universal 16S rRNA gene primer pair (44), with three nucleotides unique to the two amplicons for both the forward and the reverse primers. The real-time PCR was initiated with a denaturation step at 95°C for 10 min.…”

Section: Methodsmentioning

confidence: 99%

“…DNA was purified from both the supernatant and the pellet fractions with a DNeasy tissue kit, following the recommendations of the manufacturer (QIAGEN, Hilden, Germany). Each DNA purification was done in triplicate, adding a competitor in three different concentrations (10-fold dilutions) prior to the purifications (44).…”

Section: Methodsmentioning

confidence: 99%

“…We applied species-specific detection (44) to quantify free DNA released during autolysis. The concept was demonstrated in pure and mixed sample cultures.…”

mentioning

confidence: 99%

See 3 more Smart Citations

Use of DNA Quantification To Measure Growth and Autolysis of Lactococcus and Propionibacterium spp. in Mixed Populations

Treimo

Vegarud

Langsrud

et al. 2006

Appl Environ Microbiol

View full text Add to dashboard Cite

Autolysis is self-degradation of the bacterial cell wall that results in the release of enzymes and DNA. Autolysis of starter bacteria, such as lactococci and propionibacteria, is essential for cheese ripening, but our understanding of this important process is limited. This is mainly because the current tools for measuring autolysis cannot readily be used for analysis of bacteria in mixed populations. We have now addressed this problem by species-specific detection and quantification of free DNA released during autolysis. This was done by use of 16S rRNA gene single-nucleotide extension probes in combination with competitive PCR. We analyzed pure and mixed populations of Lactococcus lactis subsp. lactis and three different species of Propionibacterium. Results showed that L. lactis subsp. lactis INF L2 autolyzed first, followed by Propionibacterium acidipropionici ATCC 4965, Propionibacterium freudenreichii ISU P59, and then Propionibacterium jensenii INF P303. We also investigated the autolytic effect of rennet (commonly used in cheese production). We found that the effect was highly strain specific, with all the strains responding differently. Finally, autolysis of L. lactis subsp. lactis INF L2 and P. freudenreichii ISU P59 was analyzed in a liquid cheese model. Autolysis was detected later in this cheese model system than in broth media. A challenge with DNA, however, is DNA degradation. We addressed this challenge by using a DNA degradation marker. We obtained a good correlation between the degradation of the marker and the target in a model experiment. We conclude that our DNA approach will be a valuable tool for use in future analyses and for understanding autolysis in mixed bacterial populations.The present tools for measuring autolysis are not easily adaptable to analysis of mixed populations. This severely limits our understanding of autolysis in microbial populations, such as in cheese. Autolysis is self-degradation of the bacterial cell wall due to hydrolysis of the peptidoglycan network by autolysins (32,34,36,37), and the result is leakage of intracellular components (9, 27). Lactococcus lactis subsp. lactis and Propionibacterium freudenreichii are bacteria essential to the ripening of Norwegian Jarlsberg cheese (17). The propionibacteria produce propionic acid, acetic acid, and CO 2 , with the latter responsible for the typical eye formation. In addition, this secondary starter contributes to the characteristic sweet, nutty flavor, provided mainly by free proline (20)(21)(22). Autolysis of lactococci is important for cheese ripening, in which the release of intracellular enzymes plays an important role in cheese flavor development (9, 40, 48). The role and contribution of intracellular enzymes from propionibacteria in the ripening process are poorly understood (11,12,23,27,40). Proteolytic enzymes, such as chymotrypsin and pepsin, are reported to modify autolytic activity (28,29,38,39). Rennet, consisting of chymosin and bovine pepsin, hydrolyzes the -casein in milk and thus is a vital factor in ...

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…We applied species-specific detection (44) to quantify free DNA released during autolysis. The concept was demonstrated in pure and mixed sample cultures.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Use of DNA Quantification To Measure Growth and Autolysis of Lactococcus and Propionibacterium spp. in Mixed Populations

Treimo

Vegarud

Langsrud

et al. 2006

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…However, somewhat higher background signals may occur when low-quality coatings are used. Substrates based on amino silane surface chemistry are widely used for deposition of PCR products [40,68,141,145,167,175]. …”

Section: Slides With Amino Silane Coatingmentioning

confidence: 99%

Introduction to Microarray‐Based Detection Methods

Schrenzel

Kostić²,

Bodrossy

et al. 2009

Detection of Highly Dangerous Pathogens

View full text Add to dashboard Cite

Microarrays consist of an orderly arrangement of probes (oligonucleotides, DNA fragments, proteins, sugars or lectins) attached to a solid surface. The main advantages of microarray technology are high throughput, parallelism, miniaturization, speed and automation. Despite the fact that microarray analysis is a relatively novel technology, with the publication of the first microarray studies in 1995 [101,152], it is now broadly applied and the milestone of nearly 5000 published microarray papers was recorded in 2004 [80]. The scientific and technological background discussed here will be limited to DNA microarrays, excluding the new evolving fields of protein microarrays and glycomics [140].Analogous to antigen-antibody interactions on immunoarrays, DNA microarrays rely on sequence complementarity of the two strands. They put in practice the fundamentals of complementary base-pairing (hybridization) that were first described by Ed Southern [162]. In general, the strategy of microarray hybridization is reversed to that of a standard dot-blot, leading to recurring confusion in the nomenclature. Therefore, it has been suggested to describe tethered nucleic acid as the probe and free nucleic acid as the target [134].Earlier studies on duplex melting and reformation, carried out on DNA solutions, have provided the basic knowledge about the reaction kinetics. Furthermore, a method for computational determination of the melting point as a function of nucleic acid composition and salt concentration was established [6,148,149]. Much of the pioneering work can be linked to the use of nitrocellulose membranes [69], dot-blots [84] and Southern blots [162]. Development of cDNA or oligonucleotide arrays was possible by combined innovations in micro-engineering, molecular biology [33,120,123,156,163,164] and bioinformatics [59]. The real breakthrough in microarray technology was initiated by two key innovations: the use of non-porous solid supports (such as glass and silicon) and the development of methods for highdensity synthesis of oligonucleotides directly onto the microarray surface [59].

show abstract

Oligonucleotide and DNA Microarrays: Versatile Tools for Rapid Bacterial Diagnostics

Kostić¹,

François

Bodrossy³

et al. 2008

Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems

View full text Add to dashboard Cite

Total bacterial and species-specific 16S rDNA micro-array quantification of complex samples

Abstract: This is to our knowledge the first time quantification of the total bacterial DNA and the species-specific DNA compositions of mixed populations have been achieved in the same assay.

Cited by 11 publications

References 43 publications

Use of DNA Quantification To Measure Growth and Autolysis of Lactococcus and Propionibacterium spp. in Mixed Populations

Use of DNA Quantification To Measure Growth and Autolysis of Lactococcus and Propionibacterium spp. in Mixed Populations

Introduction to Microarray‐Based Detection Methods

Oligonucleotide and DNA Microarrays: Versatile Tools for Rapid Bacterial Diagnostics

Contact Info

Product

Resources

About