Days For Conventional Methods Research Articles

Development of qualitative methods of bifidobacterium bifidum in probiotic with Real-time PCR method

Bifidobacterium strains with probiotic effects have been widely used in dairy products, food additives and pharmaceuticals. Especially, Bifidobacterium bifidum (B. bifidum) is usually presented into food products such as functional food. However, it is difficult to detect, and quantify B. bifidum in a sample with a combination of different probiotics. In Vietnam, there is no official standard method to identify and quantify B. bifidum in the sample with the mix of probiotic species. To fulfil the requirements of a robust quality management, we have developed a quantitative real-time PCR assay based on groEL gene for accurate identification and quantification of Bifidobacterium bifidum. The developed assay allows an unambiguous speciesspecific detection. We built the real-time PCR method to detect and identify B. bifidum in functional and supplemented food with specific up to 100% and reproducibility (SR<0.25) suitable with Annex F AOAC: 2016. This real-time PCR method is rapidly and effectively than conventional method. It takes only 24 hours to detect and identify B. bifidum in compare with at least a period of 3-5 days for conventional methods. The low quantitative limit is 105 CFU/g/mL, which is consistent with probiotic and powdered milk products with a declared quality of more than 106 CFU/g/mL.

Modern microbiology – a quiet revolution with many benefits

In the clinical microbiology laboratory, classical culture and identification methods are rapidly giving way to molecular techniques with many benefits for clinicians and patients. Building on the discovery of the structure of DNA and the genetic code, four main scientific advances have been made which underpin these techniques (hybridisation probes, polymerase chain reaction, the observation that the microbial species signature can be read in the ribosomal genes and also in the proteins). Early discoveries have paved the way for new diagnostic methods, which are rapid, highly sensitive and specific. Automation has provided high throughput for large numbers of clinical specimens combined with reasonable cost. The benefits for the clinician and patient include confirmation of clinical diagnoses and information about antimicrobial susceptibility within hours compared to days for conventional methods. In resource-poor settings, molecular techniques and automated systems may seem unaffordable but new public-private partnerships, initiatives by the World Health Organization and new, innovative laboratory methods offer the promise of benefit for all.

Identification of Staphylococcus aureus and Escherichia coli isolated from Egyptian food by conventional and molecular methods

Staphylococcus aureus and Escherichia coli were enumerated and isolated from ready-to-eat vegetables salad and meat luncheon on their selective media (Baird-parker and Macconkey agar, respectively). Twenty suspicious colonies of each (10 from each product) were randomly chosen and identified using conventional based on morphological and physiological characteristics. S. aureus and E. coli isolates which gave the highest pathogenicity were chosen for identification and confirmation with molecular method based on 16S rRNA gene. The PCR amplification method of 16S rRNA gave the same identification results as conventional method, but it was sensitive and fast. This molecular method takes about 48 h in comparison with 6 days for conventional method. The 16S rRNA of S. aureus and E. coli were deposited in the Genebank database under accessions (AB599719.1 and AB599716.1, respectively).

Quantification of Listeria monocytogenes in minimally processed leafy vegetables using a combined method based on enrichment and 16S rRNA real-time PCR

Modern lifestyle markedly changed eating habits worldwide, with an increasing demand for ready-to-eat foods, such as minimally processed fruits and leafy greens. Packaging and storage conditions of those products may favor the growth of psychrotrophic bacteria, including the pathogen Listeria monocytogenes. In this work, minimally processed leafy vegetables samples ( n = 162) from retail market from Ribeirão Preto, São Paulo, Brazil, were tested for the presence or absence of Listeria spp. by the immunoassay Listeria Rapid Test, Oxoid. Two L. monocytogenes positive and six artificially contaminated samples of minimally processed leafy vegetables were evaluated by the Most Probable Number (MPN) with detection by classical culture method and also culture method combined with real-time PCR (RTi-PCR) for 16S rRNA genes of L. monocytogenes. Positive MPN enrichment tubes were analyzed by RTi-PCR with primers specific for L. monocytogenes using the commercial preparation ABSOLUTE™ QPCR SYBR ® Green Mix (ABgene, UK). Real-time PCR assay presented good exclusivity and inclusivity results and no statistical significant difference was found in comparison with the conventional culture method ( p < 0.05). Moreover, RTi-PCR was fast and easy to perform, with MPN results obtained in ca. 48 h for RTi-PCR in comparison to 7 days for conventional method.

Diagnostic application of genotypic identification of mycobacteria

This study evaluated conventional methods, GLC and three molecular tests, including 16S rRNA sequencing, for the identification of mycobacteria, and the experiences of the authors with the integration of these methods into a diagnostic clinical laboratory were also recorded. Of 1067 clinical isolates of mycobacteria identified by conventional tests, 365 were tested by Accuprobe hybridization assays and PCRs specific for Mycobacterium tuberculosis (MTB) complex or Mycobacterium avium complex (MAC), 202 were tested by 16S rRNA sequencing, and 142 were tested by GLC. Three runs of all tests were performed on a weekly basis. The identifications for 209 MTB complex and 118 MAC isolates obtained by species-specific PCR were in complete agreement with AccuProbe hybridization and conventional test results. The 16S rRNA sequence-based identification, at a similarity of > or =99 %, for 132 of 142 isolates was concordant with the identifications made by the biochemical methods, and for 134 isolates was concordant with the identifications made by GLC at species, group or complex level. 16S rRNA sequencing resulted in fewer incorrectly identified or unidentified organisms than GLC or conventional tests. For the slowly growing non-tuberculous mycobacteria, the mean turnaround times for identification were 4-5 days for 16S rRNA sequencing, 14-29 days for GLC and 22-23 days for conventional methods. Considering the large proportion of some species among clinical isolates, a strategy of initial screening with species-specific PCR (or AccuProbe assays) for the MTB complex and MAC, followed by direct sequencing of the strains that yield negative results, should make 16S rRNA sequencing more affordable for routine application in diagnostic laboratories.

Fractionation and Determination of Different Lead Species in Contaminated Soils

A new approach was proposed to fractionating soil lead species differing in mobility and biological availability. Ground solid samples were retained as a stationary phase in a rotating coiled column while continuously pumping aqueous solutions of salts and complexing agents necessary for the sequential leaching of different element species. It was shown that field-flow fractionation in a rotating coiled column is suitable for separating three lead species in an automatic mode within 4 h, as compared to several days for conventional methods of sequential extraction. The study of lead elution curves at different fractionation steps provides additional information about its mobilization under changing environmental conditions.

Use of a Radiometric Technique for Rapid Sensitivity Testing of Mycobacteria in Scotland: The First Year's Experience

We report the first year's experience of the use of a radiometric technique for sensitivity testing of mycobacterial isolates in Scotland. The techniques (using the Bactec 460 instrument) was much quicker than conventional methods (for example the mean time to obtain results for 60 strains of M.tuberculosis was 14.4 days, compared with 44.9 days for conventional methods). There was good agreement between results obtained by the two methods. Preliminary results also indicated the value of the radiometric technique for rapid isolation of mycobacteria from clinical samples (mean of 10.6 days with the radiometric technique compared with 26.7 days using egg media) although for optimal recovery, both techniques should be used in parallel.

Use of a Radiometric Technique for Rapid Sensitivity Testing of Mycobacteria in Scotland: The First Year's Experience

We report the first year's experience of the use of a radiometric technique for sensitivity testing of mycobacterial isolates in Scotland. The techniques (using the Bactec 460 instrument) was much quicker than conventional methods (for example the mean time to obtain results for 60 strains of M.tuberculosis was 14.4 days, compared with 44.9 days for conventional methods). There was good agreement between results obtained by the two methods. Preliminary results also indicated the value of the radiometric technique for rapid isolation of mycobacteria from clinical samples (mean of 10.6 days with the radiometric technique compared with 26.7 days using egg media) although for optimal recovery, both techniques should be used in parallel.

THE VALUE OF LONG-CHAIN FATTY ACID COMPOSITION IN THE IDENTIFICATION OF SOME BREWERY YEASTS

The cellular long-chain fatty acids of 72 strains representing 29 species of brewery yeasts were extracted by saponification and analysed as methyl esters by gas chromatography. With this method, it was possible to distinguish between Saccharomyces cerevisiae and five other groups from the brewery industry, only four hours after they were obtained from 48 h old cultures under standard conditions. This compares favourably with the 7 to 10 days for conventional methods.