Astrovirus detection in wastewater samples

2001 ◽  
Vol 43 (12) ◽  
pp. 73-76 ◽  
Author(s):  
R. M. Pintó ◽  
C. Villena ◽  
F. Le Guyader ◽  
S. Guix ◽  
S. Calallero ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Endonuclease ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Southern France ◽  
Wastewater Samples ◽  
Restriction Fragment Length

Procedures for the detection of astroviruses in wastewater samples have been developed and evaluated. Following these methodologies, we investigated the occurrence of astroviruses in wastewater samples from three different sewage treatments plants located in Southern France and two in the Barcelona area. Some positive samples were genotyped by analysis of a fragment of the ORF1a by restriction fragment length polymorphism (RFLP) with endonuclease Dde I. The amplimers generated contain several sites for the DdeI restriction endonuclease, being the number and location of sites different between strains.

scholarly journals Resolution of Streptococcus suis Serotypes 1/2 versus 2 and 1 versus 14 by PCR-Restriction Fragment Length Polymorphism Method

2020 ◽  
Vol 58 (7) ◽  
Author(s):  
Jan Matiasovic ◽  
Monika Zouharova ◽  
Katerina Nedbalcova ◽  
Natalie Kralova ◽  
Katarina Matiaskova ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Endonuclease ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Nucleotide Substitution ◽  
Fragment Length Polymorphism ◽  
Streptococcus Suis ◽  
Serotype 1 ◽  
Restriction Fragment Length

ABSTRACT Streptococcus suis is an important pathogen of pigs but is also transmissible to humans, with potentially fatal consequences. Among 29 serotypes currently recognized, some are clinically and epidemiologically more important than others. This is particularly true for serotypes 2 and 14, which have a large impact on pig production and also on human health. Conventional PCR-based serotyping cannot distinguish between serotype 1/2 and serotype 2 or between serotype 1 and serotype 14. Although serotype 1/2 and serotype 2 have a very similar cps locus, they differ in a single-nucleotide substitution at nucleotide position 483 of the cpsK gene. Similarly, serotypes 1 and 14 have a very similar cps locus but also differ in the same nucleotide substitution of the cpsK gene. Fortunately, this cpsK 483G→C/T substitution can be detected by BstNI restriction endonuclease. A PCR-restriction fragment length polymorphism (RFLP) detection method amplifying a fragment of the cpsK gene digested by BstNI restriction endonuclease was developed and tested in reference strains of these serotypes and also in field isolates.

scholarly journals Fidelity of Select Restriction Endonucleases in Determining Microbial Diversity by Terminal-Restriction Fragment Length Polymorphism

2003 ◽  
Vol 69 (8) ◽  
pp. 4823-4829 ◽  
Author(s):  
Jeff J. Engebretson ◽  
Craig L. Moyer
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Microbial Diversity ◽  
Restriction Endonuclease ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Restriction Endonucleases ◽  
Restriction Fragment Length

ABSTRACT An evaluation of 18 DNA restriction endonucleases for use in terminal-restriction fragment length polymorphism (T-RFLP) analysis was performed by using richness and density indices in conjunction with computer simulations for 4,603 bacterial small-subunit rRNA gene sequences. T-RFLP analysis has become a commonly used method for screening environmental samples for precursory identification and community comparison studies due to its precision and high-throughput capability. The accuracy of T-RFLP analysis for describing a community has not yet been thoroughly evaluated. In this study, we attempted to classify restriction endonucleases based upon the ability to resolve unique terminal-restriction fragments (T-RFs) or operational taxonomic units (OTUs) from a database of gene sequences. Furthermore, we assessed the predictive accuracy of T-RFLP at fixed values of community richness (n = 1, 5, 10, 50, and 100). Classification of restriction endonuclease fidelity was performed by measuring richness and density for the entire database of T-RFs. Further analysis of T-RFLP accuracy for determining richness was performed by iterative, random sampling from the derived database of T-RFs. It became apparent that two constraints were influential for measuring the fidelity of a given restriction endonuclease: (i) the ability to resolve unique sequence variants and (ii) the number of unique T-RFs that fell within a measurable size range. The latter constraint was found to be more significant for estimating restriction endonuclease fidelity. Of the 18 restriction endonucleases examined, BstUI, DdeI, Sau96I, and MspI had the highest frequency of resolving single populations in model communities. All restriction endonucleases used in this study detected ≤70% of the OTUs at richness values greater than 50 OTUs per modeled community. Based on the results of our in silico experiments, the most efficacious uses of T-RFLP for microbial diversity studies are those that address situations where there is low to intermediate species richness (e.g., colonization, early successional stages, biofilm formation).

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