The inclusivity recognition and exclusivity limit of six 16S rRNA gene-based
May 11, 2017
The inclusivity recognition and exclusivity limit of six 16S rRNA gene-based genus-specific PCR assays were examined. screening programs have already been referred to (1 3 4 5 8 9 The inclusivity and exclusivity of a few of these assays continues to be analyzed before (3 5 8 however the basis of the evaluations differed significantly particularly with regards to the amounts and options of strains utilized to judge the exams. This makes a target evaluation of their efficiency very difficult. Within this research purified DNA of the assortment of 43 type and guide strains owned by different (= 21) (= 15) (= 6) and (= 1) types was used to judge the inclusivity exclusivity and recognition limit of six previously referred to genus-specific PCR IPI-493 assays (1 3 4 5 8 9 all concentrating on the 16S rRNA gene. All PCR assays had been IPI-493 performed in 25-μl amounts formulated with 2.5 μl 10× PCR buffer (Invitrogen Life Technologies Merelbeke Belgium) 0.25 μl of every primer (Operon Cologne Germany) 5 μl of deoxynucleoside triphosphate mix (final concentration 200 μM; Invitrogen Lifestyle Technology) and 1 μl of DNA design template (concentrations ranged between 3 and 200 ng DNA/μl with regards to the types). Amounts of polymerase Platinum (Invitrogen Life Technologies) MgCl2 (Invitrogen Life Technologies) and DNA-free purified water were used as appropriate for each assay (Table ?(Table1).1). Reaction mixtures were heated for 5 min at 94°C as an initial denatur-ation step. PCR cycling conditions were as described in the original studies (1 3 4 5 9 with amendments from the study of Riley et al. (8) in which 35 cycles of 30 s of denaturation at 94°C 60 s of annealing at 53°C and 90 s of elongation at 72°C were used. All assays were terminated with a 5-min extension period of 72°C and were performed with IPI-493 Mastercycler ep thermocyclers (Eppendorf Hamburg Germany). Amplicons were detected by the ethidium bromide staining of electrophoresed samples as described previously (2). All PCR assays were performed in triplicate on three individual occasions. If a positive result was obtained with a species not belonging to the genus in all six assays the obtained amplicons were purified with a QIAquick PCR purification kit (Qiagen Venlo The Netherlands) and sequenced as described before (6) using the appropriate primers (Table ?(Table1)1) to exclude the contamination of the DNA with DNA. TABLE 1. genus-specific PCR primers and assay specifications A detailed overview of the inclusivity (the percentage of strains correctly identified) exclusivity (100 minus the percentage of strains of the nontarget species giving an amplicon of the correct size) and detection limits of all assays is given in Table ?Table22. TABLE 2. Inclusivity exclusivity and detection limit of each strains were included in the initial surveys. In general the investigators chose to include DNA extracts from other bacteria commonly found in the gastric and/or intestinal flora to evaluate the specificity of their assays. Frequently tested organisms IPI-493 were spp. spp. spp. spp. and spp. Our results emphasize that more problems are encountered with the accurate discrimination of closely related taxa. Therefore it is important to make use of a strain collection that properly displays the taxonomy of the target species CSF1R to validate a novel PCR assay. In all six assays an amplicon of the correct size was obtained with DNA. The sequencing of these PCR products yielded fragments that all showed 99 to 100% similarity to the 16S rRNA gene of ATCC 29543T. Therefore the accidental contamination of the DNA with DNA leading to false-positive results could be excluded. Phylogenetically is very closely related to the genus (11). In view of this the observed cross-reaction between primers designed to be specific for and DNA is not so surprising. To determine the analytical detection limit of each PCR assay 10 serial dilutions of the genomic DNA of ATCC 26695T (starting from 200 ng DNA/μl) were used as a template in the respective PCR assays and amplicons were visualized as explained above. Additionally the clinical detection limit of each assay was determined by spiking.