Dataset Information

Polymerase chain reaction amplification of the constant and variable regions of the Bacteroides nodosus fimbrial gene.

ABSTRACT: Serogrouping of Bacteroides nodosus is based on antigenic differences in fimbriae of the different New Zealand prototype strains. Because of the time needed to isolate and grow pure cultures of B. nodosus and the difficulty in distinguishing between different serogroups because of cross-agglutination, a new DNA-based diagnostic approach based on the fimbrial gene sequence of B. nodosus was developed. Published nucleotide sequences of the fimbrial genes for serogroups A, G, D, and H showed conservation at the 5' end, coding for the N terminus, and variability at the 3' end, coding for the C terminus. The polymerase chain reaction was used to amplify both the constant and variable regions of the fimbrial genes. Constant-region oligonucleotide primers were used to amplify a 100-base-pair fragment from the constant regions of the fimbrial genes of 10 New Zealand serogroups. Serogroup-specific oligonucleotide primers for serogroups A and H allowed amplification of a 282-base-pair fragment from serogroup A and a 363-base-pair fragment from serogroup H. Thus, amplification of the constant and variable regions of the fimbrial gene allows rapid detection and grouping of B. nodosus.

SUBMITTER: John GH

PROVIDER: S-EPMC268205 | biostudies-other | 1990 Nov

REPOSITORIES: biostudies-other

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Polymerase chain reaction amplification of the constant and variable regions of the Bacteroides nodosus fimbrial gene.

John G H GH Carlson J O JO Kimberling C V CV Ellis R P RP

Journal of clinical microbiology 19901101 11

Serogrouping of Bacteroides nodosus is based on antigenic differences in fimbriae of the different New Zealand prototype strains. Because of the time needed to isolate and grow pure cultures of B. nodosus and the difficulty in distinguishing between different serogroups because of cross-agglutination, a new DNA-based diagnostic approach based on the fimbrial gene sequence of B. nodosus was developed. Published nucleotide sequences of the fimbrial genes for serogroups A, G, D, and H showed conser ...[more]

PMID: 1979330

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Project description:Background: Precise determination of amplification efficiency is critical for reliable conversion of within-sample changes in fluorescence occurring on a logarithmic scale to between-sample differences in DNA content occurring on a linear scale. This endeavor is especially challenging for the telomere length (TL) quantitative-PCR (qPCR) assay, where amplification efficiency can vary between reactions targeting telomeric repeats (T) and those targeting a single-copy gene (S) to calculate TL as the T/S ratio. Methods: We compared seven different approaches toward estimating amplification efficiency, including the standard-curve method utilized by the qPCR instrument software, and alternative approaches which estimate efficiency on a reaction-by-reaction basis using the stand-alone program LinRegPCR. After calculating T/S ratios using efficiency estimates from each approach (N = 363), we tested their relative performance on metrics of assay precision and correlates of external validity including chronological age (age range = 1-72 years), across tissues within-person (leukocyte-buccal), and between parents and offspring. Results: Estimated amplification efficiency for telomere reactions was significantly lower than estimates for single-copy gene reactions. Efficiency estimates for both reaction sets were significantly higher when estimated with the standard-curve method utilized by the qPCR instrument relative to estimates reconstructed during the log-linear phase with LinRegPCR. While estimates of single-copy gene efficiency reconstructed using LinRegPCR measured within 90% of perfect exponential doubling (E = 1.92), estimates generated using the standard-curve method were inflated beyond 100% (E = 2.10-2.12), indicating poor fidelity. Despite differences in raw value, TL measurements calculated with LinRegPCR efficiency estimates exhibited similar relationships with external validity correlates to measurements generated using the qPCR instrument software. Conclusion: Since methods to estimate amplification efficiency can vary across qPCR instruments, we suggest that future analyses empirically consider external methods of efficiency calculations such as LinRegPCR, and that already generated data be re-analyzed to glean possible improvements.

Dataset Information

Polymerase chain reaction amplification of the constant and variable regions of the Bacteroides nodosus fimbrial gene.

Publications

Polymerase chain reaction amplification of the constant and variable regions of the Bacteroides nodosus fimbrial gene.

Similar Datasets

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