Journal: Applied and Environmental Microbiology

Article Title: Development of an F57 Sequence-Based Real-Time PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Milk

doi: 10.1128/aem.71.10.5957-5968.2005

Figure Lengend Snippet: FIG. 1. Dynamics of F57 sequence and IC template coamplification under optimized LC-PCR assay conditions: graphic plot from the light cycler. The data show the results for coamplification of the F57 sequence standard dilutions and a constant amount of the IC template (2,000 copies) in the LC-PCR assay. The large graph shows M. avium subsp. paratuberculosis F57 amplification as monitored in the LC-Red 705 channel, and the inset shows IC template amplification as monitored in the LC-Red 640 channel. The inset shows that the amount of IC template used is large enough to effectively monitor amplification in the presence of small amounts of the M. avium subsp. paratuberculosis F57 target (0 to 2 103

Article Snippet: a Replicates containing different amounts (140 fg to 10 fg) of M. avium subsp. paratuberculosis genomic DNA isolated from a pure culture of the M. avium subsp. paratuberculosis reference strain (ATCC 6783) were subjected to LC-PCR amplification as outlined in Materials and Methods.

Techniques: Sequencing

Journal: Applied and Environmental Microbiology

Article Title: Development of an F57 Sequence-Based Real-Time PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Milk

doi: 10.1128/aem.71.10.5957-5968.2005

Figure Lengend Snippet: FIG. 2. Evaluation of the specificity of the M. avium subsp. paratuberculosis LC-PCR assay. Genomic DNA templates (1 ng) isolated from M. avium subsp. paratuberculosis ATCC 6783 and M. avium ATCC 19421 were amplified, and the amplicons were detected as outlined in Materials and Methods. The large graph shows F57 sequence amplification monitored by LC-Red 705 detection, and the inset shows IC amplification monitored by LC-Red 640 detection. In each reaction, the IC control template was included, and its amplification was confirmed (inset). MAP, M. avium subsp. paratuberculosis.

Article Snippet: a Replicates containing different amounts (140 fg to 10 fg) of M. avium subsp. paratuberculosis genomic DNA isolated from a pure culture of the M. avium subsp. paratuberculosis reference strain (ATCC 6783) were subjected to LC-PCR amplification as outlined in Materials and Methods.

Techniques: Isolation, Sequencing, Control

Journal: Applied and Environmental Microbiology

Article Title: Development of an F57 Sequence-Based Real-Time PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Milk

doi: 10.1128/aem.71.10.5957-5968.2005

Figure Lengend Snippet: FIG. 3. Influence of nonspecific DNA background on amplification of the F57 sequence by the LC-PCR assay. Tenfold serial dilutions of F57 sequence standard dilutions (2 102 to 2 106) and the IC template (2,000 copies) were amplified alone (large graph) or after they were spiked into reaction mixtures containing 200 ng of nonspecific DNA (inset). The inset shows the influence of a DNA background mixture consisting of DNA templates from the following non-M. avium subsp. paratuberculosis mycobacteria: M. avium, M. bovis, M. intracellulare, M. phlei, M. scrofulaceum, M. terrae, and Mycobacterium strain 2333. MAP, M. avium subsp. paratuberculosis.

Article Snippet: a Replicates containing different amounts (140 fg to 10 fg) of M. avium subsp. paratuberculosis genomic DNA isolated from a pure culture of the M. avium subsp. paratuberculosis reference strain (ATCC 6783) were subjected to LC-PCR amplification as outlined in Materials and Methods.

Techniques: Sequencing

Journal: Applied and Environmental Microbiology

Article Title: Development of an F57 Sequence-Based Real-Time PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Milk

doi: 10.1128/aem.71.10.5957-5968.2005

Figure Lengend Snippet: FIG. 4. Quantitative estimation of M. avium subsp. paratuberculosis target copy numbers in defined amounts of purified M. avium subsp. paratuberculosis genomic DNA using the LC-PCR assay. F57 sequence-based quantification standards (2 101 to 2 106 copies) and quasi-unknown samples A and B containing 15 pg and 1.5 pg of M. avium subsp. paratuberculosis DNA, respectively, were amplified. Based on theoretical calculations, samples A and B contained about 3,000 and 300 M. avium subsp. paratuberculosis genome copies, respectively. The target copy numbers in these samples were estimated from the standard curve generated from amplification of the F57 standards. The results obtained from these quantitative assays are summarized in Table 4. MAP, M. avium subsp. paratuberculosis.

Article Snippet: a Replicates containing different amounts (140 fg to 10 fg) of M. avium subsp. paratuberculosis genomic DNA isolated from a pure culture of the M. avium subsp. paratuberculosis reference strain (ATCC 6783) were subjected to LC-PCR amplification as outlined in Materials and Methods.

Techniques: Sequencing, Generated

Journal: Journal of Clinical Microbiology

Article Title: Definitive Differentiation between Single and Mixed Mycobacterial Infections in Red Deer ( Cervus elaphus ) by a Combination of Duplex Amplification of p34 and f57 Sequences and Hpy188I Enzymatic Restriction of Duplex Amplicons

doi: 10.1128/jcm.43.9.4640-4648.2005

Figure Lengend Snippet: FIG. 1. Multiple nucleotide sequence alignment of M. avium subsp. avium (Maa), M. avium subsp. paratuberculosis (Map) and M. bovis (Mbo) us-p34 genes. Gaps between sequences are indicated by dots. Vertical bars indicate identity across sequences. The start codon (ATG) for the p34 open reading frame is shown in bold. Primer positions are indicated by boxes. The horizontal arrows indicate directions of transcription. The Hpy188I restriction site is indicated by a boldly outlined box. The single nucleotide substitution between M. avium subsp. paratuberculosis and M. avium subsp. avium within the restriction site is highlighted with an arrow and a bold nucleotide. Nucleotides shown in bold italics in the myc3 sequence were degenerated in the corresponding primer (see Table 1).

Article Snippet: Reference strains included two M. avium subsp. avium strains (ATCC 25291 and DSMZ 44156), three M. avium subsp. paratuberculosis strains (ATCC 19698, ATCC 43015, known as strain Linda [2], and the vaccinal strain 2E [33]), and one M. bovis strain (ATCC 19210).

Techniques: Sequencing

Journal: Journal of Clinical Microbiology

Article Title: Definitive Differentiation between Single and Mixed Mycobacterial Infections in Red Deer ( Cervus elaphus ) by a Combination of Duplex Amplification of p34 and f57 Sequences and Hpy188I Enzymatic Restriction of Duplex Amplicons

doi: 10.1128/jcm.43.9.4640-4648.2005

Figure Lengend Snippet: FIG. 2. Nucleotide sequence of M. avium subsp. paratuberculosis f57 segment (29a). Primer positions are indicated by boxes. The horizontal arrows indicate directions of transcription. Hpy188I restriction sites are shown in bold.

Article Snippet: Reference strains included two M. avium subsp. avium strains (ATCC 25291 and DSMZ 44156), three M. avium subsp. paratuberculosis strains (ATCC 19698, ATCC 43015, known as strain Linda [2], and the vaccinal strain 2E [33]), and one M. bovis strain (ATCC 19210).

Techniques: Sequencing

Journal: Journal of Clinical Microbiology

Article Title: Definitive Differentiation between Single and Mixed Mycobacterial Infections in Red Deer ( Cervus elaphus ) by a Combination of Duplex Amplification of p34 and f57 Sequences and Hpy188I Enzymatic Restriction of Duplex Amplicons

doi: 10.1128/jcm.43.9.4640-4648.2005

Figure Lengend Snippet: FIG. 3. Banding patterns of undigested us-p34–f57 amplicons. (A) Theoretical banding patterns before Hpy188I digestion (, Hpy188I restriction site). (B) Amplified DNA fragments from M. bovis (lane 3), M. avium subsp. paratuberculosis (lane 4), M. avium subsp. avium (lane 5), a mixture of M. avium subsp. avium and M. bovis (lane 6), a mixture of M. avium subsp. avium and M. avium subsp. paratuberculosis (lane 7), and a mixture of M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. bovis (lane 8) were separated by electrophoresis in a 2% agarose gel. The lengths (in base pairs) of the amplified fragments and of the 123-bp DNA ladder (lane 1) and 50-bp DNA ladder (lane 2) (molecular weight markers) are indicated on the right and left sides of the gel, respectively. Hatched boxes represent the p34 gene.

Article Snippet: Reference strains included two M. avium subsp. avium strains (ATCC 25291 and DSMZ 44156), three M. avium subsp. paratuberculosis strains (ATCC 19698, ATCC 43015, known as strain Linda [2], and the vaccinal strain 2E [33]), and one M. bovis strain (ATCC 19210).

Techniques: Electrophoresis, Agarose Gel Electrophoresis, Molecular Weight

Journal: Journal of Clinical Microbiology

Article Title: Definitive Differentiation between Single and Mixed Mycobacterial Infections in Red Deer ( Cervus elaphus ) by a Combination of Duplex Amplification of p34 and f57 Sequences and Hpy188I Enzymatic Restriction of Duplex Amplicons

doi: 10.1128/jcm.43.9.4640-4648.2005

Figure Lengend Snippet: FIG. 4. Hpy188I digestion profiles of duplex us-p34–f57 amplicons. (A) Theoretical banding patterns after Hpy188I digestion. (B) Banding patterns of M. bovis (lane 3), M. avium subsp. paratuberculosis (lane 4), M. avium subsp. avium (lane 5), a mixture of M. avium subsp. avium and M. bovis (lane 6), a mixture of M. avium subsp. avium and M. avium subsp. paratuberculosis (lane 7), and a mixture of M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. bovis (lane 8). The lengths (in base pairs) of the amplified fragments and of the 123-bp DNA ladder (lane 1) and the 50-bp DNA ladder (lane 2) (molecular weight markers) are indicated on the right and left sides of the gel, respectively.

Article Snippet: Reference strains included two M. avium subsp. avium strains (ATCC 25291 and DSMZ 44156), three M. avium subsp. paratuberculosis strains (ATCC 19698, ATCC 43015, known as strain Linda [2], and the vaccinal strain 2E [33]), and one M. bovis strain (ATCC 19210).

Techniques: Molecular Weight