Pathogen detection in milk samples by ligation detection reaction-mediated universal array method

Schematic of the grouping and clustering of the 16S rRNA gene sequences. For each node, the number of sequences used from the Ribosomal Database Project II database (length >1,200 bp and “good” quality) is reported. The dimension of the triangles is proportional to the number of sequences that were clustered together. The phylogenetic tree was built on the 15 consensus sequences by using the neighbor-joining algorithm and Phylip tree type. The scale represents the number of differences between sequences (e.g., 0.1 means 10% differences between 2 sequences). NAS=nonaureus staphylococci.

A) Universal array scheme. Each ZipCode is spotted in quadruplicate; oligonucleotides associated with actual probe pairs are highlighted in gray. ZipCodes 66 and 63 are the hybridization and ligation controls, respectively. Blank spots are negative controls (no oligo; only spotting buffer). B) Table specifying the bacterial groups with the corresponding ZipCode. C) Specificity results of the amplified 16S rRNA gene from Staphylococcus aureus (ATCC 19095, ZipCode 2), Salmonella enteritidis (ATCC 13076, ZipCode 5), Campylobacter coli (ATCC 1061, ZipCodes 8 and 9), Bacillus cereus (ATCC 14579, ZipCode 10), Streptococcus agalactiae (ATCC BAA611D, ZipCode 15), Streptococcus uberis (ATCC 9927, ZipCode 19), Mycoplasma bovis (ATCC 25523, ZipCode 20), and Escherichia coli O157 (ATCC 700927, ZipCode 28). Each bacterial group has 4 replicate spots. ZipCode numbers of the specific probes are reported under each replicate. Lighted spots at the corners represent the hybridization and ligation controls (spots 66 and 63), as in the universal array scheme (A). NAS=nonaureus staphylococci.

The signal-to-noise ratios (SNR; top, black line) and the unadjusted P-values (bottom, gray line, logarithmic scale) of the serial dilutions of Mycoplasma bovis (ATCC 25523), Streptococcus pyogenes (ATCC 12344), Staphylococcus aureus (ATCC 19095), and Streptococcus agalactiae (ATCC BAA611D). The x-axis reports the quantity of PCR product (fmol). The threshold for significance (P<0.01) is drawn as a dashed line.

Heat map of unadjusted P-values for ligation detection reaction experiments on 50 milk samples. The map (on the left) indicates the P-value of the one-sided t-tests. No multiple comparisons correction has been made. The white color indicates a P-value of 0.005 or less, whereas the black color indicates a P-value of 0.05 or more. Threshold value of 0.01 is highlighted in light gray. Microbiological characterization and source of the samples are provided on the right. NAS=nonaureus staphylococci.