1. Multiplex Detection of Ehrlichia and Anaplasma Species Pathogens in Peripheral Blood by Real-Time Reverse Transcriptase-Polymerase Chain Reaction 
Kamesh R. Sirigireddy, Roman R. Ganta 
The Journal of Molecular Diagnostics 
Volume 7, Issue 2, Pages (May 2005)
DOI: /S (10)
Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

2. Figure 1
Sequence alignment of the 16S rRNA genes and the design of RT-PCR primers, capture primer, and TaqMan probes. The 16S rRNA gene sequences available in the GenBank database for Ehrlichia and Anaplasma species were analyzed using the Genetic Computer Group programs, PILEUP and PRETTY.25 GenBank numbers for the sequences are M73222, E. chaffeensis; M73221, E. canis; M73227, E. ewingii; X61659, E. ruminantium; M82801, A. platys; M73220, A. phagocytophilum; and M60313, A. marginale. The complete sequence of E. chaffeensis from nucleotides 1 to 151 and 426 to 482 are presented. The sequences for the other species are shown only when they differ from the E. chaffeensis sequence. A dot indicates identity with the E. chaffeensis sequence. Dashes refer to the gaps introduced by the PILEUP program to obtain the best alignment. The PCR primers and the capture primer were identified with their names. Lines with arrowheads refer to the orientation of the primers. The genera- and species-specific TaqMan probes are identified with shaded text.
The Journal of Molecular Diagnostics 2005 7, DOI: ( /S (10) )
Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

3. Figure 2
Schematic representation of the molecular strategy used to generate E. ewingii-positive control. Alignment shown in Figure 1 reveals that E. ewingii 16S rRNA sequence differs from E. chaffeensis mostly at the central variable region where the TaqMan probes were designed. The schematic representation of the sequences in this figure shows variable sequences as different shaped lines. To generate an E. ewingii 16S rRNA gene segment, a long forward primer was designed. It contains the Ehrlichia species common sequence at the 5′ and 3′ ends and E. ewingii-specific sequence in the middle. This primer and a genera-specific reverse primer, together with the E. chaffeensis 16S rRNA gene segment as the template were used in the PCR to generate amplicons containing E. ewingii sequence. The amplicons were cloned into a plasmid. A similar strategy was also used to clone A. platys-positive control plasmid using the A. phagocytophilum DNA segment as the template.
The Journal of Molecular Diagnostics 2005 7, DOI: ( /S (10) )
Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

4. Figure 3
Schematic representation of the 16S rRNA capture method. A sample containing Ehrlichia/Anaplasma species organisms is lysed in the GTC buffer and added to the conjugate of the avidin-coated magnetic particles coupled with the biotin-linked capture primer. A magnetic stand is used to capture and purify 16S rRNA from the lysed sample.
The Journal of Molecular Diagnostics 2005 7, DOI: ( /S (10) )
Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

5. Figure 4
Multiplex molecular test's detection sensitivity and linearity with RNA concentration. Serial 10-fold dilutions of the RNA transcripts, made from the positive control plasmids, were analyzed at equivalent molar concentrations of the three Ehrlichia species (A) and two Anaplasma species (B). The fluorescent emission from serial dilution templates for one species each (E. canis and A. platys) is shown in the insets. The average Ct values from three independent experiments for each template concentration of the three Ehrlichia species and two Anaplasma species were plotted against the log number of RNA molecules in A and B, respectively.
The Journal of Molecular Diagnostics 2005 7, DOI: ( /S (10) )
Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions