Multiplex Detection of Ehrlichia and Anaplasma Species Pathogens in Peripheral Blood by Real-Time Reverse Transcriptase-Polymerase Chain Reaction
2005; Elsevier BV; Volume: 7; Issue: 2 Linguagem: Inglês
10.1016/s1525-1578(10)60559-4
ISSN1943-7811
AutoresKamesh R. Sirigireddy, Roman R. Ganta,
Tópico(s)Vector-Borne Animal Diseases
ResumoTick-borne infections are responsible for many emerging diseases in humans and several vertebrates. These include human infections with Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii. Because single or co-infections can result from tick bites, the availability of a rapid, multiplex molecular test will be valuable for timely diagnosis and treatment. Here, we describe a multiplex molecular test that can detect single or co-infections with up to five Ehrlichia and Anaplasma species. The test protocol includes the magnetic capture-based purification of 16S ribosomal RNA, its enrichment, and specific-pathogen(s) detection by real-time reverse transcriptase-polymerase chain reaction. We also report a unique cloning strategy to develop positive controls in the absence of a pathogen's genomic DNA. The test was assessed by examining blood samples from dogs suspected to be positive for ehrlichiosis. The dog was chosen as the model system because it is susceptible to acquire infections with up to five pathogens of the genera Ehrlichia and Anaplasma. The test identified single infections in the canine host with E. chaffeensis, E. canis, E. ewingii, A. phagocytophilum, and A. platys and co-infection with E. canis and A. platys. The multipathogen detection and novel positive control development procedures described here will be valuable in monitoring infections in people, other vertebrates, and ticks. Tick-borne infections are responsible for many emerging diseases in humans and several vertebrates. These include human infections with Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii. Because single or co-infections can result from tick bites, the availability of a rapid, multiplex molecular test will be valuable for timely diagnosis and treatment. Here, we describe a multiplex molecular test that can detect single or co-infections with up to five Ehrlichia and Anaplasma species. The test protocol includes the magnetic capture-based purification of 16S ribosomal RNA, its enrichment, and specific-pathogen(s) detection by real-time reverse transcriptase-polymerase chain reaction. We also report a unique cloning strategy to develop positive controls in the absence of a pathogen's genomic DNA. The test was assessed by examining blood samples from dogs suspected to be positive for ehrlichiosis. The dog was chosen as the model system because it is susceptible to acquire infections with up to five pathogens of the genera Ehrlichia and Anaplasma. The test identified single infections in the canine host with E. chaffeensis, E. canis, E. ewingii, A. phagocytophilum, and A. platys and co-infection with E. canis and A. platys. The multipathogen detection and novel positive control development procedures described here will be valuable in monitoring infections in people, other vertebrates, and ticks. Several rickettsial agents of the family Anaplasmataceae cause severe, tick-borne pathogen infections in a wide range of vertebrate host species.1Dumler JS Barbet AF Bekker CP Dasch GA Palmer GH Ray SC Rikihisa Y Rurangirwa FR Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia phagocytophila.Int J Syst Evol Microbiol. 2001; 51: 2145-2165Crossref PubMed Scopus (1634) Google Scholar They include emerging infections in humans with Ehrlichia chaffeensis, Ehrlichia ewingii, and Anaplasma phagocytophilum and canine infections with E. canis, E. ewingii, E. chaffeensis, A. platys, and A. phagocytophilum.1Dumler JS Barbet AF Bekker CP Dasch GA Palmer GH Ray SC Rikihisa Y Rurangirwa FR Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia phagocytophila.Int J Syst Evol Microbiol. 2001; 51: 2145-2165Crossref PubMed Scopus (1634) Google Scholar, 2Dawson JE Anderson BE Fishbein DB Sanchez CY Goldsmith CY Wilson KH Duntley CW Isolation and characterization of an Ehrlichia sp. from a patient diagnosed with human ehrlichiosis.J Clin Microbiol. 1991; 29: 2741-2745PubMed Google Scholar, 3Maeda K Markowitz N Hawley RC Ristic M Cox D McDade JE Human infection with Ehrlichia canis, a leukocytic rickettsia.N Engl J Med. 1987; 316: 853-856Crossref PubMed Scopus (408) Google Scholar, 4Chen SM Dumler JS Bakken JS Walker DH Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease.J Clin Microbiol. 1994; 32: 589-595Crossref PubMed Google Scholar, 5Buller RS Arens M Hmiel SP Paddock CD Sumner JW Rikhisa Y Unver A Gaudreault-Keener M Manian FA Liddell AM Schmulewitz N Storch GA Ehrlichia ewingii, a newly recognized agent of human ehrlichiosis.N Engl J Med. 1999; 341: 148-155Crossref PubMed Scopus (351) Google Scholar, 6Breitschwerdt EB Hegarty BC Hancock SI Sequential evaluation of dogs naturally infected with Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia equi, Ehrlichia ewingii, or Bartonella vinsonii.J Clin Microbiol. 1998; 36: 2645-2651PubMed Google Scholar, 7French TW Harvey JW Canine infectious cyclic thrombocytopenia (Ehrlichia platys infection in dogs).in: Woldehiwet Z Ristic M Rickettsial and Chlamydial Diseases of Domestic Animals. 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51: 2145-2165Crossref PubMed Scopus (1634) Google Scholar Co-infections with two or more rickettsiales and other tick-transmitted pathogens are common in vertebrate and tick hosts.6Breitschwerdt EB Hegarty BC Hancock SI Sequential evaluation of dogs naturally infected with Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia equi, Ehrlichia ewingii, or Bartonella vinsonii.J Clin Microbiol. 1998; 36: 2645-2651PubMed Google Scholar, 9Paddock CD Childs JE Ehrlichia chaffeensis: a prototypical emerging pathogen.Clin Microbiol Rev. 2003; 16: 37-64Crossref PubMed Scopus (379) Google Scholar, 11Kordick SK Breitschwerdt EB Hegarty BC Southwick KL Colitz CM Hancock SI Bradley JM Rumbough R Mcpherson JT MacCormack JN Coinfection with multiple tick-borne pathogens in a Walker Hound kennel in North Carolina.J Clin Microbiol. 1999; 37: 2631-2638PubMed Google Scholar, 12Hoskins JD Breitschwerdt EB Gaunt SD French TW Burgdorfer W Antibodies to Ehrlichia canis, Ehrlichia platys, and spotted fever group rickettsiae in Louisiana dogs.J Vet Intern Med. 1988; 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146: 186-190Crossref PubMed Scopus (85) Google Scholar, 21Kieser ST Eriks IS Palmer GH Cyclic Rickettsemia during persistent Anaplasma marginale infection of cattle.Infect Immun. 1990; 58: 1117-1119PubMed Google Scholar Because rickettsiales are able to infect a broad range of hosts, and multiple pathogens can co-exist in both vertebrate and invertebrate hosts, the availability of a rapid, highly sensitive, and specific test that can diagnose one or more pathogens, including co-infections, in a test sample will be valuable for timely diagnosis and treatment. Such a test will be useful for monitoring and controlling the spread of infections from ticks. Moreover, a multiplex molecular test will be valuable in studies to assess the impact of co-infections on the disease outcome. Similarly, it will be useful in studies to evaluate vaccines and therapeutics. In this study, we described the development of a rapid, two-step, species-specific multiplex molecular test to detect one or more infections with three Ehrlichia and two Anaplasma species. We also reported a novel cloning strategy to generate the positive controls needed to establish the test. The molecular test was used to detect natural infections, including co-infections in dogs with E. chaffeensis, E. canis, E. ewingii, A. platys, and A. phagocytophilum. The dog was chosen as the model system to evaluate the test utility because it is known to acquire infections with up to five pathogens of the genera Ehrlichia and Anaplasma. E. chaffeensis Arkansas isolate and E. canis Oklahoma isolate were grown in a canine macrophage cell line, DH82, as described previously.22Chen SM Popov VL Feng HM Walker DH Analysis and ultrastructural localization of Ehrlichia chaffeensis proteins with monoclonal antibodies.Am J Trop Med Hyg. 1996; 54: 405-412PubMed Google Scholar, 23Reddy GR Sulsona CR Barbet AF Mahan SM Burridge MJ Alleman AR Molecular characterization of a 28 kDa surface antigen gene family of the tribe Ehrlichiae.Biochem Biophys Res Commun. 1998; 247: 636-643Crossref PubMed Scopus (71) Google Scholar A 0.5-ml sample of a culture pellet was centrifuged at 13,000 × g for 15 minutes at 4°C and used to isolate total RNA using the Tri-reagent method (Sigma Chemical Co., St. Louis, MO) as per the manufacturer's instructions. The RNA recovered was dissolved in 100 μl of nuclease-free water and stored at −70°C in the presence of 40 U of RNase inhibitor, RNasin (Ambion Inc., Austin, TX). Total RNA from blood samples was also isolated according to the Tri-reagent method by using 0.25 ml of blood collected in ethylenediamine tetraacetic acid (EDTA) and the final pellet was resuspended in 100 μl of nuclease-free water. A capture method was also used to isolate Ehrlichia/Anaplasma species 16S ribosomal RNA (rRNA) (described below). Five ml of 80 to 100% Ehrlichia-infected DH82 culture was used to isolate genomic DNA by the sodium dodecyl sulfate, proteinase K, phenol, chloroform, isoamyl alcohol method.24Maniatis T Fritsch EF Sambrook J Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor1982Google Scholar A. phagocytophilum genomic DNA, isolated from in vitro cultures, was provided by Dr. J. Stephen Dumler, The Johns Hopkins Medical Institutions, Baltimore, MD. The 16S rRNA gene sequences for several Ehrlichia/Anaplasma species, available in the GenBank nucleotide sequence database, were downloaded and aligned by using the University of Wisconsin's Genetic Computer Group programs Pileup and Pretty.25Devereux J Genetics computer group sequence analysis software package, version 6.1.Nucleic Acids Res. 1984; 12: 387-395Crossref PubMed Scopus (12895) Google Scholar Genera-specific regions were used to design a capture primer to facilitate capturing of 16S rRNA of all Ehrlichia and Anaplasma species from a sample. PCR primers for the real-time RT-PCR assay development were also designed from the genera-specific region (Figure 1 and Table 1). Species-specific regions were used from the alignment to design TaqMan probes for use in the real-time, species-specific pathogen detection of E. canis, E. chaffeensis, E. ewingii, A. phagocytophilum, and A. platys. Fluorescent reporter dyes and quencher molecules on the TaqMan probes were carefully selected to facilitate the multiplex assay (Table 1). The primers and TaqMan probes were custom synthesized from Nucleic Acid Facility, University of Pennsylvania, Philadelphia, PA, or from Integrated DNA Technology Inc., Coralville, IA.Table 1Primers and Probes Used in the Multiplex Molecular Test DevelopmentSequenceLengthTemperature (°C)Primers5′ ctcagaacgaacgctgg1754.6 Ehrlichia/Anaplasma TaqMan forward primer Ehrlichia TaqMan reverse primer5′ catttctaatggctattcc1951.5 Anaplasma TaqMan reverse primer5′catttctagtggctatccc1953 Ehrlichia/Anaplasma reverse primer5′gtattaccgcggctgctggcac2272 E. ewingii long forward primer5′ctcagaacgaacgctggcggcaagcctaacacatgcaa gtcgaacgaacaattcctaaatagtctctgactatttagata gttgttagtggcagac9689.9 A. platys long forward primer5′ctcagaacgaacgctggcggcaagcttaacacatgcaag tcgaacggatttttgtcgtagcttgctatgataaaaattagtgg cagac8891.5 Ehrlichia/Anaplasma capture primer5′Biotin−ccccccccccccggacttcttctrtrggtaccgtc3574TaqMan probes E. chaffeensis5′TET/cttataaccttttggttataaataattgttag/TAMRA*Sequence with reporter and quencher molecules.3255.6 E. canis5′FAM/tatagcctctggctataggaaattgttag/TAMRA*Sequence with reporter and quencher molecules.2960.7 E. ewingii5′ROX/ctaaatagtctctgactatttagatagttgttag/BQH2*Sequence with reporter and quencher molecules.3459.5 A. platys5′FAM/cggatttttgtcgtagcttgctatgat/DABCYL*Sequence with reporter and quencher molecules.2760.1 A. phagocytophilum5′TET/ttgctataaagaataattagtggcagacg/DABCYL*Sequence with reporter and quencher molecules.2958.3 Ehrlichia/Anaplasma common5′ROX/taacacatgcaagtcgaacgga/BQH2*Sequence with reporter and quencher molecules.2258.3* Sequence with reporter and quencher molecules. Open table in a new tab Genomic DNA of E. chaffeensis, E. canis, and A. phagocytophilum were used as the templates to amplify a 0.48-kb 16S rRNA gene segment by using Ehrlichia/Anaplasma common TaqMan forward primer and Ehrlichia/Anaplasma common reverse primer. The PCRs were performed with 200 ng of genomic DNA using the Ampli TaqPCR reagent kit (Applied Biosystems, Foster City, CA). The thermal cycles include the initial denaturation for 3 minutes at 94°C, 45 cycles of 94°C for 30 seconds, 52°C for 30 seconds, and 72°C for 60 seconds, followed by one cycle of 72°C for 3 minutes. The PCR products were resolved on a 1% agarose gel in 1× Tris-acetate EDTA buffer (40 mmol/L Tris-acetate, 1 mmol/L EDTA, pH 8.0) containing 0.1 μg/ml of ethidium bromide, and were visualized under UV light.24Maniatis T Fritsch EF Sambrook J Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor1982Google Scholar The amplicons were blunt-ended by using T4 DNA polymerase and were ligated into the EcoRV site of the plasmid, pBlue Script SK+. Subsequently, the ligation mix was transformed into E. coli, XL1 Blue (Stratagene, La Jolla, CA), the transformants containing the recombinant plasmids were selected and plasmid DNA was isolated and used for sequencing with a Thermo-sequencing reaction kit (USB Corp., Cleveland, OH).24Maniatis T Fritsch EF Sambrook J Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor1982Google Scholar Because our attempts to obtain genomic DNA for A. platys and E. ewingii were unsuccessful, we could not use the approach just described for developing positive controls for these two organisms. A novel method for generating positive control plasmids without the need of genomic DNA for these two species was designed (Figure 2). The regions of the 16S rRNA gene segment selected for preparing the positive control plasmid shares extensive homology between E. ewingii and E. chaffeensis, except for one variable region located in the middle (Figure 1). Similarly, A. phagocytophilum and A. platys differ mostly at the central variable region (Figure 1). This information was used to design long forward primers having central variable regions specific for each species, plus Ehrlichia/Anaplasma conserved 5′ and 3′ overhangs. E. ewingii-specific primer was 96 bases, whereas the A. platys-specific primer was 88 bases in length (Table 1). These primers were used in combination with the Ehrlichia/Anaplasma common reverse primer in the PCRs, with E. chaffeensis or A. phagocytophilum-positive control plasmids as the templates (Figure 2). Because the long primers of E. ewingii and A. platys anneal to the E. chaffeensis and A. phagocytophilum templates only at the 5′ and 3′ ends, the amplicons are expected to contain E. ewingii- and A. platys-specific sequences, respectively. The PCR products were cloned into the plasmid, Blue Script, and the insert sequences were verified by performing DNA sequence analysis as previously described. In vitro transcripts from the inserts of all five plasmids were prepared for use in the molecular test development. The recombinant plasmids were digested with BamHI restriction enzyme (a site for BamHI is located at the 3′ end of the insert in the multiple-cloning sites region of the plasmid). BamHI was chosen because it does not have a recognition sequence within the inserts, thus allowing the plasmids to linearize downstream to inserts to facilitate the synthesis of the transcripts by using T7 polymerase. BamHI-digested plasmid DNAs (∼3 μg) were used to generate recombinant transcripts by using the T7 MEGAscript high-yield transcription kit as outlined in the kit protocol (Ambion Inc.). The recombinant transcripts were purified free of plasmid DNA by treating with DNase I and using the RNA purification kit, MEGAclear (Ambion Inc.). The quality, quantity, and length of the transcripts were determined in a Bio-analyzer (Agilent Technologies, Palo Alto, CA). A magnetic capture technique was developed to isolate Ehrlichia/Anaplasma 16S rRNA from in vitro cultures or blood by following a strategy similar to the one reported for Chlamydia trachomatis,26Stefano JE Genovese L An Q Lu L McCarty J Du Y Stefano K Burg JL King W Lane DJ Rapid and sensitive detection of Chlamydia trachomatis using a ligatable binary RNA probe and Q beta replicase.Mol Cell Probes. 1997; 11: 407-426Crossref PubMed Scopus (5) Google Scholar but with several modifications (Figure 3). An Ehrlichia and Anaplasma genera-specific capture primer was designed from the complementary sequence of 16S rRNA that is conserved in all known Ehrlichia/Anaplasma species. A 12-nucleotide-long dC tail and biotin molecule were added to the capture primer at the 5′ end to facilitate the capturing of rRNA (Table 1). The capture primer, in combination with streptavidin-coated magnetic beads and a magnetic separation rack (New England Biolabs Inc., Beverly, MA), was used to isolate the Ehrlichia/Anaplasma species 16S rRNA. Magnetic beads coated with 170 pmol of streptavidin were washed twice with a wash buffer (0.5 mol/L NaCl, 20 mmol/L Tris-HCl, pH 7.5, 1 mmol/L EDTA) and were separated with a magnetic rack. The washed beads were incubated at room temperature for 5 minutes with 25 μl of 0.8 μmol/L capture primer. Affinity between biotin on the capture primer and streptavidin on the magnetic beads allowed the beads and probe to form a complex. The complex was washed twice with the wash buffer to remove excess unbound capture primer. Twenty-five μl in vitro culture of E. canis or E. chaffeensis (5 to 10 million of 80 to 90% infected DH82 cells/ml) and 25 μl of uninfected canine blood were mixed, lysed by adding an equal volume of GTC buffer (0.4 mol/L Tris-HCl, pH 7.85 containing 5 mol/L guanidinium thiocyanate, 0.5 mol/L sarkosyl, and 20 mmol/L EDTA), incubated at 65°C for 5 minutes, transferred to a tube containing the magnetic beads-capture primer complex, and stored at room temperature for 10 minutes. This step allows the formation of Ehrlichia/Anaplasma 16S rRNA and capture primer hybrids (Figure 3). The mixture was then washed twice with the wash buffer and once with a low-salt wash buffer (0.15 mol/L NaCl, 20 mmol/L Tris-HCl, pH 7.5, and 1 mmol/L EDTA). Ehrlichia 16S rRNA was eluted by adding 50 μl of preheated (70°C) TE buffer (10 mmol/L Tris, 1 mmol/L EDTA, pH 8.0) and was stored at −80°C in the presence of RNasin until further use. TaqMan-based real-time amplification27Mackay IM Real-time PCR in the microbiology laboratory.Clin Microbiol Infect. 2004; 10: 190-212Crossref PubMed Scopus (573) Google Scholar, 28Szollosi J Damjanovich S Matyus L Application of fluorescence resonance energy transfer in the clinical laboratory: routine and research.Cytometry. 1998; 34: 159-179Crossref PubMed Scopus (190) Google Scholar was performed by using the Smart Cycler system (Cepheid, Sunnyvale, CA). Because the Smart Cycler system has the capability to detect the fluorescent emission from only four unique probes, the test procedure to detect the five pathogens was split into two parts. Part 1 of the test included the detection format for E. chaffeensis, E. canis, and E. ewingii. Part 2 of the test was designed to detect A. platys and A. phagocytophilum. The mixture for PCR assay is 25 μl in volume, containing 10 pmol each of the TaqMan forward and reverse primers, 10 nmol of dNTPs, 125 nmol MgCl2, 4 U of platinum TaqDNA polymerase (Invitrogen Technologies, Carlsbad, CA) and varying concentrations of TaqMan probes for each pathogen. They are 7.5 pmol for E. chaffeensis; 3.75 pmol for E. canis; 8.75 pmol for E. ewingii; 6.0 pmol for A. platys; and 3.75 pmol for A. phagocytophilum. The concentrations of the TaqMan probes were chosen after the standardization experiments to yield optimal results. The temperature cycles used for the assay are: initial heating for 3 minutes at 95°C, followed by 45 cycles of 95°C for 15 seconds, 50°C for 30 seconds, and 60°C for 60 seconds. The PCR product formation was monitored in real-time by measuring the emitted fluorescence in the extension phase of the PCR cycles with the Smart Cycler system. The machine qualifies a reaction positive for the presence of a template when it detects 10 fluorescent units for each fluorescent emission channel. The PCR cycle at which this occurs is regarded as the Ct value and it is template concentration-dependent. Similarly, the real time RT-PCR was performed in a 25-μl reaction, but containing 1 μl of SS-III and Taq mix (SuperScript-III, one-step RT-PCR system with platinum TaqDNA polymerase; Invitrogen Technologies). Thermal cycles for RT-PCR included an additional initial step at 48°C for 30 minutes to generate the cDNA. The optimal assay conditions for species specificity and multipathogen detection were established by using the plasmid DNA of cloned 16S rRNA gene segments. Ten-fold serial dilutions of the positive control plasmids or in vitro synthesized transcripts (ranging from 1 billion to 1 molecule) were made from the known quantities of the plasmid DNA or RNA. The samples were used in real-time PCR and RT-PCR analysis to determine the Ct values to establish detection limits of the multiplex molecular test. Clinicians from several regions within the Unites States were contacted by phone, fax, and/or mail to collect blood samples from clinically suspected canine ehrlichiosis cases. The criterion of a clinical ehrlichiosis in a dog was at the discretion of the clinician examining a case. The case reports were also received and archived at the K-State diagnostic laboratory. A total of 95 samples were collected in EDTA tubes during 2003 from Arkansas, Arizona, Connecticut, Florida, Georgia, Kansas, Kentucky, Missouri, New Mexico, New York, and US Virgin Islands. Typically, the samples were received in ice packs by overnight shipment. Within 3 days of receipt, plasma was separated and stored at −80°C for indirect fluorescent antibody (IFA) analysis. RNA from 0.25 ml of the plasma-free blood was isolated and resuspended in 100 μl of nuclease-free water by following the Tri-reagent RNA isolation method (described above). RNA also was isolated from 50 μl each of clinically suspected canine blood samples by the magnetic capture. The final RNA was eluted in 50 μl of TE buffer. RNA recovered from an equivalent of 6.5 μl of whole blood was used for evaluating samples for the presence of Ehrlichia/Anaplasma species. The assays included reaction-positive and -negative controls. Similarly, the RNA purification method included a cross contamination control that followed through all extraction procedures. The plasma samples were assessed for antibodies against E. canis by the IFA test29Waner T Harrus S Jongejan F Bark H Keysary A Cornelissen AW Significance of serological testing for ehrlichial diseases in dogs with special emphasis on the diagnosis of canine monocytic ehrlichiosis caused by Ehrlichia canis.Vet Parasitol. 2001; 95: 1-15Crossref PubMed Scopus (136) Google Scholar with a commercial kit per the protocol using fluorescein isothiocyanate-conjugated secondary antibody (VMRD Inc., Pullman, WA). E. canis IFA titers were determined by using 1:128 or higher (up to 1:4096) diluted plasma. A limited number of samples were also tested for the presence of E. canis antibodies by SNAP 3Dx test (IDEXX Laboratories, Westbrooke, ME). Paired t-test analysis was performed using the Statview statistical software package (SAS Institute Inc., Cary, NC). A P value <0.01 is considered significant. The 16S rRNA was selected as the target for the Ehrlichia and Anaplasma species multiplex pathogen diagnosis because it is expected to be present in multiple copies in a cell. Genera- and species-specific sequences were identified from the16S rRNA gene segments of Ehrlichia and Anaplasma species to prepare primers and probes used for the molecular test development (Figure 1). To aid in establishing the test, recombinant plasmids containing the 16S rRNA gene segments were prepared using conventional cloning strategies for E. chaffeensis, E. canis, and A. phagocytophilum. A novel molecular strategy was used to generate positive control plasmids for E. ewingii and A. platys pathogens (Figure 2). A TaqMan-based, quantitative, real-time RT-PCR method is used in developing the multiplex Ehrlichia/Anaplasma molecular test. The species-specific probes for the five pathogens detected templates only from their respective species and did not cross-react with templates of the other four species. Serial dilution of the 16S rRNA recombinant transcripts made from the positive control plasmids aided in determining the analytical sensitivity and linearity (Figure 4). The minimum number of transcripts detected by the test was 100 molecules for all five pathogens. The test was also linear with differing concentrations of transcripts up to 1 billion molecules (Figure 4). To examine whether nonequivalent molar ratios can be similarly detected a fixed concentration of one of the three species of Ehrlichia or Anaplasma recombinant transcripts and differing concentrations of the other transcripts were tested for the detection throughout a range of 1000 to 100,000 molecules. The assay identified the transcripts when the difference in the concentration among the templates is up to 100-fold. Beyond this, only the template having the highest concentration was tested positive. The sensitivity of detection by real-time RT-PCR assay for the RNA recovered by the magnetic capture method was also compared with a commercially available Tri-reagent RNA isolation method. RNA was isolated from either cultured E. chaffeensis or E. canis organisms, or from plasma-free or whole blood from a healthy
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