Viral and Rickettsial Zoonoses Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
Received 25 February 2003/ Returned for modification 24 April 2003/ Accepted 7 May 2003
A real-time PCR assay was developed for the detection of
Ehrlichia chaffeensis. The assay is species specific and provides quantitative
results in the range 10 to 10
10 gene copies. The assay is not
inhibited by the presence of tick, human, or mouse DNA and is
compatible with high sample throughput. The assay was compared
with previously described assays for
E. chaffeensis.
Ehrlichia chaffeensis is the causative agent of human monocytic
ehrlichiosis, a potentially fatal disease (
11). The bacterium,
first isolated in 1991 (
4), is a tick-borne zoonotic pathogen
classified within the order
Rickettsiales, family
Anaplasmataceae (
6). The pathogen is maintained in a natural transmission cycle
between the Lone Star tick (
Amblyomma americanum) and mammalian
hosts (
1,
7,
9). Experimental studies of the acquisition and
transmission of
E. chaffeensis between mammals and ticks require
a quantitative assay for the organism that is compatible with
high sample throughput.
Existing PCR assays for E. chaffeensis include a direct PCR assay for the 16S rRNA gene (2) and a nested PCR assay that amplifies the variable-length PCR target (VLPT) (13). These PCR assays can detect the presence of pathogen DNA but do not provide quantitative data. Nested PCR is typically more sensitive than direct PCR but requires more handling of amplified PCR products, decreasing throughput capacity and increasing the risk of sample cross-contamination.
Real-time (TaqMan) PCR. The single-copy 16S rRNA gene of E. chaffeensis was selected for the development of the real-time TaqMan PCR assay (10). Primers that amplify an 81-bp region of the gene (bases 17 to 97, GenBank accession no. U86665) were selected. The assay was optimized with a Brilliant quantitative-PCR core reagent kit (Stratagene, La Jolla, Calif.), with a final reaction volume of 25 µl. The reaction mixture contained a 200 nM concentration of the forward primer ECH16S-17 (5'-GCGGCAAGCCTAACACATG-3'), an 800 nM concentration of the reverse primer ECH16S-97 (5'-CCCGTCTGCCACTAACAATTATT-3'), a 100 nM concentration of the probe ECH16S-38 (5'-6-carboxyfluorescein-AGTCGAACGGACAATTGCTTATAACCTTTTGGT-3'),and 3.0 µM magnesium chloride. Real-time PCRs and fluorescence detection were performed using an iCycler thermal cycler and iQ software (Bio-Rad Laboratories, Hercules, Calif.). The optimized thermal cycler program was 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 57°C for 1 min.
Sensitivity and specificity. Quantitative results were based on a 10-fold dilution series of a plasmid encoding the 16S rRNA gene of E. chaffeensis. The specificity of the assay was determined by testing genomic DNA from three strains of E. chaffeensis, several closely related organisms, uninfected ticks, and uninfected mammalian blood and tissues. E. chaffeensis isolates (Arkansas, St. Vincent, and Jax) were obtained from clinical samples, as described previously (4, 12). All three strains of E. chaffeensis were detected by the TaqMan PCR, and agarose gel electrophoresis revealed a single band of the appropriate size. The TaqMan assay did not amplify genomic DNA from Ehrlichia muris (AS145 strain) (15), Ehrlichia canis (Oklahoma strain) (5), Neorickettsia sennetsu (formerly Ehrlichia sennetsu, Miyayama strain) (14), or Rickettsia prowazekii (Breinl strain). As revealed by agarose gel electrophoresis, the primers used for the TaqMan PCR assay amplified the 16S rRNA genes from Ehrlichia ewingii (human clinical sample) (3) and Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila, USG3 strain) (16). However, neither A. phagocytophilum nor E. ewingii was detected during real-time amplification and fluorescence detection, indicating that the TaqMan probe is species specific for E. chaffeensis (data not shown).
Genomic DNA was extracted from uninfected ticks, blood, and tissue samples using the IsoQuick nucleic acid extraction kit (Orca Research, Inc., Bothell, Wash.). The TaqMan PCR assay did not amplify genomic DNA from mouse blood, spleen, or liver (BALB/c and C57BL/6 strains), human or guinea pig blood, or uninfected ticks (Amblyomma americanum, Dermacentor variabilis, or Ixodes scapularis).
The specificity of the TaqMan PCR assay was further tested using DNA extracted from human clinical samples submitted to the Centers for Disease Control and Prevention (Atlanta, Ga.). Three E. chaffeensis-positive samples and three E. chaffeensis-negative samples (one A. phagocytophilum-positive blood sample, one negative blood sample, and one negative lymph node sample) were tested. The TaqMan PCR was positive for all three samples containing E. chaffeensis DNA but was negative for two samples lacking E. chaffeensis DNA and for the sample containing A. phagocytophilum DNA.
Effect of background DNA. The effect of background DNA on the efficiency and sensitivity of the TaqMan PCR assay was determined. Genomic DNA was pooled from 10 adult female Amblyomma americanum ticks (35.3 µg of DNA/ml) and 10 uninfected BALB/C mice (19.9 µg of DNA/ml), and human genomic DNA (25 µg of DNA/ml) was obtained from Promega (Madison, Wis.). Reaction efficiencies were compared using a fivefold dilution series of genomic E. chaffeensis DNA in water, tick DNA, mouse DNA, or human DNA. As summarized in Table 1, the presence of background DNA did not decrease the efficiency of the TaqMan PCR. The effect of host DNA on sensitivity was determined by comparing the threshold cycles in which genomic E. chaffeensis DNA was detected in the presence of water, tick DNA, mouse DNA, or human DNA. E. chaffeensis DNA was tested at a dilution of approximately 620 bacteria per µl of blood, corresponding with a moderate degree of ehrlichemia (unpublished results). The sensitivity of the assay in the presence of background DNA was not significantly different from that in water (Table 1).
fig.ommitted |
TABLE 1. Effect of host DNA on the efficiency and sensitivity of the real-time TaqMan PCR assay for E. chaffeensisa
| |
Comparison with other assays. The analytical sensitivity and specificity of the TaqMan PCR
were compared with those of three previously described PCR assays
for
E. chaffeensis: a direct PCR assay for the 16S rRNA gene
using primers HE1 and HE3 (
2), a nested PCR assay for the VLPT
of
E. chaffeensis (
13), and a real-time PCR assay for the 16S
rRNA gene, based on the SYBR Green detection system (
8). All
PCR assays were performed in a 25-µl final volume. For
the SYBR Green assay, real-time fluorescence detection was performed
by use of an ABI 7900HT sequence detection system (Applied Biosystems,
Foster City, Calif.).
The sensitivities of all four PCR assays were compared by testing a fivefold dilution series of E. chaffeensis genomic DNA (Table 2). All assays were repeated to ensure reproducible results. The sensitivity of the TaqMan assay was significantly greater than that of the direct (HE1/HE3) assay and comparable to those of the real-time SYBR Green and nested VLPT assays.
fig.ommitted |
TABLE 2. Relative sensitivities of four different PCR assays for E. chaffeensis DNA
| |
The specificity of the TaqMan PCR was compared with those of
the other three PCR assays for
E. chaffeensis (Table
3). The
HE1/HE3 direct and VLPT nested PCR assays have been evaluated
by using genomic DNA from several species of
Ehrlichia and
Anaplasma and have been demonstrated to be species specific for
E. chaffeensis (
2,
13). As reported above, the TaqMan PCR is species specific
for
E. chaffeensis. However, the SYBR Green assay detected
E. canis,
E. ewingii,
E. muris, and
A. phagocytophilum, as well
as
E. chaffeensis.
fig.ommitted |
TABLE 3. Specificities of four different PCR assays for E. chaffeensis DNA
| |
Conclusions. In conclusion, the real-time PCR assay that we have developed
for
E. chaffeensis is very sensitive (10 gene copies), is species
specific, is suitable for high-throughput applications, and
is not inhibited by the presence of human, mouse, or tick DNA.
The assay was found to be superior to three assays previously
described for the detection of
E. chaffeensis based on one or
more criteria. It is more sensitive than the HE1/HE3 direct
PCR assay, more specific than the SYBR Green real-time PCR assay,
and has a higher throughput capacity and less sample handling
than the VLPT nested PCR assay. Most importantly, the real-time
TaqMan PCR assay provides quantitative data, allowing for simultaneous
detection of the pathogen and determination of the infectious
loads in ticks and mammals. This assay provides a powerful tool
for examining the kinetics of infection with
E. chaffeensis and the transmission of the pathogen between mammalian hosts
and arthropod vectors.
We thank John W. Sumner (Centers for Disease Control and Prevention)
for providing clinical specimens. We thank Gregory A. Dasch
for his review of the manuscript. We acknowledge the Biotechnology
Core Facility of the National Center for Infectious Diseases,
Centers for Disease Control and Prevention, for the synthesis
of oligonucleotides.
This research was supported in part by the Association of Public Health Laboratories, through an appointment of the Emerging Infectious Diseases Fellowship Program funded by the Centers for Disease Control and Prevention.
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作者:
Amanda D. Loftis Robert F. Massung and Michael L 2007-5-10