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Vol. 29, No. 11JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1991, p. 2628-2630
0095-1137/91/112628-03$02.00/0
Copyright 1991, American Society for Microbiology
Specific Amplification of Rickettsia tsutsugamushi DNA from
Clinical Specimens by Polymerase Chain Reaction
YUMIKO FURUYA,1* YOSHIYA YOSHIDA,' TAKASHI KATAYAMA,' FUMIHIKO KAWAMORI,2
SEIGO YAMAMOTO,3 NORIO OHASHI,4 AKIRA TAMURA,4 AND AKIYOSHI KAWAMURA, JR.S
Division of Virology, Kanagawa Prefectural Public Health Laboratory, Asahi-ku, Yokohama 241,' Shizuoka Prefectural
Institute ofPublic Health and Environmental Science, 4-27-2 Kitaando, Shizuoka 420,2 Miyazaki Prefectural Institute
for Public Health and Environment, 2-3-2 Kibanadainishi, Gakuen, Miyazaki 889-21,3 Niigata College of
Pharmacy, Niigata 950-2l,4 and 1-17-16-306, Kamiosaki, Shinagawa-ku, Tokyo 14l,5 Japan
Received 22 April 1991/Accepted 5 August 1991
Polymerase chain reaction (PCR) was used to detect Rickettsia tsutsugamushi-specific DNA in clinical
specimens. The primer pair used for PCR was designed from the nucleotide sequence of the gene encoding the
56-kDa antigen of the Gilliam strain. Theses primers led to a 78-bp fragment by amplifying the genomic DNAs
from five serovariants, i.e., the Gilliam, Karp, Kato, Kawasaki, and Kuroki strains of R. tsutsugamushi, and
also the DNA from blood clots of patients with scrub typhus, even at the early stage of onset of the disease. This
indicates that this method is suitable for the diagnosis of scrub typhus.
About 700 to 900 patients per year in Japan suffer from
tsutsugamushi disease, a rickettsiosis transmitted by the
chigger mite. Microimmunofluorescence and immunoperoxidase techniques are generally used to diagnose this
disease. However, diagnosis is sometimes difficult in the
early stage of the illness, when the antibody titers are not yet
high enough to be detected.
Recently, the application of polymerase chain reaction
(PCR) to the diagnosis of some infectious diseases has been
reported. This method is excellent in circumstances in which
immunological techniques or isolation of the causative agent
is difficult. In tsutsugamushi disease, PCR showed rickettsial
infection during the acute rickettsemia phase, which occurs
before the antibody titer increases. Thus, diagnosis will be
possible in the early stage of the illness. Here, we describe
the use of PCR in diagnosing tsutsugamushi disease.
Rickettsia tsutsugamushi Gilliam, Karp, Kato, Shimokoshi, Kawasaki, and Kuroki strains used in our previous
studies (4, 6, 7), R. rickettsii Bitterroot strain from Denka
Seiken Co., Tokyo, Japan, and R. sibirica ATCC VR-151
donated by N. Tachibana, Miyazaki Medical College, were
propagated in L929 cells as described previously (4, 7).
Monolayers of infected cells in a 200-ml culture bottle were
homogenized with a Dounce homogenizer (Kontes Glaso
Co., N.J.) in 5 ml of 10 mM Tris-HCl buffer (pH 8.0)
containing 0.1 mM EDTA (TE buffer), and the DNA was
extracted from homogenate supernatants obtained after centrifugation at 300 x g for 10 min. In some experiments,
Gilliam strain rickettsiae purified by Percoll density gradient
centrifugation (5) were suspended in a small amount of TE
buffer and used for DNA preparation. Blood clots obtained
from patients with tsutsugamushi disease were stored at
-80°C until use. From portions of the clots, rickettsiae were
isolated in mice or L929 cells, and the rickettsial serotype
was identified with strain-specific monoclonal antibodies as
described previously (1). DNA was extracted from blood
clots which corresponded to about 0.5 ml of blood and which
had been homogenized in 1.0 ml of distilled water with a
mortar.
* Corresponding author.
To extract DNA preparations from these samples, the
rickettsial suspensions or blood clot homogenates were
mixed with 1/10 volume of 10% sodium dodecyl sulfate
(SDS) (final concentration of SDS, 1%) and incubated at 4°C
for 16 h. After the addition of 1/10 volume of 10-foldconcentrated TE buffer, the mixture was further incubated
with 3x crystallized chicken egg white lysozyme (Sigma
Chemical Co., St. Louis, Mo.) at a final concentration of 2
mg/ml for 30 min in an ice bath and then with proteinase K
(Merck & Co., Inc., Rahway, N.J.) at a final concentration
of 0.2 mg/ml for 1 h at 55°C. The DNA in this lysate was
purified by three extractions with an equal volume of a
phenol-chloroform (1:1) mixture, followed by precipitation
with 2 volumes of ethanol and resuspension in 50 ,ul of TE
buffer. A pair of primers (primer 1, ATAGAATTGGGTGAG
GAAGGAGGATTAGAG; primer 2, ACCAGTAATCATTC
CTCCAACGATTCCAAC) for PCR were synthesized by an
Applied Biosystems DNA synthesizer. Primer 1 was the
nucleotide sequence corresponding to 10 amino acids at the
N terminus of a mature 56-kDa protein in R. tsutsugamushi
(2), and primer 2 was complementary to the region 18 bp
downstream from the first primer. These primers amplified a
78-bp fragment. The PCR amplification mixture (total volume, 50 ,ul) contained 1.5 mM MgCl2, 50 mM KCl, 10 mM
Tris-HCl (pH 8.3), 0.001% (wt/vol) gelatin, 200 1±M (each)
dATP, dCTP, dGTP, and dTTP, 1 ,uM each primer, 1.25 U of
AmpliTaq polymerase (Perkin-Elmer Cetus, Norwalk,
Conn.), and 5 ,ul of template DNA. The mixture was placed
in a thermal cycler (Perkin-Elmer Cetus), and the temperature was denatured at 94°C for 30 s, annealed at 570C for 2
min, and then chain extended at 70°C for 2 min. This cycle
was repeated 30 times. In some experiments, 5 ,ul of the
amplified mixture was added into a fresh PCR reaction
mixture and further amplified through 30 cycles (a total of 60
cycles). The amplified sample (5 ,ul) was electrophoresed in
an 8% polyacrylamide gel, and the DNA bands were stained
with 0.5 ,ug of ethidium bromide per ml. For Southern
blotting analysis, the 78-bp DNA probe labeled with digoxigenin was prepared by incorporating digoxigenin-11-dUTP
during amplification of rickettsial DNA (Gilliam strain) with
the primers. DNA obtained by amplification of clinical
specimens was electrophoresed, transferred to a nylon mem2628
NOTES 2629
A
8 0 -
8 -
li ji --78
1 ? A 1rI fh f
1 2 3 4 5 6 7 8
FIG. 1. Polyacrylamide gel electrophoresis of amplified DNA by
PCR after 30-cycle amplification with template DNA from the
strains Gilliam (lane 2), Karp (lane 3), Kato (lane 4), Kawasaki (lane
5), Kuroki (lane 6), and Shimokoshi (lane 7) and L929 cells (lane 8).
Lane 1, restriction endonuclease digests of plasmid pHY as size
markers (Takara Shuzo Co., Ltd., Kyoto, Japan). The sizes of the
fragments are 4,870, 2,016, 1,360, 1,107, 926, 658, 489, 267, and 80
bp. Numbers on the figure are sizes (in base pairs).
brane by electrophoresis, and hybridized with the digoxigenin-labeled DNA probe, under highly stringent conditions in
solution containing 0.02% SDS and 5x SSC (lx SSC is 0.15
M NaCI and 0.015 M sodium citrate) at 68°C for 10 h. After
hybridization, the membrane was washed twice with O.lx
SSC containing 0.1% SDS at 68°C for 30 min and allowed to
react with alkaline phosphatase-conjugated antidigoxigenin
antibody. Detailed DNA labeling with digoxigenin and the
detection ofDNA bands containing the drug were performed
according to the manufacturer's instructions for the Genius
DNA Labeling and Detection kit (Boehringer GmbH, Mannheim, Germany).
After the PCR reaction with the primers, the predicted
78-bp DNA fragment was identified as a sharp band in
polyacrylamide gel electrophoresis, by using template DNA
from the Gilliam, Karp, Kato, Kawasaki, and Kuroki
strains, but no amplified band was seen when the DNA from
the Shimokoshi strain was used (Fig. 1). The enzymatic
amplifications were not seen in the tests with the DNA of R.
rickettsia, R. sibirica, Escherichia coli, Proteus vulgaris, and
Proteus mirabilis (data not shown) and with that of the host
cell line (L929). These results indicate that amplification with
the primers is specific for R. tsutsugamushi among the five
antigenic variants. In our preliminary tests of PCR, we
ascertained that amplified DNA was detectable when more
than 1 ng of genomic DNA from the purified rickettsia was
used as a template.
We also tested PCR in the diagnosis of tsutsugamushi
TABLE 1. Patient blood used for PCR
Patient Age Days after Serotype of
no. onset isolated rickettsiaa
1 69 1 Kawasaki
2 39 7 Karp
3 52 9 Karp
a Serotype of rickettsia isolated was identified by an immunofluorescence
technique with strain-specific antibodies as described previously (6).
FIG. 2. (A) Electrophoresis of amplified DNA by PCR after
60-cycle amplification with the template DNA from blood from
healthy humans (lanes 2 to 4) and patient blood (as shown in Table
1) (lanes 5 to 7). Lane 1, molecular size markers as described in the
legend to Fig. 1. (B) Southern blot of DNA from panel A with
amplified digoxigenin-labeled Gilliam 78-bp DNA probe. Numbers
on the figure are sizes (in base pairs).
disease. The blood of patients from which Kawasaki- or
Karp-type rickettsiae were isolated was used (Table 1). In
PCR tests of the DNAs from the blood of these three
patients, weakly stained bands were seen after a 30-cycle
amplification (data not shown). However, by an additional
30-cycle amplification (a total of 60 cycles), clear bands were
observed at the position of 78 bp, together with other minor
bands (Fig. 2A). There were no detectable bands in the
amplification tests of DNA from the blood of three healthy
donors. In the hybridization test with the amplified digoxigenin-labeled Gilliam 78-bp DNA probe, only the 78-bp
bands from the patient specimens hybridized, indicating that
the 78-bp bands are DNA fragments specific to rickettsia
(Fig. 2B).
As described above, we amplified the small amount of
rickettsial DNA in patient blood by PCR. The results described above may be because the amplified sequence, 78
bp, is short, which is a benefit for rapid and correct polymerization even through a 60-cycle amplification. The primers used here were designed from the DNA sequence corresponding to the N-terminal area of the 56-kDa protein in the
Gilliam strain, which we previously described (2). DNA of
R. tsutsugamushi of the Gilliam, Karp, Kato, Kawasaki, and
Kuroki strains was amplified by PCR with these primers.
This indicates that the nucleotide sequence of the primer
regions is well conserved among these strains. The DNA
from the Shimokoshi strain was not amplified by PCR,
indicating that these primers are not appropriate to the DNA
of this strain. However, infection with Shimokoshi-type
rickettsiae (5) is very rare, and almost all rickettsiae isolated
from the patients with tsutsugamushi disease in Japan belong
to the other five serotypes (3, 6, 7). Therefore, PCR with the
primers described above is practical for diagnosis in Japan.
Furthermore, the applicability of this method to clinical
specimens from acute-phase patients indicated its usefulness
in the diagnosis of tsutsugamushi disease.
We thank K. Oda (Kanagawa Prefectural Public Health
Laboratory) for his helpful advice.
REFERENCES
1. Furuya, Y., S. Yamamoto, M. Otu, Y. Yoshida, N. Ohashi, M.
Murata, N. Kawabata, A. Tamura, and A. Kawamura, Jr. 1991.
VOL. 29, 1991
1 :'. . 1 ') 1.
J. CLIN. MICROBIOL.
Use of monoclonal antibodies against Rickettsia tsutsugamushi
Kawasaki for serodiagnosis by enzyme-linked immunosorbent
assay. J. Clin. Microbiol. 29:340-345.
2. Ohashi, N., H. Nashimoto, H. Ikeda, and A. Tamura. 1990.
Cloning and sequencing of the gene (tsg56) encoding a typespecific antigen from Rickettsia tsutsugamushi. Gene 91:119122.
3. Ohashi, N., A. Tamuraj H. Sakurai, and S. Yamanloto. 1990.
Characterization of a new antigenic type, Kuroki, of Rickettsia
tsutsugamushi isolated from a patient in Japan. J. Clin. Microbiol. 28:2111-2113.
4. Tamura, A., K. Takahashi, T. Tsuruhara, H. Urakami, S. Miyamura, H. Sekikawa, M. Kenmotsu, M. Shibata, S. Abe, and H.
Nezu. 1984. Isolation of Rickettsia tsutsugamushi antigenically
different from Kato, Karp and Gilliam strains from patients.
Microbiol. Immunol. 28:873-882.
5. Tamura, A., H. Urakami, and T. Tsuruhara. 1982. Purification of
Rickettsia tsutsugamushi by percoll density gradient centrifugation. Microbiol. Immunol. 26:321-328.
6. Yamamoto, S., N. Kawabata, K. Ooura, M. Murata, and Y.
Minamishima. 1989. Antigenic types of Rickettsia tsutsugamushi
isolated from patients with Tsutsugamushi fever and their distribution in Miyazaki Prefecture. J. Jpn. Assoc. Infect. Dis. 63:109117. (In Japanese.)
7. Yamamoto, S., N. Kawabata, A. Tamura, H. Urakami, N. Ohashi,
M. Murata, Y. Yoshida, and A. Kawamura, Jr. 1986. Immunological properties of Rickettsia tsutsugamushi, Kawasaki strain,
isolated from a patient in Kyushu. Microbiol. Immunol. 30:611620.
2630 NOTES

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