Journal List > J Bacteriol Virol > v.38(4) > 1033908

Park, Lee, Jin, Jang, Park, Kook, and Lee: Rapid Identification of Rickettsiae using the Real-Time PCR

Abstract

In this study, new real-time PCR method based on the groEL gene was developed and investigated. Four spotted fever group (SFG) strains, four typhus group (TG) strains, and four scrub typhus group (STG) strains were easily differentiated as a distinct entity. This PCR assay was applied to detect Rickettsia DNA from 100 ticks. Twelve Haemaphysalis longicornis ticks were found positive and identified as spotted fever group Rickettsia. This real-time PCR method could simultaneously perform the rapid identification of rickettsiae and the differential diagnosis of SFG, TG, and STG in a single reaction.

REFERENCES

1). Chung HY. Rickettsial Infections. Infect. 17:89–92. 1985.
2). Chung MH., Lee SH., Kim MJ., Lee JH., Kim ES., Kim MK., Park MY., Kang JS. Japanese spotted fever, South Korea. Emerg Infect Dis. 12:1122–1124. 2006.
crossref
3). Fournier PE., Roux V., Raoult D. Phylogenetic analysis of spotted fever group rickettsiae by study of the outer surface protein rOmpA. Int J Syst Bacteriol. 48:839–849. 1998.
crossref
4). Kim KA., Lee SH., Jang WS., Oh MD., Kim I., Choe K. Two cases of tsutsugamushi disease in the spring. Korean J Infect Dis. 31:46–49. 1999.
5). Lee JH., Park HS., Jang WJ., Koh SE., Kim JM., Shim SK., Park MY., Kim YW., Kim BJ., Kook YH., Park KH., Lee SH. Differentiation of rickettsiae by groEL gene analysis. J Clin Microbiol. 41:2952–2960. 2003.
6). Lee JH., Park HS., Jung KD., Jang WJ., Koh SE., Kang SS., Lee IY., Lee WJ., Kim BJ., Kook YH., Park KH., Lee SH. Identification of the spotted fever group rickettsiae detected from Haemaphysalis longicornis in Korea. Microbiol Immunol. 47:301–304. 2003.
7). Lee MK. Real-time polymerase chain reaction (PCR) in microbiology. Infect Chemother. 36:105–113. 2004.
8). Marston EL., Sumner JW., Regnery RL. Evaluation of intraspecies genetic variation within the 60 kDa heat-shock protein gene (groEL) of Bartonella species. Int J Syst Bacteriol. 49:1015–1023. 1999.
9). Raoult D., Dasch GA. Immunoblot cross-reactions among Rickettsia, Proteus spp. and Legionella spp. in patients with Mediterranean spotted fever. FEMS Immunol Med Microbiol. 11:13–18. 1995.
10). Raoult D., Roux V. Rickettsioses as paradigms of new or emerging infectious diseases. Clin Microbiol Rev. 10:694–719. 1997.
crossref
11). Ririe KM., Rasmussen RP., Wittwer CT. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem. 245:154–160. 1997.
crossref
12). Roux V., Fournier PE., Raoult D. Differentiation of spotted fever group rickettsiae by sequencing and analysis of restriction fragment length polymorphism of PCR-amplified DNA of the gene encoding the protein rOmpA. J Clin Microbiol. 34:2058–2065. 1996.
crossref
13). Roux V., Raoult D. Phylogenetic analysis of the genus Rickettsia by 16S rDNA sequencing. Res Microbiol. 146:385–396. 1995.
14). Roux V., Rydkina E., Eremeeva M., Raoult D. Citrate synthase gene comparison, a new tool for phylogenetic analysis, and its application for the rickettsiae. Int J Syst Bacteriol. 47:252–261. 1997.
crossref
15). Song HJ., Seong SY., Huh MS., Park SG., Jang WJ., Kee SH., Kim KH., Kim SC., Choi MS., Kim IS., Chang WH. Molecular and serologic survey of Orientia tsutsugamushi infection among field rodents in southern Cholla Province, Korea. Am J Trop Med Hyg. 58:513–518. 1998.

Figure 1.
Amplification of groEL DNA from rickettsial strains using the real-time PCR. Lanes; jbv-38-221f4.tif SFG (R japonica, R. conorii, R. sibirica, R akari), jbv-38-221f5.tif: TG (R. typhi, R. typhi 87~91, R. typhi 87~100, R prowazekii), jbv-38-221f6.tif STG (O. tsutsugamushi Karp, O. tsutsugamushi Kato, O. tsutsugamushi Boryong, O. tsutsugamushi Gilliam), jbv-38-221f7.tif: DW
jbv-38-221f1.tif
Figure 2.
Amplification of groEL DNA from rickettsial strains and tick DNA using the real-time PCR. Lanes; jbv-38-221f4.tif: SFG (R. japonica, R. conorii, R sibirica, R akari), jbv-38-221f5.tif: TG (R. typhi, R. typhi 87~91, R. typhi 87~100, R. prowazekii), jbv-38-221f6.tif: STG (O. tsutsugamushi Karp, O. tsutsugamushi Kato, O. tsutsugamushi Boryong, O. tsutsugamushi Gilliam), ————: tick (Tick no. 22, 28, 33, 36, 48, 52, 55, 61, 65, 68, 77, 92) jbv-38-221f7.tif: DW
jbv-38-221f2.tif
Figure 3.
The sensitivity of real-time PCR using the R. typhi plasmid. Lanes; (A) R. typhi 106, (B) R. typhi 105, (C) R. typhi 104, (D) R. typhi 103, (E) R. typhi 102, (F) R. typhi 101, (G) R. typhi 1, (H) Negative control.
jbv-38-221f3.tif
Table 1.
Rickettsial strains used in this study
Species Strain Source Geographic location Origin (collection)a GenBank Acession No.
groEL
O. tsutsuga mushi Karp Human New Guinea ATCC VR-150 M31887
  Kato Human Japan ATCC VR-609 AY191586
  Gilliam Human Burma NIH, Korea AY191585
  Boryong Human Korea NIH, Korea AY059015
R. typhi Wilmington Human USA ATCC VR-148 AY191590
R. typhi 87~91 Blood (human) Korea   AY191591
R.typhi 87~100 Blood (human) Korea    
R. prowazekii Breinl Human Poland ATCC VR-142 Y15783
R. akari MK Blood (human) USA ATCC VR-148 AY059013
R. japonica YH Blood (human) Japan ATCC VR-1363 AF432181
R. conori Indian Tick Typhus Rhipiceohaluss anguineus India ATCC VR-597 AY059012
R. sibirica 246 Dermacentor nuttalli Russia ATCC VR-151 AY059014

a ATCC; American type culture collection, Manassas, VA, USA, NIH; National Institute of Health, Korea

Table 2.
Real-time PCR condition
Program Name Cycles Analysis Mode Target [°C] Hold [h:m:s] Ramp Rate [°C/s] Acquisition Mode
Pre-incubation 1 None 95 00:10:00 20 None
      95 00:00:10 20 None
Amplification 40 Quantification 56 00:00:05 20 None
      72 00:00:20 20 Single
      95 00:00:00 20 None
Melting curve analysis 1 Melting curves 65 00:00:15 20 None
      95 00:00:00 0.1 Continuous
Cooling 1 None 40 00:00:30 20 None
TOOLS
Similar articles