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


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.


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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
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
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.
Table 1.
Rickettsial strains used in this study
Species Strain Source Geographic location Origin (collection)a GenBank Acession No.
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
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