Rapid Identification of Rickettsiae using the Real-Time PCR

Hyo-Soon Park; Jung-Hee Lee; Kwang-Hoon Jin; Won-Jong Jang; Kyung-Hee Park; Yoon-Hoh Kook; Seung-Hyun Lee

doi:10.4167/jbv.2008.38.4.221

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

Original Article

J Bacteriol Virol 2008;38(4):221-226.

Published online: 18 January 2008

DOI: https://doi.org/10.4167/jbv.2008.38.4.221

Rapid Identification of Rickettsiae using the Real-Time PCR

Hyo-Soon Park¹, Jung-Hee Lee¹, Kwang-Hoon Jin¹, Won-Jong Jang¹, Kyung-Hee Park¹, Yoon-Hoh Kook², Seung-Hyun Lee^1,^∗

¹Department of Microbiology, College of Medicine, Konkuk University, Chugju, Chungchungbuk-Do, Korea

²Department of Microbiology, Seoul National University College of Medicine, Seoul, Korea

^∗Corresponding author: Seung-Hyun Lee. Department of Microbiology, College of Medicine, Konkuk University, Chugju, Chungchungbuk-Do 380-701, Korea. Phone: +82-43-840-3725, Fax: +82-43-851-9329 e-mail: shlee@kku.ac.kr

^∗∗ This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2004-E00080).

Revised 9 December 200812 December 2008 Accepted 12 December 2008

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Keywords: groEL gene; Rickettsia; Real-time PCR

REFERENCES

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Figure 1.

Amplification of groEL DNA from rickettsial strains using the real-time PCR. Lanes;

SFG (R japonica, R. conorii, R. sibirica, R akari),

: TG (R. typhi, R. typhi 87~91, R. typhi 87~100, R prowazekii),

STG (O. tsutsugamushi Karp, O. tsutsugamushi Kato, O. tsutsugamushi Boryong, O. tsutsugamushi Gilliam),

: DW

Figure 2.

Amplification of groEL DNA from rickettsial strains and tick DNA using the real-time PCR. Lanes;

: SFG (R. japonica, R. conorii, R sibirica, R akari),

: TG (R. typhi, R. typhi 87~91, R. typhi 87~100, R. prowazekii),

: 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)

: 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.
Species	Strain	Source	Geographic location	Origin (collection)^a	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

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