Detection of Enterovirus in Cerebrospinal Fluid by Real-Time Nested Reverse Transcription Polymerase Chain Reaction

Se Ran Heo; Sun Kyung Jin; Ho Eun Chang; Kyoung Un Park; Junghan Song; Eui Chong Kim

doi:10.3343/kjlm.2006.26.1.9

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Heo, Jin, Chang, Park, Song, and Kim: Detection of Enterovirus in Cerebrospinal Fluid by Real-Time Nested Reverse Transcription Polymerase Chain Reaction

Original Article

Korean J Leg Med 2006;26(1):9-13.

Published online: 10 February 2006

DOI: https://doi.org/10.3343/kjlm.2006.26.1.9

Detection of Enterovirus in Cerebrospinal Fluid by Real-Time Nested Reverse Transcription Polymerase Chain Reaction

Se Ran Heo¹, Sun Kyung Jin¹, Ho Eun Chang¹, Kyoung Un Park, M.D.^1,², Junghan Song, M.D.^1,², Eui Chong Kim, M.D.²

¹Department of Laboratory Medicine, Seoul National University Bundang Hospital, Seoul, Korea

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

Received 20 September 2005 Revised 9 January 2006 Accepted 16 January 2006

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

Background

Enterovirus is a common cause of aseptic meningitis, respiratory disease and nonspecific febrile illness. The conventional methods for laboratory diagnosis of enterovirus infections have been virus culture and serotyping by an immunofluorecent test. We studied a new and more rapid approach for enterovirus detection in cerebrospinal fluid (CSF) by real-time nested PCR.

Methods

This study was performed on 50 CSF specimens from patients suspected of aseptic meningitis. Enterovirus was detected in CSF by PCRs for 3 different targets and real-time nested PCR. Enterovirus culture was also performed in 44 CSF specimens.

Results

The positive rate of PCRs for each of the 3 different targets was 26.0%, 40.0%, or 46.0%, and that of real-time nested PCR was 86.0%. Only 6.8% were positive in culture. Thus, the positive rate of real-time nested PCR was much higher than other methods.

Conclusions

Our study revealed that the real-time nested PCR should be useful for diagnosis of enterovirus infections because of a high sensitivity and rapid detection.

Keywords: Enterovirus, Real-time polymerase chain reaction, Nested PCR, Reverse Transcription PCR, Aseptic meningitis

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Fig. 1.

Detection of enterovirus by RT-PCR methods for 3 different targets using CSF specimens. (A) The amplified products of first method using EV1-F and EV1-R primers are 114 bp. (B) The amplified products of second method using EV2-F and EV2-R primers are 154 bp. (C) The amplified products of third method using EV3-F and EV3-R primers are 149 bp. Lane 1, negative control; lane 2, positive control; lane M, 100 bp DNA size marker; lane 3–12, specimens.

Table 1.

Oligonucleotide primers used to detect enterovirus and reaction conditions

Methods		PCR^*
1	Primers	EV1-F: 5′-ACA CGG ACA CCC AAA GTA GTC GGT TCC-3′
		EV1-R: 5′-TCC GGC CCC TGA ATG CGG CTA ATC C-3′
	Conditions Product size	95°C 45 sec-58°C 45 sec-72°C 45 sec (35 cycles) 114 bp
2	Primers	EV2-F: 5′-CCT CCG GCC CCT GAA TGC GGC TAA T-3′
		EV2-R: 5′-ATT GTC ACC ATA AGC AGC CA-3′
	Conditions Product size	90°C 45 sec-46°C 45 sec-72°C 45 sec (37 cycles) 154 bp
3	Primers	EV3-F: 5′-CCC CTG AAT GCG GCT AAT CC-3′
		EV3-R: 5′-CAA TTG TCA CCA TAA GCA GCC A-3′
	Conditions Product size	95°C 45 sec-64.5°C 45 sec-72°C 45 sec (35 cycles) 149 bp

^* Reverse transcription PCR.

Table 2.

Comparison between the results of real-time PCR and PCRs for 3 different targets

Real-time PCR^§	PCR 1^*		PCR 2^†		PCR 3^‡
Real-time PCR^§	Negative (74.0%)	Positive (26.0%)	Negative (60.0%)	Positive (40.0%)	Negative (54.0%)	Positive (46.0%)
Negative (14.0%)	7	0	7	0	7	0
Positive (86.0%)	30	13	23	20	20	23

^* First RT-PCR method using EV1-F and EV1-R primers;

^† Second RT-PCR method using EV2-F and EV2-R primers;

^‡ Third RT-PCR method using EV3-F and EV3-R primers;

^§ Real-time nested RT-PCR.

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