Detection of 12 Respiratory Viruses with Two-set Multiplex Reverse Transcriptase-PCR Assay Using a Dual Priming Oligonucleotide System

Soo Jin Yoo; Eun-Young Kuak; Bo-Moon Shin

doi:10.3343/kjlm.2007.27.6.420

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Yoo, Kuak, and Shin: Detection of 12 Respiratory Viruses with Two-set Multiplex Reverse Transcriptase-PCR Assay Using a Dual Priming Oligonucleotide System

Original Article

Korean J Leg Med 2007;27(6):420-427.

Published online: 14 February 2007

DOI: https://doi.org/10.3343/kjlm.2007.27.6.420

Detection of 12 Respiratory Viruses with Two-set Multiplex Reverse Transcriptase-PCR Assay Using a Dual Priming Oligonucleotide System

Soo Jin Yoo, M.D., Eun-Young Kuak, M.T., Bo-Moon Shin, M.D.

Department of Laboratory Medicine, Sanggye Paik Hospital, School of Medicine, Inje University, Seoul, Korea

Received 11 July 200710 September 2007 Accepted 11 September 2007

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

We intended to evaluate the diagnostic usefulness of a multiplex reverse transcriptase-PCR (mRT-PCR) assay kit under dual priming oligonucleotide system (DPO) for the childhood acute respiratory tract infections.

Methods

Two hundred nasopharyngeal aspirates were taken from children ≤5 yr old admitted due to acute respiratory infections in 2004. Direct fluorescent antibody (FA) assays were performed with fresh specimens; then, mRT-PCRs for the detection of 12 respiratory viruses (Seeplex RV detection kit, SeeGene, Seoul, Korea) were tested with frozen specimens.

Results

FA assays for five common respiratory viruses showed positive results in 66 patients (33.0%), while mRT-PCR detected causative viruses in 112 patients (56.0%), including 16 co-infected cases (8.0%). A total of 129 viruses were identified: respiratory syncytial virus A/B (38.0%/7.8%), influenza virus A/B (10.1%/5.4%), parainfluenza virus 1/2/3 (7.0%/3.1%/7.8%), coronavirus 229E or NL63 (6.2%), human metapneumovirus (4.7%), adenovirus (4.7%), rhinovirus (3.9%), and coronavirus OC43 (1.6%).

Conclusions

DPO-based mRT-PCR was found as a sensitive tool for the detection of the viruses that cause childhood respiratory infections. Clinical significances of the agents detected by mRT-PCR need further evaluations.

Keywords: Respiratory tract infections, Reverse transcriptase polymerase chain reaction, Dual priming ologonucleotide system

REFERENCES

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Fig. 1.

Two sets (A set in the left and B set in the right) of mRT-PCR using dual priming oligonucleotide systems in 6 clinical specimens. Lane MA, Size marker of A set; MB, Size marker of B; N, negative control; 1-6, Patients 1-6; Patient 1, Positive for RSV B; Patient 2, coronavirus 229E/NL63 and rhinovirus; Patient 3, influenza A; Patient 4, RSV B, Patient 5, negative; Patient 6, influenza A. Each lane presents internal control band (719 bp). Adv, Adenovirus (534 bp); MPV, Human metapneumovirus (469 bp); 229E, Corovirus 229E/NL63 (375 bp); PIV1, Parainfluenza virus 1 (324 bp); PIV2, Parainfluenza virus 2 (264 bp); PIV3, Parainfluenza virus 3 (219 bp); Flu A, Influenza A virus (516 bp); Flu B, Influenza B virus (455 bp); RSV B, Respiratory syncytial virus B (391 bp); Rhino, Human rhinovirus A (340 bp); RSV A, Respiratory syncytial virus A (273 bp); OC43, Coronavirus OC43 (231 bp).

Abbreviation: mRT-PCR, multiple reverse transcriptase PCR.

Fig. 2.

Monthly distribution of 12 respiratory viruses isolated from children with acute respiratory tract infections in 2004 (Co-infected cases were excluded).

Table 1.

Viruses identified in 200 nasopharyngeal aspirates obtained from children with acute respiratory tract infections by mRT-PCR and fluorescent antibody assays

Virus identified	N (%) of positive specimens, by the indicated method of detection (N=200)		Concordance of mRT-PCR and fluorescent Ab assay
Virus identified	mRT-PCR	Fluorescent Ab assay	Both (+)	PCR (+) Ab (–)	PCR (–) Ab (+)	Concordance (%)^∗
RSV A	49 (24.5)	45 (22.5)^†	43	6	2	84.3
RSV B	10 (5.0)	2 (1.0)^†	2	8	0	20.0
Influenza virus A	13 (6.5)	6 (3.0)	5	8	1	35.7
Influenza virus B	7 (3.5)	2 (1.0)	0	7	2	0
Parainfluenza virus 1	9 (4.5)	2 (1.0)^†	2	7	0	22.2
Parainfluenza virus 2	4 (2.0)	0^†	0	4	0	0
Parainfluenza virus 3	10 (5.0)	7 (3.5)^†	7	3	0	70.0
Adenovirus	6 (3.0)	2 (1.0)	2	4	0	33.3
Coronavirus 229E/NL63	8 (4.0)	Not tested
Coronavirus OC43	2 (1.0)	Not tested
Human metapneumovirus	6 (3.0)	Not tested
Rhinovirus	5 (2.5)	Not tested
Total	129 (56.0)^†	66 (33.0)	61	47	5	54.0

^∗ Corcordance rate (%)=(N of positive results by both of the two methods^∗100)/(N of positive results by both of the two methods + N of positive results in multiplex RT-PCR only+N of positive results in fluorescent Ab assay only);

^† The direct fluorescent antibody assay was designed not to discriminate respiratory syncytial virus A and B or parainfluenza virus 1, 2, and 3. The results were classified according to the results of RT-PCR;

^‡ A total of 129 viruses were identified from 112 specimens (56.0%) by multiplex RT-PCR.

Abbreviations: mRT-PCR, multiple reverse transcriptase PCR; RSV, Respiratory syncytial virus.

Table 2.

Disease entities in 200 children with acute respiratory tract infections and their causative agents detected by mRT-PCR^∗

Respiratory virus	N (%) isolated virus in each disease entities
Respiratory virus	Bronchitis	Croup	Bronchiolitis	Pneumonia	Asthma exacerbation
RSV A	1 (11.1)	0	13 (26.5)	20 (18.3)	4 (25.0)
RSV B	0	0	1 (2.0)	7 (6.4)	0
Influenza A	3 (33.3)	3 (17.6)	0	6 (5.5)	0
Influenza B	1 (11.1)	1 (5.9)	0	1 (0.9)	2 (12.5)
Parainfluenza virus 1	1 (11.1)	1 (5.9)	0	1 (0.9)	0
Parainfluenza virus 2	0	0	0	2 (1.8)	0
Parainfluenza virus 3	0	2 (11.8)	2 (4.1)	3 (2.8)	1 (6.3)
Adenovirus	0	1 (5.9)	1 (2.0)	2 (1.8)	0
Coronavirus 229E/NL63	0	5 (29.4)	2 (4.1)	0	0
Coronavirus OC43	0	0	2 (4.1)	0	0
Metapneumovirus	1 (11.1)	0	0	2 (1.8)	0
Rhinovirus	0	0	2 (4.1)	2 (1.8)	0
Co-infection	2 (22.2)	0	5 (10.2)	9 (8.3)	0
Negative	0	4 (23.5)	21 (42.9)	54 (49.5)	9 (56.3)
Total	9	17	49	109	16

^∗ Co-infected cases were excluded in each causative virus count.

Abbreviations: See Table 1.

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