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

• Yoo SJ
• Kuak EY
• Shin BM

• KMID: 1781546
• DOI: http://doi.org/10.3343/kjlm.2007.27.6.420

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 < or = 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 mRTPCR need further evaluations.

Keyword

Respiratory tract infections; Reverse transcriptase polymerase chain reaction; Dual priming ologonucleotide system

MeSH Terms

Child, Preschool
DNA, Viral/analysis
Fluorescent Antibody Technique, Direct
Humans
Infant
Infant, Newborn
Oligonucleotide Probes
Reproducibility of Results
Respiratory Tract Infections/*diagnosis/epidemiology/virology
Reverse Transcriptase Polymerase Chain Reaction/*methods
Virus Diseases/*diagnosis/epidemiology/virology
Viruses/genetics/*isolation & purification

Figure

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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).

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  Fig. 2. Monthly distribution of 12 respiratory viruses isolated from children with acute respiratory tract infections in 2004 (Co-infected cases were excluded).

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