Korean J Lab Med.  2009 Dec;29(6):594-600. 10.3343/kjlm.2009.29.6.594.

Application of a Diagnostic Method Using Reverse Transcription-PCR ELISA for the Diagnosis of Enteroviral Infections

• Park K
• Lee K
• Baek K
• Jung E
• Park S
• Cho Y
• Song J
• Ahn G
• Cheon DS

• KMID: 1781616
• DOI: http://doi.org/10.3343/kjlm.2009.29.6.594

Abstract

BACKGROUND
Enteroviruses are known as major pathogen for aseptic meningitis. Although rapid diagnosis for enteroviruses is very essential to exclude bacterial infections in patients with meningitis, classical diagnostic method based on virus isolation is not practicable for timely treatment of patients due to its laborious and time-consuming procedure. Recently molecular methodologies as alternatives are routinely used for rapid and sensitive diagnosis for enteroviruses infections. METHODS: Reverse transcription (RT)-PCR ELISA kit for targeting 5'non-coding region (NCR) with highly conserved genetic identity among all genotypes of enteroviruses was introduced in this investigation. RT-PCR ELISA was evaluated about sensitivity and specificity through virus isolation using clinical specimens from patients suspected of enteroviral infections and enteroviral isolates comparing with conventional RT-PCR identifying them. RESULTS: The detection limit of the RT-PCR ELISA was up to 10-100 folds higher than virus isolation using cell culture and conventional RT-PCR. On comparison between above two methods, the detection rate of RT-PCR ELISA for clinical specimens from patients with aseptic meningitis was 7% higher than that of conventional RT-PCR targeting 5'NCR (P=0.016). CONCLUSIONS: Our results suggest that RT-PCR ELISA developed in this study could be an alternative diagnostic method for the detection of enteroviral genome with high sensitivity and specificity.

Keyword

Enterovirus; Aseptic meningitis; Conventional RT-PCR; RT PCR-ELISA

MeSH Terms

5' Untranslated Regions
Adolescent
Child
Child, Preschool
Enterovirus/genetics/*isolation & purification
Enterovirus Infections/*diagnosis
*Enzyme-Linked Immunosorbent Assay
Humans
Infant
Meningitis, Aseptic/diagnosis
RNA, Viral/analysis
*Reverse Transcriptase Polymerase Chain Reaction
Rotavirus/genetics
Rotavirus Infections/diagnosis
Sensitivity and Specificity

Figure

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  Fig. 1. The schematic design of PCR ELISA reaction applied in this study Abbreviations: HRP, horseradish peroxidase; Yellow P, phosphate; B, biotin; Purple P, horseradish peroxidase.

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  Fig. 2. The results from PCR ELISA using amplified PCR product for enteroviruses.

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  Fig. 3. The comparative results of sensitivity for various assays for the detection of enteroviruses. ⬇, Detection limit; +, Relative low; ++, Moderate; +++, High. Abbreviations: TCID50, Tissue Culture Infections Dose 50; RT-PCR, reverse transcription-PCR; OD, optical density.

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