A neutralization assay for respiratory syncytial virus using a quantitative PCR-based endpoint assessment

Virology Journal

Haruhiko Murata¹  Judy Beeler³  Keith Peden¹  Susette Audet³  Thembi Mdluli²  R Lynne Crim³  Belete Teferedegne¹  Jan C Varada³ 

[1]Laboratory of DNA Viruses, Division of Viral Products, OVRR, CBER, FDA, Bethesda, MD 20892, USA

[2]Present affiliation: Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, USA

[3]Laboratory of Pediatric and Respiratory Viral Diseases, Division of Viral Products, OVRR, CBER, FDA, Bethesda, MD 20892, USA

关键词: SYBR Green;    Quantitative PCR;    RNA;    Reverse transcription;    Antibody;    Neutralization;    Respiratory syncytial virus;   

Others  :  1149686
DOI  :  10.1186/1743-422X-10-195

 received in 2013-04-17, accepted in 2013-06-11,  发布年份 2013

【 摘 要 】

Background

Few studies have used quantitative polymerase chain reaction (qPCR) as an approach to measure virus neutralization assay endpoints. Its lack of use may not be surprising considering that sample nucleic acid extraction and purification can be expensive, labor-intensive, and rate-limiting.

Methods

Virus/antibody mixtures were incubated for one hour at 37°C and then transferred to Vero cell monolayers in a 96-well plate format. At 24 (or 48) hours post-infection, we used a commercially available reagent to prepare cell lysates amenable to direct analysis by one-step SYBR Green quantitative reverse transcription PCR using primers specific for the RSV-N gene, thereby obviating the need for cumbersome RNA extraction and purification. The neutralization titer was defined as the reciprocal of the highest dilution needed to inhibit the PCR signal by 90% when compared with the mean value observed in virus control wells in the absence of neutralizing antibodies.

Results

We have developed a qPCR-based neutralization assay for human respiratory syncytial virus. Due to the sensitivity of qPCR in detecting virus replication, endpoints may be assessed as early as 24 hours post-infection. In addition, the dynamic range of qPCR provides a basis for the assay to be relatively robust to perturbations in input virus dose (i.e., the assay is in compliance with the Percentage Law).

Conclusions

This qPCR-based neutralization assay is suitable for automated high-throughput applications. In addition, our experimental approach may be generalizable for the rapid development of neutralization assays for other virus families.

【 授权许可】

   
2013 Varada et al.; licensee BioMed Central Ltd.

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