Quantitative fluorogenic real-time PCR assay for respiratory syncytial virus detection

Abstract

Abstract: Objective To Establish a rapid and objective quantitative fluorogenic real-time PCR assay for early detection of human respiratory syncytial virus (hRSV). Methods Two pairs of primers and one TaqMan Fluorogenic probe that are specific for the recognition of the most conservative N gene of hRSV for virus detection with LighCycler PCR in 93 nasopharyngeal secretion specimens collected from infants and young children.The assary was compared with virus isolation,routine PCR,nested PCR,and enzyme-linked immunosorbent assay (ELISA). Results This TaqMan assay had a sensitivity of 1×10² cDNA copies/μl with a dynamic range between 1×10² and 1×10⁷ cDNA copies/μl,which was the same as that of nested PCR,but 10 times more sensitive than routine PCR.The specificity of the assay was evaluated by comparing hRSV with polivirus type 1,coxsackie virus type 2,influenza A,influenza B and adenovirus type 7.A PCR product of the expected size (195 bp) was produced and fluorescence signal detected for hRSV,but not for any of the other viruses.The results in LightCycler and Rotor-Gene instrument were consistent.Forty-four specimens (43.9%) were hRSV-positive with this assay and 4 (4/93,4.3%) were hRSV-positive with ELISA,showing rather low correlation between the two methods.No visible relation was found between the concentration of hRSV RNA and severity of the disease. Conclusion This assay is rapid,sensitive,specific and quantitative,and has the potential of wide application for early diagnosis of hRSV infection and evaluation of the therapeutic effect.

CLC Number:

ZHANG Qi-wei¹, YOU Shang-you², SUN Ji-min², WU Qi², YU Chun-hua², ZHANG Chu-yu¹. Quantitative fluorogenic real-time PCR assay for respiratory syncytial virus detection[J]. Journal of Southern Medical University, 2005, 25(07): 847-852.

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