[Quantitative fluorogenic real-time PCR assay for respiratory syncytial virus detection].
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 assay was compared with virus isolation, routine PCR, nested PCR, and enzyme-linked immunosorbent assay (ELISA).


RESULTS
This TaqMan assay had a sensitivity of 1 x 10(2) cDNA copies/microl with a dynamic range between 1 x 10(2) and 1 x 10(7) cDNA copies/microl, 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.


CONCLUSIONS
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.
Zhang
Qi-wei
QW
Ministry of Education Key Laboratory of Virology, Wuhan University, Wuhan 430072, China. wisy98@163.com
You
Shang-you
SY
Sun
Ji-min
JM
Wu
Qi
Q
Yu
Chun-hua
CH
Zhang
Chu-yu
CY
D002675:Child, Preschool; D006801:Humans; D007223:Infant; D009305:Nasopharynx / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000032:analysis*; D018357:Respiratory Syncytial Virus Infections / Q000821:virology*; D018113:Respiratory Syncytial Virus, Human / Q000302:isolation & purification*
D012367:RNA, Viral