[Detection of respiratory syncytial virus in nasopharyngeal aspirates of children by using real-time polymerase chain reaction].
OBJECTIVE
Human respiratory syncytial virus (hRSV) is the leading cause of acute upper and lower respiratory tract infections in infants and young children worldwide. Pediatric RSV disease claims more than 1 million lives annually. With the rapid development of specific anti-RSV agents and the spread of respiratory infections, RSV detection techniques with higher sensitivity, specificity and quicker performance are badly needed. This study was designed to develop a real-time polymerase chain reaction (PCR) for detection of RSV in nasopharyngeal aspirates.


METHODS
(1) The TaqMan probe and primers of real-time PCR for RSV subgroup A and subgroup B detection were designed from the conserved region in N protein encoding gene, respectively. The sensitivity of real-time PCR was evaluated by using the virus with known amount of PFU. The specificity of real-time PCR for RSV detection was assessed by cross testing 10 isolates of strains A, 10 isolates of strains B, and by testing a variety of other respiratory viruses positive samples. (2) Sixty-one stored RSV positive respiratory samples and 103 nasopharyngeal aspirates were detected by real-time PCR, virus isolation, immunofluorescence assay (IFA), and nested-PCR.


RESULTS
(1) The sensitivity of the real-time PCR developed in this study for RSV subgroup A detection was 5.25 pfu, and for subgroup B was 3.75 pfu, the same as that of nested-PCR. (2) No positive results were found in cross testing of other viruses positive specimens. (3) Twenty-seven out of 30 (90%) of RSV A stored samples and 27 out of 31 (87.1%) of RSV B stored samples were positive by the real-time PCR. (4) Thirty-five (34.0%) out of the 103 specimens were found RSV positive by real-time PCR (7 of them were subgroup A and 28 subgroup B); 31 (30.1%) specimens were positive by nested-PCR (6 of them were subgroup A and 25 subgroup B); 22 (21.4%) were found positive for RSV with IFA (5 of them were subgroup A and 17 subgroup B); RSV was isolated from 9 (8.7%) specimens (6 of them were subgroup A and 3 subgroup B). All the specimens found to be negative by real-time PCR were negative by rest of the methods used in this study.


CONCLUSIONS
The real time PCR method developed in this project with the TaqMan probe and primers is sensitive and specific for detecting RSV subgroup A and B in nasopharyngeal aspirates.
Sun
Yu
Y
Laboratory of Virology, Beijing Municipal Laboratory of Infection and Immunity, Capital Institute of Pediatrics, Beijing 100020, China.
Zhu
Ru-nan
RN
Deng
Jie
J
Zhao
Lin-qing
LQ
Wang
Fang
F
Qian
Yuan
Y
D002648:Child; D018076:DNA, Complementary / Q000302:isolation & purification; D005455:Fluorescent Antibody Technique; D006801:Humans; D009305:Nasopharynx / Q000378:metabolism / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000302:isolation & purification; D018357:Respiratory Syncytial Virus Infections / Q000235:genetics*; D018113:Respiratory Syncytial Virus, Human / Q000235:genetics / Q000302:isolation & purification*
D018076:DNA, Complementary; D012367:RNA, Viral