[Detection and identification of human metapneumovirus by real time reverse transcription PCR].
OBJECTIVE
To develop a rapid, sensitive and specific real time reverse transcription PCR for detecting and identifying human metapneumovirus.


METHODS
The Hmpv-L gene of human metapneumovirus was chosen as target gene, the primers and TaqMan probe were designed, and the PCR reaction was optimized systematically. The total RNA was extracted from respiratory specimens, and reverse transcription was performed through random primer. The cDNA was detected by using real time PCR. The specificity, sensitivity and reproducibility of real time PCR were estimated. The real time PCR was applied to detect 180 clinical respiratory specimens.


RESULTS
The human metapneumovirus can be detected using real time reverse transcription PCR accurately and quickly, and the sensitivity was 1 copy/microl. The coefficient of variation of intra-assay and inter-assay was less than 5%. Among those 180 specimens, 28 (15.56%) were positive for human metapneumovirus, the clinical diagnoses for these 28 patients were pneumonia (15.60%, 17/109) and bronchiolitis (15.49%, 11/71). 21 positive specimens were from patients under 2 years of age, and 6 positive specimens were from patients between 2 and 5 years of age, only 1 positive specimens was from patients over 5 years.


CONCLUSIONS
It is demonstrated that real time reverse transcription PCR is a reliable, accurate and feasible assay for human metapneumovirus, which has become one of the most important pathogens induced acute respiratory infections in pediatric patients.
Xing
Jian-ming
JM
Department of Laboratory Medicine, Huzhou Maternity and Child Care Hospital, Zhejiang Huzhou 313000, China.
Weng
Xue-jun
XJ
Zhang
Su
S
Yuan
Xin-hua
XH
Shen
Cui-fen
CF
Zhang
Ya-qin
YQ
Cheng
Hong-ling
HL
Li
Gang
G
Yao
Li-hui
LH
D002675:Child, Preschool; D005240:Feasibility Studies; D006801:Humans; D029121:Metapneumovirus / Q000235:genetics / Q000302:isolation & purification*; D012141:Respiratory Tract Infections / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D048348:Reverse Transcription; D012680:Sensitivity and Specificity