|
Background: In 2001, the Dutch scholar Van Den Hoogen first report from a new virus isolated from samples of infants suffering from respiratory tract infection, according to the sequence homology and gene cluster analysis, classification of the virus in the paramyxovirus family, lung virus subfamily, Metapneumovirus genera, and named human metapneumovirus virus (human metapeumovirus HMPV). Since its discovery in 2001 so far, HMPV in Europe (the Netherlands, Sweden, Italy, Bulgaria), the Americas (Uruguay, United States), Asia (Singapore, China, Japan), Africa (South Africa), another reported that HMPV was global epidemic and prompts HMPV is a common cause of community-acquired respiratory tract infections. HMPV detection rate of respiratory tract infections in children around the quite different, lower by 2.5% in Japan, Bulgaria, up to 16%. There are three HMPV detection methods: antigen antibody detection, virus cell culture and reverse transcription polymerase chain reaction (RT-PCR). Virus cell culture is more difficult, more time was needed, the lower detection rate of clinical specimens. Although serological methods is not difficult to use, but the existing serological detection reagents used to detect low sensitivity of HMPV clinical specimens. HMPV antigen test used in the clinic there immunofluorescence, immune chromatography (IC) and enzyme immunoassay method (EIA), the sensitivity and specificity is still inadequate. RT-PCR technology is the primary means of diagnosis of HMPV infection. According to the HMPV the nucleocapsid protein gene N matrix genes M, the fusion gene F polymerase gene L protein gene P gene sequence primers were designed for detection of HMPV real-time RT-PCR has a higher sensitivity, more economic and time-saving advantages, the lower the possibility of environmental pollution in the amplification. Objective: To observe the real-time fluorescence quantitative RT-PCR and direct immunofluorescence detection technology detects respiratory specimens HMPV specificity, sensitivity, and is designed to explore the the HMPV method of real-time quantitative RT-PCR, children clinical application prospects. Methods:] ONAstar than HMPV whole genome sequence analysis, looking for available for PCR amplification of the conserved fragment, primers were designed: primerF: 5'-GTCTCTTCAAGGGATTCACC-3 and primerR 5'-GTTGTTGTGCCTACATCTC-3, and probes the TaqMan 5 ' -FAM-CATGCTATATTA AAAGAGTCTCA-TAMRA-3 '. probe 5' end is labeled with FAM, 3 'Duanyou TAMRA tag. Establish a method to detect the HMPV fluorescence quantitative PCR. Were used to establish the real-time quantitative RT-PCR and direct immunofluorescence detection technology to detect 623 cases of Children 's Hospital of Zhejiang University School of Medicine, Department of Respiratory Medicine ward from December 2009 to March 2010 lower respiratory tract infections in children hospitalized HMPV infection. Two the method detection specificity and sensitivity of the HMPV children. Results: 1 to HMPV as a template for quantitative PCR to obtain positive results, but other common respiratory viruses (adenovirus, influenza A virus, influenza B virus, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, and respiratory syncytial virus) and other viruses PCR were negative. The sample of 623 cases direct immunofluorescence detection HMPV infection of 10 cases, the detection rate of 1.61%. Quantitative PCR detection HMPV infection of 28 cases, the detection rate of 4.49%, the detection rate of the two methods have significant difference (χ2 = 16.05, P lt; 0.01). CT value is less than 28 direct immunofluorescence to detection HMPV. 3. Direct immunofluorescence positive 10 were PCR positive, 595 were negative for both the 18 specimens by direct immunofluorescence negative PCR positive, showing a good correlation (r = 0.589, P lt; 0.01). Children with asthma in HMPV detection rate was 15.4% (4/26), a significant senior cadres pneumonia group (20/515, 3.9%), bronchiolitis group (4/46, 8.7%), tracheobronchitis group (0/36, 0%) statistically significant difference (χ2 = 11.217, P = 0.011) 5.HMPV detection rate of age-related ,0-1 positive rate of 2.2% (9/404), 13.1% in 3 years old (16/122), 6 years old 3.4% (2/58), gt; 6 years old 2.6% (1/39), including 1-3 age group children with HMPV detection rate was significantly higher than the other groups, statistically differences significantly with (χ2 = 26.443, P = 0.000) Conclusion: select HMPV N gene conservative region its own design of one pair of primers and a TaqMan probe, to establish the detection of HMPV fluorescent quantitative PCR method; 2 fluorescence quantitative RT-PCR detection of HMPV The sensitivity was significantly higher than the direct immunofluorescence method has the advantages of fast and accurate; 3 children with asthma HMPV detection rate was significantly higher than that in children with pneumonia; 4 HMPV infection of the highest incidence of children aged 1-3 years.
|