Detection Of Human Metapneumovirus In Respiratory Specimen By Fluorescent Quantitative Pcr

Background:In 2001, Holland Scholar Van Den Hoogen et al, first reported that a new type of virus was isolated from a infant with respiratory infection. According to sequence homology and gene cluster analysis, it was classified into paramyxoviridae, pneumovirinae, metapneumovirus and called human metapeumovirus (HMPV). From 2001 up to now. HMPV has been reported in Europe(Holland, Sweden, Italy, Bulgaria), America (Uruguay, American), Asia (Singapore, China, Japan) and Africa (South African), indicating global epidemics of HMPV. And it was also indicated thatt HMPV is one of the most common etiological agent of acquired character respiratory infection. However, there was a great discrepancy in the detecting rates of HMPV in respiratory infection among different areas, with the lowest (2.5%) in Japan, and the highest (16%) in Bulgaria.At present, there are three kinds of detecting methods of HMPV:including antigen-antibody detection, virus cell culture and reverse transcription-polymerase chain reaction (RT-PCR). It is difficult for clinic to culture virus, because of few sensitive cells to culture, time-consuming, and low sensitivity. Even though serological method is easy, currently-available serological detection kit showed a low sensitivity when used in clinic. HMPV antigen detecting methods include immunofluorescence, immunochromatography (IC), enzyme immunoassay (EIA). But its sensitivity and specificity is not good. Now, RT-PCR technique is the main method to diagnose HMPV infection. Primers were designed according to different gene order from nucleocapsid protein N, ground substance gene M, fusion gene F, pol gene L and protein gene P in HMPV. RT-PCR has showed high sensitivity with less time and lower environmental pollution.Objective:Through comparing the sensitivity, specificity and practicability of detecting HMPV by fluorescent quantitative RT-PCR and direct immunofluorescence, clinical value of fluorescent quantitative RT-PCR in detecting HMPV was explored.Methods:After searching conservative DNA segment for PCR amplification by DNAstar comparing and analyzing whole genome sequence of series of HMPV, primers were designed as follows: Primer F:5'-GTCTCTTCAAGGGATTCACC-3 Primer R 5'-GTTGTTGTGCCTACATCTC-3.Primer probe:TaqMan 5'-FAM-CATGCTATATTAAAAGAGTCTCA-TAMRA-3', with FAM marking 5 prime end, and use of TAMRA marking 3 prime end, then established the method of RT-PCR to detect HMPV.HMPV in 623 clinical specimens from hospitalized children n Children's Hospital Zhejiang University school of medicine from December in 2009 to March in 2010 were detected by fluorescent quantitative RT-PCR and direct immunofluorescence simultaneously. The sensitivity, specificity and clinical value between the two methods were compared.Results:1. Taking HMPV as template to carry out fluorescent quantitative PCR we got positive result, but other respirovirus (adenovirus, influenza A virus, influenza B virus, parainfluenza virus typel, parainfluenza virus type 2, parainfluenza virus type 3, respiratory syncytial virus) and other viruses showed negative results.2. By direct immunofluorescence,10 cases showed HMPV positive (positive rate: 1.61%), and by fluorescent quantitative PCR method 28 cases showed positive(rate: 4.49%). Statistical significance between the two methods was found(x2=16.05, P<0.01). Only when CT value was less 28, we could detected HMPV by direct immunofluorescence.3. Of 10 cases with positive direct immunofluorescence, PCR all showed positive, and 595 cases detected by the two methods all showed negative. A good correlation was found. The other 18 PCR-positive cases showed negative by direct immunofluorescence method (r=0.589, P<0.01)4. The positive rate of HMPV in asthma was 15.4%(4/26), much higher than the pneumonia group (3.9%(20/515)), the bronchiolitis group (8.7%(4/46)) and the tracheobronchitis group (0%(0/36)), existing statistical significance (x2=11.217, P=0.011)5. The detection rate correlated with age. The positive rate in 0-1y group was 2.2% (9/404),-3y group was 13.1% (16/122),3-6y group was 3.4%(2/58), and larger than 6 y group was 2.6%(1/39). The detection rate of HMPV in 1-3 y group was much higher than other groups, existing statistical significance (x2=26.443, P=0.000)Conclusions:1. Chosing conservative area of HMPV N gene to design a pair of primers and single band of TaqMan probe, the method of fluorescent quantitative RT-PCR was bulit up to detect HMPV.2. The sensitivity of fluorescent quantitative RT-PCR detecting HMPV was much higher than that of direct immunofluorescence.3. The detection rate of HMPV in asthma was obviously higher than that of pneumonia. 4. The infection rate of HMPV was highest in children 1-3 years old..