摘要
研究背景：
2001年,荷兰学者Van Den Hoogen等首先报告从患呼吸道感染的婴幼儿标本中分离到一种新的病毒,根据序列同源性和基因群分析,该病毒分类为副粘病毒科、肺病毒亚科、偏肺病毒属,并命名为人偏肺病毒(human metapeumovirus,HMPV)。自2001年发现至今,HMPV已在欧洲(荷兰、瑞典、意大利、保加利亚),美洲(乌拉圭、美国),亚洲(新加坡、中国、日本)非洲(南非)等地陆续报道,表明HMPV呈全球流行,并提示HMPV是社区获得性呼吸道感染的常见病原。但HMPV在呼吸道感染患儿中的检出率各地差异较大,日本较低2.5%,保加利亚高达16%。
目前有三种HMPV的检测方法：包括抗原抗体检测、病毒细胞培养和逆转录聚合酶链反应(RT-PCR)。病毒细胞培养较难,所需时间较长,临床标本检出率较低。虽然血清学方法运用并不难,但现有血清学检测试剂用于检测临床标本HMPV敏感性低。HMPV的抗原检测临床常用的有免疫荧光法、免疫色谱法(IC)和酶免疫分析法(EIA),但敏感性和特异性仍嫌不足。目前RT-PCR技术是诊断HMPV感染的主要手段。可根据HMPV的核壳体蛋白基因N、基质基因M、融合基因F、多聚酶基因L、蛋白基因P等不同基因序列设计引物用于检测HMPV.而即时RT-PCR具有敏感性更高、更为经济省时等优点,扩增时环境污染的可能性更低。
目的：
观察比较实时荧光定量RT-PCR与直接免疫荧光检测技术检测呼吸道标本HMPV的特异性、灵敏度,旨在探讨实时荧光定量RT-PCR检测儿童HMPV方法的临床应用前景。
方法：
用]ONAstar比对分析HMPV的全基因组序列,寻找可供PCR扩增的保守片断,分别设计引物：primerF:5'-GTCTCTTCAAGGGATTCACC-3和primerR 5'-GTTGTTGTGCCTACATCTC-3,探针TaqMan 5'-FAM -CATGCTATATTA AAAGAGTCTCA-TAMRA-3'.探针的5'端由FAM标记,3'端由TAMRA标记。建立检测HMPV荧光定量PCR的方法。
分别用建立的实时荧光定量RT-PCR与直接免疫荧光检测技术检测623例浙江大学医学院附属儿童医院呼吸内科病房2009年12月至2010年3月住院下呼吸道感染患儿HMPV的感染情况。比较两种方法检测儿童HMPV的特异性与灵敏度。
结果：
1.以HMPV作为模板进行荧光定量PCR获得阳性结果,但对其它常见呼吸道病毒(腺病毒,A型流感病毒,B型流感病毒,副流感病毒1型,副流感病毒2型,副流感病毒3型,呼吸道合胞病毒)及其它病毒PCR均阴性。
2.在623例样本中直接免疫荧光法检出HMPV感染共10例,检出率1.61%。荧光定量PCR检出HMPV感染共28例,检出率4.49%,两种方法检出率有统计学差异(χ2=16.05,P<0.01)。CT值小于28直接免疫荧光法才能检出HMPV。
3.直接免疫荧光法阳性者10份PCR均阳性,595份两者均阴性,18份标本直接免疫荧光法阴性PCR呈阳性,显示很好的相关性(r=0.589,P<0.01)。
4.哮喘患儿中HMPV检出率为15.4%(4/26),显著高干肺炎组(20/515,3.9%)、毛细支气管炎组(4/46,8.7%),气管支气管炎组(0/36,0%)差异具有统计意义(χ2=11.217,P=0.011)
5.HMPV检出率与年龄有关,0-1岁阳性率2.2%(9/404), -3岁13.1%(16/122),-6岁3.4%(2/58),>6岁2.6%(1/39),其中1-3岁年龄组患儿HMPV检出率明显高于其它组,统计学差异显著(χ2=26.443,P=0.000)
结论：
1选择HMPV N基因保守区域自行设计了一对引物和一条TaqMan探针,建立了检测HMPV荧光定量PCR的方法；
2荧光定量RT-PCR检测HMPV敏感性明显高于直接免疫荧光法,具有快速、准确的优点；
3哮喘患儿中HMPV检出率明显高于肺炎患儿；
4 HMPV感染以1-3岁幼儿发生率最高。
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..
引文
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[2]Kahn JS. Epidemiology of human metapneumovirus. Clin Microbiol Rev [J],2006, 19(3):546-557.
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[4]Li XY, Chen JY, Kong M, et al. Prevalence of human metapneumovirus in hospitalized children with respiratory tract infections in Tianjin, China.Arch Virol,2009,154(11):1831-1836
[5]Matsuzaki Y, Itagaki T, Abiko C, et al. Clinical impact of human metapneumovirus genotypes and genotype-specific seroprevalence in Yamagata, Japan.J Med Virol, 2008,80(6):1084-1089.
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[7]Pavlova S, Hadzhiolova T, Abadjieva P, et al. Application of RT-PCR for diagnosis of respiratory syncytial virus and human metapneumovirus infections in Bulgaria, 2006-7 and 2007-8.Euro Surveill,2009,14(23):19233.
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