15种呼吸道病原体的实时荧光定量PCR检测方法的建立及应用

Establishment and application of real-time fluorescent quantitative PCR assay for detecting 15 species of respiratory pathogens

目的建立15种呼吸道常见病原体的实时荧光定量PCR检测方法,用于新兵发热病因的快速检测。方法根据生物信息学分析结果选定15种常见呼吸道病原体的检测靶基因,建立实时荧光定量PCR方法。采用15种病原体的培养物和正常人的基因组对检测方法的敏感性和特异性进行测试,并对健康和发热新兵各50例的咽拭子进行检测。结果所建立的荧光定量PCR方法的有效检测线性范围为1×102～1×109拷贝/ml,最低检测限为5～50拷贝/ml,敏感性和特异性均较高。在50例发热新兵咽拭子中,24.0%嗜肺军团菌阳性,30.0%乙型流感病毒阳性,24.0%肺炎支原体阳性,8.0%甲型流感病毒阳性,对照组上述病原的阳性率依次为20.0%、6.0%(P<0.05)、22.0%和0.0%,而腺病毒、麻疹病毒、SARS相关冠状病毒、人冠状病毒、流行性腮腺炎病毒、呼吸道合胞病毒、甲型流感病毒H5N1、风疹病毒、猪链球菌、肺炎衣原体和肺炎链球菌均阴性。18例(36%)流感病毒感染者急性期血清IgM和IgG阳性率分别为88.9%(16/18)和72.2%(13/18),在恢复期则分别为16.8%(3/18)和100%(18/18)。健康新兵的两次血清抗体检测无临床意义。在咽拭子嗜肺军团菌及肺炎支原体阳性个体,发热期和恢复期血清抗体阳性率分别为8.3%和8.3%、8.3%和16.7%。结论成功建立可同时进行15种呼吸道病原体早期诊断的实时荧光定量PCR方法,该方法检测结果表明此次新兵营集训新兵发热病因以流感病毒感染为主。

Objective To establish a real-time fluorescent quantitative PCR assay for rapid detection of 15 species of common respiratory pathogens in the recruits with fever. Methods A real time fluorescent quantitative PCR assay was established with 15 species of respiratory pathogenic genes, selected based on their relevant reference strains by bioinformatics analysis, as target genes. The sensitivity and specificity of the assay was tested with the cultures of the 15 species of pathogens and the genome of normal person. The throat swabs and blood samples were collected for etiological study from 50 conscripts who were in acute stage of infection and with fever, and 50 conscripts who were in convalescent stage and without fever. Results The linear range of effective detection of the established PCR assay was 1 × 10 2-1 × 10 9 copies/ml, the lowest detection limit was 5--50 copies/m l, both sensitivity and specificity were higher. Out of the 50 throat swabs from the recruits with fever, 12 （24%） were positive for Legionella pneumophila, 15 （30%） for influenza B virus, 12 （24%） for Mycoplasma pneumoniae and 4 （8%） for influenza A virus. The positive rates of Legionella pneumophila, influenza B virus, Mycoplasma pneumoniae and influenza A virus in control group were 20.0%, 6. 0% （P〈0. 05）, 22. 0% and 0. 0%, respectively. The detection results were negative for adenovirus, measles virus, severe acute respiratory syndrome （SARS） associated coronavirus, human coronavirus, epidemic mumps virus, human respiratory syncytial virus, influenza A virus H5N1, rubella virus, Streptococcus suis, Chlamydia pneumoniae and Streptococcus pneumoniae. In the 18 cases （36%） infected with influenza A or B virus, the positive rates of serum IgM and IgO were 88. 9% （16/18） and 72. 2% （13/18） in febrile stage, and 16. 7% （3/18） and 100% （18/18） in convalescence, respectively. While the two detection results of serum antibody in healthy recruits showed no clinical significance. In the individuals, from whom the throat swabs were positive for Legionella pneumophila, the positive rate of serum antibody was 8. 3% both in febrile stage and convalescence; while in the individuals, from whom the throat swabs were positive for Mycoplasma pneurnoniae, the positive rates of serum antibody were 8. 3% and 16. 7%, respectively, in febrile stage and convalescence. Conclusions The real-time PCR assay for detecting 15 species of respiratory pathogens has been successfully established. It is shown that the main cause of outbreak of fever in conscripts is influenza viral infection.