急性呼吸道感染患儿人副流感病毒3型检测及临床研究

Nested RT-PCR detection and clinical study of human parainfluenza virus-3 in children with acute respiratory tract infection

目的建立细胞培养联合套式逆转录聚合酶链反应(Nested RT-PCR)检测临床样本人副流感3型病毒(HPIV3)基因的方法,研究兰州地区HPIV3的感染现状及特征。方法收集2005年7月至12月,2006年10月至2007年3月兰州大学第一附属医院300例急性呼吸道感染患儿鼻咽部分泌物标本(NPS),所有标本接种于vero细胞,用套式RT-PCR的方法检测NPS中HPIV3基因。结果300例患儿中HPIV3阳性46例,阳性率为15.33%。在2005年11月和2006年1月、2月出现感染高峰。随机选取11份阳性标本送检测序,测得的核苷酸序列经过Blast软件比较,与Gene Bank中的已知的HPIV3序列同源性在91.0%～99.6%之间。结论HPIV3在兰州地区呼吸道感染患儿中所占比例较高,是一个很重要的呼吸道病原,其所致呼吸道感染以3岁以下儿童多见,尤以1岁以下为多,但男女感染比例差异无统计学意义,在上下呼吸道感染中感染率差异无统计学意义。在所检标本中,每个月均有HPIV3的检出。但于冬春出现感染高峰。用套式RT-PCR检测用于HPIV3的基因诊断具有特异性强的优点。兰州地区HPIV3 HN基因比较稳定,变异小。

Objective To establish a method with nested reverse transcription-polymerase chain reaction （ nested RT- PCR ） for detecting specific fragment of human parainfluenza virus-3 （ HPIV3 ） RNA and to analyze the sequence of amplicons in the hope that a better understanding of viral etiology of acute respiratory tract infection （ ARI） in children in Lanzhou area will be achieved. Methods NPS of children with acute respiratory tract was collected in Lanzhou area from July to December of 2005 and October of 2006 to March of 2007. All of them were inoculated vero cells, and HPIV3 was detected by nested RT-PCR. Results Forty-six （ 15.33% ） of 300 samples were HPIV-3 positive on nested RTPCR. Positive specimens could be detected in each month of every year. The peak of HPIV-3 infectious cases of its prevalence was November of 2005 ,and January and February in 2007： Through a comparative study with the sequence of the HPIV in the Gene Bank,the sequence of 11 amplicons shared high homology with them to the degree of 91% -99.6%. Conclusion HPIV3 infections occupy an important status in children with ARI in Lanzhou. HPIV3 infection is common in children under 3 years old and much more in those younger than 12 months of age. There is no significant difference in the infectious rate between the boys and the girls （ χ^2 = 0. 3856 P 〉 0.05 ） , as well as URI and LRI （ χ^2 = 0. 7303 P 〉 0. 05 ）. Positive specimens can be detected in each month of the year,but the peak of its prevalence is in spring and winter. This nested RT-PCR technique is highly specific and convenient for the detection of HPIV3 RNA in NPS of patients with ARI. HPIV3 HN genome in Lanzhou area is stable.