副粘病毒Tianjin株RT-PCR检测方法的建立及初步应用  

Development and Application of Reverse Transcriptase-Polymerase Chain Reaction for the Diagnosis of Paramyxovirus Tianjin Strain

作　　者：石立莹[1] 李梅[1] 何健民[1] 李晓眠[1] 

机构地区：[1]天津医科大学基础医学院微生物学教研室,天津300070

出　　处：《热带医学杂志》2009年第3期265-268,共4页Journal of Tropical Medicine

基　　金：国家自然科学基金(No.30471530);天津医科大学科学基金(No.2007ky43)。

摘　　要：目的建立检测副粘病毒Tianjin株的RT-PCR方法,为临床标本的检测提供参考依据。方法根据副粘病毒Tianjin株F蛋白的基因序列,设计出一对特异性引物,以Tianjin株总RNA逆转录的cDNA为模板进行PCR扩增,然后对该方法的敏感性、特异性进行检测。并将该方法应用于临床84例下呼吸道感染患儿支气管肺泡灌洗液(BALF)标本的检测。结果通过此方法成功扩增出约500bp的预期条带。将Tianjin株鸡胚尿囊液(血凝效价为1:1280)稀释到320倍时仍可出现阳性条带,对新城疫病毒、甲型流感病毒鼠肺适应株、人副流感病毒Ⅰ型、柯萨奇病毒B3m株、单纯疱疹病毒Ⅰ型及鸡胚尿囊液的PCR检测结果均呈阴性。84例下呼吸道感染患儿BALF标本中,其中2例阳性,阳性率为2.38%,与本实验室建立的双抗体夹心ELISA法检测结果相接近。结论建立的副粘病毒Tianjin株RT-PCR检测方法是一种快速、灵敏、特异的方法,为患病儿童中Tianjin株感染情况的调查奠定了重要的基础。Objective To establish a reverse transcriptase polymerase chain reaction （RT-PCR） method for paramyxovirus Tianjin strain and to utilize the RT-PCR for detection of Tianjin strain in clinical samples. Methods PCR primers were designed according to F gene of paramyxovirus Tianjin strain （GenBank No：EF679198）. The genomic RNA of Tianjin strain was extracted and used as template for reverse transcription. The 500 bp fragment was amplified by PCR. Then the sensitivity and specificity were determined. Bronchoalveolar lavage fluid （BALF） samples from young children with severe lower respiratory tract infection were detected by RT-PCR. Results The results showed that the 500 bp PCR product was obtained. The Tianjin strain virus could be detected by PCR even with the HA titer 1：1280 was diluted to 1：320 in the allantoic fluid of chick embryos. The PCR detection was not cross to other viruses, such as Newcastle disease virus, mouse-adapted influenza A virus, human parainfluenza virus type 1, Coxsackie virus B3m and herpes simplex virus type l, and allantoic fluid of chick embryos. 2 samples were positive in 84 BALF samples by RT-PCR method. The positive rate was 2.38% （2/84）, which was similar to the result obtained by double antibody sandwich ELISA method established by our lab. Conclusion The developed RT-PCR was a rapid, reliable, sensitive and specific method for paramyxovirus Tianjin strain. The result might lay the foundation for the detection of Tianjin strain from the clinical samples.

关 键 词：副粘病毒Tianjin株 仙台病毒 RT—PCR 

分 类 号：R373.9[医药卫生—病原生物学]