CAJ | 학술논문

目的建立一种快速、准确、特异的实时荧光逆转录聚合酶链反应方法(PCR),以检测人类偏肺病毒.方法根据Hmpv-L基因序列设计引物和探针并对实时荧光PCR反应体系进行优化,提取总RNA,通过随机引物进行反转录反应;产生的cDNA通过实时荧光PCR进行鉴定.进一步评价实时荧光反转录PCR方法的特异性、灵敏度、重复性,对180例临床样本进行检测.结果本实验所建立的实时荧光反转录PCR方法可准确、特异地检测人类偏肺病毒;该方法的灵敏度可达到1拷贝/μl;检测的批间和批内的变异系数均小于5%.180例肺炎和支气管炎患儿痰标本中共检测出28例人类偏肺病毒阳性,阳性率为15.56%(28/180);肺炎患儿检出率为15.60%(17/109);支气管炎患儿检出率为15.49%(11/71).男性患儿检出率18.56%(18/97),女性患儿检出率12.05%(10/83).患儿年龄小于2岁者检出率22.34%(21/94),2～5岁者检出率8.70%(6/69),大于5岁者检出率5.88%(1/17).结论本研究建立的人类偏肺病毒实时荧光逆转录PCR方法快速、准确,结果可靠,实用性强;人类偏肺病毒已成为小儿呼吸道感染的重要病原体之一.
목적 건립일충쾌속、준학、특이적실시형광역전록취합매련반응방법(PCR),이검측인류편폐병독.방법 근거Hmpv-L기인서렬설계인물화탐침병대실시형광PCR반응체계진행우화,제취총RNA,통과수궤인물진행반전록반응;산생적cDNA통과실시형광PCR진행감정.진일보평개실시형광반전록PCR방법적특이성、령민도、중복성,대180례림상양본진행검측.결과 본실험소건립적실시형광반전록PCR방법가준학、특이지검측인류편폐병독;해방법적령민도가체도1고패/μl;검측적비간화비내적변이계수균소우5%.180례폐염화지기관염환인담표본중공검측출28례인류편폐병독양성,양성솔위15.56%(28/180);폐염환인검출솔위15.60%(17/109);지기관염환인검출솔위15.49%(11/71).남성환인검출솔18.56%(18/97),녀성환인검출솔12.05%(10/83).환인년령소우2세자검출솔22.34%(21/94),2～5세자검출솔8.70%(6/69),대우5세자검출솔5.88%(1/17).결론 본연구건립적인류편폐병독실시형광역전록PCR방법쾌속、준학,결과가고,실용성강;인류편폐병독이성위소인호흡도감염적중요병원체지일.
Objective To develop a rapid,sensitive and specific real time reverse transcription PCR for detecting and identifying human metapneumovirus. Methods The Hmpv-L gene of human metapneumovirus was chosen as target gene,the primers and TaqMan probe were designed,and the PCR reaction was optimized systematically. The total RNA was extracted from respiratory specimens,and reverse transcription was performed through random primer. The cDNA was detected by using real time PCR. The specificity,sensitivity and reproducibility of real time PCR were estimated. The real time PCR was applied to detect 180 clinical respiratory specimens. Results The human metapneumovirus can be detected using real time reverse transcription PCR accurately and quickly,and the sensitivity was 1 copy/μl.The coefficient of variation of intra-assay and inter-assay wasless than 5%. Among those 180 specimens,28(15.56%) were positive for human metapneumovirus,the clinical diagnoses for these 28 patients were pneumonia ( 15.60%,17/109) and bronehiolitis ( 15.49%,11/71 ) .21 positive specimens were from patients under 2 years of age,and 6 positive specimens were from patients between 2 and 5 years of age,only 1 positive specimens was from patients over 5 years. Conclusion It is demonstrated that real time reverse transcription PCR is a reliable,accurate and feasible assay for human metapneumovirus,whieh has become one of the most important pathogens induced acute respiratory infections in pediatric patients.