应用实时聚合酶链反应技术检测儿童呼吸道标本中的呼吸道合胞病毒

Detection of respiratory syncytial virus in nasopharyngeal aspirates of children by using real-time polymerase chain reaction

目的建立实时聚合酶链式反应（PCR）技术检测儿科呼吸道标本中呼吸道合胞病毒（RSV）的方法.方法（1）根据RSV N基因序列设计合成分别用于扩增RSV A亚型和B亚型的TaqMan探针和引物,建立实时PCR方法,进行灵敏度和特异性检测.（2）采用实时PCR检测61例RSV检测阳性的冻存标本和103例新收集的呼吸道标本,并与病毒分离、间接免疫荧光、巢式PCR结果相比较.结果（1）实时PCR对A亚型RSV检测的灵敏度为5.25 pfu,B亚型为3.75 pfu,与巢式PCR相同.（2）实时PCR检测其他呼吸道病毒阳性标本,结果均为阴性;A亚型和B亚型之间无交叉.（3）实时PCR检测其他方法检测过的RSV阳性的冻存标本61例,A亚型（30例）阳性为27例（90%）,B亚型（31）阳性为27例（87.1%）.（4）103例新鲜标本中,实时PCR检出RSV 35例（A亚型7,B亚型28）,阳性率34.0%,其中巢式PCR阳性31例,阳性率30.1%;间接免疫荧光阳性22例,阳性率21.4%;病毒分离阳性9例,阳性率8.7%.实时PCR阴性的68例,其他方法均为阴性.实时PCR与三种方法的一致性检验,一致率在74.76%～96.12%（P均＜0.01）;差异性检验结果说明,实时PCR阳性检出高于间接免疫荧光和病毒分离（P均＜0.01）,而与巢式PCR差异无统计学意义（P＞0.05）.结论实时PCR检测儿科鼻咽分泌物标本中RSV的方法,敏感性、特异性和可重复性好,可用于急性呼吸道感染患儿标本中的RSV检测.

Objective Human respiratory syncytial virus （hRSV） is the leading cause of acute upper and lower respiratory tract infections in infants and young children worldwide. Pediatric RSV disease claims more than 1 million lives annually. With the rapid development of specific anti-RSV agents and the spread of respiratory infections, RSV detection techniques with higher sensitivity, specificity and quicker performance are badly needed. This study was designed to develop a real-time polymerase chain reaction （PCR） for detection of RSV in nasopharyngeal aspirates. Methods （ 1 ） The TaqMan probe and primers of real-time PCR for RSV subgroup A and subgroup B detection were designed from the conserved region in N protein encoding gene, respectively. The sensitivity of real-time PCR was evaluated by using the virus with known amount of PFU. The specificity of real-time PCR for RSV detection was assessed by cress testing 10 isolates of strains A, 10 isolates of strains B, and by testing a variety of other respiratory viruses positive samples. （2） Sixty-one stored RSV positive respiratory samples and 103 nasopharyngeal aspirates were detected by real-time PCR, virus isolation, immunofluorescence assay （ IFA）, and nested-PCR. Results （ 1 ） The sensitivity of the real-time PCR developed in this study for RSV subgroup A detection was 5. 25 pfu, and for subgroup B was 3. 75 pfu, the same as that of nested-PCR. （2） No positive results were found in cress testing of other viruses positive specimens. （3） Twenty-seven out of 30 （90%） of RSV A stored samples and 27 out of 31 （ 87. 1% ） of RSV B stored samples were positive by the real-time PCR. （4） Thirty-five （34. 0% ） out of the 103 specimens were found RSV positive by real-time PCR （7 of them were subgroup A and 28 subgroup B） ; 31 （ 30. 1% ） specimens were positive by nested-PCR （ 6 of them were subgroup A and 25 subgroup B ） ; 22 （ 21.4% ） were found positive for RSV with IFA （ 5 of them were subgroup A and 17 subgroup B） ; RSV was isolated from 9 （8. 7% ） specimens （6 of them were subgroup A and 3 subgroup B）. All the specimens found to be negative by real-time PCR were negative by rest of the methods used in this study. Conclusion The real time PCR method developed in this project with the TaqMan probe and primers is sensitive and specific for detecting RSV subgroup A and B in nasopharyngeal aspirates.