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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TaqM an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 To establish a real - time polymerase chain reaction (PCR) method for detecting respiratory syncytial virus (RSV) in pediatric respiratory specimens. Methods (1) According to the sequence of RSV N gene, TaqMa probes and primers for amplifying RSV A subtypes and B subtypes were designed and synthesized respectively. Real-time PCR was performed to detect the sensitivity and specificity. (2) Real-time PCR was used to detect 61 RSV-positive frozen specimens and 103 freshly collected airway specimens, which were compared with the results of virus isolation, indirect immunofluorescence and nested PCR. Results (1) The sensitivity of real-time PCR to RSV detection of subtype A was 5.25 pfu and that of subtype B was 3.75 pfu, which was the same as that of nested PCR. (2) Real-time PCR detection of other respiratory virus positive specimens, the results were negative; A and B subtypes no cross. (3) In real-time PCR, 61 cases of RSV-positive frozen-thaw specimens detected by other methods were positive in 27 cases (90%) in group A subtype (30 cases) and 27 cases (87.1%) in group B subtype . (4) Of the 103 fresh samples, 35 cases of RSV were detected by real-time PCR (type A 7 and type B 28), the positive rate was 34.0%. Among them, 31 were positive by nested PCR and the positive rate was 30.1%. Indirect immunofluorescence 22 cases, the positive rate of 21.4%; 9 cases of positive virus isolation, the positive rate of 8.7%. Real-time PCR negative in 68 cases, other methods were negative. The concordance rate between real-time PCR and three methods was 74.76% ~ 96.12% (P <0.01). The results of the difference test showed that the positive rate of real-time PCR was higher than that of indirect immunofluorescence and virus isolation ), But no significant difference with nested PCR (P> 0.05). Conclusion The real-time PCR method for detection of RSV in pediatric nasopharyngeal secretions has good sensitivity, specificity and repeatability and can be used for the detection of RSV in children with acute respiratory infection.