一种快速诊断呼吸道合胞病毒感染的方法

Establishment of a rapid diagnosis method for syncytial virus infection in respiratory tract

应用引物设计原则，选择呼吸道合胞病毒（ＲＳＶ）基因组中高度保守的Ｆ基因作为扩增靶序列，独立设计合成了两对引物，建立了一个逆转录套式聚合酶链反应（ＰＣＲ）检测ＲＳＶＲＮＡ的方法。结果：对引物进行敏感度实验，其检测敏感度为每ｍｌ１０ＴＣＩＤ５０ｓ（５０％ｔｉｓｓｕｅｃｕｌｔｕｒｅｉｎｆｅｃｔｉｖｅｄｏｓｅ）的病毒量；用副流感病毒１型、甲型流感病毒、乙型流感病毒、新城疫病毒、腮腺炎病毒和腺病毒２型作对照，均无预期扩增产物出现，证实了设计引物的特异性；用建立的逆转套式ＰＣＲ方法对５例急性下呼吸道感染患儿的鼻咽部分泌物标本进行检测，发现３例阳性，２例阴性；同时用病毒培养法检测，结果完全相同。提示：该方法具有快速、敏感、特异和观察结果客观等优点。

Using the principles of primer design,the author selected the highly conservative fusion(F)gene of the respiratory syncytial virus(RSV)as the target sequence.Two pairs of primers were designed and synthesized in this study.A complete set of methodology for the detection of RSV RNA with the reverse transcriptase-polymerase chain reaction(RT-PCR)and the Nested polymerase chain reaction(Nested PCR)was established.The results were as follows:1.The sensitivity of the RT-PCR and the Nested PCR was examined by analyzing serial 10-fold dilution(100-to 108-fold)of an RSV long strain-infected Hep-2 lysate.The total amount of virus in this reaction was predicted to be approximately 104TCID50s/L.2.The specificity of the primers was examined by comparing RSV with parainfluenza type 1,influenza A,influenza B,New-Castle disease virus,mumps virus and adenovirus type 2.A Nested PCR product of the expected size(215bp)was for RSV,but not for any of the other viruses.The RT-PCR and the Nested PCR were tested on 5 nasopharyngeal secretion samples collected from children with acute lower respiratory tract infection,3 were positive and 2 negative.Meanwhile,the result of virus culture was the same.The results indicate that this method is rapid,sensitive,specific and objective.