呼吸道合胞病毒实时荧光定量PCR检测

目的 建立一种快速、准确诊断人呼吸道合胞病毒(hRSV)早期感染的实时荧光定量PCR检测方法。方法 以hRSV最保守的N基因序列为参考,设计两对特异的引物和一条TaqMan荧光探针,在LightCycler定量PCR检测仪上对93例下呼吸道感染患儿进行hRSVRNA检测,并与病毒分离培养、常规PCR、巢式PCR方法和ELISA法相比较。结果 以PCR模板浓度来定义,该方法线形范围为1×10²~1×10⁷cDNAcopies/μl,灵敏度达1×10²cDNAcopies/μl,和巢式PCR相同,比常规PCR高10倍。用人脊髓灰质炎病毒Ⅰ型、柯萨奇病毒2型、甲型、乙型流感病毒、腺病毒7型作对照,均无预期扩增产物和荧光的产生;该方法在LightCycler和Rotor-Gene两种仪器上的定量结果具有一致性;采用该法对93例患儿行FQ-PCR检测出阳性44例(43.9%),ELISA方法检测阳性4例(4.3%),两者相关性不高。hRSV浓度的高低与患儿临床症状之间的关系不是很明显。结论 该方法可快速、灵敏、特异、定量检测hRSV,在hRSV感染的早期诊断和疗效监测方面具有良好的应用前景。

Quantitative fluorogenic real-time PCR assay for respiratory syncytial virus detection

Objective To Establish a rapid and objective quantitative fluorogenic real-time PCR assay for early detection of human respiratory syncytial virus (hRSV). Methods Two pairs of primers and one TaqMan Fluorogenic probe that are specific for the recognition of the most conservative N gene of hRSV for virus detection with LighCycler PCR in 93 nasopharyngeal secretion specimens collected from infants and young children.The assary was compared with virus isolation,routine PCR,nested PCR,and enzyme-linked immunosorbent assay (ELISA). Results This TaqMan assay had a sensitivity of 1×10² cDNA copies/μl with a dynamic range between 1×10² and 1×10⁷ cDNA copies/μl,which was the same as that of nested PCR,but 10 times more sensitive than routine PCR.The specificity of the assay was evaluated by comparing hRSV with polivirus type 1,coxsackie virus type 2,influenza A,influenza B and adenovirus type 7.A PCR product of the expected size (195 bp) was produced and fluorescence signal detected for hRSV,but not for any of the other viruses.The results in LightCycler and Rotor-Gene instrument were consistent.Forty-four specimens (43.9%) were hRSV-positive with this assay and 4 (4/93,4.3%) were hRSV-positive with ELISA,showing rather low correlation between the two methods.No visible relation was found between the concentration of hRSV RNA and severity of the disease. Conclusion This assay is rapid,sensitive,specific and quantitative,and has the potential of wide application for early diagnosis of hRSV infection and evaluation of the therapeutic effect.