实时荧光定量RT-PCR快速检测星状病毒方法的研究

Analysis of rapid astrovirus detection method by RT-PCR

目的建立以特异性荧光探针为特点的Taq Man荧光定量RT-PCR检测星状病毒。方法根据星状病毒基因组保守序列设计引物和探针,建立并分析荧光定量RT-PCR的重复性、特异性、敏感性;以所建立方法对128例病毒性腹泻患者粪便标本进行检测,同时以基因测序进行验证。结果所建立的荧光定量RT-PCR检测方法对星状病毒具有很好的特异性和重复性,灵敏度达102copy/μl。128例粪便标本中星状病毒的检出率为3.1%,与基因测序比对结果相符。结论构建的星状病毒荧光定量RT-PCR方法具有快速、特异且灵敏的特点,可用于临床病原诊断和流行病学调查。

Objective To establish a fluorescent quantity real- time PCR method based on specific fluorescent Taq Man for detection of astrovirus. Methods According to the conserved genome sequence design and probes of astrovirus,a Taqman RT- PCR was constructed,and the repetitiveness and specificity and sensitivity of it were evaluated. Fecal samples from 128 cases of patients with viral diarrhea were detected by constructed method and the results were compared with gene sequencing. Results The constructed Taqman RT- PCR was specific and repetitive for astrovirus,and the stability reached to 102 copy / μl. The detection rate of astrovirus with the constructed Taqman RT- PCRwas 3. 1% of 128 stool specimens,which was correspond to the comparison results of gene sequencing. Conclusion This real time RT- PCR assay was rapid,specific and sensitive. It can be used for clinical diagnosis and epidemiological investigation on pathogen.