Development And Clinical Trial Of Fluorescence Real Time PCR To Detect Rubella Virus

BACKGROUND: Rubella virus is a global contagious pathogen, and depends on human beings for a living only. In the past, many countries such as Austrilia, the United States, Japan and China ect. had burst out rubella. Althoug at present rubella has been almost eradicated by the enforcement of immunization programs, while many countries, in which immunity item is set up poorly, still have the epidemic. In a word,rubella virus still endangers the human beings' health extremely. Retrace the history, a diagnosis of rubella has been made assessed by morphology, serology, Dot-blot, Liquid, Solt, PCR and so on, but none is satisfied by people fully. OBJECTIVES: To establish a novel rapid, convenient, sensitive and specific method applicable for quantitative analysis of the rubella virus extensively and to show the true state of RV infection and amplification level in patients. To provide the clinical diagnose and treatment with scientific evidence. METHODS: The envelope glycoprotein E1 gene was amplified from rubella virus, strain RB1, by RT-PCR, and recombined with the pMD 18-T vector. After amp selection and analysis of restriction enzyme, the clones with carry the E1 gene were identified. After quanified and serial diluted, quantitative analysis of the E1 gene by Real-time PCR with FAM. Standard curve of the Real-time PCR was plotted with starting cDNA concentration versus threshold cycle. Then the new method was used to measure 19 cases with suspectable RV infection. And the results of Real-time PCR assay were compared with the results obtained with ELISA assay. RESULTS: The RV-cDNA detected with quantification Real-time fluorescence PCR can show the RV replication level in patients. The linear range of the system was from 103 to 109copies/μl in clinical samples. The CV value was 0.94% in batch assay and 3.36% in day to day assay. The new method is more sensitive and specific than ELISA assay. CONCLUSION: For its simplicity, sensitivity, specificity and auto-digitized results, the quantification of RV-cDNA in clinical samples is available. And it will become the standard method in quantitative RV infection.