The Establishment Of A Detection Method With Fluorescence Real-time Quantitative Polymerase Chain Reaction For Enteroviruses

Objective:To construct an external standard for the detection of enteroviruses with fluorescence real-time quantitative polymerase chain reaction. And then quantify enteroviruses absolutely according to the standard curve generated by series dilution external standard.Method : To construct an external standard for detection of enteroviruses , enteroviruses cDNA standard for fluorescence real-time quantitative PCR was prepared with T-A clone . Firstly, designed and synthesized universal primers of enteroviruses. Extracted the RNA of enteroviruses and then converted them into cDNA. The best annealing temperature was selected by gradient PCR. And then purified PCR products of poliovirus-Ⅰ.,which were connected to pGEM-T-vector subsequently. After that, the connection wasre transformed into competence cells. The next step was screening and restriction Enzyme: LB / ampicillin / IPTG / X-Gal plates were used for blue-white screening. The white clones were picked,including target DNA . Positive bacterias were propagated, and then the recombinant plasmid in the bacteria was extracted. EcoRⅠwas used to restriction enzyme digest. The digested products were analyzed with electrophoresis fragment, and then sequenced by biological company. Measured the concentration of purified plasmid,on the basis of them, then copy number were obtained.For real-time PCR the concentration of primer was optimized at first.The thermal profile for PCR was 50℃for 2 min and 95℃for 10 min, followed by 40 cycles of 95℃ for 10 s and 60℃for 1 min. A post-PCR melt curve was run according to the AB7900HT User's Manual: a 15s hold at 95℃, a 20s hold at 60℃and a 20min slow ramp from 60 to95℃. The standard curves for enterovirus was generated using a dilution series of recombinant plasmid DNA and the reaction parameter as described above. This experiment was repeated to test the stability, repeatability and sensitivity of this method. The RNA of ten-fold dilution series of poliovirus, which TCID50 titer was known, were extracted , and then converted it into cDNA as the template of real time PCR. The relationship of copies by real time PCR and TCID50 by cell culture was analyzed.Result:The best annealing temperature was 58℃.The course of connection and transformation were successful. The recombinant plasmid included target products. The 300 nM concentrations of both forward and reverseprimers were found to be optimal for amplification and, therefore, for all subsequent work these primer concentrations were used. The method can detected as low as 103 copies with the linear range was 103～108 copies. There was a linear relationship of logarithm of TCID50 and logarithm of copies(r=0.92, P<0.01).Conclusion: The construction of external standard for detection of enteroviruses in water using the quantitative real-time polymerase chain reaction was successful. There was good stability, repeatability and sensitivity to quantity enteroviruses with real time PCR by recombinant plasmid as the external standard. This method can be used to quantity enteroviruses.