[Study on Application of EMA-PCR Rapid Identification of Infectious and Non-infectious Adenovirus].

OBJECTIVES

To investigate the application of cytopathic effect (EMA)-PCR to rapid identification of infectious and non-infectious adenovirus.

METHODS

The different dilutions of virus were inoculated to well-grown monolayer cells and then calculated the concentration of virus through the cytopathic effect (CPE). The adenovirus and New Castle disease virus were prepared in a fixed concentration of 106 PFU/mL then diluted in ten-time gradient and the DNA of virus were extracted and PCR were applied, then the target DNA fragments were observed through gel electrophoresis. The non-infectious adenovirus were treated with different concentrations of EMA (0 μg/mL, 70 μg/mL, 120 μg/mL and 150 μg/mL) and then detected the target DNA fragment in the same way. The different concentrations of infectious adenovirus (107 PFU/mL, 106 PFU/mL, 105 PFU/mL, 104 PFU/mL and 103 PFU/mL) were treated with 120 μg/mL EMA and then detected target DNA fragment in the same way.

RESULTS

The target DNA fragments were detected in adenovirus of 104 PFU/mL and above. The target DNA fragments were detected in all 3 types of adenovirus while not detected in New Castle disease virus. The DNA amplification of non-infectious adenovirus were suppressed by EMA at 120 μg/mL and 150 μg/mL concentration, while the DNA amplification of infectious adenovirus in concentration of 105 PFU/mL and above were not affected by 120 μg/mL EMA.

CONCLUSIONS

EMA-PCR was proved to be feasible to identify the infectious and non-infectious adenovirus, in which false positive results of PCR were effectively avoid at the same time.