ABSTRACT
@#Abstract: Objective To develop a real-time fluorescent quantitative RT-PCR (qRT-PCR) method for qualitative and quantitative Chikungunya virus (CHIKV) analysis. Methods Based on the systematic analysis of the genomic sequences of Chikungunya and its related arboviruses, the specific nucleic acid sequences for Chikungunya virus were screened and identified, and then the primers and TaqMan probe were designed. Meanwhile, the human GAPDH gene was used as an internal reference. The reaction system for qRT-PCR was systematically optimized by L9(34) orthogonal design, and a rapid detection method for Chikungunya by qRT-PCR based on TaqMan probe methods was established. The sensitivity, specificity, reproducibility, and coverage of the established method were analyzed in detail. The standard curve was made, and the absolute quantitative method was established using the cloned nucleic acid fragments as positive samples. Results A real-time fluorescent quantitative RT-PCR assay was developed for the qualitative and quantitative analysis of Chikungunya virus. The reaction system included Chikungunya virus and reference internal gene specific primers and probe, RT/Taq enzyme mixture, reaction buffer, and negative and positive reference. The established method obtained positive results with the ROSS strain of ECSA subtype, LR2006 strain of IOL branch, 181/25 strain of Asian type and Dongguan 2010 epidemic strains of Chikungunya virus, but there was no cross-reaction with other 18 arboviruses belonging to Flaviviruses, Alphaviruses and Bunyavirus. The minimum detection limit of the established method was 5.80 copies/mL, and a linear relationship was observed between the amount of input plasmid DNA and fluorescence signal value over a range of 5.80×102 copies/mL to 5.80×1010 copies/mL, and the correlation coefficient was 0.999 5. The qRT-PCR amplification efficiency was 91%, and the intra-assay variations and inter-assay variations were 0.01-0.07 and 0.03-0.11, respectively. Conclusions The TaqMan qRT-PCR method developed in this study can qualitatively and quantitatively detect Chikungunya virus rapidly with specificity and sensitivity, providing a technical method for the prevention and control of this viral disease.
ABSTRACT
<p><b>OBJECTIVE</b>To shorten the duration of seed germinating, improve seed emergence and eventurally to establish a practical method for seeding of Paris polyphylla var. yunnanensis seed.</p><p><b>METHOD</b>To study the effect of some factors (temperature, sowing time, covering-soil depth, plastic film ) on seed emergence of P. polyphylla var. yunnanensis.</p><p><b>RESULT</b>The most suitable temperature for embryonic post-maturity and seed germinating is 18-20 degrees C. Paris seed could emerge in April when it was transfer to a low temperature 0-10 degrees C for 2-4 months after being treated under 18-20 degrees C for 3-4 months. For seed emergence, the best sowing time was before April. The suitable soil depth was about 1 cm. It is a better way to cover the seedling bed with black plastic film for improving the emergence percentage.</p><p><b>CONCLUSION</b>Above results provide seedling techniques of P. polyphylla var. yunnanensis.</p>