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
Objective@#To investigate thyroid hormone concentration and associated factors among pubertal girls in Minhang District of Shanghai.@*Methods@#From January to March 2019, a stratified sampling method was used to select junior high schools from the east, south, north, and middle areas in Minhang district. A total of 386 girls of grade 6 in selected schools were included in the study. Physical examination was conducted, and their urine and blood samples were collected to determine urinary iodine concentration (UIC) and thyroid function. Puberty Development Self-rating Scale (PDS) was used to define the pubertal stage. Logistic regression models were conducted to analyze the associations between pubertal stage and thyroid function.@*Results@#The median urinary iodine concentration was 163.57(106.57, 232.96) μg/L. The geometric mean values of TSH, TT3, FT3 and FT4 were 0.29 mU/L, 0.26 nmol/L, 0.68 pmol/L and 1.18 pmol/L.The mean value of TT4 was 91.64 nmol / L. The abnormal rates of TGAb and TPOAb were 6.22% and 4.15%. The rate of abnormal TGAb combined with abnormal TPOAb was 3.68%. Girls in puberty and post-puberty had the lower level of TT4 (OR=0.47,0.43) as compared with girls in pre-puberty stage. Obese girls had higher level of TT3(OR=9.08, 95%CI=1.52-54.07). With the increase of exercise time(0.5-1, >1 h/d), FT4 level was increased (OR=2.45, 2.19). TSH levels were significantly higher in girls with higher TGAb and TPOAb. Girls had higher TT4 or FT4 levels if their TGAb levels were higher and TPOAb levels were normal.@*Conclusion@#There is an association between pubertal stage, obesity, exercise and thyroid function in school-aged girls during puberty in iodine sufficient areas. TSH, FT4 and TT4 levels are correlated with TGAb and TPOAb levels.
ABSTRACT
This study examined the effect of IL-10 on immunoglobulin-like transcript (ILT4) expression of human monocytic leukemic cell line THP-1, especially the role of the ILT4 promoter activity. ILT4 promoter area was amplified by PCR, and was cloned into the eukaryotic expressing vector pGL3-Basic. The pGL3-ILTP obtained was tested by double endonuclease digestion and sequencing. Then, the recombinant plasmid was transfected into THP-1 cells by using lipofectamine. After culture with IL-10 for 12 h, the mRNA extracted from THP-1 cells was detected by RT-PCR and the protein was detected by FACS. The dual-luciferase reporter assay system was employed to detect the activity of ILT4 promoter with or without IL-10. The results showed that the activity of pGL3-ILTP was significantly increased and was more than ten times that ofpGL3-Basic cells. After culture with IL-10 for 12 h, the expression of ILT4 protein and its mean fluorescence intensity (MFI) were increased. Moreover, the mRNA was remarkably higher than that of the control group. Dual-luciferase reporter assay revealed that ILT4 promoter was much more activated after being treated with IL-10.We were led to conclude that pGL3-ILTP containing ILT4 promoter was constructed successfully.The expression of ILT4 could be up-regulated by IL-10 both at the transcriptional and translational level. Furthermore, ILT4 promoter could be much more active after addition of IL-10. This study suggests that IL-10 up-regulates ILT4 expression on monocytes via increasing ILT4 gene promoter activity, which may have implication for inducing transplantation tolerance in clinical practice.