ABSTRACT
Objective:To establish a real-time quantitative PCR assay to detect the copy number of IS711 transposase gene (orfA) in Brucella genome, and the assay is applied to identify the species and biovars of Brucella. Methods:To establish an orfA gene copy number detection system based on Taqman real-time quantitative PCR technique. Primers and probes of bcsp31 and orfA genes were designed, the contents of bcsp31 gene and orfA gene in the same strain with the same DNA concentration were simultaneously detected by real-time quantitative PCR assay, and cycle number (CT value) of the two genes were obtained. According to the differences of CT values of bcsp31 gene and orfA gene, the copy number of orfA gene in Brucella genome was calculated. At the same time, the DNA of Brucella 16M strain was double decreasing dilution to verify the stability of the detection system. Results:A real-time quantitative PCR assay was used to detect bcsp31 gene and orfA gene simultaneously, when the DNA concentration difference of 16M strain was 2 times, the mean difference of CT values measured was 1.00, 95% confidence interval was 0.95-1.05, standard deviation was 0.17, and coefficient of variation was 0.17. The orfA gene copy number of 30 Brucella strains was detected by this detection system. It was found that there were 6, 9, and 7 copy numbers in the biovars 1-3 of Brucella melitensis, respectively. The strain of Brucella suis biovar 2 had 10 copy numbers, which were different from those of the other 4 strains of biovars 1, 3-5. There were 37 copy numbers in Brucella ovis strain. The copy numbers were stable at 5-6 copies in 8 biovars (1-7, 9) of Brucella abortus strains. Conclusions:A real-time quantitative PCR assay for detection of orfA gene copy number in Brucella DNA has been established. This method could identify some Brucella species and biovars strains.
ABSTRACT
The copy numbers of exogenous gene in transgenic animals is always regarded as an important information of transgenic animals.Thus,simple and sensitive methods are required for the detection of the copy numbers of exogenous gene.Three kinds of transgenic Shanbei white cashmere goats,containing Tβ4-GFP,FGF5s-GFP and VEGF164-GFP,has been obtained by using PiggyBac(PB) transposon system.Fluorescence quantitative PCR was carried out to detect the copy numbers of copGFP.Using Gluc as reference gene,the double standard curves of exogenous gene and reference gene were mapped and the genomic DNA of transgenic goats were analysized by real-time fluorescence quantitative PCR.Moreover,the copGFP/Gluc ratio in the samples was calculated as the copy numbers of copGFP.In addition,Tβ4-GFP transgenic cashmere goats were selected to detect the integration sites by using the genomic walking kit.The results showed that the standard curve equation of copGFP was y=-3.230 6x+39.216 (R2 =0.998 8) and the standard curve equation of Gluc was y=-3.564 8x+38.440 (R2 =0.996 0).The copy numbers of exogenous gene in the transgenic cashmere goats were obtained and the numbers of integration sites in the selected Tβ4-GFP transgenic goats were consistent with the copy numbers of copGFP.As a conclusion,the high throughput,fast and sensitive real-time fluorescence quantitative PCR is an efficient and convenient method for the copy number of exogenous gene in transgenic cashmere goats.
ABSTRACT
Objective To investigate the correlation between the human chemokine type 1 chemokine ligand 3 (CCL3L1) and chemokine ligand 4 (CCL4L) gene expression and the immune reconstitution of acquired immunodeficiency syndrome (AIDS) patients after antiretroviral therapy.Methods The gene copy numbers of CCL3L1 and CCL4L were detected by real time polymerase chain reaction in 217 AIDS patients before antiretroviral therapy.And the correlation between CCL3L1 and CCL4L gene copy numbers and the level of CD4+ and CD8+ T lymphocytes were analyzed.The changes of CD4+ and CD8+ T lymphocytes were defined as mean change value per month after 48 months treatment.The change rates of CD4+ and CD8+ T lymphocytes were defined as the logarithm of the ratio of the value after 48 month to that at baseline.Comparison between groups was conducted using analysis of variance.Results The median of gene copy numbers of CCL3L1 and CCL4L were 2 (range:0-8) and 3 (range:0-7),respectively.After antiviral treatment,there were significantly different changes of CD8+ T lymphocyte level (F=3.054,P＜0.05) and change rate of CD4+/CD8+ (F=3.520,P＜0.05) among groups of high (gene copy 4-8),median (gene copy 2-3) and low (gene copy 0-1) CCL3L1 gene copy numbers.The changes of CD8+ T lymphocyte levels (P=0.023) and change rates (P=0.038) in high and low CCL3L1 gene copy groups were both significantly different.There were significant differents changes rate of ratio of CD4+/CD8+ T lymphocyte among high and median (P=0.010),high and low CCL3L1 gene copy numbers (F=4.397,P＜0.05).The significant difference of the change rates of CD4+/CD8+ were found between the gene copy 3 group and gene copy 4-7 group CCL4L (P=0.005) and between the gene copy 4-7 group and gene copy 0-2 group of CCL4L (P=0.030).The change ratio of CD4+/CD8+ T lymphocytes increased with the increase of copy numbers of CCL4L gene.Conclusions The gene expressions of CCL3L1 and CCL4L might be associated with the ability of immune reconstitution of AIDS patients after antiretroviral therapy.