实时荧光定量PCR检测VAD1 mRNA评估新型隐球菌性脑膜炎的疗效

Objective To establish a new approach for quantitative detection of VAD1 mRNA in cryptococcus neoformans by RT-FQ-PCR, and evaluate the treatment efficacy of CNM. MethodsThe primers and TaqMan probe were designed according to the published sequence of VAD1 mRNA (GenBank),and RT-FQ-PCR method to detect VAD1 mRNA was established. Cerebrospinal fluid from 25 CNM patients and 30 controls were detected and sensitivity and specificity of the method were evaluated. VAD1 mRNA concentration in cerebrospinal fluid from both acute phase and recovery phase of 25 CNM patients were also detected and significance of CNM treatment efficacy with VAD1 mRNA analysis was evaluated. Results Correlation coefficient of standard curve was - 0. 997 9 in detection of VAD1 mRNA by RT-FQ-PCR, and the detection limit was 101 copies/μl. The intra CV of plasmid standard for high, medium and low concentrations were 0. 65% ,0. 89% and 1.23% respectively, the sensitivity of cryptococcus neoformans detection by RT-FQPCR was 96% (24/25) ,while specificity was 100% (30/30). VAD1 mRNA concentration in acute phase were significant higher than that in recovery phase (3. 042 ±0. 906 vs 2. 187 ±0. 665 ,t =4. 583 ,P ＜0. 01).Conclusions The established RT-FQ-PCR method for the detection of VAD1 mRNA is provided with sound sensitivity, specificity and reproducibility, which might be fit for the detection of VAD1 mRNA. The expression level of VAD1 mRNA is relevant with the treatment efficacy of CNM.

Evaluation for the treatment efficacy for cryptococcus neoformans meningitis by VAD1 mRNA detection with real-time fluorescent quantitative polymerase chain reaction