An ultra-fast thermocycling fluorescence quantitative PCR system for detection of influenza virus: evaluation on its efficacy

Abstract

Objective To evaluate the performance and application efficacies of a ultra-fast thermocycling fluorescence quantitative PCR system for detection of influenza A virus (IAV) and influenza B virus (IBV). Methods Our constructed plasmids of IAV and IBV was employed as standards, and then the concentration was diluted gradiently. After each concentration was repeatedly detected with the ultra-fast thermocycling fluorescence quantitative PCR system for 20 times, a standard curve was drawn to verify the minimum detection limit and liner range of the system and correlative coefficient. A total of 278 clinical samples (88 IAV-positive, 87 IBV-positive, and 103 negative samples) were collected from the Biosafety Section of Center for Disease Control and Prevention, Southern Theater Command. They were isolated during 2016 and 2018, from the patients of 164 males (59%) and 114 females (41%), and at an average age of 24 years. The repeatability of the system was analyzed through the system repeatedly detect the identified 88 IAV-positive and 87 IBV-positive samples. The results of the system and traditional real-time q-PCR (RT q-PCR) were compared to evaluate the efficacy of the system. Results The results of ultra-fast thermocycling fluorescence quantitative PCR system showed that the minimum detection limit for detection of IAV and IBV plasmids was 1×100 copies/μL, and the liner range ranged from 1×100 to 1×105 copies/μL. In detection of clinical samples, this system obtained a sensitivity of 92.57%, a specificity of 100%, and negative and positive predictive values of 88.79% and 100%, respectively, with a coincidence rate of 95.32% when compared with the results of traditional RT q-PCR. There were no cross-reactions between IAV and IBV or among them with other pathogens. No interference was observed in the detection results of influenza in presence of 30 g/dL hemoglobin, 3.2 g/dL triglyceride and 100 μg/mL ceftriaxone sodium. Conclusion The system shows good detection performance in detection of clinical samples, with advantages of small in size, light in weight, short in time, and no procedure of nucleic acid extraction, and is suitable for point-of-care testing (POCT).

Keywords

• ultra-fast thermocycling quantitative pcr system
• respiratory pathogen
• point-of-care testing
• influenza a virus
• influenza b virus