A novel multiplex PCR for virus detection by melting curve analysis

Abstract

A B S T R A C TBackground: Rapid and accurate laboratory diagnoses of viral infections are crucial for the management and treatment of patients with viral infections. Conventional methods for virus detection are labourious, time consuming, and only a single virus can be analysed in one assay. Objectives: The objective of this study was to develop a novel real-time PCR method for multiple virus detection by melting curve analysis using Taqman probes in a single reaction. Study design: As a model, six respiratory viruses were detected in one tube using three fluorophores. The specificity was assessed by cross-reaction tests with other common respiratory pathogens. The analytical sensitivity was assessed by testing the limit of detection of the assay using artificial plasmids as the positive template. The clinical evaluation of the established assay was evaluated for the detection of respiratory viruses in clinical samples, and the results were compared with direct fluorescent antibody testing (DFA). Results: The six respiratory viruses were clearly distinguished by their respective melting temperature values in the corresponding fluorescence detection channels. No cross reactions were observed by cross reaction tests. The detection limits of this assay were 2 to 2 × 10 3 copies per reaction for each virus. The clinical evaluation of this assay was demonstrated by analysing 352 clinical samples, and 67(19.0%) samples were positive for at least one virus. The accordance rate between the established PCR and DFA testing was high, and ranged from 94.57% to 100%. Conclusions: Taqman probe-based melting curve analysis is well suited for detection of multiple viruses in clinical and research laboratories because of its high throughput, reliability, and cost savings.

Preparation, characterization and antioxidant activity analysis of three Maillard glycosylated bone collagen hydrolysates from chicken, porcine and bovine

“…This is similar to research of Chen et al [36] on Maillard reaction of soybean protein hydrolysates, that is, the sample after Maillard reaction has higher fluorescence intensity in the scope of 400-600 nm. A resembling phenomenon was also observed by Liu et al [37] that the fluorescence intensity enhanced with the growing time in the intermediate stage of the Maillard reaction in the fructose-histidine model system. Moreover, G-Cbcp displayed the highest fluorescence intensity, followed by G-Pbcp and G-Bbcp (P < 0.05).…”

Plant Virome Analysis by the Deep Sequencing of Small RNAs of Fritillaria thunbergii var. chekiangensis and the Rapid Identification of Viruses

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