Sample Preparation for qPCR Detection of Listeria from Food

Mester, Patrick; Witte, Anna Kristina; Rossmanith, Peter

doi:10.1007/978-1-0716-0982-8_3

Cited by 1 publication

(1 citation statement)

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“…Compared with conventional PCR, the detection and analysis process for fluorescence real-time quantitative PCR is completed automatically by the equipment in closed single tubes, and has the advantages of high automation and is an effective solution to the problem of PCR product contamination and quantitative detection of pathogen infection, and so real-time quantitative PCR is currently considered the most ideal method for pathogen detection in laboratories [ 20 , 21 , 22 ]. For supplying more suitable, sensitive and efficient detecting methods for CMNV in the NPT activities and in the monitoring pathogens of farmed crustaceans, we designed new primers and probes targeting the CMNV coat protein (CP) gene, and then established an improved one-step real-time Taqman based reverse transcription quantitative PCR detecting method (Taqman RT-qPCR) of CMNV in present study.…”

Section: Introductionmentioning

confidence: 99%

Development of a Novel RT-qPCR Detecting Method of Covert Mortality Nodavirus (CMNV) for the National Proficiency Test in Molecular Detection

Wang

Liu

Yao

et al. 2022

Viruses

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Covert mortality nodavirus (CMNV), the pathogen of viral covert mortality disease (VCMD), has caused serious economic losses of shrimp aquaculture in Southeast Asian countries and China in the past decade. In view of that the rapid and accurate laboratory detection of CMNV plays a major role in the effective control of the spread of VCMD. The national proficiency test (NPT) for the detection of covert mortality nodavirus (CMNV) started in China from 2021. In this study, a novel TaqMan real-time reverse transcription quantitative PCR (RT-qPCR) detection method for CMNV with higher sensitivity than previous reports was established based on specific primers and probe designing from the conserved regions of the CMNV coat protein gene for using molecular detection of CMNV in NPT. The optimized RT-qPCR reaction program was determined as reverse transcription at 54.9 °C for 15 min and denaturation at 95 °C for 1 min, followed by 40 cycles including denaturation at 95 °C for 10 s, and annealing and extension at 54.9 °C for 25 s. The detection limit of the newly developed RT-qPCR method was determined to be as low as 2.15 copies of CMNV plasmids template per reaction, with the correlation coefficient (R2) at above 0.99. The new method showed no cross reaction with the six common aquatic animal pathogens and could be finished in one hour, which represents a rapid detection method that can save 50% detection time versus the previously reported assay. The CMNV TaqMan probe based RT-qPCR method developed in present study supplies a novel sensitive and specific tool for both the rapid diagnosing and quantitating of CMNV in NPT activities and in the farmed crustaceans, and will help practitioners in the aquaculture industry to prevent and control VCMD effectively.

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Section: Introductionmentioning

confidence: 99%