Fujian Key Laboratory for Technology Research of Inspection and Quarantine

Fuzhou, China

Fujian Key Laboratory for Technology Research of Inspection and Quarantine

Fuzhou, China
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Ruan J.H.,Fujian Agriculture and forestry University | Wang W.J.,Fujian Agriculture and forestry University | Wang W.J.,Fujian Key Laboratory for Technology Research of Inspection and Quarantine | Zhang T.Y.,Fujian Key Laboratory for Technology Research of Inspection and Quarantine | And 6 more authors.
Current Microbiology | Year: 2017

A gyrB gene is present in the majority of bacterial species, and encodes the ATPase domain of DNA gyraseB-subunit protein, which is essential for transcription and replication of bacteria. The gyrB gene exhibits higher nucleotide sequence variability than the 16S rDNA gene and thus could be more reliable in differentiating Serratia fonticola. A species-specific primer pair and probe were designed for quantitative real-time PCR detection of S. fonticola using gyrB as the target gene. Nine members of the Serratia family (representing nine Serratia species) were chosen to verify the specificity of the primers. Additionally, two species each of Salmonella and Klebsiella, and five other species belonging to five other genera of Enterobacteriaceae, were tested for primer cross-reaction. All the tested strains gave negative results. The limit of detection for S. fonticola using the gyrB gene was 100 copies per PCR reaction. This TaqMan PCR assay provided a specific, rapid, and sensitive method to detect S. fonticola based on its gyrB gene. © 2017 Springer Science+Business Media, LLC


Jin J.,Fujian Agriculture and forestry University | Jin J.,Fujian Key Laboratory for Technology Research of Inspection and Quarantine | Shen J.G.,Fujian Key Laboratory for Technology Research of Inspection and Quarantine | Cai W.,Fujian Key Laboratory for Technology Research of Inspection and Quarantine | And 6 more authors.
Journal of Applied Microbiology | Year: 2017

Aims: Development of a multiplex TaqMan RT-qPCR assay to simultaneously detect Narcissus yellow stripe virus (NYSV) and Narcissus mosaic virus (NMV), frequently causing mixed narcissus infection. Feasibility verification was confirmed in natural samples. Methods and Results: Primers and probes were designed based on the conserved CP gene regions of NYSV or NMV and their suitability for singleplex and multiplex TaqMan RT-qPCR assays as well as for conventional RT-PCR. Conventional RT-PCR, singleplex and multiplex TaqMan RT-qPCR assays proved to be NYSV and NMV specific. P-values and coefficients of variation of TaqMan RT-qPCR assays indicated high reproducibility. Significantly increased sensitivity was achieved compared to conventional RT-PCR. The detection limit of both viruses was 103 copies with superior correlation coefficients and linear standard curve responses between plasmid concentrations and Ct values. NYSV and NMV infection of narcissus leaves, petals and bulbs could successfully be detected via our multiplex RT-qPCR method at 1·25 mg. Conclusion: Our multiplex TaqMan RT-qPCR assay provides rapid, specific, sensitive and reliable testing to simultaneously detect NYSV and NMV, supplying useful routine monitoring for different narcissus samples. Significance and Impact of the Study: Efficient identification and discrimination of the narcissus viruses provides reliable information for scientists and conventional growers. Furthermore, it enriches the information of NYSV, NMV and other narcissus viruses. © 2017 The Society for Applied Microbiology

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