Detection of Cucumber green mottle mosaic virus in low-concentration virus-infected seeds by improved one-step pre-amplification RT-qPCR.

BACKGROUND: Seeds were an important medium for long-distance transmission of plant viruses. Therefore, appropriate, more sensitive methods for detecting low concentrations of virus-infected in seeds were crucial to ensure the quality of seed lots. In this study, we have developed a one-step pre-amplification reverse transcription quantitative PCR (RT-qPCR) assay based on the TaqMan technology to detect Cucumber green mottle mosaic virus (CGMMV) in zucchini seeds. RESULT: Seed powder samples with simulated CGMMV-infected at a low concentration were prepared (the mass ratio 1:900 and 1:1000), and their uniformity were verified using one-step pre-amplification RT-qPCR. We used one-step pre-amplification RT-qPCR to detect CGMMV in low-concentration virus-infected seeds and compared this method with universal RT-qPCR and double antibody sandwich-enzyme-linked immunosorbent (DAS-ELISA) assay, the main methods used for virus detection in seeds. The minimum limit of detection (LOD) of the improved one-step pre-amplification RT-qPCR assays for simulated CGMMV-infected seeds in large lots seeds samples were 0.1%. CONCLUSIONS: One-step pre-amplification RT-qPCR assays could reliably and stably detected a single CGMMV-infected seed in 1000 seeds and demonstrated a higher detection sensitivity than universal RT-qPCR (infected seeds versus healthy seeds 1:900) and DAS-ELISA assay (infected seeds versus healthy seeds 1:500). Our improved one-step pre-amplification RT-qPCR assay have proved to be very suitable for the analysis of large seed lots.

Detection of Escherichia coli O157:H7 by multiplex PCR-DHPLC / 中华微生物学和免疫学杂志

Objective To develop the PCR-denaturing high performance liquid chromatography (DHPLC) for detection of E. Coli O157: H7. Methods The virulence genes of Shiga-like toxin(SLT) and rfbE were specifically amplified by 2 sets of primers. The target gene fragments of the PCR assay were 224 bp and 499 bp, respectively. Results Analysis of 37 strains demonstrated that this PCR system was specific. The detection limit of the PCR was 4 CFU/ml. Conclusion These results indicated that the multiplex PCR-DHPLC assay can be used for specific and sensitive detection of E. Coli O157:H7.

Detection and identification of viable Listeria monocytogenes by real-time PCR / 中华微生物学和免疫学杂志

Objective To establish a rapid,sensitive and specific assay based on real-time PCR combined with reverse transcription for detecting and identifying viable Listeria monocytogenes.Methods The hlyA gene of Listeria monocytogenes was chosen as target,and then the primers and TaqMan probe were designed.Both ends of probe were modified with two different fluorescence groups.The PCR reaction was optimized systematically.The mRNA of Listeria monocytogenes was extracted,and then reverse transcription was performed through random primer.The cDNA Was detected by real-time PCR.Then the specificity,sensitivity and reproducibility of real-time PCR were estimated.In final,real-time PCR was applied to detect 20 mocked double-blind samplea.Results Viable Listeria monocytogenes were detected by real-time PCR accurately and quickly,and meanwhile,none of other bacteria and non-viable Listeria monocytogenes could be identified.The sensitivity was 10 CFU/ml in pure culture and 103CFU/ml for mocked samples respectively.The coefficient of variation of intra-assay and inter-assay Was less than 5%.When this assay was applied directly to identify 20 mocked double-blind samples,10 of these were positive to viable Listeria monocytogenes,5 were negative to non-viable Listeria monocytogenes,and 5 were negative to other pathogens.Conclusion It is demonstrated that real-time PCR is a reliable,accurate and feasible assay for viable Listeria monocytogenes.The establishment of this assay provided complete data for analysis and diagnosis in the field of food safety and epidemiologic survey.