Food
authenticity is a critical issue associated with the economy,
religion, and food safety. Herein, we report a label-free and colorimetric
nucleic acid assay for detecting DNA barcodes, enabling the determination
of food authenticity with the naked eye. This method, termed the CRISPR-based colorimetric DNA
barcoding (Cricba) assay, utilizes CRISPR/Cas12a (CRISPR = clustered
regularly interspaced short palindromic repeats; Cas = CRISPR associated
protein) to specifically recognize the polymerase chain reaction (PCR)
products for further trans-cleavaging the peroxidase-mimicking
G-quadruplex DNAzyme. Based on this principle, the presence of the
cytochrome oxidase subunit I gene could be directly observed with
the naked eye via the color change of 3,3′,5,5′-tetramethylbenzidine
sulfate (TMB). The whole detection process, including PCR amplification
and TMB colorimetric analysis, can be completed within 90 min. The
proposed assay can detect pufferfish concentrations diluted to 0.1%
(w/w) in a raw pufferfish mixture, making it one of the most sensitive
methods for food authenticity. The robustness of the assay was verified
by testing four common species of pufferfish, including Lagocephalus inermis, Lagocephalus
spadiceus, Takifugu bimaculatus, and Takifugu alboplumbeus. The assay
is advantageous in easy signal readout, high sensitivity, and general
applicability and thus could be a competitive candidate for food authenticity.
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.
Graphical Abstract
Background
Seeds are an important medium for long-distance transmission of plant viruses; therefore, appropriate, sensitive methods for detecting low concentrations of virus in seeds are crucial to ensure the quality of seed lots. In the present study, we 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 virus infection at a low concentration were prepared (the mass ratio 1:900 and 1:1000), and their uniformity was 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 double antibody sandwich–enzyme-linked immunosorbent assay and universal RT-qPCR, which are the main methods used for virus detection in seeds. The minimum limit of detection (LOD) of the improved one-step pre-amplification RT-qPCR method for simulated CGMMV-infected seeds in a large sample of seeds is 0.1%.
Conclusions
It can reliably and stably detect a single virus-infected seed in 1000 seeds and demonstrates a higher detection sensitivity than universal RT-qPCR and double antibody sandwich–enzyme-linked immunosorbent assay. Our improved one-step pre-amplification RT-qPCR assay has proved to be very suitable for the analysis of large seed lots.
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