A confirmation method for samples in the gray zone after nucleic acid amplification
A nucleic acid amplification reaction, confirmatory technology, applied in biomass post-processing, biochemical equipment and methods, biological material pretreatment, etc. Detection process, good specificity, and the effect of avoiding contamination
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Embodiment 1
[0080] Taking the detection of a new coronavirus sample as an example to introduce a specific embodiment, the specific steps are as follows.
[0081] 1) According to the recommendation of China CDC, the gene loci for COVID-19 detection can be ORF or N gene fragments. Take the ORF gene PCR test recommended by CDC as an example.
[0082] If the Ct value of the sample is in the gray area after the PCR amplification, and it is difficult to determine whether it is a positive sample or a negative sample, the device is activated. First prepare the amplification product-specific detection reagent, its components include 0.2 micromolar per liter of specific nucleic acid probe corresponding to the PCR amplification product, 0.2 micromolar per liter of Cas protein, and 2.5 micromolar per liter of fluorescent Labeled single-stranded probes, in order to ensure better reaction, the system also includes 1×NEB buffer, 1U per microliter of RNA inhibitor. After preparation, take 25 microliter...
Embodiment 2
[0087] The specific embodiment is introduced by taking the detection of the transgene promoter CaMV335 gene in transgenic soybean as an example.
[0088] 1) Real-time quantitative PCR detection of the transgenic soybean promoter CaMV335.
[0089] If the Ct value of the sample is in the gray area after the PCR amplification, and it is difficult to determine whether it is a genetically modified soybean, the device is activated. Firstly, the amplification product-specific detection reagent is prepared. The use of Cas protein, fluorescently labeled single-stranded probe, NEB buffer, and RNA inhibitor is the same as in Example 1. The concentration of the specific nucleic acid probe corresponding to the PCR amplification product of the transgenic soybean is the same as that in Example 1.
[0090] 2) Put the PCR sample tube with the Ct value in the gray area after the PCR detection of the transgenic soybean into the PCR test tube nucleic acid amplification reaction tube rack, so th...
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