Novel coronavirus nucleic acid and mutant gene detection kit based on fluorescent quantitative PCR technology and application of kit
A virus nucleic acid and kit technology, applied in the field of new coronavirus nucleic acid and mutant gene detection kits, can solve problems such as the inability to detect ordinary new coronavirus mutant strains
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Embodiment 1
[0043] Embodiment 1 detection kit and its use
[0044] The kit includes the following components:
[0045] PCR reaction solution, enzyme mixture, ORF1ab / N / internal reference / S mutant gene multiple reaction solution, positive control 1, negative control;
[0046] The PCR reaction solution includes 10× buffer, 25mM MgCl2, 10mM dUTP and 10mM dNTPs; the enzyme mixture includes Taq enzyme, reverse transcriptase and UNG enzyme, ORF1ab / N / internal reference / S mutant gene multiple reaction solution upstream and downstream primers, and The ratio of the probes is: 4:4:1; the preferred upstream primer is 400nM, the downstream primer is 400nM, and the probe is 100nM.
[0047] The nucleotide sequences of the primers and probes of the novel coronavirus ORF1ab / N / internal reference / S mutant gene used in this example can be found in Table 1.
[0048] Table 1 Nucleotide sequence list of primers and probes of the new coronavirus ORF1ab / N / internal reference / S mutant gene in the present inventi...
Embodiment 2
[0106] Embodiment 2 detection kit and its use
[0107] The kit includes the following components:
[0108] PCR reaction solution, enzyme mixture, ORF1ab / N / internal reference gene multiple reaction solution, S mutation gene reaction solution, positive control 1, positive control 2, negative control;
[0109] The PCR reaction solution includes 10× buffer, 25mM MgCl2, 10mM dUTP and 10mM dNTPs;
[0110] Enzyme mix includes Taq enzyme, UNG enzyme, reverse transcriptase
[0111] The ratio of upstream and downstream primers and probes of ORF1ab / N / internal reference gene multiplex reaction solution is: 4:4:1; the preferred upstream primer is 400nM, the downstream primer is 400nM, and the probe is 100nM.
[0112] The ratio of upstream, downstream primers and probes in the S mutation gene reaction solution is: 4:4:2; the preferred upstream primer is 400nM, the downstream primer is 400nM, and the probe is 200nM.
[0113] The positive control includes 2 combinations, the components a...
Embodiment 3
[0149] The sensitivity test of embodiment 3 kit
[0150] Positive control reference products are the non-replicating lentivirus of the A23063T mutant sequence of the ORF1ab, N, and S genes of the new coronavirus and the non-replicating adenovirus of part of the human β-actin gene sequence, synthesized by professional institutions; 10-fold gradient dilution to 30 copies per milliliter ( figure 1 Amplification curve with jump in middle right).
[0151] The negative reference product is RNase-free water, 1000ml of RNase-free water, then sterilized in a sterilizing pot at 121°C for 20 minutes, marked and stored at room temperature.
[0152] The kit of the present invention is used for detection. The detection method is the same as in Example 1.
[0153] The test results show that the kit of the present invention has good sensitivity for the detection of the A23063T mutation sequence of ORF1ab, N, and S genes of the new coronavirus, reaching 30 copies per milliliter, and the CT ...
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