Primer combination, kit and detection method for nucleic acid mass spectrometry detection of multiple variants of novel coronavirus
A coronavirus and nucleic acid mass spectrometry technology, which is applied in the field of nucleic acid mass spectrometry detection of multiple mutant strains of the new coronavirus, can solve the problems of inability to quickly give virus detection results, ineffective typing, and high cost of sample detection, so as to reduce primer synthesis cost, improved detection and identification, and the effect of reducing false positive results
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Embodiment 1
[0048] Example 1 Design of amplification primers and quality probe extension primers
[0049] Download the full sequences of 4 new coronavirus variant strains from NCBI, download 5-10 strains each, and use the BioEditsequence Alignment Editor software to perform sequence alignment, mainly comparing key genes, such as N, ORF1ab, S of SARS-CoV-2 and so on, select the target genes that are conserved within species and specific between species for detection (see Table 3), and select human RNaseP as an internal reference. Multiplex PCR primer design was performed using the Primer3 online web page. The adjacent sites share a pair of PCR primers. The specific amplification primers are shown in Table 1 above. Mutation sites were selected to be detected, and the genotyping system software (Rongzhi Biotechnology (Qingdao) Co., Ltd.) was used to design mass probe extension (MPE) primers, as shown in Table 2 above. In particular, for the detection of Beta variant strains, a new primer ...
Embodiment 2
[0052] Embodiment 2 Detection method is established
[0054] The plasmid dry powder obtained in Example 1 was diluted with water to a concentration of 100 ng / μL and accurately quantified with Nanodrop. The number of copies contained in the plasmid was calculated based on the plasmid sequence. A variety of concentrations were used in the experiments, 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL, 10 0 copy / μL.
[0055] 2. Primer dilution and mixing.
[0056] Configure PCR primer mixture: After the PCR primer sequences are sent to biosynthesis, use water to dissolve the dry powder into a 100 μM storage solution. The stock solution was taken out and mixed, and prepared into a primer mixture with a final concentration of 0.5 μM-5 μM of primers for each site.
[0057] Configure the MPE primer mixture: After sending the mass probe extension primer sequence to biosynthesis, use water to dissolve the dry powder int...
Embodiment 3
[0090] Example 3 Specificity Experiment
[0091] 1. Primer combination template-free amplification to verify primer specificity. First, use water to replace the sample template. See Example 2 for the entire experimental process from running RT-PCR reaction to resin desalting and purification. This step is to verify that there is no dimer between multiplex PCR amplification primers and multiplex MPE primers, and no non-specific amplification. Extend the product to ensure the specificity of primer design. Experimental results see figure 1 .
[0092] 2. Verify primer specificity with other pathogens. Common causes of respiratory tract infections are other viruses (such as influenza A, influenza B, etc.), bacteria Mycoplasma pneumoniae, chlamydia and so on. In this experiment, Flu A samples, ADV samples, and Mycoplasma pneumoniae were used to verify that no new coronavirus targets were detected, thus ensuring that other pathogenic microorganisms would not report false test res...
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