Primer composition for detecting variation sites of novel coronavirus
A primer composition and mutation site technology, which is applied in the field of molecular biology detection, can solve the problems of difficulty in qualitative diagnosis and identification of single-gene mutations
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Embodiment 1
[0083] Example 1: Primer composition design
[0084] According to SARS-CoV-2, NCBI (National Center for Biotechnology information) accession number: ON373214 variant site of the S protein. Abundance amplification primers and single base extension primers are preferably designed for G339D, S371L, S373P, S375F, T376A, N440K, Q498R, Y505H, N764K, D796Y, G954K, N969K, and the following primer sets are specifically selected from several designed primers 1-3.
[0085] Specifically, primer set 1, abundance amplification primers: SEQ ID NOs: 1 to 6. Single base extension primers: SEQ ID NOs: 7 to 18. Primer Set 2, Abundance Amplification Primers: SEQ ID NOs: 19 to 24. Single base extension primers: SEQ ID NOs: 25-36. Primer Set 3, Abundance Amplification Primers: SEQ ID NOs: 37 to 42. Single base extension primers: SEQ ID NOs: 43 to 54.
[0086] Wherein, the specific sequences of SEQ ID NOs: 1 to 18 are as follows:
[0087] SEQ ID NO: 1--ATTGCCACTAGTCTCTAGTCAGT;
[0088] SEQ I...
Embodiment 2
[0105] Example 2: Detection of specific primers, single base extension reaction combined with MALDI-TOF MS
[0106] 1. Sample source
[0107] 542 samples of suspected new crown cases were nasopharyngeal swabs, and the age distribution of the cases was between 20 and 45 years old.
[0108] Nucleic acid extraction process:
[0109] Use the nucleic acid extraction or purification reagents and automatic nucleic acid extractor of Tianlong Technology Company for nucleic acid extraction. The sample volume of each sample is 200 μL. The extraction method is carried out according to the kit instructions, and the elution volume of viral nucleic acid is about 70 μL; .
[0110] 2. Detection steps
[0111] The first step: performing RT-PCR amplification reaction on the sample to be tested with SEQ ID NOs: 1-6 to obtain amplification products; wherein, the RT-PCR preparation system and amplification procedure are shown in Table 1.
[0112] Table 1: RT-PCR preparation system and amplifica...
Embodiment 3
[0136] Example 3: Traceability effect
[0137] At the beginning of June 2022, the nasopharyngeal swab samples of 1 case in area A, 1 column in area B, and 1 case in area C of a city were subjected to nucleic acid extraction, abundance amplification, dephosphorylation, and single-base extension in the detection steps of Example 2. , point chip, and mass spectrometry, through the detection of 12 specific mutation sites, the analysis is highly consistent with the clustered epidemic mutation sites, indicating that the traceability is successful.
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