Primers for rapidly detecting fragmented new coronavirus S gene point mutation and application

A fragmentation and point mutation technology, applied in the fields of biochemistry and molecular biology, can solve problems such as difficult detection and severe fragmentation

Pending Publication Date: 2021-07-30
JIANGSU OCEAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Since the Taqman probe needs to be designed between two primers, the length of the template required by the probe method for fluorescent quantitative PCR is generally not less than 100nt. If the viral nucleic acid is damaged during extraction and storage, and the degree of fragmentation is severe, it is difficult to detect

Method used

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  • Primers for rapidly detecting fragmented new coronavirus S gene point mutation and application
  • Primers for rapidly detecting fragmented new coronavirus S gene point mutation and application
  • Primers for rapidly detecting fragmented new coronavirus S gene point mutation and application

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Detect the new coronavirus S gene template with a length of only 50nt.

[0031] 1. Operation steps:

[0032] (1) The recombinant plasmid containing the D614G mutation target fragment of the new coronavirus S gene was transcribed in vitro to obtain a 50nt RNA fragment containing the D614G point mutation, which was used to simulate fragmented viral RNA.

[0033] (2) Use the RNA template obtained in (1) and the primer pair LDR-1 / LDR2 to prepare an LDR reaction mixture; set the 50nt wild-type S gene RNA obtained by the same method as in (1) as a control, and perform LDR reactions respectively. LDR reaction mixture includes: thermostable DNA ligase, RNA template, LDR primer pair, 10X reaction buffer and deionized water. The addition amount of each component in the LDR reaction mixture is shown in Table 2. Then start the LDR reaction, the reaction program is: ① 95°C for 3 minutes, ② 95°C for 30 seconds, ③ 58°C for 4 minutes. Steps ② and ③ were repeated for 35 cycles, and t...

Embodiment 2

[0042] The template length is smaller than the primer set, and the mutation can still be detected:

[0043] 1. Operation steps:

[0044] (1) Use different primers to transcribe the recombinant plasmid containing the target fragment of the new coronavirus S gene in vitro, and obtain different RNA fragments containing the D614G point mutation with a length of 50 nt, simulating the fragmented viral RNA that does not completely match the probe region of the LDR primer ( figure 2 ).

[0045] (2) Using the RNA obtained in (1) as a template, prepare an LDR reaction mixture with the primer pair LDR-1 / LDR2, and use 50nt wild-type S gene RNA as a control to perform LDR reactions respectively. The LDR reaction system and procedures are the same as Table 2 and Table 3.

[0046] (3) Using primers LDR-UF and LDR-UR to perform fluorescence quantitative PCR reaction detection on the reaction product of step (2).

[0047] 2. Result analysis:

[0048] The experimental results are attached...

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Abstract

The invention discloses a group of primers for rapidly detecting fragmented new coronavirus S gene point mutation and application. The primers comprise a group of LDR primers LDR-1 and LDR-2 and a group of PCR (polymerase chain reaction) primers LDR-UF and LDR-UR, and each primer is a single-stranded DNA (deoxyribonucleic acid) molecule. According to the present invention, the ligase detection reaction and the polymerase chain reaction are combined, such that the point mutation contained in the highly degraded fragmented RNA with the length as low as 40 nt can be detected while the existing reverse transcription-fluorescent quantitative PCR technology can only detect the RNA template with the length of 100 nt or more.

Description

technical field [0001] The invention relates to the fields of biochemistry and molecular biology, in particular to specific primers and a detection method for rapid detection of point mutations in the fragmented SARS-CoV-2 S gene. Background technique [0002] The disease (COVID-19) caused by the new coronavirus (SARS-CoV-2) is a major public health emergency with the fastest spread, the widest range of infection, and the most difficult prevention and control since the founding of New China. It is also recognized by the World Health Organization. global pandemic of infectious diseases. [0003] The new coronavirus is a single-stranded positive-sense RNA virus, which is very prone to mutations during the replication process. At present, researchers have released nearly 80,000 new coronavirus gene sequences. Although the vast majority of gene mutations will not affect the toxicity and infectivity of the virus, it is reported that the D614G mutation of the S protein of the new...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6858C12N15/11
CPCC12Q1/701C12Q1/6858C12Q2531/137C12Q2531/113C12Q2563/107C12Q2521/501Y02A50/30
Inventor 罗志丹许恒皓张新亚张建陈晓雨卢辰
Owner JIANGSU OCEAN UNIV
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