Primers and probes for detecting SARS-CoV-2 and application of primers and probes

A coronavirus and probe technology, applied in the field of medical detection, can solve the problems of heavy primer design and screening workload, long treatment cycle, high viral load, etc., achieve good primer-probe combination and reagent specificity, and simple result interpretation Ease of understanding and high detection throughput

Active Publication Date: 2021-12-28
SHENZHEN KANGMEI BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] (2) High viral load and strong infectivity: data show that the viral load in the patient's body is 100 times higher than that of ordinary strains, and the infectivity is twice as high as that of ordinary strains. It takes a long time for nucleic acid to turn negative. long treatment period
Because the primers used by ARMS are primers that can distinguish single alleles from a set of primers that are artificially added to the 3' end and near the 3' end of the sequence, and can distinguish alleles of a single site, this method The workload of primer design and screening is heavy, and the results need to be interpreted by the difference between the amplification curve of the non-variant nucleic acid amplified by the non-mutant primer and the amplification curve of the mutated nucleic acid, and the error is large

Method used

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  • Primers and probes for detecting SARS-CoV-2 and application of primers and probes
  • Primers and probes for detecting SARS-CoV-2 and application of primers and probes
  • Primers and probes for detecting SARS-CoV-2 and application of primers and probes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] Embodiment 1 Novel coronavirus detection method steps

[0119] 1.1 Template preparation

[0120] (1) For the new coronavirus Delta variant, take 200uL directly and use nucleic acid extraction reagents to extract viral RNA on an automatic nucleic acid extraction instrument.

[0121] (2) New coronavirus pseudovirus: adjust the concentration with PBS buffer to obtain 10 4 Copies / mL and 500copies / mL two concentrations, using nucleic acid extraction reagents, extract pseudovirus RNA on a fully automatic nucleic acid extraction instrument.

[0122] (3) Pharyngeal swabs of healthy volunteers and other pathogen-positive specimens or cultures: take 200uL of nucleic acid extraction reagents, and extract nucleic acid from the specimens on an automatic nucleic acid extraction instrument.

[0123] 1.2 System configuration

[0124]

[0125] 1.3 Fluorescent PCR amplification

[0126] (1) Reaction system (30 μL): 20 μL of PCR amplification reaction solution + 10 μL of template D...

Embodiment 2

[0138] Embodiment 2 minimum detection limit test experiment

[0139] Using the pseudovirus extract with a concentration of 500copies / mL as a sensitivity template, according to the reaction procedure of Example 1, carry out amplification detection, repeat 20 reactions, and the reaction results are shown in Table 1:

[0140]

[0141] The results are shown in Table 1. When the pseudovirus concentration was 500 copies / mL, all 20 repeated experiments detected the new coronavirus and mutation sites, indicating that the minimum detection limit of this reagent is 500 copies / mL.

Embodiment 3

[0142] Example 3 Capability Verification Experiment for External Quality Assessment of Nucleic Acid Detection of the New Coronavirus Delta Mutant Strain

[0143] Use the above reagents to detect the extracted nucleic acid of the novel coronavirus Delta variant strain, and the results are shown in Table 2:

[0144]

[0145] The results are shown in Table 2. The detection results of the present invention for the nucleic acid detection of the new coronavirus delta variant fully meet the reference value requirements of the National Health and Medical Commission.

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Abstract

The invention belongs to the field of medical detection, and particularly relates to primers and probes for detecting SARS-CoV-2 and application of the primers and the probes. Four groups of primers and probes are designed, the SARS-CoV-2 is detected by adopting a common TaqMan-MGB probe method, and the primers and probes can be used for detecting a SARS-CoV-2 wild type, a SARS-CoV-2 variant, a SARS-CoV-2 B.1.617 subtype variant and a SARS-CoV-2 B.1.617.2 subtype variant. Pseudovirus test results show that the sensitivity of a reagent can reach 500 copies/mL. No non-specific amplification exists in detection of 10 healthy volunteers and 4 common other respiratory pathogens, and the primer and probe combination and the reagent have good specificity.

Description

technical field [0001] The invention belongs to the field of medical detection, in particular to primers, probes and applications for detecting novel coronaviruses. Background technique [0002] The 2019 novel coronavirus SARS-CoV-2, which causes COVID-19, is the seventh known coronavirus that can infect humans. According to the official website of the World Health Organization, hundreds of variants of the new coronavirus have been discovered around the world. Among them, the new coronavirus Delta variant (B.1.617.2, first discovered in India) has spread to more than 90 countries around the world. The Delta mutant strain may become the mainstream circulating strain in the world. The new coronavirus Delta mutant strain has the following characteristics: [0003] (1) Enhanced transmissibility and short incubation period: The Delta mutant strain has the strongest transmissibility among the several "mutant strains of concern" identified by the WHO, and is 1.23 times the transm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6858C12N15/11
CPCC12Q1/701C12Q1/6858C12Q2600/156C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2561/101C12Q2563/107C12Q2545/101C12Q2545/113Y02A50/30
Inventor 柳辉毕德熙沈亚南窦静雅郑文果毕少辉
Owner SHENZHEN KANGMEI BIOTECH
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