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A primer probe set, detection kit and application thereof for detecting different novel coronavirus mutant strains based on multiplex PCR technology

A primer-probe, technical detection technology, used in recombinant DNA technology, biochemical equipment and methods, and resistance to vector-borne diseases, etc. Detection range, avoidance of false negative results, effect of high sensitivity

Active Publication Date: 2022-04-12
潮州凯普生物化学有限公司 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

T478K is located in the interaction domain between the S protein and the ACE2 receptor. This mutation may affect the affinity of the virus to human cells, which may result in enhanced infectivity

Method used

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  • A primer probe set, detection kit and application thereof for detecting different novel coronavirus mutant strains based on multiplex PCR technology
  • A primer probe set, detection kit and application thereof for detecting different novel coronavirus mutant strains based on multiplex PCR technology
  • A primer probe set, detection kit and application thereof for detecting different novel coronavirus mutant strains based on multiplex PCR technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] A kit for detecting 15 kinds of infectious disease pathogens, which includes: gene chip (containing 22 specific probe combinations and 1 DNA sequence of non-target marker biotin spot), PCR enzyme system, PCR reaction system, positive Control products, negative quality control products.

[0091] 1. The sequences of the primer probes are shown in Table 2. The gene chip includes a nylon membrane and a combination of novel coronavirus-specific probes fixed on the nylon membrane, internal reference probes and chromogenic system control probes. The gene chip includes a probe for Position markers for positioning probes. Note: The 5' ends of the primers are all labeled with biotin.

[0092] Table 2 The information of primers and probes corresponding to the detection of each mutation site of S gene

[0093]

[0094]

[0095]

[0096] 2. Preparation of Pseudovirus

[0097] Plasmids containing the S gene, ORF1ab and N gene target fragments of each mutation site were sy...

Embodiment 2

[0111] Utilize the method for testing sample detection in the detection kit in embodiment 1

[0112] (1) Extraction of nucleic acid from samples to be tested

[0113] This kit is used together with nucleic acid extraction or purification reagents (product record number: Yuechao Machinery No. 20210003) to extract samples to be tested and quality control products to obtain nucleic acid extracts.

[0114] (2) PCR amplification

[0115] The primers in Example 1 were used to perform PCR amplification on the sample to be tested. The PCR reaction solution per person was 43 μL, the PCR enzyme system was 2 μL per person, the DNA sample volume was 5 μL, and the total reaction volume per person was 50 μL. The specific reaction system of PCR amplification is shown in Table 3.

[0116] Table 3 PCR reaction system per person

[0117]

[0118] The RT-PCR amplification program was: reverse transcription at 50°C for 15 min, hot start at 95°C for 2 min, denaturation at 95°C for 30 sec, an...

Embodiment 3

[0123] Detection limit test of SARS-CoV-2 ORF1ab, N gene and 11 S protein mutation site detection kits

[0124] SARS-CoV-2 ORF1ab and N gene pseudovirus, S gene wild-type pseudovirus, S gene HV69-70del site pseudovirus, S gene N501Y site pseudovirus, S gene D614G site pseudovirus, S gene E484K Site pseudovirus, S gene E484Q site pseudovirus, S gene K417N site pseudovirus, S gene K417T site pseudovirus, S gene L452R site pseudovirus, S gene T478K site pseudovirus, S gene P681H site pseudovirus Pseudovirus, P681R site pseudovirus of S gene, and internal reference B2M pseudovirus were used as initial samples and diluted to concentrations of 1000copies / ml, 500copies / ml, and 250copies / ml, respectively, with nucleic acid extraction or purification reagents (product record number: Yuechao Machinery No. 20210003), to verify the detection limit of the kit in Example 1.

[0125] The results show that the detection of different concentrations of pseudoviruses, ORF1ab, N gene and 11 S pr...

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Abstract

The invention provides a primer probe set, a detection kit and an application thereof for detecting different novel coronavirus mutant strains based on multiplex PCR technology, and belongs to the technical field of virus detection. The present invention downloads the sequences of 14 kinds of new coronavirus mutant strains, determines the mutation sites and conserved sequences of the new coronavirus S protein with detection significance, designs multiple amplification primers, and amplifies through single-tube multiple PCR, and hybridizes the amplified products with the probes. The results of the detection of the conservative sequence of the new coronavirus determine whether the tested sample contains the detection of the new coronavirus, and according to the detection results of the mutation site of the S protein, it is judged as a mutant strain of the new coronavirus. The invention has wide detection range, strong specificity, high detection sensitivity and strong anti-interference ability, and can effectively evaluate the dissemination, pathogenicity and whether there is immune escape of the SARS-CoV-2 infected by the subject, and also helps It is used to analyze and study the mutation law of SARS-CoV-2, and inspire new ideas for epidemic control and re-epidemic prevention.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and in particular relates to a primer probe set, a detection kit and an application thereof for detecting different novel coronavirus mutant strains based on multiplex PCR technology. Background technique [0002] Novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) is a single-stranded positive-sense RNA virus with a diameter of 60-140 nm. The virus genome consists of about 29903 nucleotides, encoding four structural proteins: nuclear Protein (nucleocapsid, N), envelope (envelop, E), matrix protein (membrane, M), spike protein (spike, S), and RNA-dependent RNA polymerase (RNAdependent DNA polymerase, RdRp). S protein is an important structural protein, its main function is to promote the conversion of angiotensin between the viral envelope and host cells through the receptor binding domain (receptorbinding domain, RBD) and N-terminal domain (N-terminal RNAbindi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2537/143Y02A50/30
Inventor 卢晓丹李烈军石琳肖新换陈灿钿吴钰熙
Owner 潮州凯普生物化学有限公司
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