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Kit for detecting novel coronavirus by adopting loop-mediated transcription isothermal amplification method

An isothermal amplification and coronavirus technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/testing, can solve problems such as complex operation, high cost, and slow detection speed of new coronaviruses, and achieve High sensitivity, low cost, guaranteed high sensitivity effect

Active Publication Date: 2021-01-29
SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a test kit for detecting novel coronavirus by loop-mediated transcription isothermal amplification method, and the described test kit for detecting novel coronavirus by adopting loop-mediated transcription isothermal amplification method needs to solve the existing The new coronavirus detection speed in technology is slow, the cost is high, and the technical problems of complicated operation

Method used

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  • Kit for detecting novel coronavirus by adopting loop-mediated transcription isothermal amplification method
  • Kit for detecting novel coronavirus by adopting loop-mediated transcription isothermal amplification method
  • Kit for detecting novel coronavirus by adopting loop-mediated transcription isothermal amplification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Primer Design

[0041]The Tm value of the outer primer set should be 55-63°C and the base length should be 15-25. The Tm value of about 20 bases at the 5' end of the internal primer set is higher than that of other primer sets, controlled at 60-68°C, and the Tm value of about 20 bases at the 3' end of the internal primer set is at 55-63°C. The transcription site is between the 5' end and the 3' end of the internal primer set, and the 5' sequence and 3' sequence of the transcription region should be fine-tuned according to the secondary structure of the primer itself, so that the secondary structure of the primer is minimized, and the base length of the entire internal primer set is between Between 50-70. The Tm value of the loop primer set is 60-66°C, and the base length is 15-25. The GC content of all primers is controlled at 30%-65%. It is necessary to ensure the stability of the 5' and 3' ends of the primers and avoid the formation of dimers between the p...

Embodiment 2

[0053] Example 2 is the result of the specific detection of clinical samples using the loop-mediated transcription isothermal amplification detection kit for novel coronavirus

[0054] 1) Sample collection

[0055] Nasopharyngeal swabs were collected from patients, and the specimens were stored at -80°C.

[0056] 2) Extract sample RNA

[0057] The sample RNA was extracted using Qiagen's miRNeasy Mini kit extraction kit.

[0058] 3) LAMP detection

[0059] The novel coronavirus reaction system is 25 μL, and the consumption of each component is: 1mM dNTP mixture, 1mM NTP mixture, 20mM Tris-HCl, 10mM (NH 4 ) 2 8O 4 , 50mM KCl, 0.1% Tween20, 2mM MgSO 4 , 8U Bst DNA polymerase, 8U Mulv reverse transcriptase, 8U RNA polymerase, the concentration of outer primer F3 and outer primer B3 is 0.2μM, the concentration of inner primer FIP-transcibe and BIP-transcibe is 1.6μM, and the concentration of LF and LB is 0.8 μM., MB 0.3μM, add sterilized purified water to 25μL system. After...

Embodiment 3

[0064] Example 3 Sensitivity detection of novel coronavirus kit using loop-mediated transcription isothermal amplification

[0065] Clinical samples were subjected to RNA extraction, and then quantified sequentially into 2.5×10 8 copy / μl, 2.5×10 7 copy / μl, 2.5×10 6 copy / μl, 2.5×10 5 copy / μl, 2.5×10 4 copy / μl, 2.5×10 3 copy / μl, 2.5×10 2 copy / μl and 2.5×10copy / μl, 1μl was added to the 25μl reaction system for amplification, a total of 8 concentration gradient detections were performed, and the detection limits of the corresponding final solutions were 10 10 copy / ml, 10 9 copy / ml, 10 8 copy / ml, 10 7 copy / ml, 10 6 copy / ml, 10 5 copy / ml, 10 4 copy / ml and 10 3 copy / ml. The result is as Figure 4 , it can be seen that the detection limit of the detection kit can reach 1000copy / ml, which has high sensitivity.

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Abstract

The invention discloses a kit for detecting a novel coronavirus by adopting a loop-mediated transcription isothermal amplification method. The kit comprises an upstream external primer F3, a downstream external primer B3, an upstream internal primer FIP-transcribe, a downstream internal primer BIP-transcribe, an upstream cyclization primer LF and an upstream cyclization primer LB. Six specific primers are designed for six regions of a segment in an N gene of a novel coronavirus, an RNA polymerase recognition site is added to an inner primer group to accelerate a reaction, and a specific amplification element is constructed under a constant temperature condition by using strand displacement Bst DNA polymerase, Mulv reverse transcription, heat-resistant RNA polymerase and a special hairpin primer, so nucleic acid index amplification is realized, and high sensitivity and rapidity of amplification are ensured. The kit of the invention is simple and convenient to operate and low in cost, the changes of a fluorescence curve can be observed in real time through a nucleic acid dye, and complex problems encountered in clinical novel coronavirus detection can be well solved.

Description

technical field [0001] The present invention is used in the technical field of biological detection, and relates to a detection technology based on loop-mediated transcription isothermal amplification, specifically a kit for detecting novel coronaviruses using the loop-mediated transcription isothermal amplification method. Background technique [0002] The new coronavirus is the seventh coronavirus that has been discovered so far to infect humans. It belongs to the family Coronaviridae, the genus Coronaviridae, and is a single-stranded RNA virus. Its genome includes 11 coding sequences with a total length of about 30kb, encoding 1 nonstructural protein, 4 structural proteins and 6 auxiliary proteins. Currently, the main methods for detecting novel coronaviruses are immunization methods based on specific antibodies IgG and IgM or virus-specific antigens and RT-PCR nucleic acid detection methods. [0003] Immunity-based detection methods cannot achieve accurate detection due...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2521/119C12Q2521/101C12Q2521/107C12Q2531/119
Inventor 范列英隋国栋赵望张童宗明迟大利程佳训
Owner SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE
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