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Nucleic acid amplification kit as well as preparation method and application thereof

A kit and amplification reaction technology, which is applied in the field of nucleic acid amplification kits and its preparation, can solve the problems of decreased specificity, achieve normal activity, improve specificity, and unaffected reagent amplification efficiency

Pending Publication Date: 2022-04-29
CHENGDU ONE CHIP BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problem of specificity decline when LAMP and RT-LAMP amplification reagents are prepared into high-concentration freeze-dried microspheres, the first object of the present invention is to provide a nucleic acid amplification kit; the second object of the present invention is to provide The preparation method of the nucleic acid amplification kit; the third purpose of the present invention is to provide the application of the nucleic acid amplification kit in RNA virus detection

Method used

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  • Nucleic acid amplification kit as well as preparation method and application thereof
  • Nucleic acid amplification kit as well as preparation method and application thereof
  • Nucleic acid amplification kit as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0043] This embodiment provides a nucleic acid amplification kit, which includes primer lyophilized microspheres and LAMP amplification reaction reagent lyophilized microspheres.

[0044] Preparation of the LAMP amplification reaction reagent The preparation of the LAMP amplification reaction reagent for lyophilized microspheres is shown in Table 1.

[0045] Table 1: Reagent preparation table for LAMP amplification reaction

[0046] ingredients 5X master mix final concentration 1X use concentration Tris-HCl (mM) 5.00 1.00 KCl(mM) 250.00 50.00 (NH 4 ) 2 SO 4 (mM)

50.00 10.00 MgSO 4 (mM)

30.00 6.00 Tween 20% (m / V) 0.50 0.10 dNTPs (mM) 7.00 1.40 PEG 4K% (m / V) 10.00 2.00 Trehalose%(m / V) 5.00 1.00 Cyclodextrin%(m / V) 0.50 0.10 Bst 3.0 enzyme (U / ul) 1.60 0.32

[0047] Refer to Table 2 for the primer formulation for preparing the primer freeze-dried microspheres.

[0048] Table 2: Prim...

Embodiment 2

[0070] This embodiment provides a nucleic acid amplification kit, which includes primer freeze-dried microspheres and RT-LAMP amplification reaction reagent freeze-dried microspheres.

[0071] Preparation of the RT-LAMP amplification reaction reagent The preparation of the RT-LAMP amplification reaction reagent for lyophilized microspheres is shown in Table 3.

[0072] Table 3: RT-LAMP amplification reaction reagent preparation table

[0073] ingredients 5X master mix final concentration 1X use concentration Tris-HCl (mM) 5.00 1.00 KCl(mM) 250.00 50.00 (NH 4 ) 2 SO 4 (mM)

50.00 10.00 MgSO 4 (mM)

30.00 6.00 Tween 20% (m / V) 0.50 0.10 dNTPs (mM) 7.00 1.40 PEG 4K% (m / V) 10.00 2.00 Trehalose%(m / V) 5.00 1.00 Cyclodextrin%(m / V) 0.50 0.10 RNase Inhibitor (U / ul) 10.00 2.00 Reverse transcriptase (U / ul) 10.00 2.00 Bst 3.0 enzyme (U / ul) 1.60 0.32

[0074] Refer to Table 4 for ...

Embodiment 3

[0100] This embodiment provides a nucleic acid amplification kit, which includes in-situ drying primer solids and RT-LAMP amplification reaction reagent freeze-dried microspheres.

[0101] Preparation of the RT-LAMP amplification reaction reagent The preparation of the RT-LAMP amplification reaction reagent for lyophilized microspheres is shown in Table 5.

[0102] Table 5: Reagent preparation table for RT-LAMP amplification reaction

[0103]

[0104]

[0105] Refer to Table 6 for the primer formulation for preparing the in-situ drying primer solid.

[0106] Table 6: Primer formulation table

[0107] ingredients 8.3X master mix final concentration 1X use concentration Cyclodextrin%(m / V) 1.00 0.12 N gene-FIP(uM) 13.28 1.60 N gene-BIP(uM) 13.28 1.60 N gene-F3(uM) 1.66 0.20 N gene-B3(uM) 1.66 0.20 N gene-LF(uM) 3.32 0.40 N gene-LB(uM) 1.00 0.12

[0108] The nucleotide sequences of the primers are the same as...

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Abstract

The invention provides a nucleic acid amplification kit as well as a preparation method and application thereof. The nucleic acid amplification kit comprises a primer dried solid / freeze-dried microsphere and an LAMP / RT-LAMP amplification reaction reagent freeze-dried microsphere. According to the method, an amplification reagent and a primer are separately and independently treated, when the LAMP / RT-LAMP isothermal amplification reagent is subjected to freeze drying treatment, the primer serves as an independent part, a protective agent is added for vacuum freeze drying or in-situ drying treatment, in the later stage, a redissolving buffer solution is added together with freeze-drying balls containing enzyme and not containing the primer for redissolving, then amplification is conducted, and the LAMP / RT-LAMP isothermal amplification reagent is obtained. The nonspecific phenomenon of the LAMP / RT-LAMP amplification reagent treated by a freeze drying technology can be effectively reduced, the amplification efficiency of the reagent is not influenced, and the activity is not reduced; the treated reagent is stable in storage and normal in activity, and the amplification specificity of the reagent is effectively improved; positive and negative properties can be distinguished in unit time, so that a false positive phenomenon caused by non-specific amplification is avoided.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a nucleic acid amplification kit and its preparation method and application. Background technique [0002] Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technology in vitro. This amplification technique can be performed under stable constant temperature conditions. Compared with ordinary PCR technology, LAMP amplification technology has the advantages of simple operation, rapid detection, and low cost. RT-LAMP is a special LAMP reaction that combines LAMP and reverse transcription to directly detect RNA templates. This technology needs to add reverse transcriptase to the LAMP reaction system, and is widely used in the treatment of new crowns, influenza Detection of RNA viruses. [0003] Freeze-drying technology uses the principle of sublimation of ice crystals to directly sublimate the frozen material moisture into steam in a ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/70
CPCC12Q1/6806C12Q1/701C12Q2527/125C12Q2521/107C12Q2527/127C12Q2531/119
Inventor 常咏吴昊魏富军
Owner CHENGDU ONE CHIP BIOTECHNOLOGY CO LTD
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