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Reagent for direct fluorescent PCR amplification of oral swab and kit

A kit and swab technology, applied in the field of reagents and kits for direct fluorescent PCR amplification of oral swabs, can solve problems such as high cost and complicated operation, and achieve the effects of ensuring activity, simple operation and protecting activity

Pending Publication Date: 2020-11-20
上海酷乐生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the problem of complicated operation and high cost caused by complex nucleic acid extraction or purification of oral swab samples in the prior art when performing fluorescent PCR amplification on oral swabs, the present invention proposes a Reagents and kits for direct fluorescent PCR amplification from buccal swabs

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  • Reagent for direct fluorescent PCR amplification of oral swab and kit
  • Reagent for direct fluorescent PCR amplification of oral swab and kit
  • Reagent for direct fluorescent PCR amplification of oral swab and kit

Examples

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Embodiment 1

[0039] This embodiment provides a preferred reagent for direct fluorescent PCR amplification of buccal swabs. Extraction or purification treatment, resulting in complex and costly operations.

[0040] A reagent for direct fluorescent PCR amplification of buccal swabs employs the following strategy:

[0041] 1. Oral swab samples contain inhibitors that seriously inhibit the activity of DNA polymerase. In order to achieve direct amplification of oral swab samples without extraction, the following conditions must be met: 1) The DNA Taq enzyme used has sufficient anti-interference ability, while retaining the 5'-3' exonuclease activity of Taq enzyme; 2) The release of nucleic acid from buccal swab must achieve cell lysis, protein denaturation, and release of nucleic acid during the pre-denaturation process before the start of fluorescent PCR; 3) The PCR buffer used must not only ensure the full release of nucleic acid in a slightly alkaline environment, but also provide a strong ...

Embodiment 2

[0052] This embodiment provides a preferred kit for direct fluorescent PCR amplification of oral swabs, including the following components and their final concentrations (as shown in Table 2):

[0053] Table 2 The components of the kit for direct fluorescent PCR amplification of buccal swabs and the final concentration of each component

[0054] components Final concentration hot start DNA polymerase 0.5U / μL oligonucleotide chain 200nM 10×Buffer 1× magnesium chloride 4mM dNPTs 0.3mM SDS 0.6％ Sorbitol 0.8mM DTT 0.6mM NP40 0.6％ Tween20 0.4％ BSA 1 / mL Brij35 0.5％ Mannitol 1.5％ Trehalose 1.8％ Primer 300nM probe 100nM

[0055] Wherein, the applicable pH of the hot-start DNA polymerase is 8.5, and the sequence of the oligonucleotide chain is: CGATCATCTCAGAACATTCTTAGCGTTTTGTTCTTGTGTATGATCG.

[0056] In addition, the kit also includes a positive control substance and...

Embodiment 3

[0058] In this embodiment, taking the detection of folic acid as an example, the kit of Example 2 is used to provide a detection method for clinical oral swabs.

[0059] 1. Experimental equipment and reagents

[0060] The primer probes for folic acid detection were entrusted to Shanghai Yiyue Biotechnology Co., Ltd. to synthesize the primer probes. The fluorescent quantitative PCR instrument was Gentier96. The sequences of the primer sets and probe sets are shown in Table 3 below:

[0061] The sequence information of primer set and probe set used in table 3 folic acid detection

[0062]

[0063] The magnetic bead method used the saliva nucleic acid extraction kit from Shanghai Cooler Biotechnology Co., Ltd., and the fluorescent quantitative PCR instrument was Gentier96.

[0064] 2. Experimental steps

[0065] Step 1, Saliva Sample Processing and Experimental Design

[0066] (1) The determination of the concentration of the oral swab sensitivity sample is to divide the sa...

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Abstract

The invention relates to the field of biotechnology, and discloses a reagent for direct fluorescent PCR amplification of an oral swab and a kit. The reagent for direct fluorescent PCR amplification ofthe oral swab comprises a hot-start DNA polymerase, an oligonucleotide chain and a PCR buffer solution, and the sequence of the oligonucleotide chain is CGATCATCTCAGAACATTCTTAGCGTTTTGTTCTTGTGTATGATCG. The reagent for direct fluorescent PCR amplification of the oral swab and the kit provided by the invention are good in sensitivity and repeatability, and stable in performance; in addition, the oral swab nucleic-acid-extraction-free method is adopted, direct sample loading and amplification are achieved, complex nucleic acid extraction and purification steps are omitted, a large amount of experiment consumables and labor cost are saved, operation is easy, and the reagent and the kit are suitable for large-scale clinical use.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a reagent and kit for direct fluorescent PCR amplification of oral swabs. Background technique [0002] Fluorescent quantitative PCR (also known as FQ-PCR) is a technique developed in the late 1990s. FQ-PCR introduces a specific fluorescent probe, which is homologous to a sequence inside the PCR primer pair. The 5' end of the probe is marked with a reporter fluorescence, while the 3' end is marked with a quencher fluorescence. When the probe remains intact, the emission wavelength emitted by the reporter fluorescence is absorbed by the excitation wavelength of the quenched fluorescence, and cannot be detected at this time. The fluorescent signal of the reporter fluorescence; during the PCR process, the 5'-3'exonucleic acid activity of Taq DNA polymerase can cut the fluorescent probe into a single base, so that the reporter fluorescence is no longer affected by the quenched fluoresce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686
CPCC12Q1/686C12Q2527/125C12Q2563/107
Inventor 刘荣兵徐赛涛李杨
Owner 上海酷乐生物科技有限公司
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