A nucleic acid extraction releasing agent for multiplex pathogenic microorganism detection and a method of use thereof

Abstract

This invention provides a nucleic acid extraction and release agent for the detection of multiple pathogenic microorganisms, comprising the following components: Tween 20, proteinase K, EDTA, and KOH. The rapid nucleic acid extraction and release agent of this invention, after incubation with the sample, completes the nucleic acid extraction and release process, improving extraction efficiency, reducing reagent and sample usage, simplifying operation steps, and lowering labor costs. It has advantages such as high efficiency, low cost, and high quality.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a nucleic acid extraction and release agent and its application method. Background Technology

[0002] Nucleic acid extraction is an essential step before nucleic acid detection, and the efficiency of nucleic acid template extraction and purification directly affects the sensitivity and accuracy of the analysis. Since molecular diagnostic techniques, primarily RT-PCR, require the participation of one or more enzymes, they are highly sensitive to various substances in actual samples that inhibit enzymatic amplification. Careful sample processing, repeated sample loading and centrifugation, and removal of inhibitors are typically necessary to complete nucleic acid extraction, obtain purified nucleic acid for testing, and proceed to the amplification and detection step. This complex sample processing makes it difficult to deploy nucleic acid detection methods in locations with relatively scarce experimental resources, such as airports, clinics, and centralized quarantine sites, hindering rapid diagnosis and timely prevention of virus transmission.

[0003] Conventional nucleic acid extraction methods, primarily magnetic bead extraction and column extraction, involve numerous steps, are time-consuming, and are prone to cross-contamination, posing challenges to the research and integration of in-situ detection technologies for multiple pathogens. Furthermore, the genetic material of some pathogens is RNA, which is more easily degraded than DNA, placing higher demands on nucleic acid extraction. Summary of the Invention

[0004] To overcome the problems of the prior art, the present invention provides a nucleic acid extraction and release agent and its application method for rapid on-site detection of multiple pathogenic microorganisms.

[0005] This invention provides the following technical solutions: A nucleic acid extraction and release agent for the detection of multiple pathogenic microorganisms comprises the following components: Tween 20, proteinase K, EDTA, and KOH, wherein the volume content of Tween 20 is 0.5% to 1.5%, the concentration of proteinase K is 1 mg / mL to 4 mg / mL, the concentration of EDTA is 1 mM to 10 mM, and the concentration of KOH is 0.001 mg / mL to 10 mg / mL.

[0006] Furthermore, it also includes the following ingredients: Tween 20, proteinase K, EDTA, KOH, sodium dodecyl sulfate, trehalose, and guanidine isothiocyanate.

[0007] Furthermore, the Tween 20 contains 0.5%–1.5% by volume, the proteinase K concentration is 1 mg / mL–4 mg / mL, the EDTA concentration is 1 mM–10 mM, the KOH concentration is 0.001 mg / mL–10 mg / mL, the sodium dodecyl sulfate concentration is 0.5%–1.5% by volume, the trehalose concentration is 0.1 mM–0.5 mM, and the guanidine isothiocyanate concentration is 5 mM–50 mM.

[0008] It is applied to the release of DNA or RNA genetic material from ambient air or water samples. The processed samples can be directly used for real-time quantitative PCR detection.

[0009] A method for using a nucleic acid extraction and release agent for the detection of multiple pathogenic microorganisms includes the following steps: 1) The nucleic acid release agent is mixed with the sample at a ratio of 1:1 to 3; 2) Nucleic acid release: Incubate the mixture at 25℃~90℃ for 5~10 min; 3) Take 10 μL of the above mixture and add it to the RT-PCR or RPA reaction system for amplification and detection.

[0010] By adopting the above technical solution, the present invention has the following beneficial effects: 1. The rapid nucleic acid release agent for multiple pathogenic microorganisms of the present invention can complete the nucleic acid release process of the sample simply by mixing it with the sample and incubating it. This improves the extraction efficiency, reduces the amount of reagents and samples used, simplifies the operation steps, reduces labor costs, and eliminates the dependence on special equipment. It has the advantages of high efficiency, low cost, and high quality.

[0011] 2. The rapid nucleic acid release agent of this invention can be used for the release of RNA from cells and viruses while protecting the RNA from degradation. Samples treated with the rapid nucleic acid release agent of this invention do not require further purification and can be directly used for RT-PCR detection or RPA isothermal rapid detection. Attached Figure Description

[0012] Figure 1 This is the RT-PCR amplification curve of the sample treated with the rapid nucleic acid extraction and release agent used for multiple pathogen detection in Example 1.

[0013] Figure 2 This is the RPA amplification curve of the sample treated with the rapid nucleic acid extraction and release agent used for multiple pathogen detection in Example 2. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the structural diagrams and specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0015] Example 1 This invention provides a nucleic acid extraction and release agent for the detection of multiple pathogenic microorganisms, characterized in that it comprises the following components: Tween 20, proteinase K, EDTA, and KOH, wherein the volume content of Tween 20 is 0.5% to 1.5%, the concentration of proteinase K is 1 mg / mL to 4 mg / mL, the concentration of EDTA is 1 mM to 10 mM, and the concentration of KOH is 0.001 mg / mL to 10 mg / mL.

[0016] It also includes the following components: Tween 20, proteinase K, EDTA, KOH, sodium dodecyl sulfate, trehalose, and guanidine isothiocyanate. Specifically, Tween 20 comprises 0.5%–1.5% by volume, proteinase K comprises 1 mg / mL–4 mg / mL, EDTA comprises 1 mM–10 mM, KOH comprises 0.001 mg / mL–10 mg / mL, sodium dodecyl sulfate comprises 0.5%–1.5% by volume, trehalose comprises 0.1 mM–0.5 mM, and guanidine isothiocyanate comprises 5 mM–50 mM.

[0017] The nucleic acid extraction and release agent of this invention for the detection of multiple pathogenic microorganisms uses a certain concentration of surfactant and strong alkali to destroy the cell membrane or viral envelope. Triton X-100 can prevent RNA molecule degradation in an alkaline environment. Bovine serum albumin acts as an enzyme stabilizer to ensure amplification efficiency. Sucrose is used to maintain the osmotic pressure inside and outside the cell, playing a role in nucleic acid protection.

[0018] It is applied to the release of DNA or RNA genetic material from ambient air or water samples. The processed samples can be directly used for real-time quantitative PCR detection.

[0019] 5. A method for using a nucleic acid extraction and release agent for the detection of multiple pathogenic microorganisms, comprising the following steps: 1) The nucleic acid release agent is mixed with the sample at a ratio of 1:1 to 3; 2) Nucleic acid release: Incubate the mixture at 25℃~90℃ for 5~10 min; 3) Take 10 μL of the above mixture and add it to the RT-PCR or RPA reaction system for amplification and detection.

[0020] The rapid nucleic acid extraction and release agent of the present invention can achieve rapid release of cellular or viral RNA without affecting the subsequent amplification and detection process. The processed sample does not require further extraction and purification and can be directly added to the reaction system for RT-PCR detection or RPA isothermal rapid detection. Example 2

[0021] Samples treated with a rapid nucleic acid release agent for multiple pathogen detection were subjected to RT-PCR detection of the GAPDH housekeeping gene.

[0022] The samples used were six negative pharyngeal swab samples for COVID-19 nucleic acid collected at Shijiazhuang Zhengding Airport between April 13, 2021 and May 3, 2021. The negative control was nuclease-free water.

[0023] The rapid nucleic acid release agent used includes the surfactant Surfactin, sucrose, Triton X-100, bovine serum albumin, and NaOH. The concentration of Surfactin is 5 mg / mL, the concentration of sucrose is 80 mg / mL, the volume content of Triton X-100 is 2%, the concentration of bovine serum albumin is 40 mg / mL, and the pH value is 9.5.

[0024] A method for preparing a rapid nucleic acid extraction and release agent includes: (1) Add Tween 20, proteinase K, EDTA and KOH, wherein Tween 20 is 0.5% to 1.5% by volume, proteinase K is 1 mg / mL to 4 mg / mL, EDTA is 1 mM to 10 mM and KOH is 0.001 mg / mL to 10 mg / mL, and add nuclease-free water to 0.9 times the total volume.

[0025] (2) Add 40 mg / mL NaOH solution to adjust the pH value to 9.5, and add nuclease-free water to the full volume.

[0026] Methods for RT-PCR detection using rapid nucleic acid release agents include: (1) Mix the sample to be tested with the rapid nucleic acid release agent thoroughly, with a mixing ratio of 1:1 between the nucleic acid release agent and the sample.

[0027] (2) The mixture of sample and nucleic acid release agent is incubated at 95°C for 10 min to complete nucleic acid release.

[0028] (3) Take 5 μL of the above mixture and add it to the RT-PCR reaction system for amplification and detection.

[0029] In addition to the mixture described above, the RT-PCR reaction system also contains the following components: 2×RT-PCR Buffer 12.5μL 10 μM upstream primer 1 μL 10 μM downstream primer 1 μL 0.3 μL of 10 μM fluorescent probe 25×RT-PCR Enzyme Mix 1μL 4.2 μL of nuclease-free water The 2×RT-PCR Buffer and 25×RT-PCR Enzyme Mix were from ABI's AgPath-IDTMOne-Step RT-PCR Kit. The upstream primer, downstream primer, and fluorescent probe were all designed in-house and synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0030] The reaction procedure for RT-PCR detection is as follows: The reaction was carried out at 45℃ for 10 min; at 95℃ for 10 min; at 95℃ for 15 s; at 60℃ for 45 s, and the cycle was repeated 45 times. Fluorescence was read at 60℃.

[0031] The results are as follows Figure 1 As shown, all samples treated with the rapid nucleic acid extraction and release agent tested positive for the GAPDH housekeeping gene, indicating that the nucleic acid was effectively released and not degraded. The negative control showed no amplification signal. The results were 100% consistent with those obtained by performing nucleic acid extraction followed by detection. Example 3

[0032] Samples treated with a nucleic acid extraction and release agent used for the detection of multiple pathogens were used to perform rapid RPA isothermal detection of norovirus GII.

[0033] The samples used were norovirus GII type pseudovirus standards, diluted 7 times at different concentrations, and the negative control was nuclease-free water.

[0034] The rapid nucleic acid release agent used includes the surfactant Surfactin, sucrose, Triton X-100, bovine serum albumin, and NaOH. The concentration of Surfactin is 1 mg / mL, the concentration of sucrose is 10 mg / mL, the volume content of Triton X-100 is 1%, the concentration of bovine serum albumin is 5 mg / mL, and the pH value is 9.5.

[0035] A method for preparing a nucleic acid extraction and release agent for the detection of multiple pathogenic microorganisms includes: (1) Mix Tween 20, proteinase K, EDTA and KOH, wherein Tween 20 is 0.5% to 1.5% by volume, proteinase K is 1 mg / mL to 4 mg / mL, EDTA is 1 mM to 10 mM and KOH is 0.001 mg / mL to 10 mg / mL, and then add nuclease-free water to 0.9 times the total volume.

[0036] (2) Add 40 mg / mL NaOH solution to adjust the pH value to 9.5, and add nuclease-free water to the full volume.

[0037] Methods for RT-PCR detection using rapid nucleic acid extraction and release reagents include: (1) Mix the sample to be tested with the rapid nucleic acid release agent thoroughly. The mixing ratio of the nucleic acid release agent to the sample is 3:5.

[0038] (2) The mixture of sample and nucleic acid release agent is incubated at 95°C for 10 min to complete nucleic acid release.

[0039] (3) Take 5 μL of the above mixture and add it to the RPA reaction system for isothermal rapid amplification detection.

[0040] In addition to the mixture mentioned above, the RPA reaction system also contains the following components: Freeze-dried enzyme powder A Buffer 29.4μL 10 μM upstream primer 2 μL 10 μM downstream primer 2 μL 0.6 μL of 10 μM fluorescent probe B Buffer 2.5μL 8.5 μL of nuclease-free water The lyophilized enzyme powder, Buffer A, and Buffer B were obtained from the RNA isothermal rapid amplification kit (fluorescent type) from Anpu Future Biotechnology Co., Ltd. The upstream primer, downstream primer, and fluorescent probe were all designed in-house and synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0041] The reaction procedure for rapid isothermal detection of RPA is as follows: react at 42℃ for 20 min, and read the fluorescence every 30 s.

[0042] The results are as follows Figure 2 As shown, samples treated with the rapid nucleic acid release agent of this invention all tested positive for norovirus GII nucleic acid, indicating that the nucleic acid was effectively released and not degraded, while the negative control showed no amplification signal. The detection results were 100% consistent with those obtained by extraction followed by detection.

[0043] Furthermore, it is shown that the rapid nucleic acid release agent of the present invention can be used not only for the detection of intracellular RNA, but also for the detection of viral RNA.

[0044] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A nucleic acid extraction and release agent for the detection of multiple pathogenic microorganisms, characterized in that, It includes the following components: Tween 20, proteinase K, EDTA, and KOH, wherein the volume content of Tween 20 is 0.5% to 1.5%, the concentration of proteinase K is 1 mg / mL to 4 mg / mL, the concentration of EDTA is 1 mM to 10 mM, and the concentration of KOH is 0.001 mg / mL to 10 mg / mL.

2. The nucleic acid extraction and release agent according to claim 1, characterized in that, It also includes the following ingredients: Tween 20, proteinase K, EDTA, KOH, sodium dodecyl sulfate, trehalose, and guanidine isothiocyanate.

3. The nucleic acid extraction and release agent according to claim 1, characterized in that, The components included Tween 20 (by volume) of 0.5%–1.5%, proteinase K (by volume) of 1 mg / mL–4 mg / mL, EDTA (by volume) of 1 mM–10 mM, KOH (by volume) of 0.001 mg / mL–10 mg / mL, sodium dodecyl sulfate (by volume) of 0.5%–1.5%, trehalose (by volume) of 0.1 mM–0.5 mM, and guanidine isothiocyanate (by volume) of 5 mM–50 mM.

4. The nucleic acid extraction and release agent according to any one of claims 1 to 3, characterized in that, It is applied to the release of DNA or RNA genetic material from ambient air or water samples. The processed samples can be directly used for real-time quantitative PCR detection.

5. A method of using the nucleic acid extraction and release agent for the detection of multiple pathogenic microorganisms according to claim 4, characterized in that, Includes the following steps: 1) The nucleic acid release agent is mixed with the sample at a ratio of 1:1 to 3; 2) Nucleic acid release: Incubate the mixture at 25℃~90℃ for 5~10 min; 3) Take 10 μL of the above mixture and add it to the RT-PCR or RPA reaction system for amplification and detection.

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