Nucleic acid hand-free reagent and application and use method thereof in nucleic acid detection

文档序号：872149 发布日期：2021-03-19 浏览：4次中文

阅读说明：本技术 一种核酸免提取试剂及其在核酸检测中的应用和使用方法 (Nucleic acid hand-free reagent and application and use method thereof in nucleic acid detection ) 是由闫亚平张鑫于 2020-12-09 设计创作，主要内容包括：本发明公开了一种核酸免提取试剂及其在核酸检测中的应用和使用方法,属于核酸检测技术领域,该核酸免提取试剂是由海藻糖、PEG8000、Tris-HCl、EDTA、Triton X-100及核酸释放剂组成的混合液体；本发明通过Triton X-100和核酸释放剂的协同作用,能够高效裂解病原微生物,快速释放其中的DNA或RNA。一方面,通过EDTA能够鳌和金属离子,降低DNase和RNase的酶活,另一方面,通过海藻糖和PEG8000在RNA和DNA表面形成保护层,使其能够在溶液中稳定存在。最后通过Tris稳定该试剂的pH,以确保RNA或DNA均可稳定保存。通过上述组份的协同作用,本发明的核酸免提取试剂能够在室温条件下直接完成核酸的释放,避免了加热、离心、洗涤等繁琐操作,所得产物可以直接用于PCR扩增。(The invention discloses a nucleic acid hands-free reagent and an application and a using method thereof in nucleic acid detection, belonging to the technical field of nucleic acid detection, wherein the nucleic acid hands-free reagent is a mixed liquid consisting of trehalose, PEG8000, Tris-HCl, EDTA, Triton X-100 and a nucleic acid releasing agent; the invention can efficiently crack pathogenic microorganisms and quickly release DNA or RNA in the pathogenic microorganisms through the synergistic action of the Triton X-100 and the nucleic acid releasing agent. On one hand, EDTA can chelate metal ions and reduce the enzyme activity of DNase and RNase, and on the other hand, trehalose and PEG8000 form a protective layer on the surfaces of RNA and DNA, so that the DNA can stably exist in a solution. Finally, the pH of the reagent is stabilized by Tris to ensure that RNA or DNA can be stably stored. Through the synergistic effect of the components, the nucleic acid taking-free reagent can directly complete the release of nucleic acid at room temperature, avoids the complicated operations of heating, centrifuging, washing and the like, and can be directly used for PCR amplification.)

1. A nucleic acid extraction-free reagent is characterized in that the reagent is a mixed liquid consisting of trehalose, PEG8000, Tris-HCl, EDTA, Triton X-100 and a nucleic acid releasing agent;

wherein the concentration of trehalose is 0.1-1M, the concentration of Tris-HCl is 1-10mM, and the concentration of EDTA is 0.1-5 mM; PEG8000 accounts for 0.1-2% of the total mass of the mixed liquid, Triton X-100 accounts for 0.1-1% of the total mass of the mixed liquid, and nucleic acid releasing agent accounts for 0.001-0.1% of the total mass of the mixed liquid.

2. The reagent of claim 1, wherein the pH of the reagent is 6.5-7.5.

3. The reagent for nucleic acid isolation according to claim 1, wherein the reagent for nucleic acid isolation is stably present at-80 ℃ to 100 ℃.

4. The nucleic acid extraction-free reagent according to claim 1, wherein the nucleic acid releasing agent is dodecyl dimethyl benzyl ammonium chloride or dodecyl dimethyl benzyl ammonium bromide.

5. The use method of the nucleic acid non-extraction reagent according to any one of claims 1 to 4, characterized in that the nucleic acid non-extraction reagent is mixed with a sample, placed at room temperature for 1 to 10min, and then 1 to 10 μ l is taken as a template for nucleic acid detection.

6. The method of using the nucleic acid isolation reagent according to claim 5, wherein the nucleic acid detection is a PCR method, a fluorescent quantitative PCR method, a loop-mediated isothermal amplification method, or a recombinase polymerase amplification method.

7. Use of the nucleic acid isolation reagent according to any one of claims 1 to 4 as a nucleic acid detection reagent.

8. The use of claim 7, wherein the nucleic acid detection reagent is used to detect buccal swabs, saliva, serum, cerebrospinal fluid, genital tract secretions or urine.

Technical Field

The invention belongs to the technical field of nucleic acid detection, and relates to a nucleic acid hand-free reagent, and application and a use method thereof in nucleic acid detection.

Background

The PCR-based detection of pathogenic microorganisms has the characteristics of good specificity, high sensitivity, simplicity and rapidness. The quality of nucleic acid extraction of pathogenic microorganisms is the key of all downstream nucleic acid detection, and the quality of the obtained nucleic acid directly influences research or diagnosis results. However, the traditional nucleic acid extraction methods, including column extraction methods, magnetic bead methods, etc., all require very complicated processes such as lysis, multiple washing, elution, etc., which not only takes a long time, but also is prone to cross contamination. Meanwhile, a large amount of nucleic acid is easily lost in the nucleic acid extraction process, and particularly, the nucleic acid loss ratio is higher in the nucleic acid extraction process of a trace sample, and even the omission occurs. When the sample to be detected is RNA virus, the degradation of RNA is also easily caused by a long extraction process. These factors greatly affect the accuracy of nucleic acid detection results.

Chinese patent 200910309980.2 discloses a one-step method for fluorescence quantitative PCR detection of pathogen nucleic acid, which is characterized in that specific primers are designed aiming at housekeeping genes of pathogens to be detected, a nucleic acid quick release agent is adopted to extract the pathogen nucleic acid, and then the pathogen nucleic acid is directly used for PCR amplification to realize PCR fluorescence quantitative detection. However, the nucleic acid releasing agent contains a certain amount of SDS, Surfactin, etc., and has a serious inhibitory effect on PCR amplification.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a nucleic acid extraction-free reagent and an application and a use method thereof in nucleic acid detection.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

the invention discloses a nucleic acid extraction-free reagent, which is a mixed liquid consisting of trehalose, PEG8000, Tris-HCl, EDTA, Triton X-100 and a nucleic acid releasing agent;

Preferably, the pH value of the nucleic acid non-extraction reagent is 6.5-7.5.

Preferably, the nucleic acid extraction reagent is stable at-80 ℃ to 100 ℃.

Preferably, the nucleic acid releasing agent is dodecyl dimethyl benzyl ammonium chloride or dodecyl dimethyl benzyl ammonium bromide.

The invention also discloses a use method of the nucleic acid hands-free reagent, which comprises the steps of uniformly mixing the nucleic acid hands-free reagent with a sample, standing at room temperature for 1-10min, and then taking 1-10 mu l as a template for nucleic acid detection.

Preferably, the nucleic acid detection is a PCR method, a fluorescent quantitative PCR method, a loop-mediated isothermal amplification method or a recombinase polymerase amplification method.

The invention also discloses application of the nucleic acid hand-free reagent as a nucleic acid detection reagent.

Preferably, the nucleic acid detection reagent is used for detecting oral swabs, saliva, serum, cerebrospinal fluid, genital secretion or urine.

Compared with the prior art, the invention has the following beneficial effects:

the nucleic acid non-extraction reagent disclosed by the invention can efficiently crack pathogenic microorganisms and quickly release DNA or RNA in the pathogenic microorganisms through the synergistic action of Triton X-100 and a nucleic acid releasing agent. On one hand, EDTA can chelate metal ions and reduce the enzyme activity of DNase and RNase, and on the other hand, trehalose and PEG8000 form a protective layer on the surfaces of RNA and DNA, so that the DNA can stably exist in a solution. Finally, the pH of the reagent is stabilized by Tris to ensure that RNA or DNA can be stably stored. Through the synergistic effect of the components, the nucleic acid taking-free reagent can directly complete the release of nucleic acid at room temperature, avoids the complicated operations of heating, centrifuging, washing and the like, and can be directly used for PCR amplification.

Furthermore, the nucleic acid non-extraction reagent has high thermal stability and can stably exist under the conditions of low temperature of 80 ℃ below zero and high temperature of 100 ℃.

Further, the nucleic acid releasing agent of the present invention may be dodecyl dimethyl benzyl ammonium chloride (also known as benzalkonium chloride, DDBAC) or dodecyl dimethyl benzyl ammonium bromide (also known as benzalkonium bromide). Under the synergistic effect of Triton X-100 and dodecyl dimethyl benzyl ammonium chloride or dodecyl dimethyl benzyl ammonium bromide, pathogenic microorganisms can be efficiently cracked, and DNA or RNA in the pathogenic microorganisms can be rapidly released.

The detection method of the nucleic acid hands-free reagent disclosed by the invention only needs to uniformly mix the nucleic acid hands-free reagent with a sample, place the mixture at room temperature for 1-10min, and then take 1-10 mul as a template to carry out nucleic acid detection. The traditional extraction of pathogenic microorganism nucleic acid needs complicated purification steps, while the nucleic acid extraction-free reagent of the invention can complete template preparation within 10min at normal temperature. Meanwhile, the method enables the nucleic acid detection of the pathogenic microorganism to be more convenient, rapid and accurate.

The nucleic acid extraction-free reagent has wide applicability, and can be suitable for various samples such as oral swabs, saliva, serum, cerebrospinal fluid, genital secretion, urine and the like.

Drawings

FIG. 1 shows the amplification results after processing high concentration samples by different methods;

FIG. 2 shows the amplification results after different methods for treating low concentrations of Escherichia coli 16S rRNA;

FIG. 3 shows the amplification results after different methods for treating low concentrations of Escherichia coli 16S rRNA (high temperature resistant);

FIG. 4 shows the amplification results after different methods for treating high concentration samples in saliva;

FIG. 5 shows the amplification results after different methods for treating high concentration samples in serum;

FIG. 6 shows the amplification results after treating high concentration samples in cerebrospinal fluid by different methods;

FIG. 7 shows the amplification results after treating high concentration samples in urine by different methods;

FIG. 8 shows the amplification results of Staphylococcus aureus treated by different methods;

FIG. 9 shows the amplification results after different methods for treating Cryptococcus;

FIG. 10 shows the results of amplification after treatment of lentiviruses by different methods;

FIG. 11 LAMP amplification results after treatment of lentiviruses by different methods.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the accompanying drawings:

the nucleic acid hand-free reagent comprises 0.1-1M trehalose, 0.1-2% PEG8000, 1-10mM Tris-HCl (pH6.5-7.5), 0.1-5mM EDTA, 0.1-1% Triton X-100, and 0.001-0.1% nucleic acid releasing agent. The nucleic acid releasing agent may be dodecyl dimethyl benzyl ammonium chloride (also known as benzalkonium chloride) or dodecyl dimethyl benzyl ammonium bromide (also known as benzalkonium bromide).

The nucleic acid releasing agent can act together with Triton X-100, effectively cracks pathogenic microorganisms and releases nucleic acid at an extremely low concentration, has an extremely low working concentration, and has no interference on PCR, RT-PCR, LAMP, RPA and other nucleic acid amplification technologies. Tris-HCl (pH6.5-7.5) can stabilize pH, and EDTA can chelate metal ions to reduce DNase and RNase activity. In addition, trehalose and PEG8000 form a protective layer on the surface of DNA and RNA, and can stabilize DNA and RNA to exist stably in a solution.

The nucleic acid release process does not need operations such as heating, centrifugation and the like, avoids the loss of nucleic acid in the extraction and purification process, and improves the positive rate of the nucleic acid detection result.

Example 1

The nucleic acid extraction reagent comprises: 0.1M trehalose, 2% PEG8000, 5mM Tris-HCl (pH6.5-7.5), 5mM EDTA, 0.5% Triton X-100, 0.001% dodecyl dimethyl benzyl ammonium chloride.

Preparation of a detection sample: repeatedly wiping with oral swab from buccal mucosa at oral cavity inner side for more than 20 times, respectively adding into 1ml of the nucleic acid hands-free reagent of the present invention and 1ml of physiological saline as interfering substances, and respectively adding 0.1ml of 10-concentration solution⁷copy/ml Escherichia coli. Standing at room temperature for 5min, and taking 5 μ l as a detection template.

The reaction system is as follows: 12.5. mu.l of 2 XPCR Mix, 1. mu.l of 10. mu.M primer F, 1. mu.l of 10. mu.M primer R, 1. mu.l of 25 XSSYBR Green I, 4.5. mu.l of ddH₂O, 5. mu.l of cleavage product as template. The reaction conditions are as follows: 1min at 95 ℃; reacting at 95 ℃ for 15s and 55 ℃ for 30s for 35 cycles; at 25 ℃ for 10 s.

Wherein the primer F: CGGATGTGCCCAGATGGGAT (shown in SEQ. ID. NO. 1);

and (3) primer R: GGGTAACGTCAATGAGCAAAGGTATTA (shown in SEQ. ID. NO. 2).

The detection results are shown in FIG. 1, in which A. the amplification results after treatment with the nucleic acid hands-free kit of the present invention, B. the amplification results of the sample after treatment with physiological saline, and C. the negative control. The amplification time of Escherichia coli in the nucleic acid extraction reagent is obviously shortened compared with that of Escherichia coli in physiological saline. Escherichia coli in physiological saline can also be amplified because high temperature of 95 ℃ can denature part of the strains, but the amplification time is significantly later.

Example 2 detection of Escherichia coli 16S rRNA (Low concentration)

The nucleic acid extraction reagent comprises: 1M trehalose, 1% PEG8000, 10mM Tris-HCl (pH6.5-7.5), 2mM EDTA, 0.1% Triton X-100, 0.1% dodecyldimethylbenzyl ammonium bromide.

Preparation of a detection sample: repeatedly wiping with oral swab from buccal mucosa at oral cavity inner side for more than 20 times, respectively adding into 1ml of the nucleic acid hands-free reagent of the present invention and 1ml of physiological saline as interfering substances, and respectively adding 0.1ml of 10-concentration solution⁵copy/ml Escherichia coli. Standing at room temperature for 5min, and taking 1 μ l as a detection template.

The reaction system is as follows: 12.5. mu.l of 2 XPCR Mix, 1. mu.l of 10. mu.M primer F, 1. mu.l of 10. mu.M primer R, 1. mu.l of 25 XSSYBR Green I, 8.5. mu.l of ddH₂O, 1. mu.l of cleavage product was used as template. The reaction conditions are as follows: 1min at 95 ℃; reacting at 95 ℃ for 15s and 55 ℃ for 30s for 40 cycles; at 25 ℃ for 10 s.

Wherein the primer F: CGGATGTGCCCAGATGGGAT (shown in SEQ. ID. NO. 1);

and (3) primer R: GGGTAACGTCAATGAGCAAAGGTATTA (shown in SEQ. ID. NO. 2).

The detection results are shown in FIG. 2, A. the amplification results after treatment with the nucleic acid extraction-free reagent of the present invention, and B. the amplification results of the sample after treatment with physiological saline. The detection result of Escherichia coli placed in the nucleic acid non-extraction reagent is positive, and the detection result of Escherichia coli placed in physiological saline is negative, because Escherichia coli in the reagent can be quickly cracked to release nucleic acid, and Escherichia coli in physiological saline cannot release nucleic acid, and simultaneously, due to the low concentration of a sample, sufficient nucleic acid cannot be released in the heating process.

Example 3 detection of Escherichia coli 16S rRNA (high temperature resistant)

In order to verify the high temperature resistance and repeated freeze-thaw characteristics of the nucleic acid extraction-free reagent, the reagent is placed in a boiling water bath for heating for 20min, then placed at-20 ℃ for 30min, repeated for 10 times, and then the cracking effect of the reagent is verified.

The nucleic acid extraction reagent comprises: 0.5M trehalose, 1% PEG8000, 5mM Tris-HCl (pH6.5-7.5), 5mM EDTA, 1% Triton X-100, 0.05% dodecyl dimethyl benzyl ammonium chloride.

Preparation of a detection sample: repeatedly wiping with oral swab from buccal mucosa of oral cavity for more than 20 times, respectively placing in 1ml of nucleic acid hands-free reagent subjected to 10 times of repeated freeze thawing, neutralizing with 1ml of physiological saline, and adding 0.1ml of 10-concentration interfering substances⁵copy/ml Escherichia coli. Standing at room temperature for 5min, and taking 5 μ l as a detection template.

Wherein the primer F: CGGATGTGCCCAGATGGGAT (shown in SEQ. ID. NO. 1);

and (3) primer R: GGGTAACGTCAATGAGCAAAGGTATTA (shown in SEQ. ID. NO. 2).

The detection results are shown in FIG. 3, wherein A is the amplification result of the nucleic acid extraction-free reagent after repeated heating and freezing, B is the amplification result of the nucleic acid extraction-free reagent without repeated heating and freezing, and C is the amplification result of the sample after normal saline treatment. The detection result of Escherichia coli in the nucleic acid non-extraction reagent is positive, and the detection result of Escherichia coli in physiological saline is negative.

In this example, except that the composition of the nucleic acid extraction-free reagent is different from that in example 2, the nucleic acid extraction-free reagent of the present invention is subjected to 10 times of repeated freeze thawing, which indicates that the reagent has high thermal stability.

In this example, except that the composition of the nucleic acid extraction-free reagent is different from that in example 1, the concentration of Escherichia coli in the sample is only 1% of that in example 1, which shows that the positive rate of the detection result of the low-concentration sample can be significantly improved by using the nucleic acid extraction-free reagent of the present invention.

EXAMPLE 4 different interference samples

This example differs from example 1 in that the type of interfering substance in the sample preparation process was saliva, and the effect of the saline-treated sample was not verified. The results are shown in FIG. 4, A. amplification results after treatment with the nucleic acid removal kit of the present invention, and B. negative control. The detection result after the Escherichia coli is treated by the nucleic acid hands-free reagent is positive, and the negative control is not amplified.

Example 5

The example differs from example 1 in that the kind of interfering substance in the sample preparation process was rat serum, and the effect of the saline-treated sample was not verified. The results are shown in FIG. 5, A. amplification results after treatment with the nucleic acid removal kit of the present invention, and B. negative control. The detection result after the Escherichia coli is treated by the nucleic acid hands-free reagent is positive, and the negative control is not amplified.

Example 6

This example differs from example 1 in that the type of interfering substance in the sample preparation process was rabbit cerebrospinal fluid and the effect of the saline treatment of the samples was not verified. The results are shown in FIG. 6, A. amplification results after treatment with the nucleic acid removal kit of the present invention, and B. negative control. The detection result after the Escherichia coli is treated by the nucleic acid hands-free reagent is positive, and the negative control is not amplified.

Example 7

This example differs from example 1 in that the type of interfering substance in the sample preparation process was urine, and the effect of the saline treatment on the sample was not verified. The results are shown in FIG. 7, A. amplification results after treatment with the nucleic acid removal kit of the present invention, and B. negative control. The detection result after the Escherichia coli is treated by the nucleic acid hands-free reagent is positive, and the negative control is not amplified.

Example 8

The present example differs from example 1 in that the pathogenic microorganism to be detected was staphylococcus aureus and the effect of the saline-treated sample was not verified.

The primers used were F: GCGCAAGCTATGATCAATTTGGAC (shown in SEQ. ID. NO. 3);

r: CCAGGCTTTGCACCATCACC (shown in SEQ. ID. NO. 4).

The results of detection are shown in FIG. 8, A. the amplification results after treatment with the nucleic acid removal kit of the present invention, and B. the negative control. The detection result of the staphylococcus aureus treated by the nucleic acid extraction-free reagent is positive, and the negative control is not amplified. The nucleic acid hand-free reagent has stronger lysis capacity on gram-positive bacteria.

Example 9

The present example differs from example 1 in that the pathogenic microorganism to be detected is cryptococcus, and the effect of the saline treatment of the sample is not verified. The primers used were F: GGGGTGTGTGCTGTGCGA (shown in SEQ. ID. NO. 5), R: GGCGTCTTACCCCAAGTCCC (shown in SEQ. ID. NO. 6). The results of detection are shown in FIG. 9, A. amplification results after treatment with the nucleic acid removal kit of the present invention, and B. negative control. The detection result of the cryptococcus treated by the nucleic acid extraction-free reagent is positive, and the detection result of the negative control is negative. The nucleic acid taking-free reagent of the invention also has stronger cracking capability to fungi.

Example 10

This example differs from example 1 in that the pathogenic microorganism to be detected is a lentivirus containing SARS-CoV 2E gene and the effect of the saline-treated sample is not verified.

The primers used were:

f: GGGGTGTGTGCTGTGCGA (shown in SEQ. ID. NO. 7);

r: GGCGTCTTACCCCAAGTCCC (shown in SEQ. ID. NO. 8).

The results of detection are shown in FIG. 10, A. amplification results after treatment with the nucleic acid removal kit of the present invention, and B. negative control. The detection result of the lentivirus treated by the nucleic acid extraction-free reagent is positive, and the detection result of the negative control is negative. The nucleic acid taking-free reagent has stronger lytic capacity to lentivirus. The structural similarity of lentivirus and coronavirus indirectly shows that the reagent of the invention also has stronger splitting capacity to coronavirus.

Example 11

The difference between this embodiment and embodiment 10 is that Loop-mediated Isothermal Amplification (LAMP) is used for detecting pathogenic microorganisms, and the specific reaction system is as follows: 20mM Tris-HCl, 10mM (NH)₄)₂SO₄，50mM KCl，8mM MgSO₄0.1% Tween-20, 0.8M betaine, 1.4mM dNTP, 1 XSSYBR Green I, 0.8. mu.M FIP and BIP, 0.4. mu.M LF and LB, 0.2. mu. M F3 and B3, 0.32U/. mu.l Bst DNA Polymerase, 0.2U/. mu.l AMV.

The primer sequence is as follows:

f3: GTACTCATTCGTTTCGGAAGA (shown in SEQ. ID. NO. 9);

b3: CTCTAGAAGAATTCAGATTTTTAA (shown in SEQ. ID. NO. 10);

FIP: TGGCTAGTGTAACTAGCAAGAATCAGGTACGTTAATAGTTAATAGC (shown in SEQ. ID. NO. 11);

and (3) BIP: TGCGCTTCGATTGTGTGCGTAGAGTAAACGTAAAAAGAAGGTTT (shown in SEQ. ID. NO. 12);

LF: CACGAAAGCAAGAAAAAGAAGT (shown in SEQ. ID. NO. 13);

LB: GCTGCAATATTGTTAACGTGAGT (shown in SEQ. ID. NO. 14).

Results see fig. 11, a. amplification results after treatment with the nucleic acid non-sampling reagent of the present invention, b. negative control.

The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Sequence listing

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