阅读说明：本技术 核糖核酸酶的检测方法与试剂盒及其应用 (Ribonuclease detection method and kit and application thereof ) 是由 林钰琼 于 2019-09-26 设计创作，主要内容包括：本发明涉及生物技术领域,具体而言,提供了一种核糖核酸酶的检测方法与试剂盒及其应用。该检测方法是在待测核糖核酸酶和RNA酶抑制剂存在的条件下,在逆转录扩增体系中对RNA模板进行逆转录扩增,根据待测核糖核酸酶逆转录扩增Ct值确定待测核糖核酸酶的浓度。该方法可实现核糖核酸酶的高通量检测、无放射性污染、广谱性强、定量或相对定量检测。(The invention relates to the technical field of biology, and particularly provides a ribonuclease detection method, a kit and application thereof. The detection method comprises the steps of carrying out reverse transcription amplification on an RNA template in a reverse transcription amplification system in the presence of ribonuclease to be detected and an RNase inhibitor, and determining the concentration of the ribonuclease to be detected according to the Ct value of the reverse transcription amplification of the ribonuclease to be detected. The method can realize high-throughput detection of ribonuclease, no radioactive pollution, strong broad-spectrum property, and quantitative or relative quantitative detection.)

1. A method for detecting ribonuclease for non-disease diagnosis and treatment is characterized by that under the condition of existence of ribonuclease to be detected and RNase inhibitor, the RNA template is reverse-transcribed and amplified in reverse transcription-amplification system, and the concentration of ribonuclease to be detected is determined according to the Ct value of reverse-transcription and amplification of ribonuclease to be detected.

2. The detection method according to claim 1, wherein the RNA template contains a target gene, and the reverse transcription amplification system comprises a detection primer and a detection probe for detecting the target gene.

3. The detection method according to claim 2, wherein the target gene comprises a conserved sequence;

preferably, the conserved sequence comprises an internal reference gene;

preferably, the reference gene comprises GAPDH, β -Actin, β -tubulin, 18sRNA, 28sRNA, SDHA or HPRT 1.

4. The detection method according to claim 3, wherein the target gene is GAPDH;

preferably, the detection primer and detection probe for GAPDH comprise:

an upstream detection primer: 5'-TGGAGAAACCTGCCAAGTATGATA-3' (SEQ ID NO. 1);

downstream detection primers: 5'-CAGTGGGAGTTGCTGTTGAAGTC-3' (SEQ ID NO. 2);

detecting a probe: 5'-ATCAAGAAGGTGGTGAAGCAGGCAT-3' (SEQ ID NO. 3).

5. The detection method according to claim 1, wherein the reverse transcription amplification system comprises reverse transcriptase, DNA polymerase, dNTPs and buffer;

preferably, the reverse transcription amplification system further comprises at least one of magnesium ions, ammonium ions, potassium ions and surfactants.

6. The detection method according to any one of claims 1 to 5, wherein the ribonuclease comprises RNase A, RNase B or RNase C.

7. The detection method according to claim 6, further comprising a step of preparing a standard curve, wherein the Ct value of reverse transcription amplification of the ribonuclease to be detected is compared with the standard curve to obtain the concentration of the ribonuclease to be detected;

the standard curve comprises a corresponding relation between the concentration of the ribonuclease standard substance and the reverse transcription amplification Ct value of the ribonuclease standard substance, wherein the reverse transcription amplification Ct value of the ribonuclease standard substance is obtained by performing reverse transcription amplification detection on an RNA template by a reverse transcription amplification system.

8. A ribonuclease assay kit comprising an RNase inhibitor.

9. The kit of claim 8, wherein the kit further comprises a reverse transcriptase and a DNA polymerase;

preferably, the kit further comprises dNTPs and a buffer;

preferably, the kit further comprises at least one of magnesium ions, ammonium ions, potassium ions, and a surfactant;

preferably, the kit also comprises a detection primer and a detection probe for detecting the target gene;

preferably, the detection primer and detection probe comprise:

an upstream detection primer: 5'-TGGAGAAACCTGCCAAGTATGATA-3' (SEQ ID NO. 1);

downstream detection primers: 5'-CAGTGGGAGTTGCTGTTGAAGTC-3' (SEQ ID NO. 2);

detecting a probe: 5'-ATCAAGAAGGTGGTGAAGCAGGCAT-3' (SEQ ID NO. 3);

preferably, the kit further comprises a ribonuclease standard.

10. Use of the detection method according to any one of claims 1 to 7 or the kit according to claim 8 or 9 in a) or b) as follows:

a) detecting the content or relative content of the ribonuclease to be detected for non-disease diagnosis and treatment purposes;

b) the amounts or relative amounts of two or more ribonuclease samples to be tested are compared for non-disease diagnostic and therapeutic purposes.

Technical Field

The invention relates to the technical field of biology, in particular to a ribonuclease detection method, a kit and application thereof.

Background

Ribonucleases (rnases) are a class of nucleases that catalyze the degradation of RNA into small fragments. The RNase family includes RNase A, RNase B, RNase C, RNase H, S-RNase, RNase P, RNase T and the like. Wherein, RNase A is a widely used endonuclease, RNase A efficiently and specifically catalyzes cleavage of phosphodiester bond on single-stranded RNA chain skeleton at 3 ' end of pyrimidine nucleotide residues C and U to form oligonucleotide with 2 ', 3 ' -cyclic phosphate derivative.

Ribonucleases are ubiquitous environmental pollutants in laboratories, and monitoring of ribonuclease activity is a routine Quality Control (QC) step, however, few methods are currently available for ribonuclease activity detection. Mainly comprises a radioisotope method, a spectrophotometry method, a fluorescence quenching method and the like, however, the method is difficult to realize high flux, simultaneously needs to synthesize specific oligoribonucleic acid molecules as acting substrates, and needs to design and synthesize specific modified RNA probes, so that the cost is higher; or radioactive contamination is easy to generate, or the steps are multiple and the period is long.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention is to provide a method for detecting ribonuclease for non-disease diagnosis and treatment, which can realize high-throughput, modular and rapid detection of ribonuclease concentration.

The second purpose of the invention is to provide a ribonuclease detection kit, which can rapidly and efficiently detect the concentration of ribonuclease.

The third purpose of the invention is to provide the application of the detection method or the kit.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a method for detecting ribonuclease for non-disease diagnosis and treatment features that under the existance of ribonuclease to be detected and RNase inhibitor, the RNA template is reverse-transcribed and amplified in reverse transcription-amplification system, and the concentration of ribonuclease to be detected is determined according to the Ct value of reverse transcription-amplification of ribonuclease.

Furthermore, the RNA template contains a target gene, and the reverse transcription amplification system comprises a detection primer and a detection probe for detecting the target gene.

Further, the target gene comprises a conserved sequence;

preferably, the conserved sequence comprises an internal reference gene;

preferably, the reference gene comprises GAPDH, β -Actin, β -tubulin, 18sRNA, 28sRNA, SDHA or HPRT 1.

Further, the target gene is GAPDH;

preferably, the detection primer and detection probe for GAPDH comprise:

an upstream detection primer: 5'-TGGAGAAACCTGCCAAGTATGATA-3' (SEQ ID NO. 1);

downstream detection primers: 5'-CAGTGGGAGTTGCTGTTGAAGTC-3' (SEQ ID NO. 2);

detecting a probe: 5'-ATCAAGAAGGTGGTGAAGCAGGCAT-3' (SEQ ID NO. 3).

Further, the reverse transcription amplification system comprises reverse transcriptase, DNA polymerase, dNTPs and buffer;

preferably, the reverse transcription amplification system further comprises at least one of magnesium ions, ammonium ions, potassium ions and surfactants.

Further, the ribonuclease includes RNase a, RNase B or RNase C.

Further, the detection method also comprises a step of preparing a standard curve, wherein the reverse transcription amplification Ct value of the ribonuclease to be detected is compared with the standard curve to obtain the concentration of the ribonuclease to be detected;

the standard curve comprises a corresponding relation between the concentration of the ribonuclease standard substance and the reverse transcription amplification Ct value of the ribonuclease standard substance, wherein the reverse transcription amplification Ct value of the ribonuclease standard substance is obtained by performing reverse transcription amplification detection on an RNA template by a reverse transcription amplification system.

A ribonuclease assay kit comprising an rnase inhibitor.

Further, the kit further comprises a reverse transcriptase and a DNA polymerase;

preferably, the kit further comprises dNTPs and a buffer;

preferably, the kit further comprises at least one of magnesium ions, ammonium ions, potassium ions, and a surfactant;

preferably, the kit also comprises a detection primer and a detection probe for detecting the target gene;

preferably, the detection primer and detection probe comprise:

an upstream detection primer: 5'-TGGAGAAACCTGCCAAGTATGATA-3' (SEQ ID NO. 1);

downstream detection primers: 5'-CAGTGGGAGTTGCTGTTGAAGTC-3' (SEQ ID NO. 2);

detecting a probe: 5'-ATCAAGAAGGTGGTGAAGCAGGCAT-3' (SEQ ID NO. 3);

preferably, the kit further comprises a ribonuclease standard.

The application of the detection method or the kit in a) or b) is as follows:

a) detecting the content or relative content of the ribonuclease to be detected for non-disease diagnosis and treatment purposes;

b) the amounts or relative amounts of two or more ribonuclease samples to be tested are compared for non-disease diagnostic and therapeutic purposes.

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

the ribonuclease concentration detection method for non-disease diagnosis and treatment provided by the invention realizes the concentration detection of the ribonuclease to be detected through the quantification of the RNA template. Because the ribonuclease can rapidly degrade RNA, in order to realize accurate detection, the inventor utilizes the principle that the ribonuclease can form a 1:1 complex with an RNase inhibitor, partially inhibits the activity of the ribonuclease in the detection process, delays the speed of the ribonuclease acting on an RNA substrate, enables the ribonuclease to accurately detect the concentration, excludes inactive enzyme from being quantitatively detected, and avoids the occurrence of false positive and false negative. The detection method can realize the modularization of detecting the ribonuclease, namely, the ribonuclease to be detected can be detected only by putting the ribonuclease into a detection system.

The detection method comprises the following steps: (1) the kit can realize high-flux detection, can detect a large amount of samples, has small reaction volume and high detection speed (usually 1 hour), and is suitable for routine quality control of a laboratory; (2) real-time observation can be realized; (3) no radioactive pollution and high safety; (4) the broad spectrum property is strong, and the residual activity of different types of ribonuclease in different samples can be detected; (5) the amount or relative amount of enzyme can be quantified, the amount of enzyme in an unknown sample can be calibrated at a level of known concentration of enzyme, and comparison of ribonuclease activity in different samples can also be achieved.

The ribonuclease detection kit provided by the invention contains the RNase inhibitor in the detection method, and the actual detection adopts the detection method provided by the invention, so that the ribonuclease detection kit has the advantages of the detection method, is low in cost, can be produced in batches and has wide application scenes.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a graph showing the amplification curves of RNase A standards at different known concentrations in example 3;

FIG. 2 is a standard curve prepared in example 3;

FIG. 3 is an amplification curve of sample 1 and sample 2 in example 4;

FIG. 4 is a reverse transcription assay amplification curve in the absence of RNase inhibitor in example 5.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.

A method for detecting ribonuclease for non-disease diagnosis and treatment features that under the existance of ribonuclease to be detected and RNase inhibitor, the RNA template is reverse-transcribed and amplified in reverse transcription-amplification system, and the concentration of ribonuclease to be detected is determined according to the Ct value of reverse transcription-amplification of ribonuclease.

The ribonuclease concentration detection method for non-disease diagnosis and treatment provided by the invention realizes the concentration detection of the ribonuclease to be detected through the quantification of the RNA template. Because the ribonuclease can rapidly degrade RNA, in order to realize accurate detection, the inventor utilizes the principle that the ribonuclease can form a 1:1 complex with an RNase inhibitor, partially inhibits the activity of the ribonuclease in the detection process, delays the speed of the ribonuclease acting on an RNA substrate, enables the ribonuclease to accurately detect the concentration, excludes inactive enzyme from being quantitatively detected, and avoids the occurrence of false positive and false negative. The detection method can realize the modularization of detecting the ribonuclease, namely, the ribonuclease to be detected can be detected only by putting the ribonuclease into a detection system.

The detection method has the following advantages: (1) the kit can realize high-flux detection, can detect a large amount of samples, has small reaction volume and high detection speed (usually 1 hour), and is suitable for routine quality control of a laboratory; (2) real-time observation can be realized; (3) no radioactive pollution and high safety; (4) the broad spectrum property is strong, and the residual activity of different types of ribonuclease in different samples can be detected; (5) the amount or relative amount of enzyme can be quantified, the amount of enzyme in an unknown sample can be calibrated at a level of known concentration of enzyme, and comparison of ribonuclease activity in different samples can also be achieved.

It is well known to those skilled in the art that ribonucleases work very efficiently on single-stranded RNA, e.g., RNaseA, 1. mu.g of RNA requires only 1ng of RNaseA and can be completely digested in about 30 min. In order to delay the speed of the ribonuclease acting on a substrate, the RNase inhibitor is introduced into a detection system, so that inactive enzyme can be excluded from being quantified, and the enzyme reaction speed is better controlled, thereby realizing accurate quantification. It is understood that the amount of RNase inhibitor in the reverse transcription amplification system can be adjusted according to actual needs, as long as the reverse transcription reaction can be detected reasonably and the concentration of RNase can be detected.

In a preferred embodiment, the RNA template contains a target gene, and the reverse transcription amplification system comprises a detection primer and a detection probe for detecting the target gene. In the present invention, the detection primer and the detection probe are not particularly limited as long as the reverse transcription amplification detection of the target gene in the RNA template can be achieved.

In a preferred embodiment, the gene of interest comprises a conserved sequence, preferably an internal reference gene, more preferably GAPDH, β -Actin, β -tubulin, 18sRNA, 28sRNA, SDHA or HPRT 1. It should be noted that, in the present invention, the target gene is preferably a template containing a conserved sequence, which avoids the tedious operation of designing a specific substrate in the ribonuclease detection in the prior art, and reduces the cost. In addition, it is understood that the RNA template may include the target gene, that is, may further include other substances as long as specific amplification of the target gene can be achieved, for example, the RNA template given in the present embodiment is total RNA of extracted mammalian cells.

In a preferred embodiment, the gene of interest is GAPDH. It is understood that the detection template may contain single-stranded mRNA of GAPDH gene, that is, the detection template may be pure single-stranded mRNA of GAPDH gene, or may contain other substances, for example, the RNA template given in the examples of the present invention is total RNA of extracted mammalian cells.

In a preferred embodiment, the detection primers and detection probes for GAPDH comprise:

an upstream detection primer: 5'-TGGAGAAACCTGCCAAGTATGATA-3' (SEQ ID NO. 1);

downstream detection primers: 5'-CAGTGGGAGTTGCTGTTGAAGTC-3' (SEQ ID NO. 2);

detecting a probe: 5'-ATCAAGAAGGTGGTGAAGCAGGCAT-3' (SEQ ID NO. 3).

In a preferred embodiment, the reverse transcription amplification system comprises a reverse transcriptase, a DNA polymerase, dNTPs and a buffer. The reverse transcriptase can be AMV reverse transcriptase, MMLV reverse transcriptase and the like; the DNA polymerase may be Hotstart DNA polymerase, Antart DNA polymerase, etc.; the buffer may be Tris-HCl or the like.

In a preferred embodiment, the reverse transcription amplification system further comprises at least one of magnesium ions, ammonium ions, potassium ions, and surfactants. Examples of the reverse transcription amplification system include, but are not limited to, magnesium ions, ammonium ions, potassium ions, and surfactants, magnesium ions, and ammonium ions, or magnesium ions, ammonium ions, potassium ions, and surfactants, and the like; the magnesium ion may be MgSO₄、MgCl₂Etc., the ammonium ion may be (NH)₄)₂SO₄、NH₄Cl, etc., and the potassium ion can be KCl or K₂SO₄And the surfactant may be tween-20 or the like.

In preferred embodiments, the ribonuclease comprises RNase a, RNase B or RNase C.

In a preferred embodiment, the detection method further includes a step of preparing a standard curve, and comparing the reverse transcription amplification Ct value of the ribonuclease to be detected with the standard curve to obtain the concentration of the ribonuclease to be detected, wherein the standard curve includes a corresponding relationship between the concentration of the ribonuclease standard substance and the reverse transcription amplification Ct value of the ribonuclease standard substance, and the reverse transcription amplification Ct value of the ribonuclease standard substance is obtained by performing reverse transcription amplification detection on the RNA template by a reverse transcription amplification system. It is understood that the precise quantitative detection of the ribonuclease to be detected can be achieved by preparing a standard curve using known concentrations of ribonuclease standards, such as: the method is characterized in that the RNase inhibitor with proper amount is utilized to realize incomplete inhibition on RNase standards with different gradients and known concentrations, under the condition of an RNA template with certain concentration, the RNase degrades the RNA in a gradient manner, the reverse transcription one-step real-time fluorescence quantitative PCR is utilized to detect, the concentration of the residual RNA template can be monitored in real time, a standard curve can be made according to the concentration of a substrate (namely Ct value) and the concentration of the RNase standard, and the concentration of a sample with unknown concentration can be quantified. That is, the detection method of the present invention can realize: when the standard curve is known, the same reverse transcription reaction system is utilized to realize the modular detection of the ribonuclease to be detected.

A ribonuclease assay kit comprises an RNase inhibitor. The RNase inhibitor in the detection method is contained, and the detection method provided by the invention is adopted for actual detection, so that the method has the advantages of the detection method, and is low in cost, capable of realizing batch production and wide in application scene.

In a preferred embodiment, the kit further comprises a reverse transcriptase and a DNA polymerase; preferably, dNTPs and a buffer are also included; further preferably, the kit further comprises at least one of magnesium ions, ammonium ions, potassium ions and surfactants; still more preferably, the kit further comprises a detection primer and a detection probe for detecting the RNA template.

In a preferred embodiment, the detection primer and the detection probe in the kit are GAPDH detection primers and detection probes, specifically:

an upstream detection primer: 5'-TGGAGAAACCTGCCAAGTATGATA-3' (SEQ ID NO. 1);

downstream detection primers: 5'-CAGTGGGAGTTGCTGTTGAAGTC-3' (SEQ ID NO. 2);

detecting a probe: 5'-ATCAAGAAGGTGGTGAAGCAGGCAT-3' (SEQ ID NO. 3);

in preferred embodiments, the kit further comprises a ribonuclease standard.

The invention provides the application of the detection method or the kit in the following a) or b):

a) detecting the content or relative content of the ribonuclease to be detected for non-disease diagnosis and treatment purposes;

b) the amounts or relative amounts of two or more ribonuclease samples to be tested are compared for non-disease diagnostic and therapeutic purposes.

The detection method or kit provided by the invention can be applied to, but is not limited to, the following applications: qualitatively detecting whether the target sample contains the ribonuclease or not; indirectly quantifying the ribonuclease content by preparing a standard curve; comparing how much ribonuclease content is between two or more samples, and so forth.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

Example 1 reverse transcription primer design

Selecting a segment of conserved sequence RNA, in particular to the mRNA sequence of mouse GAPDH gene, designing a detection primer:

detecting an upstream primer: 5'-TGGAGAAACCTGCCAAGTATGATA-3' (SEQ ID NO. 1);

detecting a downstream primer: 5'-CAGTGGGAGTTGCTGTTGAAGTC-3' (SEQ ID NO. 2);

detecting a probe: 5'-ATCAAGAAGGTGGTGAAGCAGGCAT-3' (SEQ ID NO. 3).

Example 2 ribonuclease assay kit

The kit comprises the following components, taking 50 mul of PCR reaction liquid sample as an example:

(1) the final concentration of the RNase inhibitor was 17U;

(2)60mM Tris-HCl(pH8.2)，50mM KCl，12.5mM(NH₄)₂SO₄，3.3mM MgSO₄0.01 w/v% tween-20, a final concentration of 3U of Hostart Taq enzyme, 80U of MMLV reverse transcriptase;

and (3) the detection primer in example 1: 10 μ M upstream detection primer, 10 μ M downstream detection primer and 5 μ M Taqman detection probe.

Example 3 preparation of a Standard Curve

The commercial RNase A is diluted by RNase A diluent in a concentration gradient manner, RNase A standard substances with final concentrations of 30 ng/mu l, 32 ng/mu l, 35 ng/mu l and 40 ng/mu l are sequentially added into the PCR reaction solution sample in the embodiment 2, a single-stranded RNA template (extracted mouse cell total RNA) with final concentration of 100ng is added, the incubation is carried out for 5min at room temperature, and the reverse transcription one-step amplification detection is carried out by a fluorescence quantitative PCR instrument.

The reaction program is 50 ℃ and 15 min; at 95 ℃ for 10 min; (94 ℃, 15 s; 55 ℃, 40 s). times.45.

And (3) obtaining Ct values of various concentrations of the RNase A standard product by monitoring through a real-time fluorescent quantitative PCR instrument, and preparing a linear standard curve by taking the Ct values as abscissa and the RNaseA concentration values as ordinate according to the amplification Ct values and concentration values (ng grade) of the RNase A standard product with known concentration, wherein the amplification curve of the RNase A standard product is shown in figure 1, and the prepared standard curve is shown in figure 2.

EXAMPLE 4 detection of the ribonuclease to be detected

The sample 1 and the sample 2 to be detected are genetic engineering recombinant RNaseA enzymes, are both purified from recombinant Escherichia coli, and carry RNaseA genes from bovine pancreas.

The test kit in the embodiment 2 is used for testing the sample 1 to be tested and the sample 2 to be tested respectively, the test conditions are the same as the test conditions of the standard substance in the embodiment 3, the Ct values of the obtained sample 1 to be tested and the sample 2 to be tested are respectively substituted into the standard curve, and the RNaseA enzyme concentration values are calculated to be respectively 28.86 ng/mu l of the sample 1 to be tested and 38.70 ng/mu l of the sample 2 to be tested. The amplification curves of the sample 1 and the sample 2 are shown in FIG. 3.

Comparative example detection without RNase inhibitor

Taking 30 ng/. mu.l RNaseA standard as an example, the kit in the example 2 is referred, but the PCR reaction solution sample does not contain RNase inhibitor, the single-stranded RNA template is added to the PCR reaction solution sample with the final concentration of 100ng, and the reaction program is 50 ℃ and 15 min; at 95 ℃ for 10 min; (94 ℃, 15 s; 55 ℃, 40 s). times.45), and carrying out reaction detection.

As shown in FIG. 4, it can be seen that when no RNase inhibitor is added into the system, 30 ng/. mu.l of RNaseA standard substance can cut 100ng of RNA template into the RNA template quickly, the time required for the action is short, while the time for the fluorescence quantitative PCR reaction is usually 1 hour, if no RNase inhibitor is added, the ribonuclease cannot be detected by a reverse transcription detection method, and it is difficult to accurately calibrate the RNase A activity.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

SEQUENCE LISTING

<110> Fenpeng biological products Ltd

GUANGDONG FEIPENG BIOLOGICAL Co.,Ltd.

<120> ribonuclease detection method and kit and application thereof

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<170> PatentIn version 3.5

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