CN112553295B - Ribonuclease detection method, kit and application thereof - Google Patents
Ribonuclease detection method, kit and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biology, and particularly provides a ribonuclease detection method, a ribonuclease detection kit and application of ribonuclease detection kit. In the detection method, RNA template is subjected to reverse transcription amplification in a reverse transcription amplification system in the presence of ribonuclease to be detected and RNase inhibitor, and the concentration of the ribonuclease to be detected is determined according to the Ct value of reverse transcription amplification of the ribonuclease to be detected. The method can realize high-flux detection of ribonuclease, no radioactive pollution, strong broad spectrum, and quantitative or relative quantitative detection.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a ribonuclease detection method, a ribonuclease detection kit and application of ribonuclease detection kit.
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, etc. Among them, RNaseA is a widely used endonuclease, and RNase A catalyzes cleavage of a phosphodiester bond on a single-stranded RNA strand backbone at the 3' -ends of pyrimidine nucleotide residues C and U with high efficiency and specificity to form an oligonucleotide having a 2',3' -cyclic phosphate derivative.
Ribonucleases are environmental pollutants commonly found in laboratories, and monitoring ribonuclease activity is a routine Quality Control (QC) step, however, at present, there are few methods for ribonuclease activity detection. The method mainly comprises a radioisotope method, a spectrophotometry, a fluorescence quenching method and the like, however, the method is difficult to realize high flux, meanwhile, a specific oligoribonucleic acid molecule needs to be synthesized as an action substrate, and a specific RNA probe with modification needs to be designed and synthesized, so that the cost is high; or is easy to produce radioactive pollution, or has multiple steps and long period.
In view of this, the present invention has been made.
Disclosure of Invention
The first object of the present invention is to provide a method for detecting the purpose of diagnosis and treatment of ribonuclease non-disease, which can realize high-throughput, modularized and rapid detection of ribonuclease concentration.
The second object of the present invention is to provide a ribonuclease assay kit that can rapidly and efficiently detect the concentration of ribonuclease.
A third object of the present invention is to provide the use of the above detection method or kit.
In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:
a method for detecting the purpose of non-disease diagnosis and treatment of ribonuclease includes such steps as reverse transcription amplifying RNA template in reverse transcription amplifying system in the presence of ribonuclease to be detected and RNase inhibitor, and determining the concentration of ribonuclease to be detected according to the Ct value of reverse transcription amplifying ribonuclease to be detected.
Further, 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 gene of interest includes a conserved sequence;
preferably, the conserved sequence comprises a reference gene;
preferably, the reference gene comprises GAPDH, β -action, β -tubulin, 18sRNA, 28sRNA, SDHA or HPRT1.
Further, the target gene is GAPDH;
preferably, the detection primer and detection probe for GAPDH comprise:
upstream detection primer: 5'-TGGAGAAACCTGCCAAGTATGATA-3' (SEQ ID NO. 1);
downstream detection primer: 5'-CAGTGGGAGTTGCTGTTGAAGTC-3' (SEQ ID NO. 2);
detection probe: 5'-ATCAAGAAGGTGGTGAAGCAGGCAT-3' (SEQ ID NO. 3).
Further, the reverse transcription amplification system comprises reverse transcriptase, DNA polymerase, dNTPs and buffer solution;
preferably, at least one of magnesium ion, ammonium ion, potassium ion and surfactant is also included in the reverse transcription amplification system.
Further, the ribonuclease comprises RNase A, RNase B or RNase C.
Further, the detection method further comprises the step of preparing a standard curve, and comparing the Ct value of reverse transcription amplification of the ribonuclease to be detected with the standard curve to obtain the concentration of the ribonuclease to be detected;
the standard curve comprises the 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 carrying out reverse transcription amplification detection on the 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 ion, ammonium ion, potassium ion and surfactant;
preferably, the kit further comprises a detection primer and a detection probe for detecting the target gene;
preferably, the detection primer and the detection probe comprise:
upstream detection primer: 5'-TGGAGAAACCTGCCAAGTATGATA-3' (SEQ ID NO. 1);
downstream detection primer: 5'-CAGTGGGAGTTGCTGTTGAAGTC-3' (SEQ ID NO. 2);
detection probe: 5'-ATCAAGAAGGTGGTGAAGCAGGCAT-3' (SEQ ID NO. 3);
preferably, the kit further comprises a ribonuclease standard.
Use of the above detection method or kit in a) or b) as follows:
a) Detecting the content or relative content of ribonuclease to be detected for non-disease diagnosis and treatment purposes;
b) The non-disease diagnosis is compared with the content or relative content of two or more ribonuclease samples to be tested for therapeutic purposes.
Compared with the prior art, the invention has the beneficial effects that:
the detection method for the purpose of non-disease diagnosis and treatment of the ribonuclease provided by the invention realizes the concentration detection of the ribonuclease to be detected by quantifying the RNA template. Because ribonuclease can rapidly degrade RNA, in order to realize accurate detection, the inventor utilizes the principle that 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 RNA substrates, enables the ribonuclease to accurately detect the concentration of the ribonuclease, eliminates the quantitative detection of inactive enzyme, and avoids the occurrence of false positives and false negatives. The detection method can realize the modularization of the detection of the ribonuclease, namely, the detection can be realized only by putting the ribonuclease to be detected into a detection system.
The detection method comprises the following steps: (1) The high-flux detection can be realized, a large amount of samples can be detected, the reaction volume is small, the detection speed is high (usually 1 hour), and the method is suitable for the conventional quality control of a laboratory; (2) real-time observation can be realized; (3) no radioactive pollution and high safety; (4) The broad spectrum is strong, and the residual activity of different ribonucleases in different samples can be detected; (5) The enzyme content or relative content can be quantified, the enzyme content of an unknown sample can be calibrated at the enzyme content level of a known concentration, and comparison of ribonuclease activities in different samples can be realized.
The ribonuclease detection kit provided by the invention has the advantages of the detection method, low cost, mass production and wide application field because the ribonuclease inhibitor in the detection method is contained in the detection method and the detection method is adopted in actual detection.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows amplification curves for RNase A standard at various known concentrations in example 3;
FIG. 2 is a standard curve prepared in example 3;
FIG. 3 is an amplification curve of the sample 1 to be tested and the sample 2 to be tested in example 4;
FIG. 4 is a reverse transcription assay amplification curve without the presence 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 for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer.
Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any method or material similar or equivalent to those described may be used in the present invention.
A method for detecting the purpose of non-disease diagnosis and treatment of ribonuclease includes such steps as reverse transcription amplifying RNA template in reverse transcription amplifying system in the presence of ribonuclease to be detected and RNase inhibitor, and determining the concentration of ribonuclease to be detected according to the Ct value of reverse transcription amplifying ribonuclease to be detected.
The detection method for the purpose of non-disease diagnosis and treatment of the ribonuclease provided by the invention realizes the concentration detection of the ribonuclease to be detected by quantifying the RNA template. Because ribonuclease can rapidly degrade RNA, in order to realize accurate detection, the inventor utilizes the principle that 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 RNA substrates, enables the ribonuclease to accurately detect the concentration of the ribonuclease, eliminates the quantitative detection of inactive enzyme, and avoids the occurrence of false positives and false negatives. The detection method can realize the modularization of the detection of the ribonuclease, namely, the detection can be realized only by putting the ribonuclease to be detected into a detection system.
The detection method has the following advantages: (1) The high-flux detection can be realized, a large amount of samples can be detected, the reaction volume is small, the detection speed is high (usually 1 hour), and the method is suitable for the conventional quality control of a laboratory; (2) real-time observation can be realized; (3) no radioactive pollution and high safety; (4) The broad spectrum is strong, and the residual activity of different ribonucleases in different samples can be detected; (5) The enzyme content or relative content can be quantified, the enzyme content of an unknown sample can be calibrated at the enzyme content level of a known concentration, and comparison of ribonuclease activities in different samples can be realized.
As is well known to those skilled in the art, ribonucleases work on single stranded RNA rapidly, for example, RNaseA only requires 1ng of RNaseA for 1. Mu.g of RNA, and can be digested completely in about 30 minutes. In order to delay the speed of the ribonuclease acting on the substrate, the invention introduces the RNase inhibitor into the detection system, can exclude the inactive enzyme from being quantified, and better controls the enzyme reaction speed, thereby realizing accurate quantification. It will be appreciated that the amount of RNase inhibitor in the reverse transcription amplification system can be adjusted as desired, as long as a reasonable detection of the reverse transcription reaction can be achieved, and the concentration of ribonuclease is reacted.
In a preferred embodiment, the RNA template contains the gene of interest and the reverse transcription amplification system includes a detection primer and a detection probe for detecting the gene of interest. The detection primer and the detection probe in the present invention 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 gene, further preferably GAPDH, β -action, β -tubulin, 18sRNA, 28sRNA, SDHA or HPRT1. In the invention, the target gene is preferably a template containing a conserved sequence, so that the complex operation of designing a specific substrate in ribonuclease detection in the prior art is avoided, and the cost is reduced. In addition, it is understood that the RNA template may include the target gene, that is, may contain other substances as long as specific amplification of the target gene can be achieved, for example, the RNA template given in the embodiment of the present invention is total RNA of the extracted mammalian cells.
In a preferred embodiment, the gene of interest is GAPDH. It will be appreciated that the detection template may be single-stranded mRNA of the GAPDH gene, i.e., the detection template may be single-stranded mRNA of the pure GAPDH gene, or may contain other substances, such as the RNA template described in the examples of the present invention is total RNA of the extracted mammalian cells.
In a preferred embodiment, the detection primer and detection probe for GAPDH comprises:
upstream detection primer: 5'-TGGAGAAACCTGCCAAGTATGATA-3' (SEQ ID NO. 1);
downstream detection primer: 5'-CAGTGGGAGTTGCTGTTGAAGTC-3' (SEQ ID NO. 2);
detection probe: 5'-ATCAAGAAGGTGGTGAAGCAGGCAT-3' (SEQ ID NO. 3).
In a preferred embodiment, reverse transcriptase, DNA polymerase, dNTPs and buffer are included in the reverse transcription amplification system. The reverse transcriptase may be AMV reverse transcriptase, MMLV reverse transcriptase, etc.; the DNA polymerase may be Hotstart DNA polymerase, anstart DNA polymerase, etc.; the buffer may be Tris-HCl or the like.
In a preferred embodiment, at least one of magnesium ion, ammonium ion, potassium ion and surfactant is also included in the reverse transcription amplification system. Examples of reverse transcription amplification systems include, but are not limited to, magnesium,ammonium ion, potassium ion and surfactant, magnesium ion and ammonium ion, or magnesium ion, ammonium ion, potassium ion and surfactant, etc.; the magnesium ion may be MgSO 4 、MgCl 2 Etc., the ammonium ion may be (NH) 4 ) 2 SO 4 、NH 4 Cl, etc., the potassium ion can be KCl, K 2 SO 4 Etc., the surfactant may be tween-20, etc.
In preferred embodiments, the ribonuclease comprises RNase A, RNase B or RNase C.
In a preferred embodiment, the detection method further comprises 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 comprises the corresponding relation between the concentration of the ribonuclease standard and the reverse transcription amplification Ct value of the ribonuclease standard, and the reverse transcription amplification Ct value of the ribonuclease standard is obtained by carrying out reverse transcription amplification detection on the RNA template by a reverse transcription amplification system. It can be understood that, the standard curve can be prepared by using standard substances with known concentration of ribonuclease, so as to realize accurate quantitative detection of ribonuclease to be detected, for example: the method is characterized in that a proper amount of RNase inhibitor is utilized to realize incomplete inhibition on RNase standard products with known concentrations of different gradients, the RNase is subjected to gradient degradation on RNA under an RNA template with a certain concentration, the concentration of the residual RNA template can be monitored in real time by utilizing a reverse transcription one-step method for real-time fluorescence quantitative PCR detection, a standard curve can be made by the concentration of a substrate (namely Ct value) and the concentration of the RNase standard product, the concentration of a sample with unknown concentration is quantified, and it is understood that for the accuracy of a result, a PCR reaction liquid sample of the standard curve and a PCR reaction liquid sample for detecting the RNase to be detected and the detection procedures of the two should be the same. That is, the detection method of the present invention can be implemented: when the standard curve is known, the same reverse transcription reaction system is utilized to realize the modularized detection of the ribonuclease to be detected.
A ribonuclease assay kit comprising an rnase inhibitor. The detection method provided by the invention is adopted for actual detection due to the fact that the RNase inhibitor in the detection method is contained, so that the detection method has the advantages of low cost, mass production and wide application range.
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 ion, ammonium ion, potassium ion and surfactant; 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 primers and detection probes in the kit are those of GAPDH, in particular:
upstream detection primer: 5'-TGGAGAAACCTGCCAAGTATGATA-3' (SEQ ID NO. 1);
downstream detection primer: 5'-CAGTGGGAGTTGCTGTTGAAGTC-3' (SEQ ID NO. 2);
detection probe: 5'-ATCAAGAAGGTGGTGAAGCAGGCAT-3' (SEQ ID NO. 3);
in a preferred embodiment, the kit further comprises a ribonuclease standard.
The invention provides the use of the above detection method or kit in a) or b) as follows:
a) Detecting the content or relative content of ribonuclease to be detected for non-disease diagnosis and treatment purposes;
b) The non-disease diagnosis is compared with the content or relative content of two or more ribonuclease samples to be tested for therapeutic purposes.
The detection method or kit provided by the invention can be applied to the following applications, for example and without limitation: qualitatively detecting whether the target sample contains ribonuclease; indirectly quantifying the ribonuclease content by preparing a standard curve; comparing the amount of ribonuclease content between two or more samples, and the like.
The invention is further illustrated by the following specific examples, however, it should be understood that these examples are for the purpose of illustration only in greater detail and are not to be construed as limiting the invention in any way.
EXAMPLE 1 reverse transcription primer design
Selecting a segment of conserved sequence RNA, specifically an mRNA sequence of a mouse GAPDH gene, and designing a detection primer:
detecting the upstream primer: 5'-TGGAGAAACCTGCCAAGTATGATA-3' (SEQ ID NO. 1);
detecting the downstream primer: 5'-CAGTGGGAGTTGCTGTTGAAGTC-3' (SEQ ID NO. 2);
detection probe: 5'-ATCAAGAAGGTGGTGAAGCAGGCAT-3' (SEQ ID NO. 3).
Example 2A ribonuclease assay kit
The kit comprises the following components, taking 50 μl of PCR reaction liquid sample as an example:
(1) The final concentration of rnase inhibitor was used in an amount of 17U;
(2)60mM Tris-HCl(pH8.2)，50mM KCl，12.5mM(NH 4 ) 2 SO 4 ，3.3mM MgSO 4 0.01w/v% tween-20, hostart Taq enzyme at a final concentration of 3U, 80U MMLV reverse transcriptase;
and (3) the detection primer in example 1: 10. Mu.M upstream detection primer, 10. Mu.M downstream detection primer and 5. Mu.M Taqman detection probe.
EXAMPLE 3 preparation of a Standard Curve
Commercial RNase A was subjected to concentration gradient dilution with RNase A diluent, RNaseA standard substances with final concentrations of 30 ng/. Mu.l, 32 ng/. Mu.l, 35 ng/. Mu.l and 40 ng/. Mu.l were sequentially added to the PCR reaction liquid sample in example 2, and then 100ng of single-stranded RNA template (extracted total RNA of mouse cells) was added thereto, and incubated at room temperature for 5min, and the amplification detection was performed by a reverse transcription one-step method using a fluorescent quantitative PCR instrument.
The reaction procedure was 50℃for 15min;95 ℃ for 10min; (94 ℃,15s;55 ℃,40 s). Times.45.
And (3) monitoring by a real-time fluorescence quantitative PCR instrument to obtain the Ct value of each concentration of the RNase A standard substance, and preparing a linear standard curve by taking the Ct value as an abscissa and the RNaseA concentration value as an ordinate according to the amplified Ct value and the concentration value (ng level) of the RNase A standard substance with known concentration, wherein the amplified curve of the RNase A standard substance is shown in figure 1, and the prepared standard curve is shown in figure 2.
Example 4 detection of ribonuclease to be detected
The sample 1 and the sample 2 to be tested are genetically engineered recombinant RNaseA enzymes, and are purified from recombinant escherichia coli, and the strain carries RNaseA genes from bovine pancreas.
And (3) respectively detecting the sample 1 to be detected and the sample 2 to be detected by adopting the kit in the embodiment 2, wherein the detection conditions are the same as those of the standard product in the embodiment 3, substituting Ct values of the sample 1 to be detected and the sample 2 to be detected into standard curves respectively, and calculating RNaseA enzyme concentration values of 28.86 ng/. Mu.l of the sample 1 to be detected and 38.70 ng/. Mu.l of the sample 2 to be detected. The amplification curves of the sample 1 and the sample 2 are shown in FIG. 3.
Comparative example detection of RNase inhibitor-free
Taking 30 ng/. Mu.l RNaseA standard as an example, referring to the kit in example 2, the PCR reaction liquid sample does not contain RNase inhibitor, 100ng of single-stranded RNA template is added, and the reaction procedure is 50 ℃ for 15min;95 ℃ for 10min; (94 ℃,15s;55 ℃,40 s). Times.45, the reaction was examined.
As shown in FIG. 4, when no RNase inhibitor is added to the system, 30 ng/. Mu.l of RNaseA standard product can cut 100ng of the RNA template quickly, the action time is short, and the typical fluorescent quantitative PCR reaction time is 1 hour, if no RNase inhibitor is added, the detection of ribonuclease by reverse transcription detection cannot be realized, and it is difficult to accurately mark the RNaseA activity.
While particular embodiments of the present invention have been illustrated and described, it will be appreciated 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> Phenant biological Co., ltd
GUANGDONG FEIPENG BIOLOGICAL Co.,Ltd.
<120> ribonuclease detection method and kit and application thereof
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 24
<212> DNA
<213> artificial sequence
<400> 1
tggagaaacc tgccaagtat gata 24
<210> 2
<211> 23
<212> DNA
<213> artificial sequence
<400> 2
cagtgggagt tgctgttgaa gtc 23
<210> 3
<211> 25
<212> DNA
<213> artificial sequence
<400> 3
atcaagaagg tggtgaagca ggcat 25
Claims (15)
1. A detection method for the purpose of ribonuclease non-disease diagnosis and treatment is characterized in that under the existence of ribonuclease to be detected and an RNase inhibitor, reverse transcription amplification is carried out on a conserved sequence of an RNA template in a reverse transcription amplification system, and the concentration of the ribonuclease to be detected is determined according to a Ct value of reverse transcription amplification of the ribonuclease to be detected;
the detection method further comprises the 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;
the standard curve comprises the 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 carrying out reverse transcription amplification detection on the RNA template by a reverse transcription amplification system.
2. The method according to claim 1, wherein the reverse transcription amplification system comprises a detection primer and a detection probe for detecting a conserved sequence.
3. The method of claim 2, wherein the conserved sequence comprises a reference gene.
4. The method according to claim 3, wherein the reference gene comprises GAPDH, beta-action, beta-tubulin, 18sRNA, 28sRNA, SDHA or HPRT1.
5. The method of claim 2, wherein the conserved sequence is GAPDH.
6. The method according to claim 5, wherein the GAPDH detection primer and probe include:
upstream detection primer: 5'-TGGAGAAACCTGCCAAGTATGATA-3' (SEQ ID NO. 1);
downstream detection primer: 5'-CAGTGGGAGTTGCTGTTGAAGTC-3' (SEQ ID NO. 2);
detection probe: 5'-ATCAAGAAGGTGGTGAAGCAGGCAT-3' (SEQ ID NO. 3).
7. The method of claim 1, wherein the reverse transcription amplification system comprises reverse transcriptase, DNA polymerase, dNTPs and buffer.
8. The method according to claim 7, wherein the reverse transcription amplification system further comprises at least one of magnesium ion, ammonium ion, potassium ion and surfactant.
9. The method of any one of claims 1-8, wherein the ribonuclease comprises RNase a, RNase B or RNase C.
10. The ribonuclease detection kit is characterized by comprising an RNase inhibitor, a detection primer for detecting a conserved sequence, a detection probe, a reverse transcription amplification system and a ribonuclease standard.
11. The kit of claim 10, wherein the reverse transcription amplification system further comprises a reverse transcriptase and a DNA polymerase.
12. The kit of claim 10, wherein the reverse transcription amplification system further comprises dNTPs and a buffer.
13. The kit of claim 10, wherein the reverse transcription amplification system further comprises at least one of magnesium ions, ammonium ions, potassium ions, and surfactants.
14. The kit of claim 10, wherein the detection primer and the detection probe comprise:
upstream detection primer: 5'-TGGAGAAACCTGCCAAGTATGATA-3' (SEQ ID NO. 1);
downstream detection primer: 5'-CAGTGGGAGTTGCTGTTGAAGTC-3' (SEQ ID NO. 2);
detection probe: 5'-ATCAAGAAGGTGGTGAAGCAGGCAT-3' (SEQ ID NO. 3).
15. Use of the detection method of any one of claims 1-9 or the kit of any one of claims 10-14 in a) or b) as follows:
a) Detecting the content or relative content of ribonuclease to be detected for non-disease diagnosis and treatment purposes;
b) The non-disease diagnosis is compared with the content or relative content of two or more ribonuclease samples to be tested for therapeutic purposes.
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