CN105567826A - Detection of has-miR-29b-3p - Google Patents

Abstract

The invention relates to the field of molecular biology, and discloses a kit and method for detecting has-miR-29b-3p. The kit comprises a specificity primer pair for specifically amplifying has-miR-29b-3p. Primers have the continuous nucleotide sequence shown in SEQ ID No:1 and the continuous nucleotide sequence shown in SEQ ID No:2. The kit further comprises an RNA reverse transcription primer which has the sequence shown in SEQ ID No:3. The kit is disclosed for the first time, so when has-miR-29b-3p in serum of a blood bacterium infected patient is increased, the blood bacterium infection situation can be rapidly screened and checked. The number of required blood samples is small, and the detection cycle is short; pains of the patient can be relieved, and a doctor can easily interfere with blood bacterium infection in the early period.

Description

Detection of has-miR-29b-3p

technical field

The invention relates to the field of biotechnology, in particular to the technical field of rapid detection indicators, in particular to a kit and method for detecting microRNAhas-miR-29b-3p.

Background technique

Bloodstream infection is one of the most serious clinical manifestations of infectious diseases. Patients with bloodstream infection will lead to prolonged hospitalization, increased hospitalization costs, and a high mortality rate. The diagnosis of bloodstream infection is traditionally identified by blood culture, but it has the disadvantages of requiring a large amount of blood samples and taking a long time to report.

Macrophages in the human body play a huge role in the host's defense against pathogenic infection by recognizing, phagocytizing, and finally clearing bacteria. has-miR-29b-3p is a microRNA that is highly expressed in macrophages when they are infected by bacteria, and it can be detected in the serum of patients with positive blood cultures compared with the control group.

Contents of the invention

The purpose of the present invention is to provide a kit for detecting has-miR-29b-3p and establish a method for detecting has-miR-29b-3p. The method has the advantages of quickness, accuracy, simplicity and low cost, and is useful for clinical use and popularization.

In order to solve these problems in the prior art, the technical scheme provided by the invention is as follows:

A kit for detecting has-miR-29b-3p, characterized in that the kit includes a pair of specific primers for specific amplification of has-miR-29b-3p, and the primers have SEQ ID No: 1 and a contiguous nucleotide sequence having SEQ ID No: 2;

The kit also includes an RNA reverse transcription primer whose sequence is SEQ ID No: 3.

SEQ ID No: 1:5'GGGTAGCACCATTTGAAA3'

SEQ ID No: 2:5'TGCGTGTCGTGGAGTC3'

SEQ ID No: 3 is shown below:

5'GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACAACACTG3'

The preferred technical solution is: the kit also includes Pfu buffer, dNTPs, MgSO ₄ , PfuDNA polymerase, template DNA, and fluorescent dyes that form a PCR reaction system with primer sequences; the final concentration of the PCR reaction system is composed of:

The preferred technical scheme is: the fluorescent dye can be selected from DNA saturating dyes including EvaGreen dyes, PLUS dye, ResoLight dye, SYTO9 dye, SYBRgreen dye; the dNTP mixture includes a mixture of 10mMdATP, 10mMdCTP, 10mMdTTP, and 10mMdGTP.

A second aspect of the present invention provides a method for using the aforementioned kit, comprising the following steps:

(1) Extraction of serum total RNA: including taking the blood sample and centrifuging, taking the supernatant, adding TRIzolLSReagent reagent and glacial acetic acid, vigorously shaking and mixing for homogenization; adding chloroform for extraction after incubation, taking the aqueous phase and adding isopropanol to precipitate RNA, Centrifuge and precipitate RNA, remove the supernatant, add ethanol to wash the precipitated RNA, and finally air dry, add RNase-free water to dissolve the RNA for later use;

(2) Reverse transcription: react under the action of reverse transcriptase of SEQ ID No: 3 and the presence of RNase inhibitors, the reaction conditions are 16°C for 30min, 42°C for 40min, 85°C for 5min, and finally stand on ice or Store at -20°C;

(3) Fluorescent quantitative PCR (RT-PCR): use the primer of SEQIDNo: 1 and the primer of SEQIDNo: 2 to carry out fluorescent quantitative PCR reaction,

(4) Establish a standard curve, and analyze the results according to the absolute quantitative method.

In the present invention, the term "kit" refers to a mixture for PCR reaction, which includes reaction buffer (buffer), dNTP mixture, primers, fluorescent dyes, MgCl ₂ , and PfuDNA polymerase.

In the present invention, the term "primer" refers to an oligonucleotide, which can be natural or synthetic, and it can be used as a starting point for inducing DNA synthesis under certain conditions. The amplification product of primers with complementary strands, that is, in the presence of four different deoxyribonucleic acid triphosphates and a polymerization reagent (i.e., DNA polymerase or reverse transcriptase), in a suitable buffer and at a suitable temperature The above synthesis was carried out below. Preferred primers are single-stranded oligodeoxyribonucleotides. The appropriate length of a primer depends on the intended use of the primer, but is generally between 15 and 25 nucleotides, with shorter primer molecules generally requiring lower temperatures to form sufficiently stable hybrid complexes with the template. Primers do not have to reflect the exact sequence of the template, but must be sufficiently complementary to have hybridized to the template and prime DNA synthesis.

The present invention adopts real-time fluorescent quantitative PCR to detect the expression level of has-miR-29b-3p in the patient's serum sample, so as to quickly screen patients with bloodstream bacterial infection.

Compared with the prior art, the present invention has the following advantages and effects:

The present invention reveals for the first time that has-miR-29b-3p is elevated in serum of patients with bloodstream bacterial infection, and can be used as an indicator for rapid screening of bloodstream bacterial infection. The invention requires a small amount of blood samples and a short detection period (only a few hours). It can not only reduce the pain of patients, but also help doctors intervene in bloodstream bacterial infection in the early stage.

Description of drawings

Figure 1 is a graph showing the results of has-miR-29b-3p expression in macrophages detected by real-time fluorescent quantitative PCR after bacterial infection.

Fig. 2 Real-time fluorescent quantitative PCR detection results of has-miR-29b-3p expression in serum of healthy adults and patients.

detailed description

The above solution will be further described below in conjunction with specific embodiments. It should be understood that these examples are used to illustrate the present invention and not to limit the scope of the present invention. The implementation conditions used in the examples can be further adjusted according to the conditions required by specific applications, and the unspecified implementation conditions are usually the conditions in routine experiments.

Materials and Instruments

TRIzolLSReagent reagent, glacial acetic acid, and RNase inhibitor were all purchased from Takara Company;

Real-time fluorescence quantitative RT-PCR instrument-PE company (USA).

Example 1

1 Extraction of serum total RNA

1.1 homogenate

Plasma samples were centrifuged at 12,000×g at 4°C for 10 minutes to remove possible impurities. Take 250ul of plasma liquid and transfer it to a 1.5ml centrifuge tube, add 750μl of TRIzolLSReagent reagent and 20μl of glacial acetic acid, shake the tube vigorously by hand to mix well.

1.2 Two-phase separation

After homogenization, the samples were incubated at 15 to 30°C for 5 minutes to allow the complete dissociation of nucleic acid-protein complexes. Add 0.2ml of chloroform to every 750μl of TRIzolLSReagent reagent homogenized sample, and close the cap tightly. Shake the tube vigorously by hand for 15 seconds, and incubate at 15 to 30°C for 2 to 3 minutes. Centrifuge at 12,000 x g for 15 min at 4°C. After centrifugation, the mixed liquid will be divided into the red phenol chloroform phase of the lower layer, the colorless aqueous phase of the middle layer and the upper layer. The RNA was all partitioned into the aqueous phase. The volume of the aqueous phase was approximately 60% of the TRIzolLSReagent added during homogenization.

1.3 RNA precipitation

Transfer the aqueous phase to a new centrifuge tube, add 500 μl isopropanol, and mix well to precipitate the RNA therein. After mixing, incubate at 15 to 30°C for 10 minutes, then centrifuge at 12,000×g for 10 minutes at 4°C. At this point the RNA pellet that was not visible prior to centrifugation will form a gelatinous pellet on the bottom and sides of the tube.

1.4 RNA cleaning

Remove the supernatant, add at least 1 ml of 75% ethanol to the homogenized sample of 750 μl TRIzolLSReagent reagent, and wash the RNA pellet. After shaking, centrifuge at 7,500 x g for 5 minutes at 4°C.

1.5 Redissolving the RNA pellet

Remove the ethanol solution, dry the RNA precipitation in the air for 5-10 minutes, and dissolve the RNA by adding RNase-free water and pipetting several times with a gun, then incubate at 55 to 60°C for 10 minutes. The obtained RNA solution can be used for subsequent experiments.

2 reverse transcription

2.1 Reverse transcription primer

5'GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACAACACTG3'

2.2 Reaction system:

dNTP (2.5mMeach) 2ul

10XRTBuffer2ul

RT specific primer (1uM) 0.3ul

Total RNA800ng

MMLV reverse transcriptase (200U/ul) 0.2ul

RNase inhibitor (40u/ul) 0.3ul

RNase-free water upto20ul

Total volume 20ul

2.3 Reaction conditions:

16°C 30min; 42°C 40min; 85°C 5min

After the reaction, it was kept on ice or stored at -20°C.

3 Real-time fluorescence quantitative PCR (RT-PCR)

3.1 Primer: F: 5'GGGTAGCACCATTTGAAA3';

R: 5'TGCGTGTCGTGGAGTC3'

3.2 The system configuration is as follows:

2×Master Mix 5μl

10uM PCR-specific primer F0.5μl

10uM PCR-specific primer R0.5μl

Add water to a total volume of 8 μl

3.3 Sample addition

a. Add 8ul of the mixture to each corresponding well of the RT-PCR plate.

b. Then add the corresponding 2μl cDNA.

c. Apply parafilm carefully and centrifuge briefly to mix.

c. Place the prepared PCR plate on ice before setting up the PCR program.

3.4 Place the above RT-PCR plate on the Realtime PCR instrument for PCR reaction.

Reaction conditions:

95°C, 10 min; 40 PCR cycles (95°C, 10 seconds; 60°C, 60 seconds (collection of fluorescence)).

In order to establish the melting curve of the PCR product, after the amplification reaction, press (95°C, 10 seconds; 60°C, 60 seconds; 95°C, 15 seconds); and slowly heat from 60°C to 99°C (automatically performed by the instrument-RampRate is 0.05°C/sec).

3.5 Result Calculation Method

A standard curve was established, and the results were analyzed by the absolute quantitative method.

The fluorescent dye used in real-time fluorescent PCR is SYBRgreen, which is a DNA double-strand binding dye that emits fluorescence when it binds to a DNA double-strand, and its signal intensity represents the number of DNA double-strand molecules. Except that fluorescent dyes need to be added to the system, the real-time fluorescent quantitative PCR reaction system is exactly the same as the ordinary PCR mediated by high-fidelity enzymes.

The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the industry should understand that the present invention is not limited by the above-mentioned examples. What are described in the above-mentioned examples and descriptions are only to illustrate the principles of the present invention. The present invention also has various changes without departing from the spirit and scope of the present invention. These changes and improvements all fall within the scope of the claimed invention. The protection scope of the present invention is defined by the appended claims and their equivalents.

Claims (4)

1. one kind is detected the test kit of has-miR-29b-3p, it is characterized in that, described test kit comprises the Auele Specific Primer pair for specific amplification has-miR-29b-3p, and described primer is have the continuous nucleotide sequence of SEQIDNo:1 and have the continuous nucleotide sequence of SEQIDNo:2;

Also comprise RNA reverse transcriptase primer in described test kit, its sequence is SEQIDNo:3.

2. test kit according to claim 1, is characterized in that, described test kit also comprise form PCR reaction system with primer sequence Pfu damping fluid, dNTPs, MgSO ₄, PfuDNA polysaccharase, template DNA, fluorescence dye; The final concentration of described PCR reaction system consists of:

3. test kit according to claim 2, is characterized in that, described fluorescence dye can be selected from DNA saturability dyestuff comprise EvaGreen dyestuff, pLUS dyestuff, ResoLight dyestuff, SYTO9 dyestuff, SYBRgreen dyestuff; Described dNTP mixed solution comprises the mixed solution of 10mMdATP, 10mMdCTP, 10mMdTTP, 10mMdGTP.

4. a using method for test kit according to claim 1, comprises the steps:

(1) extraction of serum total serum IgE: comprise blood sampling centrifugal after, get supernatant, add TRIzolLSReagent reagent and Glacial acetic acid, thermal agitation mixing carry out homogenate; Add chloroform after hatching, water intaking is added to isopropanol precipitating RNA, centrifugation RNA, and add ethanol purge precipitated rna, last dry air after removing supernatant liquor, the water dissolution RNA added without RNA enzyme is for subsequent use;

(2) reverse transcription: react under the effect of the ThermoScript II of SEQIDNo:3 and under RNA enzyme inhibitors existent condition, reaction conditions 16 DEG C of 30min, 42 DEG C of 40min, 85 DEG C of 5min, finally in stand-by or-20 DEG C of preservations on ice;

(3) quantitative fluorescent PCR (RT-PCR): use the primer of SEQIDNo:1 and the primer of SEQIDNo:2 to carry out quantitative fluorescent PCR reaction,

(4) Criterion curve, analyzes result by absolute quantification method.