CN115725797A - Rubella virus fluorescent PCR kit and detection method and application thereof - Google Patents

Abstract

The invention discloses a rubella virus fluorescent PCR kit and a detection method and application thereof, wherein the rubella virus fluorescent PCR kit comprises the following components: MIX reaction solution 1, MIX reaction solution 2, positive control and negative control. The kit is simple and convenient to operate, can effectively prevent pollution, and the PCR fluorescence detection time (from the sample treatment) is only 1.5-3 hours. The PCR fluorescence detection is a totally enclosed operation, and after the sample extraction product and the reaction solution are added, the tube cover can not be opened any more, so that the pollution generation chance is reduced. The invention has the advantages of high sensitivity, strong repeatability, simple and convenient operation, quick and objective detection result and the like.

Description

Rubella virus fluorescent PCR kit and detection method and application thereof

Technical Field

The invention relates to a rubella virus fluorescent PCR kit and a detection method and application thereof, belonging to the field of gene detection.

Background

Rubella Virus (RV) is a single positive strand RNA virus that can transmit acute infectious diseases through the air. Rubella is similar to the symptoms of awake measles and is easily confused with the diagnosis of small DNA virus B19 in humans, scarlet fever, and the like. The position of differential diagnosis in laboratories is therefore becoming more and more important. The detection methods of the rubella laboratory which are commonly used at present mainly comprise virus isolation culture, serology detection, nucleic acid detection and the like. The Elisa method has low detection sensitivity and high false positive; the virus isolation and culture are time-consuming and labor-consuming, and have certain dangerousness. In recent years, the types of viruses detected by using a fluorescent PCR technology are more and more, and for detecting the viruses by using the fluorescent PCR technology, the design of primers is very important, and the specificity of a detection result is directly influenced. By designing a taqman probe and a primer with high specificity aiming at a specific sequence of the virus, the specificity of a detection result can be greatly ensured. Most of the existing rubella virus detection kits have the defects of poor specificity, low sensitivity and the like, and need to be improved

Disclosure of Invention

The invention mainly aims to provide a rubella virus fluorescent PCR kit, and aims to solve the technical problems.

In order to achieve the purpose, the rubella virus fluorescence PCR kit provided by the invention comprises the following components: MIX reaction solution 1, MIX reaction solution 2, positive control and negative control.

In one embodiment, the MIX reaction solution 1 comprises Taq polymerase, 10 Xbuffer, 25mM MgCL2 and 10mM dNTPs.

In one embodiment, the MIX reaction solution 2 comprises a pair of primers for detecting rubella virus and a probe for detecting rubella virus, wherein the 5 'end of the probe is marked with a fluorescent reporter group, and the 3' end of the probe is marked with a fluorescent quencher group.

In one embodiment, the sequence listing is shown in the following table:

name (R)	SEQ ID	Sequence (5 '→ 3')
			rubella-PF	1	GCGTCCGCTTTGAGT
rubella-PR	2	GCAGCGGGTACCCAGGC
			rubella-P	3	X1-CAAGATTGTGGACGGCGGCTGC-Y1

In one embodiment, the base sequence of the primer is shown in the sequence table SEQ ID NO.1 and SEQ ID NO. 2.

In one embodiment, the base sequence of the probe is shown as SEQ ID NO.3 of the sequence table.

In one embodiment, the fluorescent reporter group comprises any one of FAM, HEX, JOE, TET, CY3, CY5, VIC, ROX.

In one embodiment, the fluorescence quenching group comprises any one of BHQ1, BHQ2, BHQ3 and Dabcy 1.

In one embodiment, the positive control is a recombinant plasmid solution carrying the fragment of interest to be detected, and the negative control is sterile water.

In addition, the invention also provides a detection method adopting the rubella virus fluorescence PCR kit, which comprises the following steps:

s1, reagent preparation:

s11, taking out the MIX reaction solution 1 in the kit, melting and uniformly mixing at room temperature, and carrying out low-speed instant centrifugation. Taking out the MIX reaction solution 2 in the kit, carrying out low-speed instantaneous centrifugation, and placing on ice for later use;

s12, determining the reaction number N, wherein the reaction number N is at least 2 more than the number of detection samples and is calculated according to 10 mu LN MIX reaction solution 1, 3. Mu.L XN reaction solution two and 5. Mu.L XN ddH ₂ O, preparing a reaction system;

s13, shaking and uniformly mixing the mixed solution, instantaneously centrifuging for 5 seconds, and subpackaging the mixture into PCR tubes according to 18 mu L/tube;

s2, sample preparation:

and S21, taking 2 mu L of sample to be detected, and respectively adding a positive control and a negative control into the subpackaged reaction mixed solution. And (4) covering the PCR reaction tube cover tightly, and performing instantaneous low-speed centrifugation. Putting the PCR reaction tube on a machine for detection;

s3, PCR amplification detection:

s31, setting circulation parameters;

and S32, reading a sample detection result according to the Ct value of the sample to be detected.

In addition, the invention also provides application of the rubella virus fluorescence PCR kit, which is used for detecting rubella virus in vitro.

The kit disclosed by the invention adopts a fluorescent quantitative PCR technology, and the specific probes and primers for the rubella virus are designed, so that the rubella virus can be quickly, effectively and specifically detected. The designed primers and probes are compared in a GeneBank database of NCBI, and the specificity of the probes and the primers is detected.

The kit is simple and convenient to operate and can effectively prevent pollution, and the PCR fluorescence detection time (from the beginning of sample treatment) is only 1,5-3 hours. The PCR fluorescence detection is a totally-enclosed operation, and after the sample extraction product and the reaction solution are added, the tube cover can not be opened any more, so that the pollution generation chance is reduced.

The invention has the advantages of high sensitivity, strong repeatability, simple and convenient operation, quick and objective detection result and the like, and has great application prospect in the fields of clinical diagnosis, disease prevention and monitoring and the like.

Drawings

FIG. 1 is a graph showing the amplification of 30 different vaginal microorganisms including rubella virus using the kit of the present invention.

Detailed Description

The rubella virus fluorescence PCR kit provided by the invention, the detection method and the application thereof are further described in detail and completely by combining the embodiment. The following examples are illustrative only and are not to be construed as limiting the invention.

The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were all commercially available unless otherwise specified.

Example 1 preparation of kit

The sequence of this example is as follows:

wherein X1 is a fluorescence reporter group, and Y1 is a fluorescence quenching group.

The detection kit of the embodiment comprises the following components (25 parts by persons):

example 2

1. Reagent preparation

1.1, taking out the MIX reaction solution 1 in the kit, melting and uniformly mixing at room temperature, and carrying out low-speed instant centrifugation. And taking out the MIX reaction solution 2 in the kit, carrying out low-speed instantaneous centrifugation, and placing on ice for later use.

1.2, determining the reaction number N: and calculating the required reaction number according to the number of the detection samples, wherein if the number of the detection samples is N, the reaction number is N = (the number of the samples to be identified is N + negative quality control 1+ positive quality control 1), and configuring a reaction system according to the following table.

Reaction system configuration

1.3, dividing a reaction system into pipes: preparing a corresponding number of PCR reaction tubes, shaking and uniformly mixing the mixed solution, carrying out instantaneous centrifugation for 5 seconds, and subpackaging the mixture into the PCR tubes according to 18 mu L/tube.

2. Sample preparation R

And 2.1, extracting the sample to be detected by adopting a commercial DNA extraction kit. The specific operation was performed according to the instructions of the nucleic acid extraction kit.

2.2, taking 2 mu L of sample to be detected, and respectively adding a positive control and a negative control into the subpackaged reaction mixed solution. And (4) covering the PCR reaction tube cover tightly, and performing instantaneous low-speed centrifugation. And (4) performing machine detection on the PCR reaction tube.

3. PCR amplification detection

3.1, placing the PCR reaction tube in a fluorescent quantitative PCR instrument for amplification and detection, wherein the instrument adopted in the invention is an example of an elegant MA6000 fluorescent quantitative PCR instrument.

3.2, setting cycle parameters:

4. determination of Experimental effectiveness

In this example, the RV gene reporter fluorescence is FAM and the internal standard gene reporter fluorescence is ROX.

(1) Positive quality control:

the FAM channel has a typical S-type amplification curve and a Ct value less than or equal to 38.

The internal standard ROX channel has a typical S-shaped amplification curve and the Ct value is less than or equal to 38.

(2) Negative quality control:

the Ct value obtained by the FAM channel is more than or equal to 40 or no Ct value, the linear shape is a straight line or a slight oblique line, and the exponential increase time is avoided.

(4) Only if the conditions of 1,2 are met at the same time, the experiment is effective, otherwise, the experiment is ineffective.

5. Determination of test results

Using ABI 7500 as an example: and automatically storing the result after the reaction is finished, adjusting the Start Value, the end Value and the Threshold Value of a baseline according to the analyzed image (the user can automatically adjust according to the actual situation, the Start Value can be 3-15, the end Value can be 5-20, setting the Value of the Threshold Value in a Log map window, enabling a Threshold line to be positioned in the exponential phase of an amplification curve, and the amplification curve of a negative quality control product to be straight or lower than the Threshold line), clicking Analysis to automatically obtain the Analysis result, and reading the detection result in the Report window.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and all equivalent structural changes made by using the contents of the specification and drawings, or any other related technical fields, which are directly or indirectly applied to the present invention, are included in the scope of the present invention.

Claims (10)

1. The rubella virus fluorescent PCR kit is characterized in that the detection kit is used for qualitatively or quantitatively detecting rubella virus, and comprises the following components: MIX reaction solution 1, MIX reaction solution 2, positive control and negative control.

2. The rubella virus fluorescent PCR kit of claim 1, wherein the MIX reaction solution 1 comprises Taq polymerase, 10 Xbuffer, 25mM MgCL2 and 10mM dNTPs.

3. The rubella virus fluorescent PCR kit of claim 1, wherein the MIX reaction solution 2 comprises a pair of primers for detecting rubella virus and a probe for detecting rubella virus, wherein the 5 'end of the probe is labeled with a fluorescent reporter group, and the 3' end of the probe is labeled with a fluorescent quencher group.

4. The rubella virus fluorescent PCR kit according to claim 3, wherein the base sequence of the primer is shown in SEQ ID No.1 and SEQ ID No.2 of the sequence Listing.

5. The rubella virus fluorescent PCR kit according to claim 4, wherein the base sequence of the probe is shown as SEQ ID No.3 of the sequence table.

6. The rubella virus fluorescent PCR kit of claim 3, wherein the fluorescent reporter group comprises any one of FAM, HEX, JOE, TET, CY3, CY5, VIC, ROX.

7. The rubella virus fluorescent PCR kit of claim 3, wherein the fluorescence quenching group comprises any one of BHQ1, BHQ2, BHQ3, dabcy 1.

8. The rubella virus fluorescent PCR kit of any one of claims 1-7, wherein the positive control is a recombinant plasmid solution carrying a fragment of interest for detection and the negative control is sterile water.

9. A method of detection using the rubella virus fluorescent PCR kit of any one of claims 1-8, comprising the steps of:

s1, reagent preparation:

s11, taking out the MIX reaction solution 1 in the kit, melting and uniformly mixing at room temperature, carrying out low-speed instant centrifugation, taking out the MIX reaction solution 2 in the kit, carrying out low-speed instant centrifugation, and placing on ice for later use;

s12, determining the reaction number N, wherein the reaction number N is at least 2 more than the number of detection samples, and according to 10. Mu.L × N MIX reaction solution 1, 3. Mu.L × N reaction solution two and 5. Mu.L × N ddH ₂ O, preparing a reaction system;

s13, shaking and uniformly mixing the mixed solution, carrying out instantaneous centrifugation for 5 seconds, and subpackaging the mixture into PCR tubes according to 18 mu L/tube;

s2, sample preparation:

s21, taking 2 mu L of sample to be detected, performing positive control and negative control, respectively adding the sample to the subpackaged reaction mixed solution, tightly covering a PCR reaction tube cover, performing instantaneous low-speed centrifugation, and performing machine detection on the PCR reaction tube;

s3, PCR amplification detection:

s31, setting circulation parameters;

and S32, reading a sample detection result according to the Ct value of the sample to be detected.

10. Use of the rubella virus fluorescent PCR kit according to any one of claims 1-8 for in vitro detection of rubella virus.

Images