阅读说明：本技术 用于恒温扩增检测麻疹病毒的核酸序列、试剂盒及方法和应用 (Nucleic acid sequence, kit and method for detecting measles virus by isothermal amplification and application ) 是由 苏秀兰 温永俊 韩汶延 段蓉 郑利英 于 2019-01-08 设计创作，主要内容包括：本发明涉及病毒检测领域,具体讲,涉及一种用于恒温扩增检测麻疹病毒的核酸序列、试剂盒及检测方法和应用。本发明的核酸序列包含SEQ ID No.1和SEQ ID No.2所示的链置换引物序列、SEQ ID No.3和SEQ ID No.4所示的探针和SEQ ID No.5所示的交叉扩增引物序列。本发明试剂盒及检测方法具有特异性好、灵敏度高、步骤简单、可重复性高的技术优势。(The invention relates to the field of virus detection, in particular to a nucleic acid sequence for detecting measles virus by isothermal amplification, a kit, a detection method and application. The nucleic acid sequence of the invention comprises a strand displacement primer sequence shown by SEQ ID No.1 and SEQ ID No.2, a probe shown by SEQ ID No.3 and SEQ ID No.4 and a cross amplification primer sequence shown by SEQ ID No. 5. The kit and the detection method have the technical advantages of good specificity, high sensitivity, simple steps and high repeatability.)

1. A nucleic acid sequence for isothermal amplification detection of measles virus, comprising the strand displacement primer sequence shown in SEQ ID No.1 and SEQ ID No.2, the probe shown in SEQ ID No.3 and SEQ ID No.4 and the cross amplification primer sequence shown in SEQ ID No. 5.

2. The nucleic acid sequence of claim 1, wherein the 5 'end of the probe sequence shown in SEQ ID No.3 is linked to fluorescein, and the 5' end of the probe sequence shown in SEQ ID No.4 is linked to biotin.

3. The kit for detecting measles virus by isothermal amplification is characterized by comprising an isothermal amplification reagent, wherein the isothermal amplification reagent contains a strand displacement primer sequence shown by SEQ ID No.1 and SEQ ID No.2, a probe shown by SEQ ID No.3 and SEQ ID No.4 and a cross amplification primer sequence shown by SEQ ID No. 5.

Preferably, the 5 'end of the probe sequence shown in SEQ ID No.3 is connected with fluorescein, and the 5' end of the probe sequence shown in SEQ ID No.4 is connected with biotin.

4. The kit according to claim 3, wherein the concentrations of the strand displacement primers shown in SEQ ID No.1 and SEQ ID No.2 are 0.1. mu.M, and the concentrations of the cross amplification primers shown in SEQ ID No.3 and SEQ ID No.4 are 0.3-0.5. mu. M, SEQID No.5 are 0.6. mu.M.

5. The kit of claim 4, wherein the isothermal amplification reagent further comprises 1 × BstBuffer, betaine, MgSO₄dNTPs, RNase protectant, Bst DNA polymerase and AMV reverse transcriptase;

preferably, the isothermal amplification reagent contains 20mM Tris-HCl (pH 8.8), 10mM KCl, and 10mM (NH)₄)₂SO₄、1M MgSO₄10 × Thermo pol buffer, 5M betaine, 8U/. mu.l Bst DNA polymerase, 10U AMV reverse transcriptase, 10mM dNTPs, 25 × RNA secure.

6. The kit of claim 3, further comprising an RNA extraction reagent.

7. The kit according to claim 3, wherein the kit further comprises a positive control and a negative control, wherein the positive control is an in vitro transcription product of the measles virus N gene fragment; the negative control was sterilized deionized water.

8. The kit of claim 3, further comprising a test strip;

preferably, the test strip is selected from a vertical flow nucleic acid test strip anti-pollution detection device, the vertical flow nucleic acid test strip anti-pollution detection device comprises a liner, the liner is sequentially provided with a sample pad, a colored particle conjugate pad, a fiber membrane and a water absorption filter paper pad from bottom to top, and the parts of the liner are overlapped at adjacent parts; the fiber membrane is sequentially provided with a detection line and a quality control line, the detection line is coated with an anti-fluorescein antibody, the quality control line is coated with biotin, and the colored particles on the colored particle conjugate pad are coated with avidin.

9. A method for isothermal amplification detection of measles virus comprising at least the steps of:

(1) extracting virus RNA from a sample to be detected;

(2) adding the viral RNA extracted in the step (1) into a PCR tube filled with a constant-temperature amplification reagent as a template; the isothermal amplification reagent contains strand displacement primer sequences shown by SEQ ID No.1 and SEQ ID No.2, probes shown by SEQ ID No.3 and SEQ ID No.4 and a cross amplification primer sequence shown by SEQ ID No. 5;

preferably, carrying out amplification reaction at 58-62 ℃ for 80-100 min, and carrying out enzyme inactivation at 78-82 ℃ for 2-3 min;

more preferably, the isothermal amplification conditions are: amplification reaction at 60 deg.C for 90min, and enzyme inactivation at 80 deg.C for 2 min.

(3) The detection is carried out by adopting a detection test strip, and preferably, the detection is carried out by adopting a vertical flow nucleic acid test strip anti-pollution detection device.

10. Use of a nucleic acid sequence according to claim 1 in the detection of measles virus.

Technical Field

The invention relates to the field of virus detection, in particular to a nucleic acid sequence, a kit, a method and application for detecting measles virus by isothermal amplification.

Background

Following the global elimination of polio, the World Health Organization (WHO) has targeted the elimination of measles as the next goal. With the continuous efforts of various countries, the number and incidence of measles have been decreasing in recent years, but they have not yet reached the elimination level. Measles is an acute respiratory infectious disease caused by measles virus. Measles Virus (MV) belongs to the genus measles virus of the family paramyxoviridae, is highly infectious and is often associated with manifestations of fever, rash, cough, conjunctivitis, and the like. The incidence rate is high, and measles and serious complications including giant cell pneumonia, inclusion body encephalitis and the like can be caused after the infection of a human body. Although the incidence and mortality of measles has decreased dramatically in recent years with the use of measles vaccine on a large scale, local outbreaks of measles still occur in various places. Early diagnosis and preventive measures can effectively reduce the incidence of diseases. Because measles is very similar to the symptoms related to diseases such as acute eruption, drug eruption, rubella and the like of infants, the identification difficulty is increased.

At present, the detection methods commonly used in clinic include enzyme-linked immunosorbent assay (enzyme-linked immuno sorbent assay, E L ISA), real-time fluorescence quantitative PCR, etc., wherein the E L ISA is a traditional method applied to early measles diagnosis, a patient does not produce IgM in vivo or the level of the produced IgM is low at the early stage of measles onset, and usually can rise to the peak within 10-15 days, so that a serum sample is detected by using the E L ISA method within 3 days after the measles onset, a false negative result may occur, if the result is determined, a misdiagnosis phenomenon may occur, and treatment is delayed.

Disclosure of Invention

The primary object of the present invention is to propose a nucleic acid sequence for isothermal amplification for the detection of measles virus.

The second invention aims to provide a kit for detecting measles virus by isothermal amplification.

The third invention of the present invention is to propose a method for isothermal amplification detection of measles virus.

The fourth object of the present invention is to propose the use of this nucleic acid sequence for the detection of measles virus.

In order to achieve the purpose of the invention, the technical scheme is as follows:

the invention relates to a nucleic acid sequence for detecting measles virus by isothermal amplification, which comprises a strand displacement primer sequence shown by SEQ ID No.1 and SEQ ID No.2, a probe shown by SEQ ID No.3 and SEQ ID No.4 and a cross amplification primer sequence shown by SEQ ID No. 5.

Preferably, the 5 'end of the probe sequence shown in SEQ ID No.3 is connected with fluorescein, and the 5' end of the probe sequence shown in SEQ ID No.4 is connected with biotin.

The invention also relates to a kit for detecting measles virus by isothermal amplification, which comprises an isothermal amplification reagent, wherein the isothermal amplification reagent contains a strand displacement primer sequence shown by SEQ ID No.1 and SEQ ID No.2, a probe shown by SEQ ID No.3 and SEQ ID No.4 and a cross amplification primer sequence shown by SEQ ID No. 5.

Preferably, the 5 'end of the probe sequence shown in SEQ ID No.3 is connected with fluorescein, and the 5' end of the probe sequence shown in SEQ ID No.4 is connected with biotin.

Preferably, the concentration of the strand displacement primers shown in SEQ ID No.1 and SEQ ID No.2 is 0.1 mu M, SEQ. mu.M, the concentration of the probes shown in SEQ ID No.3 and SEQ ID No.4 is 0.3-0.5. mu.M, and the concentration of the cross amplification primer shown in SEQ ID No.5 is 0.6. mu.M.

Preferably, the isothermal amplification reagent also contains 1 × Bst Buffer, betaine, MgSO4, dNTPs, RNase protective agent, Bst DNA polymerase and AMV reverse transcriptase;

preferably, the isothermal amplification reagent contains 20mM Tris-HCl(pH＝8.8)、10mM KCl、10mM(NH₄)₂SO₄、1M MgSO₄10 × Thermo pol buffer, 5M betaine, 8U/. mu.l Bst DNA polymerase, 10U AMV reverse transcriptase, 10mM dNTPs, 25 × RNA secure.

Preferably, the kit further comprises an RNA extraction reagent.

Preferably, the kit also comprises a positive control and a negative control, wherein the positive control is an in vitro transcription product of the measles virus N gene segment; the negative control was sterilized deionized water.

Preferably, the kit further comprises a test strip; preferably, the test strip is selected from a vertical flow nucleic acid test strip anti-pollution detection device, the vertical flow nucleic acid test strip anti-pollution detection device comprises a liner, the liner is sequentially provided with a sample pad, a colored particle conjugate pad, a fiber membrane and a water absorption filter paper pad from bottom to top, and the parts of the liner are overlapped at adjacent parts; the fiber membrane is sequentially provided with a detection line and a quality control line, the detection line is coated with an anti-fluorescein antibody, the quality control line is coated with biotin, and the colored particles on the colored particle conjugate pad are coated with avidin.

The invention also relates to a method for detecting measles virus by isothermal amplification, which at least comprises the following steps:

(1) extracting virus RNA from a sample to be detected;

(2) adding the viral RNA extracted in the step (1) into a PCR tube filled with a constant-temperature amplification reagent as a template; the constant temperature amplification reagent contains strand displacement primer sequences shown by SEQ ID No.1 and SEQ ID No.2, probes shown by SEQ ID No.3 and SEQ ID No.4 and a cross amplification primer sequence shown by SEQ ID No. 5;

preferably, carrying out amplification reaction at 58-62 ℃ for 80-100 min, and carrying out enzyme inactivation at 78-82 ℃ for 2-3 min;

more preferably, the isothermal amplification conditions are: amplification reaction at 60 deg.C for 90min, and enzyme inactivation at 80 deg.C for 2 min.

(3) The detection is carried out by adopting a detection test strip, and preferably, the detection is carried out by adopting a vertical flow nucleic acid test strip anti-pollution detection device.

The invention also relates to the use of the above nucleic acid sequences in the detection of measles virus.

The technical scheme of the invention at least has the following beneficial effects:

the nucleic acid sequence for detecting the measles virus by isothermal amplification has good specificity, and the nucleic acid sequence of the invention is added with two specific probes in the detection process, so the accuracy of the result is ensured.

The detection method has high sensitivity. Compared with agarose gel electrophoresis, the detection sensitivity is improved by nearly 100-200 times.

The detection method of the invention has simple steps and high repeatability: the method only needs simple operation treatment according to the instruction of the kit, and the whole reaction process does not need complex instruments.

The detection method also has the technical advantages of quick and totally-enclosed detection. The single sample takes only 2 hours from sample processing to complete detection. The PCR tube cover does not need to be opened in the whole amplification and detection process, so that the chance of amplification product pollution is reduced.

The detection method has good applicability, and can simultaneously meet the requirements of sample detection with high flux and low flux.

Description of the drawings:

FIG. 1 is a schematic structural diagram of an anti-contamination detection device of a vertical flow nucleic acid test strip;

FIG. 2 is a schematic diagram showing the principle of positive results of the anti-contamination detection device for vertical flow nucleic acid test strip;

FIG. 3 is a graph showing comparative results of example 3;

FIG. 4 is a graph showing comparative results of example 4;

FIG. 5 is a graph showing comparative results of example 5;

FIG. 6 is a graph showing the results of detection in example 6;

FIG. 7 is a graph showing the results of detection in example 7.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Detailed Description

Aiming at the defects in the prior art, the invention provides a constant-temperature amplification detection nucleic acid sequence, a kit and a method which are suitable for basic units and field application and can quickly, sensitively and accurately detect measles virus nucleic acid.

The embodiment of the invention relates to a nucleic acid sequence for detecting measles virus by isothermal amplification, which comprises a strand displacement primer sequence shown in SEQ ID No.1 and SEQ ID No.2, a probe shown in SEQ ID No.3 and SEQ ID No.4 and a cross amplification primer sequence shown in SEQ ID No. 5.

Wherein, the 5 'end of the probe sequence shown in SEQ ID No.3 is connected with fluorescein, and the 5' end of the probe sequence shown in SEQ ID No.4 is connected with biotin.

The specific nucleotide sequence of the primer sequence is shown in table 1:

table 1:

optionally, the fluorescein attached to the 5' end of the primer sequence shown in SEQ ID No.3 is selected from FITC, but is not limited thereto.

The nucleic acid sequence of the embodiment of the invention comprises two strand displacement primer sequences, one cross primer and two detection probes. The 5 oligonucleotide sequences in the embodiment of the invention depend on the high-activity strand displacement characteristic of Bst DNA polymerase, so that the self-amplification cycle of the strand displacement DNA synthesis is continuous.

The embodiment of the invention also relates to a kit for detecting measles virus by isothermal amplification, which comprises an isothermal amplification reagent, wherein the isothermal amplification reagent contains strand displacement primer sequences shown by SEQ ID No.1 and SEQ ID No.2, probes shown by SEQ ID No.3 and SEQ ID No.4 and a cross amplification primer sequence shown by SEQ ID No. 5.

Preferably, the 5 'end of the probe sequence shown in SEQ ID No.3 is connected with fluorescein, and the 5' end of the probe sequence shown in SEQ ID No.4 is connected with biotin. Optionally, the fluorescein attached to the 5' end of the primer sequence shown in SEQ ID No.3 is selected from FITC, but is not limited thereto.

Optionally, the concentrations of the strand displacement primers shown in SEQ ID No.1 and SEQ ID No.2 are both 0.1 μ M, SEQ IDNo.3 and 0.5 μ M for the probes shown in SEQ ID No.4, and 0.6 μ M for the cross amplification primer shown in SEQ ID No. 5. Further preferably, the concentration of the probes shown in SEQ ID No.3 and SEQ ID No.4 is 0.3. mu.M each. The embodiment of the invention optimizes the concentration of the primer probe, so that the detection sensitivity of the kit can reach 100 copies of molecules/reaction, thereby meeting the requirement of rapidly detecting measles virus.

Optionally, the isothermal amplification reagent further comprises 1 × Bst Buffer, betaine, MgSO₄dNTPs, RNase protectant, Bst DNA polymerase and AMV reverse transcriptase;

preferably, each 20. mu.l of isothermal amplification reagent contains 2. mu.l of 1 × Bst Buffer, 4. mu.l of betaine and MgSO₄0.6. mu.l, 0.8. mu.l dNTPs, 0.8. mu.l RNase protectant, 1. mu.l Bst DNA polymerase, 0.1. mu.l AMV reverse transcriptase.

Wherein the unit of Bst DNA polymerase activity is 8U/. mu.l, and the unit of AMV reverse transcriptase activity is 10U/. mu.l.

Wherein 1 × Bst Buffer contains 20mM Tris-HCl (pH 8.8), 10mM KCl, and 10mM (NH)₄)₂SO₄、10×Thermo pol buffer。

Preferably, MgSO₄The concentration of (A) is 1M, the concentration of dNTPs is 10mM, the concentration of betaine is 5M, and the RNase protective agent adopts 25 × RNA secure.

Preferably, the isothermal amplification reagent contains 20mM Tris-HCl (pH 8.8), 10mM KCl, and 10mM (NH)₄)₂SO₄、1M MgSO₄10 × Thermo pol buffer, 5M betaine, 8U/. mu.l Bst DNA polymerase, 10U AMV reverse transcriptase, 10mM dNTPs, 25 × RNAtech.

Optionally, the kit further comprises an RNA extraction reagent. Preferably, the German Qiagen viral RNA extraction kit (Qia-52904) is used.

Optionally, the kit further comprises a positive control and a negative control, so that the amplification accuracy can be further ensured. The positive control is an in vitro transcription product of the measles virus N gene segment, and the sequence number is MG 181208.1; the negative control was sterilized deionized water.

Optionally, the kit further comprises a test strip; preferably, the test strip is selected from a vertical flow nucleic acid test strip anti-contamination detection device, and the schematic structural diagram is shown in fig. 1. As shown in figure 1, the vertical flow nucleic acid test strip anti-pollution detection device comprises a pad 1, wherein the pad is sequentially provided with a sample pad 4, a colored particle conjugate pad 3, a fiber membrane 2 and a water absorption filter paper pad 5 from bottom to top, and the parts are partially overlapped at the adjacent parts; the fiber membrane 2 is sequentially provided with a detection line 7 and a quality control line 6, the detection line 7 is coated with an anti-fluorescein antibody, the quality control line 6 is coated with biotin, and the colored particles on the colored particle conjugate pad 3 are coated with avidin.

The schematic diagram of the positive result of the anti-pollution detection device for the vertical flow nucleic acid test strip is shown in fig. 2. As shown in FIG. 2, the positive amplification product dropped on the sample pad can be combined with the colored particles on the colored particle conjugate pad due to the biotin label and the FITC label, and under the siphon action of the absorbent filter paper pad, the positive amplification product combined with the colored particles moves upwards and is captured by FITC antibody on the detection line, and stays for color development to show positive reaction.

Further alternatively, the specific composition of the kit of the embodiment of the present invention is shown in table 2:

TABLE 2

The embodiment of the invention also relates to a method for detecting measles virus by isothermal amplification, which at least comprises the following steps:

(1) extracting virus RNA from a sample to be detected by using an RNA extraction reagent; the operation is carried out by specifically referring to the kit instruction;

(2) adding the RNA extracted in the step (1) as a template into a PCR tube filled with a constant-temperature amplification reagent;

the specific amplification system is as follows: adding 18 mul of isothermal amplification reagent into 2 mul of template for 20 mul in total for isothermal amplification;

preferably, the isothermal amplification conditions are: carrying out amplification reaction at the temperature of 58-62 ℃ for 80-100 min, and carrying out enzyme inactivation at the temperature of 78-82 ℃ for 2-3 min;

more preferably, the isothermal amplification conditions are: performing amplification reaction at 60 deg.C for 90min, and inactivating enzyme at 80 deg.C for 2 min;

(3) the detection is carried out by adopting a detection test strip, and preferably, the detection is carried out by adopting a vertical flow nucleic acid test strip anti-pollution detection device.

And judging and reading the experimental result after 5 min: when the quality control line (C line) and the detection line (T line) of the test strip are red, the test strip is positive, and the measles virus nucleic acid is contained in the sample; when the quality control line (line C) of the test strip is red and the detection line (line T) is colorless, the test strip is negative, which indicates that the sample does not contain measles virus nucleic acid; when the quality control line (line C) of the test strip is colorless, whether the detection line (line T) is red or not indicates that the experiment fails, and the sample needs to be repeatedly detected.

The 5 oligonucleotide primers in the kit of the embodiment of the invention perform continuous DNA strand displacement amplification circulation on cDNA generated by reverse transcription of AMV reverse transcriptase under the high-activity strand displacement characteristic of Bst DNA polymerase, and apply a vertical flow nucleic acid test strip detection system to the detection of a constant-temperature amplification product, thereby establishing the constant-temperature amplification detection method of measles virus nucleic acid. The measles virus isothermal amplification detection kit and the detection method thereof provided by the invention have very good signal-to-noise ratio, and the designed primers and probes have very good specificity, so that the measles virus and other genetically or clinically related viruses can be accurately distinguished.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.