CN117467804A - Primer for rapidly detecting respiratory syncytial virus nucleic acid and application thereof - Google Patents

Primer for rapidly detecting respiratory syncytial virus nucleic acid and application thereof Download PDF

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CN117467804A
CN117467804A CN202311816961.5A CN202311816961A CN117467804A CN 117467804 A CN117467804 A CN 117467804A CN 202311816961 A CN202311816961 A CN 202311816961A CN 117467804 A CN117467804 A CN 117467804A
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nucleic acid
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respiratory syncytial
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syncytial virus
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CN117467804B (en
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姚雪春
王志贤
盛长忠
周泽奇
粟艳
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Dynamiker Biotechnology Tianjin Co Ltd
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Abstract

The invention discloses a primer for rapidly detecting respiratory syncytial virus nucleic acid and application thereof. The nucleic acid sequence of the primer comprises any one group of sequences shown as SEQ ID NO.1 and SEQ ID NO.2 or SEQ ID NO.1 and SEQ ID NO. 3. The invention creatively designs the primer for rapidly detecting the nucleic acid of the respiratory syncytial virus, adopts an RT-ERA combined CRISPR/Cas12a detection system, targets the N gene of the RSV, can simultaneously detect the A type RSV and the B type RSV, can finish the nucleic acid amplification and detection at constant temperature, has higher detection sensitivity and stronger specificity, does not need professional instruments and equipment and operators, and is convenient to popularize.

Description

Primer for rapidly detecting respiratory syncytial virus nucleic acid and application thereof
Technical Field
The invention belongs to the technical field of biology, and relates to a primer for rapidly detecting respiratory syncytial virus nucleic acid and application thereof.
Background
Respiratory Syncytial Virus (RSV) is a single-stranded negative-strand RNA virus with a filiform envelope, comprising two antigenic subtypes a and B. The two subtypes of RSV are generally alternately popular or co-popular, and have no obvious difference in clinical manifestation, infection age and the like, and the treatment modes are consistent. In infants, the virus commonly causes lower respiratory tract infections, including bronchiolitis and pneumonia. Therefore, accurate early diagnosis is an effective measure for timely treatment, reduction of economic burden and reduction of mortality.
The detection methods commonly used at present include nucleic acid detection techniques based on detection of pathogen-specific genes, immunodetection techniques based on detection of pathogen antigens or antibodies, and the like. Traditional diagnostic methods, such as virus culture and direct/indirect immunofluorescence, are time consuming, laborious and limited in sensitivity; antibody detection does not allow reliable data to be obtained early in viral infection; high-sensitivity nucleic acid amplification detection is the most widely used respiratory virus infection detection means at present, however, the detection needs professional detection environment and detection personnel, so that the detection cannot be popularized to basic medical institutions.
The polymerase chain reaction (polymerase chain reaction, PCR) is a currently very popular molecular detection method with short development cycle and high sensitivity and specificity, however, this method requires expensive instrumentation and requires a trained professional to operate the whole procedure. The rise of isothermal amplification technology is undoubtedly a breakthrough in the field of pathogen nucleic acid detection. The isothermal amplification technology has the advantage that the nucleic acid in the system can be amplified and identified at a specific temperature without special instruments and equipment. However, each isothermal amplification method also has different problems, wherein the most widely used loop-mediated isothermal amplification method (LAMP) is adopted, 4 primers are required to be designed for 6 areas, the method is quite complex, and the reaction temperature is high; recombinase Polymerase Amplification (RPA), while meeting substantially all of the requirements, has a relatively high probability of false positives. The detection method for combining the recombinase polymerase amplification with the CRISPR/Cas12a provided by the invention can not only meet the requirement of simple operation and no need of complex instruments, but also has the advantage of high specificity, and is very suitable for rapid detection of pathogen nucleic acid.
CN111778357a discloses a rapid detection kit and a detection method for respiratory syncytial virus nucleic acid based on CRISPR/Cas12a, wherein the method targets RSV F gene for nucleic acid detection, and adopts RT-RAA combined CRISPR/Cas12a detection system to detect type a and type B RSV respectively. The two subtypes are separately detected, and although the two subtypes have a certain guiding effect on epidemiological analysis, the detection cost is increased, and the clinical application value is not great.
Both the CN114717362A and the CN114807435A use CRISPR/Cas13a as a detection system, the Cas13a recognizes single-stranded RNA, the amplified DNA is required to be transcribed into RNA during detection, and the system is more complex than the Cas12 a; meanwhile, although the two subtypes are not distinguished, it is not clearly pointed out that the two subtypes can be detected simultaneously, and by comparing primer sequences in the patents, the type A RSV is mainly detected, whether the type B RSV can be detected can not be determined, and the detection sensitivity is low.
In summary, the current method for detecting respiratory syncytial virus nucleic acid has the problems of incapability of accurately detecting A-type and B-type RSV simultaneously, poor sensitivity and specificity and the like. How to provide a primer and a method for rapidly detecting respiratory syncytial virus nucleic acid, which can finish nucleic acid amplification and detection at constant temperature, and improve sensitivity and accuracy, has become one of the problems to be solved in the biotechnology field at present.
Disclosure of Invention
Aiming at the defects and actual demands of the prior art, the invention provides a primer for rapidly detecting respiratory syncytial virus nucleic acid and application thereof, which solve the problems that the existing respiratory syncytial virus nucleic acid detection method cannot detect A-type and B-type RSV simultaneously, has high false positive probability, high cost, complex operation, low sensitivity and the like, can finish nucleic acid amplification and detection at constant temperature, and improves the detection accuracy and sensitivity through two-round specific signal amplification.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the invention provides a primer for rapid detection of respiratory syncytial virus nucleic acid, the nucleic acid sequence of the primer comprising any one of the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 or SEQ ID NO.1 and SEQ ID NO. 3.
The existing A-type and B-type RSV gene sequences of NCBI are compared, the N gene sequences are relatively conserved, and the A-type and B-type RSV can be simultaneously identified in one reaction system by adding two sgRNAs into the system. The invention creatively designs the primer and sgRNA for rapidly detecting the respiratory syncytial virus nucleic acid, adopts the RT-ERA combined with the CRISPR/Cas12a detection system to target the RSV N gene, can simultaneously detect the A type RSV and the B type RSV, can finish nucleic acid amplification and detection at constant temperature, has higher detection sensitivity and stronger specificity, does not need professional instruments and operators, and is convenient to popularize.
The invention comprises two main systems: the first is a recombinase polymerase isothermal amplification system, so that the first round of specific signal amplification on nucleic acid is realized; the second is a CRISPR/Cas12a detection system, the specificity of detection is further improved by utilizing the targeted cleavage activity of Cas12a, and probes (ssDNA reporters) put into the system are cut off by utilizing the accompanying cleavage activity activated by the CRISPR/Cas12a, so that the signal amplification of the second round is realized, the sensitivity of the whole detection is improved, and the detection is suitable for fluorescence detection. When a distinct fluorescent signal appears, it is indicated that the virus to be detected is present in the system. The technical schematic diagram is shown in fig. 1.
SEQ ID NO.1（RSV-N-F）：
ATGGCTCTTAGCAAAGTCAAGTTGAATGATAC。
SEQ ID NO.2（RSV-N-R1）：
ATTTATGATTAGCATCTTCTGTGATTAATAACAT。
SEQ ID NO.3（RSV-N-R2）：
CTTCCTAATCTAGACATAGCATATAACATACCT。
In a second aspect, the invention provides the use of the primer for rapid detection of respiratory syncytial virus nucleic acid according to the first aspect for preparing a product for rapid detection of respiratory syncytial virus nucleic acid.
In a third aspect, the invention provides a kit comprising the primer for rapid detection of respiratory syncytial virus nucleic acid according to the first aspect.
In a fourth aspect, the invention provides the use of the primer for rapid detection of respiratory syncytial virus nucleic acid according to the first aspect for detection of respiratory syncytial virus nucleic acid.
In a fifth aspect, the invention provides a method of detecting respiratory syncytial virus nucleic acid, the method comprising:
and (3) carrying out recombinase polymerase isothermal amplification on DNA of a sample to be detected by using the primer for rapidly detecting respiratory syncytial virus nucleic acid in the first aspect, preparing a CRISPR/Cas12a detection system, and then carrying out real-time fluorescent quantitative PCR detection.
Preferably, the CRISPR/Cas12a detection system comprises: the primer for rapid detection of respiratory syncytial virus nucleic acid according to the first aspect.
Preferably, the CRISPR/Cas12a detection system further comprises: RT-ERA amplification reaction reagent, RT-ERA reaction Buffer, magnesium acetate, cas12a enzyme, ssDNA reporter, specific sgRNA, cas12a detection Buffer and nuclease-free water.
Preferably, the concentration of the amplification primer set is 0.1 to 1. Mu.M.
Specific spot values among the above 0.1 to 1. Mu.M may be selected from 0.1. Mu.M, 0.2. Mu.M, 0.3. Mu.M, 0.4. Mu.M, 0.5. Mu.M, 0.6. Mu.M, 0.7. Mu.M, 0.8. Mu.M, 0.9. Mu.M, 1. Mu.M, etc.
Preferably, the Cas12a enzyme concentration is 10-200 nM.
Specific spot values in the above 10-200 nM may be selected from 10. Mu.M, 20. Mu.M, 30. Mu.M, 40. Mu.M, 50. Mu.M, 60. Mu.M, 100. Mu.M, 150. Mu.M, 160. Mu.M, 180. Mu.M, 200 nM, etc.
Preferably, the ssDNA reporters are present in a concentration of 20-100 nM.
Specific spot values in the above 20-100 nM may be selected from 20. Mu.M, 30. Mu.M, 40. Mu.M, 50. Mu.M, 60. Mu.M, 70. Mu.M, 80. Mu.M, 90. Mu.M, 100 nM, etc.
Preferably, the concentration of the specific sgRNA is 10-200 nM.
Specific spot values in the above 10-200 nM may be selected from 10. Mu.M, 20. Mu.M, 30. Mu.M, 40. Mu.M, 50. Mu.M, 60. Mu.M, 100. Mu.M, 150. Mu.M, 160. Mu.M, 180. Mu.M, 200 nM, etc.
Preferably, the preparation method of the specific sgRNA comprises the following steps: and (3) designing sgRNA aiming at N genes of respiratory syncytial viruses, synthesizing DNA oligo corresponding to the designed sgRNA, amplifying sgDNA containing a T7 promoter and a stem-loop sequence by using an upstream primer containing the T7 promoter sequence and a downstream primer containing a target sequence through PCR, and performing in vitro transcription to obtain the specific sgRNA.
Preferably, the specific sgrnas comprise sgRNA1 and sgRNA2.
Preferably, the nucleic acid sequences of the sgRNA1 and the sgRNA2 comprise the sequences shown in SEQ ID NO.4-SEQ ID NO. 5.
SEQ ID NO.4（RSV-N-sgRNA1）：TGCACATCATAATTAGGAGT。
SEQ ID NO.5（RSV-N-sgRNA2）：TGCACATCATAATTGGGAGT。
Preferably, the ratio of sgRNA1 to sgRNA2 is 1 (1-3).
The specific point values in the above (1-3) may be 1, 2, 3, etc.
Preferably, the nucleic acid sequence of the sgDNA comprises the sequence shown in SEQ ID NO.6-SEQ ID NO. 7.
SEQ ID NO.6（RSV-N-sgDNA1）：
GAAATTAATACGACTCACTATAGGGTAATTTCTACTAAGTGTAGATTGCACATCATAATTAGGAGT。
SEQ ID NO.7（RSV-N-sgDNA2）：
GAAATTAATACGACTCACTATAGGGTAATTTCTACTAAGTGTAGATTGCACATCATAATTGGGAGT。
Preferably, the amplification targets of the primers include RSV N genes of type a and/or RSV N genes of type B.
Preferably, the sample to be tested comprises any one of plasma, pharyngeal swab, saliva or tissue.
Compared with the prior art, the invention has the following beneficial effects:
the invention creatively designs the primer for rapidly detecting the nucleic acid of the respiratory syncytial virus, adopts an RT-ERA combined CRISPR/Cas12a detection system, targets the N gene of the RSV, can simultaneously detect the A type RSV and the B type RSV, can finish the nucleic acid amplification and detection at constant temperature, has higher detection sensitivity and stronger specificity, does not need professional instruments and equipment and operators, and is convenient to popularize.
Drawings
FIG. 1 is a schematic diagram of the present invention;
FIG. 2 is a graph showing the results of the fluorescent quantitative detection of respiratory syncytial virus in example 1;
FIG. 3 is a graph showing the results of fluorescent quantitative detection of nucleic acid obtained by detecting a throat swab in example 2;
FIG. 4 is a graph showing the sensitivity of the detection of respiratory syncytial virus in example 3;
FIG. 5 is a graph showing the results of specific detection of respiratory syncytial virus in example 3.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
RT-based nucleic acid amplification kit (ERA method) and Cas12a enzyme were purchased from Souzhou first reach Gene technologies Co., ltd; hiScribe ™ T7 rapid and efficient RNA synthesis kit and purification kit Zymo RNA Clean & Concentrator-5 were purchased from Tianjin Pruis biotechnology Co., ltd; nucleic acid extraction reagents were purchased from Jifan Biotechnology (Changzhou); nucleic acid, primer and probe synthesis was performed by biological engineering (Shanghai) Inc.
Example 1
The embodiment provides a method for detecting respiratory syncytial virus by using Cas12a protein.
In this example, the RSV gene fragment was referred to the N gene fragment corresponding to the A and B subtypes of RSV in NCBI database, and the partial sequence 600 bp (RSVA-N) of the A subtype N gene and the partial sequence 600 bp (RSVB-N) of the B subtype N gene were synthesized by the biological engineering (Shanghai) Co., ltd, and constructed into pUC57 vectors, designated pUC57-RSVA-N and pUC57-RSVB-N. The detection method specifically comprises the following steps: referring to the RT-ERA isothermal amplification operation step, reacting for 20 min at 39 ℃, and amplifying to obtain a sample to be detected; preparing a Cas12a detection system, setting the reaction temperature to be 39 ℃, and performing constant-temperature reaction on 99 cycles (1 min/cycle) in a real-time fluorescence quantitative PCR instrument.
RSVA-N（SEQ ID NO.8）：
ATGGCTCTTAGCAAAGTCAAGTTGAATGATACACTCAACAAAGATCAACTTCTATCATCCAGCAAATATACCATCCAACGGAGCACAGGAGACAGCATTGACACTCCTAATTATGATGTGCAGAAACACATTAATAAGTTATGTGGCATGTTATTAATCACAGAAGATGCTAATCATAAATTCACTGGGTTAATAGGTATGTTATATGCTATGTCTAGATTAGGAAGAGAAGACACCATAAAAATACTCAAAGATGCGGGATATCATGTTAAGGCAAATGGAGTGGATGTAACAACACATCGACAAGACATCAATGGGAAAGAAATGAAATTTGAAGTGTTAACATTAGCAAGCTTAACAACTGAAATTCAAATCAACATTGAGATAGAATCTAGAAAATCCTACAAAAAAATGCTAAAAGAAATGGGAGAGGTGGCTCCAGAATACAGGCATGACTCTCCTGATTGTGGGATGATAATATTATGTATAGCAGCATTAGTAATAACCAAATTGGCAGCAGGAGATAGATCAGGTCTTACAGCTGTGATTAGGAGAGCTAATAATGTCCTAAAAAATGAAATGAAACGTTATAAAGGTTTA。
RSVB-N （SEQ ID NO.9）：
ATGGCTCTTAGCAAAGTCAAGTTGAATGATACATTGAATAAGGATCAGCTGCTGTCATCCAGCAAATACACTATTCAACGTAGTACAGGAGATAATATTGACACTCCCAATTATGATGTGCAAAAACACCTAAACAAACTATGTGGTATGCTATTAATCACTGAAGATGCAAATCATAAATTCACAGGATTAATAGGTATGCTATATGCTATGTCCAGATTAGGAAGGGAAGACACTATTAAGATACTTAAAGATGCTGGATATCATGTTAAAGCTAATGGAGTAGATGTAACAACATATCGTCAAGATATAAATGGAAAGGAAATGAAATTCGAAGTATTAACATTATCAAGCTTGACATCAGAAATACAAGTCAATATTGAGATAGAATCTAGAAAGTCCTACAAAAAAATGCTAAAAGAGATGGGAGAAGTGGCTCCAGAATATAGGCATGATTCTCCAGACTGTGGGATGATAATACTGTGTATAGCTGCCCTTGTAATAACCAAATTAGCAGCAGGAGATAGATCAGGTCTTACAGCAGTAATTAGGAGGGCAAACAATGTCTTAAAAAACGAAATAAAACGCTACAAGGGCCTA。
ssDNA reporter：5’ 6-FAM/TTAATT/3’ BHQ1。
The RT-ERA isothermal amplification reaction system is shown in Table 1.
TABLE 1
* Finally, the activator is added to the tube cover, and the short centrifugation is carried out to enable the activator to enter the reaction system, namely the reaction is started.
The CRISPR/Cas12a detection system is as in table 2.
TABLE 2
As shown in FIG. 2, pUC57-RSVA-N and pUC57-RSVB-N are added into the amplification system respectively, after amplification, the amplification products are added into the CRISPR/Cas12a detection system and detected by adopting a real-time quantitative PCR instrument, and compared with a control group, the primers have obvious differences, so that the primers can amplify both A-type and B-type RSV simultaneously, and the mixed sgRNA added into the system can detect both A-type and B-type RSV simultaneously.
Example 2
This example provides for rapid detection of respiratory syncytial virus nucleic acid in clinical samples.
In this example, nucleic acid from clinical samples collected in the Tianjin area 2023 was rapidly detected, all samples and procedures were performed in the laboratory, in this example, nucleic acid obtained from a throat swab, and CRISPR/Cas12a detection was performed on the obtained nucleic acid. This example uses a nucleic acid extraction kit (magnetic bead method) from Jifan Biotechnology (Changzhou) limited to obtain pretreated nucleic acid. Referring to the RT-ERA isothermal amplification operation step, reacting for 20 min at 39 ℃, and amplifying to obtain a sample to be detected; preparing a Cas12a detection system, setting the reaction temperature to 39 ℃, and performing constant-temperature reaction on 99 cycles (1 min/cycle) in a real-time fluorescence quantitative PCR instrument, wherein the reaction system is identical to that in the example 1.
As shown in FIG. 3, the fluorescence value of the experimental sample is significantly enhanced compared with that of the control group (nucleic acid obtained from the throat swab of a non-infected RSV person), which indicates that the detection system can realize rapid detection of RSV nucleic acid in the clinical throat swab.
Example 3
The sensitivity and the specificity of the detection method of the respiratory syncytial virus provided by the invention are analyzed.
This example uses a positive plasmid for detection. The plasmid was quantified using a Mickey digital PCR instrument and diluted to 10 6 -10 1 The templates with different concentrations were detected by using the detection method in example 1 with copies/mL as the template to be detected, the detection results are shown in FIG. 4, 10 of which are shown 4 -10 2 The detection result of the copies/mL shows that the sensitivity of the detection method can reach 10 3 The copies/mL shows that the detection method provided by the invention has high sensitivity and can be used for clinical RSV nucleic acid detection.
This example uses influenza vaccine, neisseria meningitidis, escherichia coli, cryptococcus, staphylococcus epidermidis, streptococcus pneumoniae, staphylococcus aureus as negative samples for specificity evaluation, and the samples were tested using the test method in example 1. As shown in FIG. 5, only the positive plasmid detected obvious fluorescence value, and the negative control had no obvious fluorescence value, indicating that the detection method has high specificity.
In conclusion, the primer for rapidly detecting the respiratory syncytial virus nucleic acid is creatively designed, the RT-ERA is combined with the CRISPR/Cas12a detection system to target the RSV N gene, the A-type RSV and the B-type RSV can be detected simultaneously, the nucleic acid amplification and detection can be completed at a constant temperature, the detection sensitivity is higher, the specificity is stronger, no special instrument or equipment or operators are needed, and the popularization is convenient.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

Claims (10)

1. A primer for rapidly detecting respiratory syncytial virus nucleic acid, which is characterized in that the nucleic acid sequence of the primer comprises any one group of sequences shown as SEQ ID NO.1 and SEQ ID NO.2 or SEQ ID NO.1 and SEQ ID NO. 3.
2. Use of the primer for rapid detection of respiratory syncytial virus nucleic acid according to claim 1 for the preparation of a product for rapid detection of respiratory syncytial virus nucleic acid.
3. A kit comprising the primer for rapid detection of respiratory syncytial virus nucleic acid according to claim 1.
4. Use of the primer for rapid detection of respiratory syncytial virus nucleic acid according to claim 1 for detection of respiratory syncytial virus nucleic acid.
5. A method of detecting respiratory syncytial virus nucleic acid, the method comprising:
the primer for rapidly detecting respiratory syncytial virus nucleic acid is used for carrying out isothermal amplification of DNA of a sample to be detected by recombinase polymerase to prepare a CRISPR/Cas12a detection system, and then carrying out real-time fluorescence quantitative PCR detection.
6. The method of claim 5, wherein the CRISPR/Cas12a detection system comprises: the primer for rapid detection of respiratory syncytial virus nucleic acid of claim 1;
the CRISPR/Cas12a detection system further comprises: RT-ERA amplification reaction reagent, RT-ERA reaction Buffer, magnesium acetate, cas12a enzyme, ssDNA reporter, specific sgRNA, cas12a detection Buffer and nuclease-free water;
the concentration of the amplification primer group is 0.1-1 mu M;
the concentration of the Cas12a enzyme is 10-200 nM;
the concentration of the ssDNA reporters is 20-100 nM;
the concentration of the specific sgRNA is 10-200 nM.
7. The method of claim 6, wherein the preparation of the specific sgrnas comprises: and (3) designing sgRNA aiming at N genes of respiratory syncytial viruses, synthesizing DNA oligo corresponding to the designed sgRNA, amplifying sgDNA containing a T7 promoter and a stem-loop sequence by using an upstream primer containing the T7 promoter sequence and a downstream primer containing a target sequence through PCR, and performing in vitro transcription to obtain the specific sgRNA.
8. The method of claim 6, wherein the specific sgrnas comprise sgRNA1 and sgRNA2;
the nucleic acid sequences of the sgRNA1 and the sgRNA2 comprise sequences shown in SEQ ID NO.4-SEQ ID NO. 5;
the ratio of the sgRNA1 to the sgRNA2 is 1 (1-3);
the nucleic acid sequence of the sgDNA comprises the sequences shown in SEQ ID NO.6-SEQ ID NO. 7.
9. The method of claim 5, wherein the amplification targets of the primers comprise RSV N genes of type a and/or RSV N genes of type B.
10. The method of claim 5, wherein the sample to be tested comprises any of plasma, pharyngeal swab, saliva, or tissue.
CN202311816961.5A 2023-12-26 2023-12-26 Primer for rapidly detecting respiratory syncytial virus nucleic acid and application thereof Active CN117467804B (en)

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