CN117187417A - Primer, probe, kit and application for detecting drug-resistant gene arr2 by RAA fluorescence method - Google Patents
Primer, probe, kit and application for detecting drug-resistant gene arr2 by RAA fluorescence method Download PDFInfo
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Abstract
The application relates to the technical field of biological detection, and discloses a primer, a probe, a kit and application for detecting a drug-resistant gene arr2 by a RAA fluorescence method, wherein the primer comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO.2, and the probe comprises a probe with a sequence shown as SEQ ID NO.3 or a modified probe based on the sequence shown as SEQ ID NO. 3. The application effectively improves the detection efficiency and sensitivity of the pseudomonas aeruginosa drug resistance gene arr2.
Description
Technical Field
The application relates to the technical field of biological detection, in particular to a primer, a probe, a kit and application for detecting a drug-resistant gene arr2 by an RAA fluorescence method.
Background
In recent years, the wide occurrence of antibiotic resistance has led to an increase in resistance to pathogenic bacteria. Pseudomonas aeruginosa is a common conditional pathogenic bacteria of Pseudomonas and is also a main pathogenic bacteria of the intensive care unit infection of the trimethyl hospital, and has wide distribution, is not pathogenic under normal conditions, but when the human body has weak resistance, the infection rate of the Pseudomonas aeruginosa is increased. After the human body is infected with pseudomonas aeruginosa, the diseases such as lung, urinary tract, wound, blood (such as septicemia), eye and the like can be caused. As the pseudomonas aeruginosa can be attached on the surface of the contact lens to form a biological film, the growth capacity is extremely strong, the treatment is difficult, the cornea infection of eyes is extremely easy to occur under the condition that the contact lens is not thoroughly cleaned, and the blindness can be caused in serious cases. The rapid and efficient detection mode for the pseudomonas aeruginosa drug resistance gene is beneficial to preventing the spread of diseases and the establishment of clinical diagnosis and treatment schemes.
The detection methods of pathogenic bacteria in the laboratory and clinical platform at present comprise the following types: traditional detection methods (smear microscopy, bacterial isolation culture), immunological methods (relying on specific binding of antigen to antibody), molecular biological methods (gene chip technology, nucleic acid hybridization technology, DNA sequencing technology, various PCR-based technologies, novel molecular detection technologies, etc.). The traditional detection method can directly dye the pathogenic bacteria smear, and the microscopic examination process is simple, convenient and quick, but consumes longer time, requires complex instruments and specialized detection platforms, and is not suitable for basic experiment platforms and detection in remote areas. The novel molecular detection technique LAMP technology is a novel technology developed by Japanese researchers Notomi and the like in 2000 for in vitro isothermal amplification of specific nucleic acid fragments, and utilizes two pairs of special primers and BstDNA polymerase with strand displacement activity to enable a circular single-stranded structure to appear at the joint of the primers at two ends of a template in a circulating manner, so that the primers are smoothly combined with the template under isothermal conditions to perform strand displacement amplification reaction, but the technology requires multiple pairs of primers to perform loop isothermal amplification, thereby increasing the complexity of detection.
Based on the isothermal amplification technology RAA mediated by the recombinase, instead of the traditional polymerase chain reaction, the technology is originally developed by Cambridge Twistdx company in England, and the recombinase-assisted amplification technology developed by Jiangsu Qihai gene biotechnology Co., ltd is now becoming a molecular tool for identifying a plurality of pathogens. RAA is a detection method based on RPA, except that the key component of RPA is the recombinase UVSX of T4 phage, whereas the recombinases of RAA (SC-recA, BS-recA, rad 51) are obtained from E.coli and can be tightly bound to primer DNA at room temperature. The RAA technology utilizes single-chain binding protein, DNA polymerase and recombinase to amplify nucleic acid under isothermal (38-41 ℃) condition, and when fluorescent probes are added into a reaction system, real-time detection can be carried out, detection results can be output within 5-20 minutes, and the sensitivity is high.
Based on the above, if the RAA fluorescence method can be applied to the detection of the drug resistance gene of Pseudomonas aeruginosa, the detection efficiency and sensitivity of the drug resistance gene will be improved.
Disclosure of Invention
Therefore, the application aims to provide a primer, a probe, a kit and application for detecting the drug-resistant gene arr2 by using the RAA fluorescence method, and the detection efficiency and the sensitivity of the drug-resistant gene arr2 of pseudomonas aeruginosa are effectively improved.
The application solves the technical problems by the following technical means:
the first aspect of the application provides a primer for detecting a drug-resistant gene arr2 by a RAA fluorescence method, which comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2.
In a second aspect, the application provides a probe for detecting a drug-resistant gene arr2 by a RAA fluorescence method, which comprises a probe with a sequence shown as SEQ ID NO.3 or a modified probe based on the sequence shown as SEQ ID NO. 3.
In combination with the second aspect, in some embodiments, the modification is to mark a tetrahydrofuran base site as a recognition site I of the exonuclease at a position greater than or equal to 35nt from the 5' end of the probe sequence, add T bases on both sides, T at the 5' end adds FAM fluorophore, T at the 3' end adds quencher, and recognize and cleave THF sites by the exonuclease.
In a third aspect, the present application provides a kit for detecting a drug-resistant gene arr2 by RAA fluorescence, the kit comprising the above primer and/or the above probe.
With reference to the third aspect, in some embodiments, the kit further includes a positive quality control and a negative quality control, the positive quality control being a kit containing pseudomonas aeruginosa drug resistance gene arr2The negative quality control is ddH 2 O or purified water.
In a fourth aspect, the present application provides the use of a primer or probe or kit as described above for detecting in vitro the presence or absence of the pseudomonas aeruginosa drug resistance gene arr2 in a sample, said use being for non-diagnostic purposes.
In combination with the fourth aspect, in some embodiments, the final concentration of the primer in the reaction system is independently 10. Mu. Mol/L and the final concentration of the probe in the reaction system is 10. Mu. Mol/L.
The application has the following beneficial effects:
(1) The sensitivity of the reaction detection target DNA is high, the minimum detection limit can be as low as 10 copies/. Mu.L, and the designed primer has high specificity;
(2) The fluorescent probe is introduced into the basic reaction unit, so that the timing and quantitative visual analysis of the result can be realized, the probe is designed into a sequence in the middle of the primer, the whole reaction process is simple, convenient and quick, the variability of negative and positive changes is observed through a fluorescent amplification curve, the method is simple and convenient, and aerosol pollution caused by uncapping after the reaction is avoided;
(3) The application can be carried out under isothermal conditions when detecting the pseudomonas aeruginosa drug-resistant gene arr2, and only a constant temperature environment and a fluorescence detector capable of detecting FAM are needed, thereby reducing the labor cost and the instrument cost required by experiments;
(4) The probe contains a fluorescence report group and a quenching group, a tetrahydrofuran base site (THF) is marked at the middle position with the distance of more than or equal to 35nt from the 5' end and is used as a recognition site I of exonuclease, two T bases are added at two sides, FAM fluorescent groups are added at the T of the 5' end, the quenching group is added at the T of the 3' end, the THF site is recognized and cut by the exonuclease, when THF and a target sequence are combined and cut off by restriction endonuclease, the two groups are separated, signals are emitted, and fluorescent signals are collected and rapidly detected in an instrument with a fluorescence detection function, so that the probe has strong sensitivity.
Drawings
FIG. 1 is a graph showing quantitative amplification of the drug-resistant gene arr2 detected by the RAA fluorescence method of the present application;
FIG. 2 is a graph showing the results of detection of the sensitivity of the arr2 plasmid.
Detailed Description
The following description of the embodiments of the present application will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
The following examples were conducted under conventional conditions or conditions recommended by the manufacturer, without specifying the specific conditions. The raw materials, equipment or instruments used are conventional products commercially available without identifying the manufacturer.
The application is based on the rapid detection of pseudomonas aeruginosa drug resistance gene by recombinase-mediated isothermal amplification technology, is formed by using 1 pair of special primers and probes with fluorescence detection capability, specifically recognizes the gene on a target sequence, realizes amplification reaction under isothermal condition (39-42 ℃), overcomes the defects of single-chain template acquisition, long detection time, easy pollution, high cost and the like in the traditional PCR reaction through a thermal denaturation process, and simultaneously, the method is carried out under mild temperature condition, has simple operation, low technical quality requirements on detection personnel, and is convenient for realizing rapid screening of large samples with low cost.
At present, the related inspection of food microbiology is mainly carried out in China by using the microorganism separation culture and morphological identification and combining the biochemical identification, the serological typing identification and other general methods, the preliminary identification generally needs 2-3 days, and the final completion of the identification report needs 10-15 days, but the detection by adopting the kit only needs 8 hours from the collection of a sample to the detection completion, and the reaction of the kit can be detected in a fluorescent instrument in a short time, so that the kit is more visual and clear.
Example 1 primers and probes
In the embodiment, primers and probes for carrying out RAA fluorescence detection on the pseudomonas aeruginosa drug resistance gene arr2 are designed according to the conserved sequence of the specific gene in the pseudomonas aeruginosa drug resistance gene arr2 and are used for qualitatively and quantitatively detecting the pseudomonas aeruginosa drug resistance gene arr2 in a sample. The full-length sequence of the pseudomonas aeruginosa drug resistance gene arr2 is as follows:
Cccagaaccttttatatttgtctcagtggttgcgccggccaatgccggttttgcgttggctgaaaaataaacagccacatatgagtgccaagt
tactacgttctaacaatcggctgcacggcgaccggttttccgctgcttcgcagctacaaaccggcgcgtgagccgggcgttatgcagccaaatcc
caacaattaagggtcttaaaATGGTAAAAGATTGGATTCCCATCTCTCATGATAATTACAAGCAGGT
GCAAGGACCGTTCTATCATGGAACCAAAGCCAATTTGGCGATTGGTGACTTGCTAACCA
CAGGGTTCATCTCTCATTTCGAGGACGGTCGTATTCTTAAGCACATCTACTTTTCAGCCT
TGATGGAGCCAGCAGTTTGGGGAGCTGAACTTGCTATGTCACTGTCTGGCCTCGAGGGT
CGCGGCTACATATACATAGTTGAGCCAACAGGACCGTTCGAAGACGATCCGAATCTTAC
GAACAAAAAATTTCCCGGTAATCCAACACAGTCCTATAGAACCTGCGAACCCTTGAGAA
TTGTTGGCGTTGTTGAAGACTGGGAGGGGCATCCTGTTGAATTAATAAGGGGAATGTTG
GATTCGTTAGAGGACTTAAAGCGCCGTGGTTTACACGTCATTGAAGACTAG(SEQ ID NO.4)
and (3) selecting a conserved sequence of the pseudomonas aeruginosa drug resistance gene arr2 for RAA detection, wherein the sequence of the conserved region is as follows:
TTCCCATCTCTCATGATAATTACAAGCAGGTGCAAGGACCGTTCTATCATGGAACCAAAGCCAATTTGGCGATTGGTGACTTGCTAACCACAGGGTTCATCTCTCATTTCGAGGACGGTCGTATTCTTAAGCACATCTACTTTTCAGCCTTGATGGAGCCAGCAGTTTGGGGAGCTGAACTTGCTATGTCACTGTCTGGCCTCGA(SEQ ID NO.5)
based on the above, first, primer screening was performed by using RAA non-fluorescent method kit manufactured by Jiangsu Hope gene biotechnology Co., ltd, and the screening range is shown in Table 1. The PA-arr2-primer1-fw/PA-arr2-primer1-rv with high amplification efficiency and early peak time is screened out by the efficiency of amplifying fluorescence and the peak time in the same time and is used as a primer for detecting the drug-resistant gene arr2 by the RAA fluorescence method.
Table 1:
a fluorescent reporter group (FAM) and a fluorescent quenching group (BHQ) are adopted to modify a probe, a tetrahydrofuran base site is marked at the middle position which is more than or equal to 35nt away from the 5' end of the probe sequence and is used as a recognition site I of the exonuclease, T bases are respectively added at two sides, the FAM fluorescent group is added at the T of the 5' end, the quenching group is added at the T of the 3' end, and the THF site is recognized and cut by the exonuclease.
The sequence of the probe is as follows:
CGAGGACGGTCGTATTCTTAAGCACATCTACTCAGCCTTGATGGAGCC(SEQ ID NO.3)
the modified probes were as follows:
CGAGGACGGTCGTATTCTTAAGCACATCTAC[FAM-dT][THF][BHQ-dT]TCAGCCTTG ATGGAGCC
example 2 kit
The rapid detection kit for detecting the drug-resistant gene arr2 by using the RAA fluorescence method of the embodiment adopts a RAA fluorescence method kit produced by Jiangsu zier gene biotechnology limited company, and comprises a RAA basic fluorescence universal reaction reagent, a reaction buffer, a positive quality control product, a negative quality control product, the primers PA-arr2-primer1-fw/PA-arr2-primer1-rv screened in the embodiment 1 and a probe; the concentration of the upstream primer and the downstream primer is independently selected to be 10. Mu. Mol/L, and the concentration of the probe is preferably 10. Mu. Mol/L. Wherein, the positive quality control product preferably contains the genome DNA of the pseudomonas aeruginosa drug resistance gene arr2, and the dosage of the genome DNA of the pseudomonas aeruginosa drug resistance gene arr2 is 25 mu L; the negative quality control material is preferably ddH 2 O or purified water. The amount of each component to be provided in each reaction kit is (i.e. total volume): buffer (25. Mu.L), PA-arr2-primer1-fw (concentration 10. Mu.M, 2. Mu.L), PA-arr2-primer1-rv (concentration)10. Mu.M, 2. Mu.L), purified water (17.5. Mu.L), template (1. Mu.L), probe (concentration 10. Mu.M, 0.6. Mu.L).
The kit also comprises a RAA basic fluorescent universal reaction reagent, and the RAA basic fluorescent universal reaction reagent is preferably freeze-dried powder subjected to low-temperature freeze drying. In the embodiment of the application, the RAA basic fluorescent universal reagent is purchased from Jiangsu Qitide gene biotechnology Co., ltd, and the product number is F01006. The reaction specification of the RAA-based fluorescent universal reagent is 50 mu L, and the reaction buffer is used for redissolution before use.
The reaction buffer of the re-dissolved RAA-based fluorescent universal reagent preferably comprises the following components in content: tris-HCl (pH 7.4) buffer with a concentration of 500mmol/L, mgAc with a concentration of 240mmol/L and PEG30000 with a mass fraction of 10%. In the application, the concentrations of Tris-HCl buffer solution, mgAc and PEG30000 are all final concentrations. The sources of the Tris-HCl buffer solution, the MgAc and the PEG30000 are not particularly limited, and the conventional commercial products are adopted. Tris-HCl, and PEG30000 in the examples of the present application were purchased from Sigma-Aldrich, mgAc was purchased from Shanghai, china.
Example 3 detection method
The rapid detection method for detecting pseudomonas aeruginosa drug resistance gene arr2 by using the RAA fluorescence method provided by the embodiment comprises the following steps:
(1) According to the existing conventional method, clinical isolates are obtained from urine, feces and lungs of patients in hospital, and 160 clinical strains from the national hospitals of Shaanxi province and the Western-safety children hospital are collected in total in the experiment, and the clinical strains are separated from different samples, namely a sputum isolate 134 strain, a urine isolate 8 strain, a blood isolate 6 strain, a catheter isolate 3 strain, a bronchoalveolar lavage isolate 5 strain and a skin secretion isolate 4 strain;
(2) Separating target strain on a Pseudomonas aeruginosa separating plate, picking up full Pseudomonas aeruginosa monoclonal and placing in a liquid culture medium, placing in a constant temperature incubator at 36+ -1deg.C, culturing for 24 h+ -2 h;
(3) After the enrichment culture is completed, the microorganisms are centrifugally separated at a high speed, DNA extraction is carried out on pseudomonas aeruginosa after high-temperature pyrolysis, and the pseudomonas aeruginosa is taken as a template;
(4) Adding the primer PA-arr2-primer1-fw/PA-arr2-primer1-rv screened in the example 1 and a probe into a freeze-drying isothermal amplification agent, and adding template DNA to completely dissolve after finger flicking and mixing uniformly to obtain a reaction solution;
(5) And (3) adding magnesium acetate into the tube cover of the reaction unit tube in the step (4).
(6) To avoid contamination of the test sample, template DNA is finally added.
(7) Culturing in an isothermal water bath or incubator at 39-42 ℃ for 4min, then placing in an RAA-F1620 isothermal nucleic acid amplification detector, reacting for 20min at 39 ℃, collecting fluorescence every 30s, and collecting 40 times. The test results are shown in FIG. 1, wherein in FIG. 1, sample A04 represents patient saliva (sputum), A05 represents patient urine, B03 represents patient saliva (sputum), B04 represents patient alveolar lavage fluid, B05 represents patient catheter separation fluid, kidney drug-induced fluid, and B06 is a negative control which is established simultaneously, namely ddH 2 O or purified water; the abscissa in fig. 1 represents time, 1 represents 0.5min,2 represents 1min,3 represents 1.5min, and so on, and the ordinate represents fluorescence value (millivolts). The data in FIG. 1 shows that specific gene amplification of the drug resistance gene arr2 of Pseudomonas aeruginosa is present in clinical isolates obtained from saliva (sputum), urine, alveolar lavage fluid, catheter isolates, and kidney drug-treated fluid.
Example 4 sensitivity detection
In this example, the sensitivity of RAA detection was established by detecting the rifampicin resistance gene arr2, and the recombinant plasmid containing the target gene arr2, which was constructed in advance, was prepared according to the copy number formula copies/. Mu.L= ((6.02X10) 23 )×(ng/μL×10 -9 ) (DNA length. Times.660) to calculate the gene copy number of the synthetic plasmid DNA, gradient dilution (10) 9 COPIES/. Mu.L to 10 0 copies/. Mu.L), RAA detection was performed on DNA samples of different concentrations, respectively, and the detection results are shown in FIG. 2. The result of the fluorescence detection instrument shows that the RAA detection arr2 shows good sensitivity, and the fluorescence intensity is increased along with the increase of the plasmid concentration, so that the minimum sensitivity of the RAA detection is 10 copies/. Mu.L.
The above embodiments are only for illustrating the technical solution of the present application and not for limiting the same, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present application, which is intended to be covered by the scope of the claims of the present application. The technology, shape, and construction parts of the present application, which are not described in detail, are known in the art.
Claims (7)
- The primer for detecting the drug-resistant gene arr2 by using the RAA fluorescence method comprises an upstream primer and a downstream primer, and is characterized in that the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2.
- The probe for detecting the drug-resistant gene arr2 by using the RAA fluorescence method is characterized by comprising a probe with a sequence shown as SEQ ID NO.3 or a probe modified on the basis of the sequence shown as SEQ ID NO. 3.
- 3. The probe for detecting a drug-resistant gene arr2 by using the RAA fluorescence method according to claim 2, wherein the modification is that a tetrahydrofuran base site is marked at a middle position which is more than or equal to 35nt from the 5' end of a probe sequence and is used as a recognition site I of exonuclease, T bases are respectively added at two sides, a FAM fluorescent group is added at the 5' end, a quenching group is added at the 3' end, and the THF site is recognized and cut by the exonuclease.
- Kit for the detection of a drug resistant gene arr2 by raa fluorescence method, characterized in that the kit comprises a primer according to claim 1 and/or a probe according to claim 2 or 3.
- 5. The kit for detecting the drug-resistant gene arr2 by the RAA fluorescence method according to claim 4, further comprising a positive quality control and a negative quality control, the positive quality control product is genomic DNA containing pseudomonas aeruginosa drug resistance gene arr2, and the negative quality control product is a negative quality control productThe property control material is ddH 2 O or purified water.
- 6. Use of the primer of claim 1 or the probe of claim 2 or 3 or the kit of claim 4 or 5 for in vitro detection of the presence or absence of the pseudomonas aeruginosa drug resistance gene arr2 in a sample, said use being of non-diagnostic interest.
- 7. The use according to claim 6, wherein the final concentration of the primer in the reaction system is independently 10. Mu. Mol/L and the final concentration of the probe in the reaction system is 10. Mu. Mol/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311155783.6A CN117187417A (en) | 2023-09-08 | 2023-09-08 | Primer, probe, kit and application for detecting drug-resistant gene arr2 by RAA fluorescence method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311155783.6A CN117187417A (en) | 2023-09-08 | 2023-09-08 | Primer, probe, kit and application for detecting drug-resistant gene arr2 by RAA fluorescence method |
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