Primers, detection methods and applications for the specific detection of tet(X) positive Acinetobacter amygdalae

Abstract

This invention discloses specific primers, detection methods, and applications for the detection of tet(X)-positive Acinetobacter amysii. The specific primers include amysii-6712F / amyesii-6712R and ase-F / ase-R. Using these primers, PCR amplification of genomic DNA or single colonies of tet(X)-positive Acinetobacter amysii was performed, with band sizes of 690 bp and 318 bp, respectively. The detection accuracy of tet(X)-positive Acinetobacter amysii was 100%, demonstrating high accuracy. The limit of detection (LOD) DNA concentration was only 0.003 ng / μL, indicating high sensitivity. The total time for colony picking and detection was only about 2.5 hours. For tet(X)-positive Acinetobacter amysii, the method based on specific PCR primers for detection is simple, accurate, and rapid, and has broad application prospects and economic value.

Description

Technical Field

[0001] This invention relates to the detection of Acinetobacter amygdalae, and more specifically, to primers, detection methods, and applications for the specific detection of tet(X)-positive Acinetobacter amygdalae. Background Technology

[0002] tet(X) genes are a class of resistance genes encoding tetracycline antibiotic-degrading enzymes, such as tet(X3), tet(X4), tet(X5), and tet(X6), thereby limiting the use of tetracycline antibiotics such as tetracycline, doxycycline, minocycline, tigecycline, eracycline, and omalicycline. *Acinetobacter amyesii* is an aerobic, Gram-negative bacterium without flagella or motility, and is non-hemolytic. It can grow at temperatures ranging from 30°C to 41°C and has potential pathogenicity. Based on average nucleotide identity analysis, Acinetobacter amygdalae carrying genes such as tet(X3) and tet(X6) (e.g., YH01010, NCBI accession number: GCA_013420775.1) were detected, accounting for 14.69% of tet(X) positive Acinetobacter species, second only to Acinetobacter indicus (29.17%) and Acinetobacter towneri (15.35%).

[0003] Currently, novel pathogen detection methods, such as biosensor methods that detect biological receptors using sensors, microfluidic technologies that achieve various complex manipulation functions through microfluidic channel systems, and DNA microarray technologies that form biomolecular arrays by spotting microparticles and reacting with target molecules in the analyte sample, are costly. Traditional detection methods, such as physiological and biochemical characterization based on bacterial culture, are also time-consuming. PCR (polymerase chain reaction) detection is widely used due to its advantages of high sensitivity, strong specificity, simplicity, speed, and low purity requirements. However, there is currently a lack of PCR primers and methods for detecting tet(X)-positive Acinetobacter emetica. Summary of the Invention

[0004] To address the current lack of PCR primers and methods for detecting tet(X)-positive Acinetobacter aemeticus, this invention provides a specific detection primer, detection method, and application for tet(X)-positive Acinetobacter aemeticus. This detection primer can be used for efficient and rapid detection of this type of pathogen.

[0005] To achieve the above objectives, the present invention provides a specific detection primer for tet(X)-positive Acinetobacter amygdala, comprising the following two pairs of primers:

[0006] amyesii-6712F:5'-GCCTATTGTTTTTGACCCAAT-3'(SEQ ID NO.1)

[0007] amyesii-6712R: 5'-GCACCATAAACCAATACC-3' (SEQ ID NO. 2);

[0008] ase-F: 5'-TTACAGAAAACGGC-3' (SEQ ID NO.3)

[0009] ase-R: 5'-AACAAGTTTTCTATCCCAAA-3' (SEQ ID NO. 4).

[0010] A second aspect of the present invention provides a method for detecting tet(X)-positive Acinetobacter emetans, comprising the following steps:

[0011] S1. Use the above-mentioned specific primers to perform PCR amplification of the genomic DNA of the test strain or a single colony;

[0012] S2. If two specific DNA bands of 690bp and 318bp are found in the gel electrophoresis results, the strain is identified as tet(X) positive Acinetobacter amygdalae.

[0013] Specifically, in step S1, the lowest detection limit for tet(X)-positive Acinetobacter amygdalae in the genomic DNA is 0.003 ng / μL.

[0014] Specifically, in step S1, the PCR amplification system includes: 12.5 μL of 2×Taq Master Mix, 0.5 μL of each primer, 1 μL of PCR template, and 9.5 μL of sterile deionized water.

[0015] Further, in step S1, the PCR amplification reaction program includes: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ final extension for 5 min; storage at 16℃.

[0016] A third aspect of the present invention provides a detection kit for tet(X) positive Acinetobacter amygdalae, which includes the above-mentioned specific primers.

[0017] Through the above technical solution, the present invention achieves the following beneficial effects:

[0018] This invention provides a specific primer combination for tet(X)-positive Acinetobacter amysii, namely amysii-6712F / amyesii-6712R and ase-F / ase-R. Using these primers for PCR amplification of genomic DNA or single colonies of tet(X)-positive Acinetobacter amysii, the band sizes were 690 bp and 318 bp, respectively, with a detection accuracy of 100%, demonstrating high accuracy. The limit of detection (LOD) DNA concentration was only 0.003 ng / μL, indicating high sensitivity. The total time for colony picking and detection was only about 2.5 hours. For tet(X)-positive Acinetobacter amysii, the method based on specific PCR primers for detection is simple, accurate, and rapid, and has broad application prospects and economic value. Attached Figure Description

[0019] Figure 1 This is the PCR amplification band of the MFS transporter protein gene in Example 1 of this invention;

[0020] Figure 2 This is the PCR amplification band of the tet(X) homologous gene in Example 1 of this invention;

[0021] Figure 3 This is the result of the determination of the lowest DNA concentration detection limit in Example 3 of the present invention. Detailed Implementation

[0022] The specific embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0023] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through conventional commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods in the prior art.

[0024] Example 1: Design of specific PCR primers for tet(X) positive Acinetobacter amygdalae

[0025] 1) Using the conserved genes MFS transporter gene and tet(X) homolog gene in tet(X) positive Acinetobacter amysii as templates, PCR amplification primers for tet(X) positive Acinetobacter amysii were designed using Primer Premier 6.0 software. The primers consisted of two pairs: amysii-6712F / amyesii-6712R (as shown in SEQ ID NO.1 and SEQ ID NO.2) and ase-F / ase-R (as shown in SEQ ID NO.3 and SEQ ID NO.4).

[0026] 2) Tet(X3) positive Acinetobacter ames YH16040 was streaked onto LB agar plates (tigecycline, 2 μg / mL) and incubated at 37°C for 18 h, followed by further purification. The semi-circular purified bacterial culture was scraped and placed in 600 μL of autoclaved deionized water. Genomic DNA was extracted using the Tiangen Bacterial Genome Extraction Kit, with an initial concentration of 98.26 ng / μL.

[0027] 3) Using the genomic DNA described above as a template, the specific primers were initially validated using 2×Taq Master Mix (Novozymes). The PCR detection system (25 μL) consisted of: 12.5 μL of 2×Taq Master Mix; 1 μL of template; 0.5 μL each of forward and reverse primers; and finally, sterile deionized water to make up the difference. The PCR reaction program was as follows: 94℃ pre-denaturation for 5 min, (94℃ denaturation for 30 s, 49℃ annealing for 30 s, 72℃ extension for 30 s) × 35 cycles, final extension at 72℃ for 5 min, and storage at 16℃.

[0028] Agarose gel electrophoresis results showed that the PCR products were 690 bp in size. Figure 1 ) and 318bp ( Figure 2 The bands were clear. 5 μL of the PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for first-generation sequencing, and the results were consistent with the corresponding gene template.

[0029] Example 2: Determination of the optimal PCR annealing temperature for tet(X)-positive Acinetobacter amygdala.

[0030] Using genomic DNA from tet(X3)-positive Acinetobacter amysiella YH16040 as a template, the annealing temperature range of the PCR reaction program was 47℃~58℃ (with a gradient of 1℃). PCR amplification was performed simultaneously using the two pairs of primers mentioned above, with three replicates per group. The PCR detection system (25μL) consisted of: 12.5μL of 2×Taq Master Mix; 1μL of template; 0.5μL each of primers amysiii-6712F, amysiii-6712R, ase-F, and ase-R; and finally, sterile deionized water was added to make up the volume. The PCR reaction program was: 94℃ pre-denaturation for 5 min, (94℃ denaturation for 30 s, 47℃~58℃ annealing for 30 s, 72℃ extension for 30 s) × 35 cycles, a final extension at 72℃ for 5 min, and storage at 16℃.

[0031] After agarose gel electrophoresis, images were taken, and the grayscale values ​​of the electrophoretic bands were analyzed using ImageJ version 1.8.0. The results are shown in Table 1. The highest average grayscale value was 11328.74, corresponding to an optimal annealing temperature of 50℃.

[0032] Table 1. Determination of optimal PCR annealing temperature

[0033]

[0034] Example 3: Determination of the lowest PCR detection limit for tet(X)-positive Acinetobacter amygdala.

[0035] The genomic DNA of tet(X3)-positive Acinetobacter aemetic YH16040 was serially diluted twofold using autoclaved deionized water, with a dilution range of 2. -7 ~2 -24 Using this as a PCR template, PCR was performed at an annealing temperature of 50°C, following the PCR system and procedure described in Example 2. The amplification results are as follows... Figure 3 The display shows that the gradient of the lowest detection limit is 2. -16 That is, 0.003 ng / μL.

[0036] Example 4: Accuracy Verification of PCR Detection Based on Bacterial Genomic DNA

[0037] The tested bacterial strains included *Acinetobacter ames*, *Acinetobacter ininus*, *Acinetobacter baumannii*, *Acinetobacter lwoffii*, *Acinetobacter gandensis*, *Acinetobacter tangsii*, *Acinetobacter baylyi*, *Empedobacter falsenii*, *Escherichia coli*, *Salmonella enterica*, *Klebsiella pneumoniae*, *Campylobacter coli*, and *Campylobacter jejuni*. The presence of the tet(X) variant is shown in Table 2, totaling 96 strains. Each strain was streaked into three regions on blank LB agar plates, purified, and genomic DNA was extracted using the Tiangen Bacterial Genome Kit. Accuracy was determined according to the method described in Example 2 at an annealing temperature of 50°C. The results, as shown in Table 2, indicate that tet(X)-positive Acinetobacter emetica showed two clear bands at 690 bp and 318 bp, while other strains, including tet(X)-negative Acinetobacter emetica, yielded negative results. Therefore, the accuracy of PCR detection based on tet(X)-positive Acinetobacter emetica genomic DNA was 100%.

[0038] Table 2. Validation results of PCR detection accuracy based on genomic DNA and single colonies.

[0039]

[0040]

[0041]

[0042] Note: ++, 2 PCR bands; +, 1 PCR band; -, no band.

[0043] Example 5: Accuracy Verification of PCR Detection Based on Single Bacterial Colonies

[0044] Single colonies of the test strains from Example 4 were selected and PCR was performed at an annealing temperature of 50°C, following the method described in Example 2. The results, as shown in Table 2, indicate that tet(X)-positive Acinetobacter ames exhibited two clear bands at 690 bp and 318 bp, while other strains showed only one band or no band at 318 bp, making them clearly distinguishable. Therefore, the PCR detection accuracy based on tet(X)-positive Acinetobacter ames single colonies was 100%, with a total detection time of approximately 2.5 hours.

[0045] The preferred embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0046] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0047] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A kind tet (X) positive Acinetobacter amycin ( Acinetobacter amyesii ) Specific detection primers, characterized in that, The two pairs of primers include: amyesii-6712F: 5'-GCCTATGTTTTTGACCCAAT-3' amyesii-6712R: 5'-GCACCATAAAACCAATACC-3'; ase-F: 5'-TTACAGCAAAACGGC-3' ase-R: 5'-AACAAGTTTTCTATCCCAAA-3'.

2. A method according to claim 1, wherein the disease is selected from the group consisting of: atherosclerosis, stroke, myocardial infarction, peripheral vascular disease, heart failure, and diabetes. tet (X) A method for detecting Acinetobacter baumannii for non-disease diagnostic purposes, characterized in that, The method comprises the following steps: S1, using the specific primers in claim 1 to perform PCR amplification on the genomic DNA or single colony of the strain to be tested; S2, if there are two DNA specific bands of 690 bp and 318 bp, the strain is determined to be tet (X) positive Acinetobacter baumannii.

3. The detection method according to claim 2, characterized in that, In step S1, the genomic DNA in tet (X) The limit of detection for positive Acinetobacter baumannii was 0.003 ng / µL.

4. The method of claim 2, wherein In step S1, the PCR amplification system comprises: 2x Taq Master Mix 12.5 μL, primers 0.5 μL each, PCR template 1 μL, and sterile deionized water 9.5 μL.

5. The detection method according to claim 4, characterized in that, In step S1, the reaction program of the PCR amplification comprises: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, 50 ℃ annealing for 30 s, 72 ℃ extension for 30 s, 35 cycles; 72 ℃ final extension for 5 min; and 16 ℃ preservation.

6. A method according to any one of claims 1 to 5, wherein the method comprises the step of: (i) determining the presence of a mutation in the gene encoding the protein of interest in the sample. tet (X) A test kit for the detection of positive Acinetobacter baumannii, characterized by, The specific primers in claim 1 are included.

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