CN110184370B - Specific primer for detecting Acinetobacter johnsonii, method and application - Google Patents
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Abstract
本发明公开了一种检测约翰逊不动杆菌的特异性引物及方法和应用，所述的特异性引物如SEQ ID NO.1～2所示，通过提取待测样品基因组DNA，使用上述引物进行PCR扩增，凝胶电泳检测，于凝胶成像系统下拍照检测，存在365bp的DNA特异性条带，则确定样品中含有约翰逊不动杆菌。采用本发明的检测方法检测约翰逊不动杆菌所需时间短、特异性强、灵敏度高。本发明避免了采用传统鉴定方法操作繁琐、耗时长、准确性低、检出率低等缺点。The invention discloses a specific primer, method and application for detecting Acinetobacter johnsonii. The specific primer is shown in SEQ ID NO. 1-2, by extracting the genomic DNA of a sample to be tested, and using the primer to carry out PCR Amplification, gel electrophoresis detection, photographing and detection under a gel imaging system, and there is a DNA-specific band of 365 bp, it is determined that the sample contains Acinetobacter johnsonii. Using the detection method of the present invention to detect Acinetobacter johnsonii requires short time, strong specificity and high sensitivity. The invention avoids the disadvantages of using the traditional identification method, such as complicated operation, long time-consuming, low accuracy and low detection rate.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a specific primer for detecting Acinetobacter johnsonii, a method and application thereof.
Background
The genus acinetobacter has received increasing attention in recent years not only because of its ability to cause diseases in humans, but also because of the unique biological characteristics of each species in the genus acinetobacter. Acinetobacter johnsonii is one of the acinetobacter genera. The Acinetobacter johnsonii is a gram-negative bacterium, widely exists in nature, has extremely strong viability, can survive in a high-phosphorus or high-salt environment, is one of strains of respiratory tracts of people and animals, is an important conditional pathogen, can cause diseases of people and animals, and has been reported to develop a drug resistance mechanism continuously under the selective pressure due to the wide clinical use of antibiotic drugs, so that the drug resistance strains are increased increasingly, become important nosocomial infection pathogenic bacteria, cause the transmission of human diseases including respiratory tract transmission, contact transmission and the like, and the nosocomial infection is mostly related to invasive operations (such as intravenous infusion, tracheal intubation and indwelling ureter); meanwhile, Acinetobacter johnsonii can cause acute death of channel catfish and diseased death of wild wood frogs. Under the condition of unknown pathogenic bacteria, drugs are abused, so that the pertinence is not strong and the effect is not obvious; secondly, the drug resistance of the bacteria is enhanced. Therefore, a rapid and accurate detection means is urgently needed clinically, and the pathogeny is distinguished and is aimed at prevention and treatment.
The traditional bacteria identification method mainly classifies bacteria according to physiological and biochemical characteristics, requires a large amount of time to interpret results of physiological and biochemical reactions, and is not beneficial to diagnosing pathogeny, searching pathogeny and controlling the spread of pathogeny in time. However, such means as 16S rRNA sequencing are not suitable for routine detection. The PCR technology has the characteristics of simple and convenient operation, rapidness, high sensitivity, strong specificity and the like, and is gradually applied to the rapid detection of bacteria. Through the literature search of the prior art, no report related to the PCR detection method, nucleic acid and primer of Acinetobacter johnsonii is found. The detection method established by the invention can provide technical support for clinical rapid diagnosis of Acinetobacter johnsonii and epidemiological investigation.
Disclosure of Invention
The invention aims to provide a PCR detection primer and a PCR detection method for Acinetobacter johnsonii. The primer can be used for PCR detection of Acinetobacter johnsonii, and has the advantages of short detection time, low cost, high detection result specificity, easy result identification and strong practicability.
The invention is realized by the following technical scheme:
a specific primer for detecting Acinetobacter johnsonii comprises a forward primer and a reverse primer, and specifically comprises the following components:
a forward primer F: 5'-CAGGTCCTGCGCCAGAAGTTG-3', respectively;
reverse primer R: 5'-GATGCCATCCGTCACGGCTAAG-3' are provided.
In another aspect of the present invention, there is provided a method for detecting Acinetobacter johnsonii, comprising the steps of:
1) extracting genome DNA of a sample to be detected;
2) performing PCR amplification by using the primers;
3) and (3) gel electrophoresis detection, photographing and detecting under a gel imaging system, and determining that the sample contains Acinetobacter johnsonii if a 365bp DNA specific band exists.
Further, the sample genome is extracted by an Ezup column kit.
Further, the PCR amplification reaction system is 10 μ L: 2 × TSINGKE MasterMix (blue)5 μ L, primer pair 1 μ L, template DNA 1 μ L, double distilled water to make up to 10 μ L.
Further, the PCR amplification reaction program is as follows: pre-denaturation at 94 ℃ for 5 min; performing circulation, performing denaturation at 94 ℃ for 10s, annealing at 64 ℃ for 30s, and extending at 72 ℃ for 30s, and performing 35 cycles; extending for 10min at 72 ℃, cooling for 12 ℃, and finishing.
In addition, the invention also provides application of the specific primer in the detection of Acinetobacter johnsonii.
Meanwhile, the invention also provides a kit for detecting Acinetobacter johnsonii, wherein the kit comprises the primer pair.
The invention has the beneficial effects that:
1) the Acinetobacter johnsonii specific primer designed by the invention can effectively distinguish Acinetobacter johnsonii from various common pathogenic strains, and shows strong specificity.
2) The Acinetobacter johnsonii specific primer designed by the invention has high sensitivity, and the minimum detection concentration of the Acinetobacter johnsonii can be effectively detected to be 7.45 multiplied by 102CFU/mL。
3) The PCR detection method of Acinetobacter johnsonii in clinical samples provided by the invention enables the detection of Acinetobacter johnsonii in clinical samples to be simpler and more convenient. In clinical samples, the pathogenic bacterium Acinetobacter johnsonii can be rapidly identified once existing, and an effective detection method is provided for clinical treatment.
4) The method avoids the defects of complex operation, time consumption, low accuracy, low detection rate and the like of the traditional identification method. After the 16S rRNA universal primer is amplified, the bacterial species also needs to be determined by sequencing, and the conventional detection is not suitable. Meanwhile, the detection target of the invention has single specificity, specific detection result and easy judgment, and saves time compared with the 16S rRNA sequencing method.
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FIG. 1 shows the optimization of PCR reaction conditions for Acinetobacter johnsonii specific primers in the examples; m: marker; 1-6 annealing temperatures of 59 deg.C, 60 deg.C, 61 deg.C, 62 deg.C, 63 deg.C, 64 deg.C, respectively; 7: negative control;
FIG. 2 is a diagram showing the results of gel electrophoresis in the test for evaluating the specificity of the PCR detection method in the example; m: marker; 1: negative control; 2: serratia marcescens; 3: citrobacter bacteria; 4: a bacillus; 5: pseudomonas aeruginosa; 6: acinetobacter johnsonii; 7: acinetobacter pitegueta; 8: acinetobacter calcoaceticus; 9: acinetobacter baumannii; 10: acinetobacter seiferii; 11: e.coli; 12: streptococcus agalactiae; 13: enterococcus faecalis; 14: staphylococcus aureus bacteria; 15: staphylococcus saprophyticus; 16: streptococcus dysgalactiae;
FIG. 3 is a diagram showing the results of gel electrophoresis in the sensitivity evaluation test of the PCR detection method in the example; m: marker; 1: 7.45X 106CFU/mL；2：7.45×105CFU/mL；3：7.45×104CFU/mL；4：7.45×103CFU/mL；5：7.45×102CFU/mL；6：7.45×101CFU/mL; 7: 7.45 CFU/mL; 8: negative control;
FIG. 4 is a graph showing the results of gel electrophoresis in clinical sample detection in the examples; m: marker; 1, positive control of Acinetobacter johnsonii; 2-8 are nasal swab samples; 9: and (5) negative control.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 establishment of Acinetobacter johnsonii specific PCR detection method
(I) Acinetobacter johnsonii specific primer design
In this example, a pair of specific primers with strong specificity and high sensitivity to acinetobacter johnsonii is designed for the tyrosine protein kinase gene (stk gene) of acinetobacter johnsonii, and the specific information of the specific primer pair is as follows:
the primer sequence is as follows:
F：5’-CAGGTCCTGCGCCAGAAGTTG-3’；
R：5’-GATGCCATCCGTCACGGCTAAG-3’。
(II) Acinetobacter johnsonii DNA template preparation
2.1 extraction of Acinetobacter johnsonii DNA
Extracting DNA of Acinetobacter johnsonii by using an Ezup column genome DNA extraction kit (bacteria), and specifically comprising the following steps:
1) taking 1mL Acinetobacter johnsonii bacterial liquid cultured in a constant temperature incubator at 37 ℃ for 24h, adding the Acinetobacter johnsonii bacterial liquid into a 1.5mL centrifuge tube, centrifuging for 1min at room temperature of 8000rmp, removing supernatant, collecting thalli, adding 180 mu L Buffer Digestion, adding 20 mu L of LProteinase K solution, shaking and uniformly mixing, and carrying out water bath at 56 ℃ for 1h until cells are completely lysed. During the water bath, the mixture was inverted every 10 minutes to facilitate cell lysis.
2) Add 200. mu.L of BufferBD and mix well by inversion. After adding BufferBD, if a precipitate is generated, the mixture is bathed in water at 70 ℃ for 10 minutes.
3) Adding 200 μ L of anhydrous ethanol, fully inverting and mixing, adding anhydrous ethanol to generate translucent fibrous suspended substance, without affecting DNA extraction and application.
4) And (3) putting the adsorption column into a collecting pipe, and using a liquid transfer device to transfer the solution and the semitransparent fibrous suspended substance obtained in the step (3) without influencing the extraction and application of the DNA.
5) The adsorption column is recycled into the collection tube again, 500 mu L PW Solution10000rmp is added for centrifugation for 30s, and the Solution in the collection tube is poured out.
6) The adsorption column was replaced again to the collection tube, 500. mu.L of Wash Solution was added, 10000rmp was centrifuged for 30s, and the filtrate was decanted off.
7) The adsorption column was replaced in the collection tube, centrifuged at 12000rmp for 2min at room temperature, and the residual Wash Solution was discarded. The adsorption column is opened and placed at room temperature for a plurality of minutes to thoroughly dry the Wash Solution remained in the adsorption material, and the yield of the genome DNA and subsequent experiments are influenced by the residue of the Wash Solution.
8) Taking out the adsorption column, putting the adsorption column into a new 1.5mL centrifuge tube, adding 50-200 μ L CE Buffer, standing for 3min, centrifuging at 12000rmp at room temperature for 2min, and collecting DNA solution, namely the DNA of Acinetobacter johnsonii. The extracted DNA can be immediately subjected to the next test or stored at-20 ℃.
2.2 exploration of the optimal annealing temperature for Acinetobacter johnsonii specific primers
PCR amplification is carried out by taking the bacterial liquid of Acinetobacter johnsonii as a template, and the annealing temperatures of the target primers are set to 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃ and 64 ℃. And (3) carrying out electrophoresis on the PCR amplification product in a 1% agarose 1 × TAE buffer system, adding 5 μ L of sample in each hole, detecting the electrophoresis result by using a full-automatic gel imaging analysis system, and determining the optimal annealing temperature of the Acinetobacter johnsonii specific primer according to the gel electrophoresis result after PCR. The PCR amplification procedure is shown in Table 1.
TABLE 1
As can be seen from FIG. 1, the temperature other than 59 ℃ has no significant effect on the primer, but according to the basic principle of PCR, the higher the temperature is, the more favorable the specific binding of the primer is, so that the optimal annealing temperature is determined to be 64 ℃.
(III) establishment of specific PCR detection method for Acinetobacter johnsonii
The PCR detection system is as follows: 10 μ L reaction system is 2 × TSINGKE Master Mix (blue) is 5 μ L; the primer pair is 1 mu L; the template DNA is 1 mu L, and the DNA is finally supplemented to 10 mu L by sterilized double distilled water; and finally, supplementing 10 mu L of the solution with sterilized double distilled water.
The PCR detection reaction program is as follows: pre-denaturation at 94 ℃ for 5 min; performing circulation, performing denaturation at 94 ℃ for 10s, annealing at 64 ℃ for 30s, and extending at 72 ℃ for 30s, and performing 35 cycles; extending for 10min at 72 ℃, cooling for 12 ℃, and finishing.
(IV) determination of PCR amplification result by gel electrophoresis
The PCR amplification product is electrophoresed in a 1% agarose 1 × TAE buffer system, 5 μ L of sample is added to each hole, the electrophoresis result is detected by a full-automatic gel imaging analysis system, and if a 365bp single amplification band appears, the sample contains Acinetobacter johnsonii; otherwise, the sample does not contain Acinetobacter johnsonii.
Example 2 specificity evaluation test
The laboratory-stored Serratia marcescens, Citrobacter, Bacillus, Pseudomonas aeruginosa, Acinetobacter johnsonii, Acinetobacter pittanicus, Acinetobacter calcoaceticus, Acinetobacter baumannii, Acinetobacter seifertii, Escherichia coli, Streptococcus agalactiae, enterococcus faecalis, Staphylococcus aureus, Staphylococcus saprophyticus, and Streptococcus dysgalactiae were subjected to PCR amplification reaction according to the DNA template preparation and PCR detection methods described in example 1.
The electrophoresis result in FIG. 2 shows that only A.johnsonii shows a specific band at 365bp, but no specific band appears in other species.
Example 3 sensitivity evaluation test
Inoculating Acinetobacter johnsonii into 1mL of nutrient broth liquid culture medium, culturing at 37 deg.C in an incubator for 24h to increase the number of cells, diluting with a 10-fold gradient of sterilized nutrient broth liquid culture medium, counting by plate method to obtain the concentration of cells, extracting genomic DNA from 1mL of the culture solution according to example 1, and extracting with 10% of sterilized double distilled water0-10-6Performing 10-fold gradient dilution on the genome DNA, performing PCR amplification by using 7 gradient dilutions as templates, detecting an amplification product by gel electrophoresis, and observing a gel electrophoresis result under ultraviolet light.
As can be seen from FIG. 3, a clear band is seen in lane 5, which corresponds to a detected bacterial concentration of 7.45X 102CFU/mL, the method has better sensitivity.
EXAMPLE 4 preliminary application of the Acinetobacter johnsonii PCR method
7 nasal swab samples collected from the nasal cavity of beef cattle were tested using the Acinetobacter johnsonii specific PCR assay established in example 1.
The results are shown in FIG. 4, in which lane M is DL2000 molecular weight standard, lane 1 is Acinetobacter johnsonii positive control, lane 9 is sterilized double distilled water negative control, and lanes 2-8 are 7 nasal swab samples. As can be seen, the lane 2 strain shows a positive result.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Sequence listing
<110> Sichuan university of agriculture
<120> specific primer for detecting Acinetobacter johnsonii, method and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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caggtcctgc gccagaagtt g 21
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gatgccatcc gtcacggcta ag 22
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Effective date of registration: 20220808 Address after: 635200 group 3, Changchang village, Sanhui Town, Qu county, Dazhou City, Sichuan Province Patentee after: Quxian Caijiashan Livestock Breeding Co.,Ltd. Address before: 611130 Huimin Road, Wenjiang District, Chengdu, Sichuan Province, No. 211 Patentee before: SICHUAN AGRICULTURAL University |
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