CN110184371B - A kind of specific primer for detecting Acinetobacter pietii and its method and application - Google Patents
A kind of specific primer for detecting Acinetobacter pietii and its method and application Download PDFInfo
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Abstract
The invention discloses a specific primer for detecting acinetobacter cutaneum as well as a method and application thereof, wherein the specific primer is shown as SEQ ID No. 1-2, and the acinetobacter cutaneum is determined to be contained in a sample by extracting the genomic DNA of a sample to be detected, carrying out PCR amplification and gel electrophoresis detection by using the primer, photographing and detecting under a gel imaging system, and if a 485bp DNA specific strip exists. The detection method of the invention has the advantages of short time, strong specificity and higher sensitivity for detecting the acinetobacter cutaneus. The method avoids the defects of complex operation, long time consumption, low accuracy, low detection rate and the like of the traditional identification method.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a specific primer for detecting acinetobacter cutaneus as well as a method and application thereof.
Background
Acinetobacter pinteus (Acinetobacter pittii) belongs to the genus Acinetobacter, is a gram-negative coccobacillus which is strictly aerobic and non-lactose fermented, widely exists in nature, is distributed in respiratory tracts of human beings and animals, is generally nonpathogenic to healthy individuals, and belongs to a common flora. At present, because acinetobacter is distributed in a large amount in a hospital environment and has harm, the acinetobacter is highly valued by researchers, is one of important conditional pathogenic bacteria of hospital acquired infection, and can cause infection of multiple systems of the whole body such as a respiratory system, a urinary system, blood source, skin, wounds and the like. And along with the abuse of antibiotics, multiple drug-resistant strains and pan-drug-resistant strains gradually appear, the harmfulness is continuously increased, and the possibility of becoming super bacteria is provided. Acinetobacter cutaneum, Acinetobacter baumannii and Acinetobacter hospital (Acinetobacter nosocomialis) belong to Acinetobacter baumannii-calcium acetate complex, and have high detectable rate and harmfulness in human hospitals, the Acinetobacter cutaneum has been proved to cause fish diseases and have high fatality rate, the morbidity rate and the mortality rate of patients in hospitals are slightly lower than those of the Acinetobacter baumannii, but the morbidity rate and the mortality rate of the Acinetobacter cutaneum have a tendency of rising year by year, and the importance of researchers is attracted. And acinetobacter cutaneus, acinetobacter baumannii and acinetobacter in hospitals have similar phenotype and genetic relevance, are difficult to distinguish by conventional means, and need to be identified by molecular biology means.
Because the traditional bacteria identification method such as biochemical test not only takes long time and has low accuracy, the genus can be generally determined through the biochemical test. Further, Acinetobacter pitteri, Acinetobacter baumannii and Acinetobacter in hospitals cannot be distinguished efficiently and reliably, and therefore, detection by a molecular biological detection method is required. For a large amount of samples, the detection cost is high, and the method such as 16S rRNA sequence sequencing is not suitable for routine detection. Therefore, a PCR detection method with high speed, specificity and sensitivity to Acinetobacter pitteus is urgently needed.
Disclosure of Invention
The invention aims to provide an acinetobacter laterosporus PCR detection primer and a method. The primer can be used for PCR detection of acinetobacter cutaneum, and has the advantages of short detection time, low cost, high detection result specificity, easy result interpretation and strong practicability.
The invention is realized by the following technical scheme:
a specific primer for detecting acinetobacter cutaneus comprises PARC-F and PARC-R, and specifically comprises the following components:
PARC-F：CTCTCAGCAAAAGCAGCG；
PARC-R：CCTGATGAGCTAGCAGCAATAAG。
in another aspect of the present invention, there is provided a method for detecting acinetobacter cutaneus, 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, wherein the gel electrophoresis detection is carried out by photographing under a gel imaging system, and if a 485bp DNA specific band exists, the sample is determined to contain the acinetobacter cutaneus.
Further, the sample genome is extracted by an Ezup column genome DNA extraction kit.
Further, the PCR amplification reaction system is 10 μ L: 2 × TSINGKE Master Mix (blue)5 μ L, PARC-F0.5 μ L, PARC-R0.5 μ L, template DNA 1 μ L, double distilled water to 10 μ L.
Further, the PCR amplification reaction program is as follows: pre-denaturation at 95 ℃ for 5 min; performing circulation, namely performing denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extending at 72 ℃ for 30s for 35 cycles; extension at 72 ℃ for 10min, and storage of the PCR product at 4 ℃ to complete.
In addition, the invention also provides application of the specific primer in detecting acinetobacter cutaneus.
Meanwhile, the invention also provides a kit for detecting acinetobacter cutaneus, and the kit comprises the primer pair.
The invention has the beneficial effects that:
because acinetobacter cutaneus, acinetobacter baumannii and acinetobacter in hospitals have similar phenotypes, the traditional biochemical tests cannot be effectively distinguished, and after 16S rRNA universal primer amplification, sequencing is needed to determine the bacterial species, so that the conventional detection is not suitable. The specific primer is used for amplifying a specific fragment of a highly conserved PARC gene of acinetobacter cutaneus, and whether the acinetobacter cutaneus is detected can be directly judged according to an electrophoresis result of a PCR product. Not only has the specificity of molecular biology, but also saves time and cost compared with a 16S rRNA sequencing method.
Drawings
FIG. 1 is a diagram showing the results of gel electrophoresis in the annealing temperature evaluation test of the PCR detection method in the example; m: marker; 1-7 annealing temperatures of 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃,61 ℃ and 8: 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: acinetobacter pitegueta, 2: acinetobacter pitegueta, 3: acinetobacter johnsonii, 4: acinetobacter besii, 5: acinetobacter baumannii 6: acinetobacter torpedo, 7: escherichia coli, 8: pseudomonas aeruginosa, 9: klebsiella pneumoniae, 10: staphylococcus aureus, 11: morganella morganii, 12: serratia marcescens, 13: negative control;
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: 7.2 ng/uL; 2: 7.2X 10-1ng/uL；3：7.2×10-2ng/uL；4：7.2×10-3ng/uL；5：7.2×10-4ng/uL；6：7.2×10-5ng/uL；7：7.2×10-6ng/uL；8：7.2×10-7ng/uL; 9: negative control;
FIG. 4 is a graph showing the results of gel electrophoresis in clinical sample detection in examples, M: marker; 1: a positive control; 2-9: a suspected strain; 10: 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 specific PCR detection method for Acinetobacter pitteus
Design of specific primer of acinetobacter cutaneus
In this example, a pair of specific primers having strong specificity and high sensitivity to acinetobacter cutaneus is designed for a housekeeping gene (ParC gene) of acinetobacter cutaneus, and specific information of the specific primer pair is as follows:
the primer sequence is as follows:
F：5’-CTCTCAGCAAAAGCAGCG-3’；
R：5’-CCTGATGAGCTAGCAGCAATAAG-3’。
(II) extraction of bacterial DNA
Extracting the DNA of the bacteria by using an Ezup column genome DNA extraction kit (bacteria), and specifically comprising the following steps:
1) adding 1mL of bacterial liquid cultured in a constant temperature incubator at 37 ℃ for 24h into a 1.5mL centrifuge tube, centrifuging at room temperature of 8000rmp for 1min, discarding the supernatant, collecting the thalli, adding 180 mu L of Buffer digest, adding 20 mu L of solution of L-protease K, shaking and mixing uniformly, and carrying out water bath at 56 ℃ for 1h until the 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 Buffer BD and mix well by inversion. After adding Buffer BD, if 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 bacterial DNA. The extracted DNA can be immediately subjected to the next test or stored at-20 ℃.
(III) optimum annealing temperature of acinetobacter pitaya primer
After inoculating Acinetobacter Pittani into 1mL of a nutrient broth, culturing the culture broth in a constant temperature incubator at 37 ℃ for 24 hours for enrichment, genomic DNA was extracted as in example 1. Using the A.pittanicus DNA as a PCR template, PCR was carried out using the objective primers at reaction annealing temperatures of 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃,61 ℃ respectively, and the remaining reaction conditions were not changed. Detecting the amplification product by gel electrophoresis, and determining that the temperature of 58 ℃ is the optimal annealing temperature of the acinetobacter cutaneus specific primer according to the gel electrophoresis result after PCR by observing the gel electrophoresis result under ultraviolet light.
As shown in FIG. 1, the specific band is most evident in lane 4, and the optimal annealing temperature of the primer is 58 ℃.
Establishment of (IV) acinetobacter cutaneus specificity PCR detection method
The PCR amplification reaction system is 10 mu L: 2 × TSINGKE Master Mix (blue)5 μ L, PARC-F0.5 μ L, PARC-R0.5 μ L, template DNA 1 μ L, double distilled water to 10 μ L.
The PCR amplification reaction program is as follows: pre-denaturation at 95 ℃ for 5 min; performing circulation, namely performing denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extending at 72 ℃ for 30s for 35 cycles; extension at 72 ℃ for 10min, and storage of the PCR product at 4 ℃ to complete.
(V) determination of PCR amplification result by gel electrophoresis
The PCR product was subjected to 1% agarose gel electrophoresis, and the PCR amplification result was examined. The judgment is specifically as follows: detecting the amplification product by 1% gel electrophoresis, observing the electrophoresis result under the irradiation of an ultraviolet lamp, and if a 485bp single amplification band appears, indicating that the sample contains acinetobacter cutaneus; otherwise, the sample does not contain acinetobacter cutaneus.
Example 2 specificity evaluation test
The specificity verification test of the primer PARC-F/PARC-R was carried out using bacterial DNAs of Acinetobacter Pecteobacter (Acinetobacter pittii), Acinetobacter johnsonii (Acinetobacter johnsonii), Acinetobacter beijerinckii (Acinetobacter baylyi), Acinetobacter baumannii (Acinetobacter baumannii), Acinetobacter torula (Acinetobacter torula), Escherichia coli (Escherichia coli), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Klebsiella pneumoniae (Klebsiella penoniae), Staphylococcus aureus (Staphylococcus aureus), Morganella morganii (Morganella morganii), and Serratia marcescens (Serratia marcescens) as templates, respectively.
The extraction method of the bacterial DNA template comprises the following steps: inoculating the selected bacteria into a 5mLNB nutrient broth liquid culture medium, culturing the bacteria in a 37 ℃ constant temperature incubator for 24h for enrichment, taking 1mL of bacteria liquid, and putting the bacteria liquid into a 1.5mL sterile centrifuge tube: centrifuging at 12000r/min for 15min, discarding supernatant, adding 500 μ L of sterilized double distilled water, gently blowing and beating with a pipette, re-suspending thallus, centrifuging at 12000r/min for 15min, discarding supernatant, and collecting bacteria; adding 100 μ L of sterile double distilled water, gently blowing and beating with a pipette, resuspending the thallus, boiling in boiling water for 15min, immediately taking out, and standing at-20 deg.C for 30 min. Then unfreezing at 35 ℃, centrifuging at 12000r/min for 15min, and taking supernatant and placing at 4 ℃ for later use or storing at-20 ℃.
The PCR amplification reaction system is 10 mu L: 2 × TSINGKE Master Mix (blue)5 μ L, PARC-F0.5 μ L, PARC-R0.5 μ L, template DNA 1 μ L, double distilled water to 10 μ L.
The PCR amplification reaction program is as follows: pre-denaturation at 95 ℃ for 5 min; performing circulation, namely performing denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extending at 72 ℃ for 30s for 35 cycles; extension at 72 ℃ for 10min, and storage of the PCR product at 4 ℃ to complete.
As shown in FIG. 2, the electrophoresis results show that the specific band appears only in 485bp in lanes 1-2A. pithelius, while the specific band does not appear in other strains.
Example 3 sensitivity evaluation test
Acinetobacter pittani was inoculated into 1mL of a nutrient broth liquid medium, the medium was cultured at 37 ℃ for 24 hours to increase the amount of cells, 1mL of the cell liquid was used as in example 1 to extract genomic DNA, and the concentration of DNA was measured by a microspectrophotometer to be 7.2 ng/. mu.L. Sterilizing double distilled water by 100-10-7Performing 10-fold gradient dilution on the extracted DNA, performing PCR amplification by using 8 gradient diluents 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, corresponding to a dilution of 10 for the detected DNA-4The method has the advantages of double dilution and better sensitivity, and the concentration is 0.00072 ng/. mu.L.
Example 4 clinical sample testing
The 8 suspected strains isolated from the oral and nasal cavities of beef cattle were detected using the acinetobacter cutaneus-specific PCR detection method established in example 1.
The results are shown in FIG. 4, and lanes 4 and 8 are positive.
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 cutaneus, and method and application thereof
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Citations (3)
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|---|---|---|---|---|
| JP2014207895A (en) * | 2013-03-25 | 2014-11-06 | 愛知県 | Genotypic typing method of acinetobacter bacteria and primer set using the same |
| CN106755552A (en) * | 2017-03-20 | 2017-05-31 | 辽宁大学 | The PCR detection method of Vibrio vulnificus in a kind of aquiculture animal or aquatic food |
| CN109266658A (en) * | 2018-10-17 | 2019-01-25 | 昆明理工大学 | The specific gene and its primer of a kind of Acinetobacter bauamnnii and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2014207895A (en) * | 2013-03-25 | 2014-11-06 | 愛知県 | Genotypic typing method of acinetobacter bacteria and primer set using the same |
| CN106755552A (en) * | 2017-03-20 | 2017-05-31 | 辽宁大学 | The PCR detection method of Vibrio vulnificus in a kind of aquiculture animal or aquatic food |
| CN109266658A (en) * | 2018-10-17 | 2019-01-25 | 昆明理工大学 | The specific gene and its primer of a kind of Acinetobacter bauamnnii and application |
Non-Patent Citations (2)
| Title |
|---|
| Development and Evaluation of Species-Specific PCR for Detection of Nine Acinetobacter Species;Li X. M.等;《Ann Clin Lab Sci.》;20160531;270-278 * |
| Rapid Determination of Quinolone Resistance in Acinetobacter spp.;Hujer K. M.等;《JOURNAL OF CLINICAL MICROBIOLOGY》;20090531;1436-1442 * |
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