Specific primer and method for detecting Acinetobacter johnsonii and application

A specific, bacilli technology, applied in the field of microorganisms, can solve the problems of PCR nucleic acid and primers for Acinetobacter johnsonii that have not been invented yet, and achieve the effects of high sensitivity, time saving, and easy detection.

Active Publication Date: 2019-08-30
渠县菜家山牲畜养殖有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Through literature search to the prior art, it is found that no one has invented the relevant reports r...

Method used

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  • Specific primer and method for detecting Acinetobacter johnsonii and application
  • Specific primer and method for detecting Acinetobacter johnsonii and application
  • Specific primer and method for detecting Acinetobacter johnsonii and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Acinetobacter Johnson specific PCR detection method is established

[0029] (1) Design of specific primers for Acinetobacter johnsonii

[0030] In this embodiment, for the tyrosine protein kinase gene (stk gene) of Acinetobacter johnsonii, a pair of specific primers with strong specificity and high sensitivity to Acinetobacter johnsonis is designed, and the specific information of the specific primer pair is as follows :

[0031] The primer sequences are:

[0032] F: 5'-CAGGTCCTGCGCCAGAAGTTG-3';

[0033] R: 5'-GATGCCATCCGTCACGGCTAAG-3'.

[0034] (2) Preparation of Acinetobacter johnsonii DNA template

[0035] 2.1 Extraction of Acinetobacter johnsonii DNA

[0036] Use the Ezup column's genomic DNA extraction kit (bacteria) to extract the DNA of Acinetobacter johnsonii, and the specific steps are as follows:

[0037] 1) Take 1 mL of Acinetobacter johnsonii cultured in a constant temperature incubator at 37°C for 24 hours, add it to a 1.5 mL centrifuge tube...

Embodiment 2

[0056] Embodiment 2 Specificity evaluation test

[0057] According to DNA template preparation and PCR detection method among the embodiment 1, Serratia marcescens, Citrobacter, Bacillus, Pseudomonas aeruginosa, Acinetobacter johnsonii, Acinetobacter pite, calcium acetate preserved in this laboratory baumannii, Acinetobacter seifertii, Escherichia coli, Streptococcus agalactiae, Enterococcus faecalis, Staphylococcus aureus, Staphylococcus saprophyticus and Streptococcus dysgalactiae for PCR amplification.

[0058] figure 2 The electrophoresis results showed that only Acinetobacter johnsonii had a specific band at 365bp, while other species did not.

Embodiment 3

[0059] Embodiment 3 sensitivity evaluation test

[0060] Inoculate Acinetobacter johnsonii in 1mL nutrient broth liquid medium, place in a 37°C constant temperature incubator and culture for 24 hours to enrich the bacteria, then use 10-fold gradient dilution with sterilized nutrient broth liquid medium, and count the bacterial concentration by plate method For, take 1mL of bacterial liquid to extract genomic DNA according to Example 1, and use sterilized double distilled water to press 10 0 -10 -6 Perform 10-fold gradient dilution of genomic DNA, use 7 gradient dilutions as templates for PCR amplification, detect the amplified products by gel electrophoresis, and observe the results of gel electrophoresis under ultraviolet light.

[0061] Depend on image 3 It can be seen that a clear band can be seen in lane 5, and the corresponding detection bacteria concentration is 7.45×10 2 CFU / mL, this method has good sensitivity.

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Abstract

The invention discloses a specific primer and a method for detecting Acinetobacter johnsonii and application, wherein the specific primer is as shown in SEQ ID NO. 1 to 2; the method includes the steps of extracting genomic DNA of a sample to be detected, using the primer for PCR amplification, performing gel electrophoresis detection, and photographing under a gel imaging system for detection, finding that a 365 bp DNA-specific band exists, which can determine that the sample contains the Acinetobacter johnsonii. The detection method of the invention has the advantages of short time, high specificity and high sensitivity in detecting the Acinetobacter johnsonii. A traditional identification method that is cumbersome in operation, long time-consuming, low in accuracy and low in detection rate is not used.

Description

technical field [0001] The invention belongs to the technical field of microbes, and in particular relates to a specific primer for detecting Acinetobacter johnsonii and its method and application. Background technique [0002] The genus Acinetobacter has received increasing attention in recent years, not only because of its ability to cause human disease, but also because of the unique biological characteristics of each species in the genus Acinetobacter. Acinetobacter johnsonii is a species of Acinetobacter. Acinetobacter johnsonii is a gram-negative bacterium that exists widely in nature and has a strong ability to survive. It can survive in high-phosphorus or high-salt environments. opportunistic pathogenic bacteria, which can cause disease in humans and animals. According to current reports, due to the widespread use of antibiotics in clinical practice, the bacteria continue to develop drug-resistant mechanisms under their selection pressure, and the number of drug-res...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689
Inventor 左之才崔耀成才冬杰任志华苟丽萍胡延春王之盛马晓平余树民谢跃邹华围沈留红马志宇
Owner 渠县菜家山牲畜养殖有限公司
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