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Specific primer and method for detecting towneri acinetobacter and application

A specific, bacilli-based technology, applied in the field of microbiology, can solve problems such as nucleic acid and primers that have not been invented by anyone, and achieve the effects of simple detection, easy determination, and high sensitivity

Active Publication Date: 2019-08-30
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Through literature search to the prior art, it is found that no one has invented the relevant reports related to the PCR detection method, nucleic acid and primers of Acinetobacter towneri of the present invention.

Method used

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  • Specific primer and method for detecting towneri acinetobacter and application
  • Specific primer and method for detecting towneri acinetobacter and application
  • Specific primer and method for detecting towneri acinetobacter and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Establishment of a specific PCR detection method for Acinetobacter towneri

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

[0030] In this embodiment, for the outer membrane protein A gene (OmpA gene) of Acinetobacter towneri, a pair of specific primers with strong specificity and high sensitivity to Acinetobacter towneri are designed. The specific information of the specific primer pair is as follows :

[0031] The primer sequences are:

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

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

[0034] (2) DNA template preparation of Acinetobacter towneri

[0035] 2.1 Extraction of Acinetobacter towneri DNA

[0036] Utilize TE buffer boiling method to extract the DNA of Acinetobacter towneri, concrete steps are as follows:

[0037] 1) Take 1 mL of Acinetobacter towneri cultured in a 37°C constant temperature incubator for 24 hours in a 4 mL centrifuge tube, centrifuge (room temperature, 6000r, 3 min), and discard the...

Embodiment 2

[0050] Embodiment 2 Specificity evaluation test

[0051] According to the DNA template preparation and PCR detection method in Example 1, the Acinetobacter baumannii marcescens, Acinetobacter Pitt, Acinetobacter Johnson, Morganella, Staphylococcus aureus, E. Reedia, Klebsiella pneumoniae, Neisseria, Pasteurella, Shigella, Pseudomonas aeruginosa, Proteus vulgaris, Citrobacter kirsi, Serratia marcescens for PCR amplification Increased response.

[0052] Depend on figure 2 In the electrophoresis results, it can be seen that only Acinetobacter towneri has a clear and bright specific band at 401bp, while other bacteria have no specific band.

Embodiment 3

[0053] Embodiment 3 sensitivity evaluation test

[0054] Inoculate Acinetobacter towneri in 1mL nutrient broth liquid medium, place it 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 8.7×10 9 CFU / mL, get 1mL bacterium liquid and extract genomic DNA according to embodiment 1, press 10 with sterilized double distilled water 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.

[0055] Depend on image 3 It can be seen that a clear band can be seen in lane 4, and the corresponding detection bacteria concentration is 8.7×10 6 CFU / mL, this method has good sensitivity.

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Abstract

The invention discloses a specific primer and method for detecting towneri acinetobacter and application. The specific primer is as shown in SEQ ID NO. 1-2. The genomic DNA of a sample to be tested isextracted; PCR amplification is carried out by using the primer; gel electrophoresis detection is carried out; photographing detection is carried out under a gel imaging system; if DNA specific bandswith 401 bp exist, the towneri acinetobacter is determined to be contained in the sample. Through adopting the detection method disclosed by the invention to detect the towneri acinetobacter, time needed is short; the specificity is strong; the sensitivity is relatively high. According to the specific primer and method, the shortcomings of complex operation, long consuming time, low accuracy, lowdetection rate and the like by adopting a traditional identification method are avoided.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a specific primer for detecting Acinetobacter towneri, a method and an application. Background technique [0002] Acinetobacter (Acinetobacter) is a non-glucose-fermenting Gram-negative coccobacillus, which has many species and is widely distributed in water, soil and skin surfaces in nature. Previous studies have shown that most of Acinetobacter are nosocomial Acinetobacter isolated from hospital clinics, and it has also been reported that Acinetobacter is one of the important conditional pathogens that cause nosocomial infections, and is common in ventilator-associated pneumonia and bacteremia , wound infection, etc., usually colonize the host of the body. When the body's immune function is low, the colonized Acinetobacter may invade other parts and cause infection. According to the research in the past ten years, Acinetobacter has gradually shifted from hum...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689
Inventor 左之才岑垚王之盛才冬杰苟丽萍马志宇谢跃任志华邓俊良马晓平余树民胡延春沈留红
Owner SICHUAN AGRI UNIV
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