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Special primers for identification or assisted identification of Propionibacterium acnes, and applications thereof

A P. acnes, auxiliary identification technology, applied in the biological field, can solve the problems of long cycle, cumbersome identification methods, etc., and achieves the effects of high speed, high sensitivity and clear spectrum

Inactive Publication Date: 2016-11-30
沈阳中科赛尔生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There are many kinds of microorganisms on the surface of human skin, and there are only three species of Propionibacterium, and it is almost difficult to distinguish them only from culture and morphological observation
The current identification method for Propionibacterium acnes is cumbersome and takes a long period of time. It is necessary to culture the sample with a specific medium first, and then pick a single clone for 16sPCR amplification and sequencing to complete the identification.

Method used

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  • Special primers for identification or assisted identification of Propionibacterium acnes, and applications thereof
  • Special primers for identification or assisted identification of Propionibacterium acnes, and applications thereof
  • Special primers for identification or assisted identification of Propionibacterium acnes, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Sample to be tested: Cotton swab after wiping face or post-culture on cotton swab after wiping face

[0033] For extracting the DNA of the sample to be tested, refer to the extraction method of Gram-positive bacteria.

[0034] Specifically, 6 cases of acne patients (ranging from 1 to 4 grades of severity) facial swab samples were taken, and 3 of them were cultured on Columbia blood agar plates, and the cultures were washed and recovered from the plates with PBS buffer, and extracted for culture. total DNA. For the remaining 3 cases, the total DNA of the samples was directly extracted with the QIAamp DNA mini kit (QIAGEN); set aside.

[0035] PCR detection:

[0036] Using the DNA obtained above as templates, primer pair 12 was used for PCR amplification detection. The total volume of the PCR reaction system was 20 μL, including: 0.5 μM upstream primers, 0.5 μM downstream primers, 1 μL template, 10 μL 2×PCR mix (2×PCR Premixed reaction solution), deionized water;

[0...

Embodiment 2

[0042] Sample to be tested: sample culture detection of Propionibacterium acnes and identification of monoclonal bacteria

[0043] Take a cotton swab sample with open comedones (blackheads) on the face and spread it on Columbia blood agar plate for culture. Pick 3 single colonies (2 of which are dry and raised small colonies suspected of Propionibacterium acnes, and the other is a flat translucent colony) for expansion and culture, and set aside.

[0044] PCR detection:

[0045] Using the DAN obtained above as templates, primer pair 34 (see Table 1) was used for PCR amplification detection. The total volume of the PCR reaction system was 20 μL, including: 0.5 μM upstream primer, 0.5 μM downstream primer, 1 μL template, 10 μL 2× PCRmix (2×PCR premixed reaction solution), deionized water;

[0046] The reaction conditions are: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30s, annealing at 60°C for 30s, extension at 72°C for 45s, 35 cycles;

[0047] Electrop...

Embodiment 3

[0051] Sample to be tested: Genomic DNA of a monoclonal culture of Propionibacterium acnes confirmed by sequencing was used as a standard, and its concentration was diluted to 10 7 a / μL; 10 6 a / μL; 10 5 a / μL; 10 4 a / μL; 10 3 a / μL; 10 2 a / μL; 10 1 cells / μL; for use.

[0052] qPCR detection:

[0053] Using the DNA of different concentrations obtained above as a template, PCR amplification detection was performed using primer pair 56. The total volume of the PCR reaction system was 20 μL, including: 0.5 μM upstream primer, 0.5 μM downstream primer, 1 μL template, 10 μL 2×qPCR mix ( 2×qPCR premix reaction solution), deionized water;

[0054] The reaction conditions are: pre-denaturation at 94°C for 3 min; 40 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 45 s. Fluorescence was detected after each cycle (see Figure 4-7 );

[0055] Depend on Figure 4 Visible, 10 7 for template concentration 10 7 qPCR amplification curve o...

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Abstract

The present invention relates to the field of biotechnology, particularly to special primers for identification or assisted identification of Propionibacterium acnes, and applications thereof, wherein the primers are a primer pair formed by combining any two sequences selected from sequences represented in SEQ ID NO:1-6. According to the present invention, the special primers provide great significance for the species identification of the Propionibacterium acnes and the prevention and control of facial diseases caused by the Propionibacterium acnes infection.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a special primer for identifying or assisting in identifying Propionibacterium acnes and its application. Background technique [0002] Propionibacterium acnes is a fatty acid-loving actinomycete, Gram-positive, widely distributed on the human skin surface, and its distribution range on the skin surface varies with the degree of sebaceous gland secretion. [0003] Propionibacterium acnes is generally believed to be the main pathogenic bacteria causing acne. In addition, in surgical operations such as heart valve surgery and orthopedic device surgery, postoperative P. acnes infection is not uncommon. [0004] There are many kinds of microorganisms on the surface of human skin, and there are only three species of Propionibacterium, but it is almost difficult to distinguish them only from culture and morphological observation. The current identification method for Propionibacterium ac...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/689
Inventor 赵书阳辛成奇慈家宇郭海燕
Owner 沈阳中科赛尔生物科技有限公司
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