Biological amplification-real-time fluorescent quantitative PCR combined kit and method for rapidly detecting staphylococcus aureus bacteriophage

A real-time fluorescence quantitative, phage biological technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problem of time-consuming, achieve optimized experimental conditions, high sensitivity, low cost Effect

Pending Publication Date: 2022-05-13
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional phage biological amplification method indirectly detects the presence of pathogenic bacteria by observing the plaques formed by progeny phages on the agar plate, but the plaque formation process usually takes 3-4 hours, which takes a long time

Method used

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  • Biological amplification-real-time fluorescent quantitative PCR combined kit and method for rapidly detecting staphylococcus aureus bacteriophage
  • Biological amplification-real-time fluorescent quantitative PCR combined kit and method for rapidly detecting staphylococcus aureus bacteriophage
  • Biological amplification-real-time fluorescent quantitative PCR combined kit and method for rapidly detecting staphylococcus aureus bacteriophage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Activation of Example 1 Phage

[0054] 1. Activation of host bacteria

[0055] Take out the staphylococcus aureus ATCC 25923 stored in the glycerol tube from the refrigerator at -80°C and inoculate it on the Baird Parker solid medium, culture it at 37°C for 12 hours, pick a single colony and inoculate it in 3 mL of LB liquid medium, and inoculate it on Baird Parker solid medium. Cultivate on a shaker at 37°C and 160r / min for 8 hours, take 100 μL of the cultured bacterial solution, and measure the concentration of the bacterial solution by the dilution coating method;

[0056] Bacterial concentration (CFU / mL) = number of single colonies × dilution factor × 10;

[0057] 2. Phage Activation

[0058] Take out the phage LSA2311 stored in the glycerol tube from the -80°C refrigerator and mix it with the logarithmic phase host bacteria solution for 12-18h. Centrifuge the cultured mixture at 8000r / min for 10min at 4°C. The host bacteria were removed by filtration through a p...

Embodiment 2

[0059] The establishment of embodiment 2 phage biological amplification method

[0060] 1. Effect of host bacteria concentration

[0061] Staphylococcus aureus ATCC 25923 bacteria solution was serially diluted 10 times (10 5 -10 9 CFU / mL), 100 μL of each concentration was mixed with the same volume of phage LSA2311 (10 7 PFU / mL), the volume was made up to 1mL with LB medium, placed in a culture shaker at 37°C, shaken at 160r / min for 24h, centrifuged and sterilized by filtration, and the phage titer was determined by the double-layer plate method. The optimal concentration of host bacteria was selected by progeny phage titer.

[0062] Depend on figure 1 A Visible: 10 8 The phage titer after amplification of the host bacteria with CFU / mL concentration was significantly higher than 10 9 and 10 7 CFU / mL concentration group, so the concentration of host bacteria in the culture system was determined to be 10 8 CFU / mL.

[0063] 2. Selection of Phage Titers

[0064] Determi...

Embodiment 3

[0081] Evaluation of embodiment 3 phage biological amplification method in Staphylococcus aureus detection

[0082] 1. Specificity of phage biological amplification method

[0083] 40 strains of bacteria (see Table 2) were detected by the phage biological amplification method. At the same time, Staphylococcus aureus ATCC 25923 inactivated by heat treatment at 100°C for 10 minutes was set up as the experimental group, and live bacteria without heating were used as the control group to observe the ability of the phage biological amplification method to detect Staphylococcus aureus.

[0084] Detected by phage biological amplification method, among 40 strains of bacteria, 29 strains of Staphylococcus aureus had plaque formation; 11 non-staphylococcus aureus had no plaque formation; heat-inactivated Staphylococcus aureus ATCC 25923 had no plaque formation Plaque formation, but not inactivated plaque formation. It can be seen that the phage biological amplification method can only...

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Abstract

The invention discloses a bacteriophage biological amplification-real-time fluorescent quantitative PCR combined kit for rapidly detecting staphylococcus aureus and a method thereof. The combined kit comprises a phosphate buffer solution containing staphylococcus aureus bacteriophage LSA2311 and a primer pair tsp for real-time fluorescent quantitative PCR detection, a good detection effect can be achieved by combining phage biological amplification with a qPCR method to detect staphylococcus aureus in a food matrix, and the detection limit is 101 CFU / mL. Compared with a traditional culture method, the detection time is greatly shortened, and sample detection is completed within 2-3 h. According to the invention, an experimental basis and a theoretical basis can be provided for establishment of a staphylococcus aureus rapid detection method based on bacteriophage biological amplification.

Description

technical field [0001] The invention relates to the field of food safety detection, in particular to a combination kit for rapidly detecting Staphylococcus aureus phage biological amplification-real-time fluorescent quantitative PCR and a method thereof. Background technique [0002] Staphylococcus aureus (S. aureus) is a Gram-positive bacterium that is cocciform and tends to be arranged in clusters described as "grape-like." It is widely distributed in nature, such as air, soil, water and other environments, and also exists in the normal human flora of the skin and mucous membranes (usually the nasal cavity area) of most healthy individuals. Staphylococcus aureus is widely distributed and exists in the air, water, livestock and poultry animals such as humans and dairy cows, and their excrement, which makes food more likely to be contaminated. Foodborne illnesses due to Staphylococcus aureus contamination have been reported to be a global health problem. The disease caused...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/689C12Q1/6851C12Q1/14C12Q1/06C12N15/11
CPCC12Q1/701C12Q1/689C12Q1/6851C12Q2600/166C12Q2531/113C12Q2563/107C12Q2561/113C12Q2545/113
Inventor 王小红丁一峰王佳邵彦春陈慕潇朱文娟王吉龚云霞
Owner HUAZHONG AGRI UNIV
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