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Primer and application thereof in PCR detection of alicyclobacillus spp. in fruit juice

A technology of bacillus and alicyclic acid, which is applied in the field of PCR detection primers of alicyclic acid bacillus in fruit juice, can solve the problems of heavy workload, unanalyzed, and detection lag, and achieve good amplification specificity, high sensitivity, highly specific effect

Inactive Publication Date: 2015-12-16
NORTHWEST A & F UNIV
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  • Abstract
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Problems solved by technology

[0003] At present, the internationally accepted detection method for Alicyclobacillus is still the culture method. Although it is simple and easy to operate, it has a large workload, takes a long time, and the detection seriously lags behind the actual production.
At the same time, conventional PCR, TaqMan real-time fluorescence quantitative PCR, Fourier transform infrared spectroscopy and electronic nose detection methods developed by research and development are mainly aimed at A.acidoterrestris, and have not been really applied to the analysis of actual samples in the food industry

Method used

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  • Primer and application thereof in PCR detection of alicyclobacillus spp. in fruit juice
  • Primer and application thereof in PCR detection of alicyclobacillus spp. in fruit juice
  • Primer and application thereof in PCR detection of alicyclobacillus spp. in fruit juice

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Embodiment 1

[0059] Detection and Evaluation of Alicyclobacillus in Juice

[0060] Take 10mL of the proliferation culture solution of Alicyclobacillus standard strain (2.8×10 8 CFU / mL), with kiwi fruit juice (soluble solids ≤ 15Brix) for gradient dilution to obtain spiked juice. At the same time, the bacteria were diluted with sterile water for sample separation and detection. Then 5 mg of immunomagnetic microspheres were added to 2 mL of fruit juice sample system, immunocaptured at 37°C for 30 min, and the "immunomagnetic microspheres-bacteria" complex was obtained by separating under the condition of an external magnetic field. Take 200 μL of the sample to be tested, discard the supernatant under magnetic conditions, add 50 μL Tris-HCl-EDTA buffer solution (TEBuffer, pH 8.0) to the bacterial complex, boil in a boiling water bath for 10 minutes, and then centrifuge at 4000×g for 5 minutes. The collected supernatant is the extracted DNA sample. A nucleic acid titrator was used to measur...

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Abstract

The invention discloses a primer and an application thereof in PCR detection of alicyclobacillus spp. in fruit juice. The method comprises the following steps: acquiring the specific primer according to the design of a conserved domain of a 16S rDNA gene sequence of the alicyclobacillus spp.; establishing an SYBR Green I fluorescent quantitative PCR method system; carrying out amplification on a standard positive sample, establishing a standard curve and drawing a solubility curve; carrying out immunomagnetic separation and DNA extraction on the alicyclobacillus spp. in a fruit juice sample; and carrying out analysis and judgment on the result. According to the invention, the established fluorescent quantitative detection method can detect ninety-six samples within three hours, and has the characteristics of rapidness and simplicity, low detection cost, simplicity in realizing standard operation, good accuracy, and high sensitivity.

Description

technical field [0001] The invention belongs to the technical field of food microorganism detection, and in particular relates to a primer for PCR detection of Alicyclobacillus in fruit juice and an application thereof. Background technique [0002] Alicyclobacillus (Alicylobacillus spp.) is a general term for a class of acidophilic, heat-resistant Gram-positive bacillus, rod-shaped, spore-forming, and strictly aerobic. The pH range in which such bacteria can grow is 2.0-6.0, and the optimum pH value is 3.0-5.0; the temperature range in which they can grow is 20-60°C, and the optimum growth temperature is 45-50°C. These bacteria are also known as "acidophilic and thermostable bacteria" because they can tolerate ordinary pasteurization and can survive and even grow under low pH and high temperature conditions. Alicyclobacillus can grow and metabolize in fruit juice to produce guaiacol, 2,6-dibromophenol, 2,6-dichlorophenol and other substances, which will deteriorate the tas...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6851C12Q1/689C12Q2531/113C12Q2563/107
Inventor 王周利蔡瑞岳田利袁亚宏
Owner NORTHWEST A & F UNIV
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