Primer and probe for alicyclobacillus acidoterrestris PCR detection and applications thereof

A technology of bacillus and alicyclic acid, which is applied in the field of food microorganism detection, can solve the problems of low content, long time consumption, and has not yet been applied, and achieves the effects of strong specificity, high sensitivity and broad application prospects.

Active Publication Date: 2015-04-08
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the routine detection of A. acidoterrestris is mainly carried out by the culture method. Although this method is simple and easy to operate, it takes a long time and the detection lags behind the production.
At the same time, the content of A.acidoterrestris in beverages and foods is very low, and complex food ingredients and non-target microorganisms have a great impact on the detection of target bacteria. Conventional PCR, enzyme-linked immunosorbent assay, Fourier transform infrared spectroscopy Detection methods such as electronic nose and electronic nose need to be combined with bacterial culture enrichment to achieve the purpose of detection. Therefore, they have not been applied to the actual detection of the food industry.

Method used

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  • Primer and probe for alicyclobacillus acidoterrestris PCR detection and applications thereof
  • Primer and probe for alicyclobacillus acidoterrestris PCR detection and applications thereof
  • Primer and probe for alicyclobacillus acidoterrestris PCR detection and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Primer and probe design and synthesis

[0020] Use Primer Premier5 software to compare the 16SrDNA gene sequences of 10 different species in Alicyclobacillus spp., such as figure 1 As indicated, primers and fluorescent probes based on the conserved region of A. acidoterrestris were designed using the probe design software Primer Express, as shown in Table 1. After extensive screening, the primer sequences and probes of the following sequences were obtained, as shown in Table 2.

[0021] The upstream primer (FP) sequence of the primer is SEQ ID NO.1, and the downstream primer (RP) sequence of the primer is SEQ ID NO.2. The composition of the specific primer sequence is as follows:

[0022] SEQ ID NO.1: 5'-TGAGT AACAC GTGGG CAATC TG-3';

[0023] SEQ ID NO. 2: 5'-CTACC CGTGT ATTAT CCGGC AT-3'.

[0024] The nucleotide sequence of the fluorescent probe is: 5'-CTTTCAGACTGGAATAAC-3', the 5' end of the probe is labeled with 6-carboxyfluorescein (FAM) fluorescent dye, and the...

Embodiment 2

[0035] specificity test

[0036] The real-time fluorescent PCR detection system that adopts primer SEQ ID NO.1, SEQ ID NO.2 and probe SEQ ID NO.3 to set up in the method of the present invention detects bacterial strain as shown in table 1, evaluates primer, probe among the present invention The specificity of needle and real-time fluorescent PCR detection system.

[0037] The 30 bacterial strains shown in Table 1 were cultivated under optimal conditions, and their concentration was adjusted to 10 with sterile ultrapure water. 4 -10 5CFU / mL. Place 5 mL of the prepared bacterial cell dilution sample in a centrifuge tube, freeze and centrifuge at 4°C and 6000 rpm for 10 min, and discard the supernatant. Add 20mg / mL of lysozyme buffer (20mmol / L Tris-HCl, pH8.0, 2mmol / L EDTA and1.2%Triton X-100) and treat in a water bath at 37°C for 30min. Extract according to the instructions of the kit, and finally dissolve the extracted sample with 100 μL eluent. Adopt nucleic acid titrato...

Embodiment 3

[0041] Sensitivity experiment

[0042] Take 10mL A.acidoterrestris standard strain proliferation medium (3 × 10 7 CFU / mL), diluted 10 times with sterile ultrapure water, the concentrations were: 3×10 6 CFU / mL, 3×10 5 CFU / mL, 3×10 4 CFU / mL, 3×10 3 CFU / mL, 3×10 2 CFU / mL, 3×10 1 CFU / mL, 3×10 0 CFU / mL. Place 5 mL of the prepared bacterial cell dilution sample in a centrifuge tube, freeze and centrifuge at 4°C and 6000 rpm for 10 min, and discard the supernatant. Add 20mg / mL of lysozyme buffer (20mmol / L Tris-HCl, pH8.0, 2mmol / L EDTA and1.2%Triton X-100) and treat in a water bath at 37°C for 30min. Extract according to the instructions of the kit, and finally dissolve the extracted sample with 100 μL eluent. A nucleic acid titrator is used to measure the concentration and purity of the extracted DNA, and the PCR amplification detection of the sample is carried out according to the above-mentioned method. At the same time, according to the relationship between the sample co...

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PUM

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Abstract

The invention relates to a primer and a probe for alicyclobacillus acidoterrestris PCR detection and applications thereof. The primer and the probe are designed according to a conserved domain of an A.a cidoterrestris16S rDNA gene, and are used for quantitatively detecting the nucleic acid copy number of A.a cidoterrestris in a sample to be detected. Specifically, an upstream primer of the primer has a nucleotide sequence of EQ ID NO.1 in a sequence table, and a downstream primer has a nucleotide sequence of EQ ID NO.2 in the sequence table; the probe has a nucleotide sequence of EQ ID NO.3 in the sequence table. A related method is implemented by taking the total DNA of a sample as a template through carrying out real-time fluorescent PCR amplification by using the primer and the probe, acquiring data after each cycle is completed, and determining results according to an amplification curve after the reaction is reacted. The probe disclosed by the invention has the characteristics of good specificity, rapid detection speed, 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 primers, probes and applications in fluorescence quantitative PCR detection of Alicyclobacillus acidoterrestris (A. acidoterrestris). Background technique [0002] Alicyclobacillus spp. is a Gram-positive bacillus, rod-shaped, spore-forming, and strictly aerobic. It can survive and grow at a pH of 2.5-6.0 and a temperature of 20-60°C. Because of its heat-resistant and acid-resistant characteristics, it is difficult to kill it by ordinary pasteurization, and its growth and metabolism produce substances such as guaiacol and 2,6-dibromophenol, which deteriorate the taste and flavor of the juice and form White precipitate or cloudy turbidity, therefore, Alicyclobacillus has become an important quality indicator in the fruit juice and beverage processing industry. Because Alicyclobacillus acidoterrestris (A.acidoterrestris) is the most isolated Alicyc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/01
CPCC12Q1/04C12Q1/686C12Q2561/113C12Q2563/107
Inventor 岳田利王周利袁亚宏蔡瑞牛晨
Owner NORTHWEST A & F UNIV
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