Primer and probe for Zygosaccharomyces bailii detection and detection method thereof

A technology of Zygomyces beijerinckii and primer set, applied in the field of microbial detection, can solve the problems of undetectable damaged cells, low specificity, false positives, etc., to ensure consumer rights, high DNA purity, Guaranteed effect of specificity and sensitivity

Pending Publication Date: 2016-05-25
INSPECTION & QUARANTINE TECH CENT OF QINHUANGDAO ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The currently developed method for detecting Zygomyces baijerinferii uses the selective medium method, which relies on the growth ability of Zygomyces baijerinkoye in sorbate or other weak acid selective medium, and the propagation time usually takes 4-5 days, due to the existence of selection Damaged cells may not be detected under selective pressure such as sorbate
With the development of molecular biology technology, some foreign scholars have used SYBRGreen fluorescent dye to detect Zygomyces baijerin yeast, but the fluorescent dye relies on the combination of double-stranded DNA to generate a fluorescent signal, which has a certain false positive probability and low specificity.

Method used

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  • Primer and probe for Zygosaccharomyces bailii detection and detection method thereof
  • Primer and probe for Zygosaccharomyces bailii detection and detection method thereof
  • Primer and probe for Zygosaccharomyces bailii detection and detection method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1, the design of real-time fluorescent PCR detection method specific primer

[0041] Download the gene sequences of various microorganisms published in GenBank, and use DNAMAN software to compare and analyze, select the 26S ribosomal RNA gene sequence of Zygomyces beijerinferi to design specific primers and probes for fluorescent PCR (only Amplified DNA restriction fragments of Zygomyces baijerinferi, but not other microbial DNA restriction fragments), the primers and probes are as follows:

[0042] The upstream primer sequence is: 5'-TTTGATCAGACATGGTGTTTTGC-3';

[0043] The downstream primer sequence is: 5'-ATGCTGGCCCAGTGAACTG-3';

[0044] Probe sequence: 5'(FAM)-CCCCTCGCCTCTCGTGGGTG-3'(TAMRA).

Embodiment 2

[0045] Embodiment 2, the extraction of test sample DNA template

[0046] (2.1) Take 1mL sample and add it to a 1.5mL sterile centrifuge tube, centrifuge at 8000r / min for 5min, pour off the supernatant, add 1mL10mmol / LTris-HClpH8.0 to resuspend the precipitate, and spin at 8000r / min Centrifuge for 5 minutes, place it upside down on absorbent paper, and blot the liquid. Different samples should be blotted dry in different places on the absorbent paper;

[0047] (2.2) Yeast cell wall breaking: Add 600 μL of sorbitol buffer to the collected bacterial pellet, see Appendix A.2, add 50 U of Lyticase, shake well on a vortex shaker, and treat at 30°C for 30 minutes. Centrifuge at 4000r / min for 10min, discard the supernatant, and keep the precipitate;

[0048](2.3) Add 700 μL of 20g / LCTAB lysate to the cell pellet after breaking the wall, shake on a vortex shaker to mix well, and place in a 65°C water bath for 0.5-2 hours;

[0049] (2.4) Extraction: After cooling to room temperature, ...

Embodiment 3

[0055] Embodiment 3, real-time fluorescent PCR detection of Zygomyces baijerin

[0056] Using the specific primers and probes designed in Example 1 above, PCR amplification was performed using the extracted DNA as a template.

[0057] (3.1) The real-time fluorescent PCR reaction system is 25 μL, including 12.5 μL 2×realtimePCRBuffer (containing dNTP, Mg 2+ and Taq enzyme), 1 μL of designed upstream primer and downstream primer (final concentration of 0.2 μM), 1 μL of probe (final concentration of 0.2 μM), 2 μL of extracted product DNA template, 7.5 μL of pure water;

[0058] (3.2) The PCR reaction program is: pre-denaturation: temperature 95°C, time 10 minutes, then PCR amplification: temperature 95°C, time 15 seconds, temperature 63°C, time 1 minute, cycle 40 times;

[0059] (3.3) Use the random software of the fluorescent quantitative PCR instrument to set the reaction parameters, save the file after the reaction, open the analysis software, and analyze the experimental res...

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Abstract

The invention relates to a quick qualitative detection technology on Zygosaccharomyces bailii in grape wine and fruit juice and in particular relates to a primer and a probe which are used for Zygosaccharomyces bailii detection and a detection method thereof. According to the invention, a 26S ribosome RNA gene sequence of Zygosaccharomyces bailii is selected, a specific primer and probe set is designed, and simple and quick detection on Zygosaccharomyces bailii in the grape wine and fruit juice can be realized by adopting the primer and the probe and a real-time fluorescent PCR technology.

Description

technical field [0001] The invention relates to the detection of microorganisms, in particular to a primer and a probe for detection of Zygomyces baijerin yeast and a detection method thereof. Background technique [0002] Zygosaccharomyces bailii is an important class of microorganisms that cause food spoilage. In the food and beverage industry, especially wine and fruit juice, its raw materials and production processes are destined to have a great probability of contamination. Zygomyces baijerinferi can grow under weakly acidic preservatives such as benzoic acid and sorbic acid (PittandHocking, 1999), and the growth density of Zygomycetes baijirin is very low-dependent, and the amount of cells required for food spoilage is very small, studies have shown Five cells are enough to spoil a carbonated drink (Pitt and Hocking, 1999). Zygomyces baijerinferi widely exists in the natural environment, and raw grapes, water, and air may introduce Zygomycetes baijerinferi. The compl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895C12Q1/686
Inventor 钱云开高飞崔宗岩王海洋王飞肖艳霞吴曦张进杰曹彦忠刘永明李学民
Owner INSPECTION & QUARANTINE TECH CENT OF QINHUANGDAO ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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