Fluorescence quantitative polymerase chain reaction (PCR) method for detecting bacillus coagulans quickly
A technology of Bacillus coagulans and fluorescence quantification, which is applied in the field of rapid detection of fluorescence quantitative PCR, to achieve the effects of improving sensitivity, fast detection speed and reducing detection cost
Inactive Publication Date: 2013-03-20
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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Problems solved by technology
However, so far, there have been no reports in China that are conducive to the rapid detection of Bacillus coagulans by fluorescent quantitative PCR.
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[0017] First, take 1g of the sample to be tested by aseptic operation, add it into a sterilized container filled with 9ml of acidic broth (acid canned food) or bromcresol purple glucose broth (low-acid canned food and other samples to be tested), at 55 ° C ± 1 ℃, cultivated under aerobic conditions for 48h. Take the cultured bacterial solution and add it to a centrifuge tube, centrifuge at 12000rpm for 1min, discard the supernatant, use bacterial cell precipitation to extract genomic DNA and dilute it for later use.
[0018] Then perform fluorescent quantitative PCR reaction, the total volume of each fluorescent quantitative PCR reaction is 25 μl, including extracted genomic DNA, 2 μl; 10×PCR Buffer, 2.5 μl; Mg 2+ 1.5mmol / L; Tag enzyme 1u; UNG enzyme 1u; dNTP 0.1mmol / L; Bacillus coagulans-specific primers and probes 10pmol each; add ddH at the end 2 0 to 25 μl; set the program parameters of the fluorescent quantitative PCR instrument as follows: 37°C for 2min; pre-denaturati...
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Abstract
The invention relates to a fluorescence quantitative polymerase chain reaction (PCR) method for detecting bacillus coagulans quickly. The fluorescence quantitative polymerase chain reaction (PCR) method comprises the following steps of: extracting deoxyribonucleic acid (DNA) of a genome of enrichment liquid of a sample to be detected, and diluting for later use; designing specific primers by utilizing a conservative DNA sequence of a genome of the bacillus coagulans; performing fluorescence quantitative PCR amplification and detection on the DNA of a genome of a suspected bacterial colony on a separated agar plate of the sample to be detected by using the primers; adding 25 micro liters of double distilled H2O (dd H2O); setting process parameters of a fluorescence quantitative PCR instrument, namely the temperature is 37 DEG C and the time is 2 minutes; pre-modifying at the temperature of 95 DEG C for 30 seconds, modifying at the temperature of 95 DEG C for 5 seconds, annealing at the temperature of 60 DEG C for 40 seconds, collecting carboxy fluorescein (FAM) fluorescence, and circulating for 40 times; and storing at the temperature of 4 DEG C, wherein in sample loading parameters, the total volume of each fluorescence quantitative PCR reaction is 25 micro liters, and each fluorescence quantitative PCR reaction uses 2 micro liters of DNA of the extracted genome, 2.5 micro liters of 10*PCR Buffer, 1.5 mmol / L of Mg<2+>, 1 u of Tag enzyme, 1 u of glycosylation (UNG) enzyme, 0.1 mmol / L of deoxynucleotide triphosphate (dNTP), 10 pmol of specific primers of the bacillus coagulans and 10 pmol of probe. The fluorescence quantitative PCR method has the advantages of quick detection speed, high sensitivity and low cost.
Description
technical field [0001] The invention relates to a rapid detection method of fluorescent quantitative PCR for Bacillus Coagulans. Background technique [0002] Microorganisms that cause canned food to become rancid but not fat (that is, produce acid but not gas) are called flat acid bacteria in the canning industry. It is a group of high-temperature types in the family of Aerobic Bacillus, with the characteristics of thermophile and heat resistance, and its suitable growth temperature is 45°C to 60°C. Bacillus coagulans is an important microorganism responsible for the rancidity of canned food. Bacillus coagulans is also a probiotic, and it is one of the microbial grade feed additives approved by the Ministry of Agriculture in 2004. Because of its advantages of regulating intestinal dysfunction, maintaining the balance of intestinal flora, improving the health of the body, and avoiding problems such as drug resistance and double infection caused by taking antibiotics, it ha...
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IPC IPC(8): C12Q1/68C12Q1/04C12R1/07
Inventor 刘云国祝素珍王英超孙涛马维兴唐静张健马云
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU