Pcr Technology For Rapid Detection Of Beer-spoilage Baeteria

In the brewery, Beer-spoilage baeteria are those that harm the quality of beer, destruction of beer flavor and biological stability, which is a class of strictly anaerobic microorganisms. The bacteria is usually a class of Gram-positive, cata-lase-negative and produce lactic acid. Beer-spoilage baeteria Appear in beer. At present, the beer-spoilage baeteria of beer brewery detection method is that the sample make membrane filtration,and than take the membrane to the surface of AGAR medium or fluid nutrient medium, in anaerobic environment, according to the strains of growth in the medium, Such as growth speed, the special colony morphol-ogy and physiological reaction (such as special substrates of chromogenic reaction), and whether there produce spores or appear opacity which detect the living bacteri-um. The detection of Beer-spoilage baeteria is at least 5～7d, and the testing of Lb.lindneri need at least 7d, even longer. To resolve this problem, rapid detection technique is expected. Detecting and identify them specially and rapidly can not only guarantee the quality of beer, but also indicate the level of technology in the com-pany, As the development of modern biology technology, the ways of detecting b-eer-spoilage bacterium become more variously. Current research more rapid detec-tion methods including ATP, immunology,and molecular biological detection meth-od, etc.This paper concerned with Polymerase chain reacrion (PCR) can specifically amplify target sequence millions times in several hours,So PCR provides a rapid,sensitive and specific means to detect microorganisms.In this study,the selective culture mediums are used to isolate and purifacation the beer-spoilage bacteria and 5 strains are obtained,Which are named after their characteristic as G9181,G9182,G9251,G9252, G9253.By the means of API50CHL kits the strains above are identificated as L.brevis, L.brevis, L.Plantarum, L.brevis, L.buchneri.To identify the L.brevis,L.Plantarum,L.buchneri DNA as template, according to ribosomal DNA (rDNA) of highly conserved universal primers designed so that the primers can also amplify the amplification of the three bacteria product. The purpose of this paper is to design a rational species-specific primers, and then designed primers and PCR condition optimization, the final design of primers to determine the optimal amplification conditions. L.brevis as experimental subject, to make pre-enrichment, DNA extraction methods,PCR amplification such conditional optimization.The result of the studies are listed as follows: 1,Bacteria contamination in beer were isolated, purified, through a series of physiological and biochemical reactions and API50CHL reagent strain identification, the results show that contamination in beer is Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus buchneri.2,Pre-enrichment study the optimization of culture medium, compared with MRS medium, as well as improved MRS1 medium (glucose were replaced with sol-uble starch, inulin) for effect of bacteria. The results showed that inulin on the gro-wth of lactic acid bacteria have a certain role in promoting. Sensitivity of PCR dete-ction of Lactobacillus brevis to 18 CFU/mL, as bacterial cells arranged in sin-gle, double and into the beam, made of bundles of cells can be as high as 10, sug-gesting that the sensitivity of PCR detection of up to 18～180 CFU/mL.When the initial cell concentration 10CFU/mL, through the use of MRS 1 to cell enrichment culture, the proliferation of the time needed for the 24～30h. With sample preparation, template extraction, PCR amplification and electrophoresis observation time 8 h, detection of harmful strains of the total time 32～38h, shorter than the traditional plate culture method 3～5d, greatly increased the efficiency of detection.3,Template extraction, studied the CTAB method, boiling method, lysozyme method, proteinase K method, Lysozyme and proteinase K enzyme mixture cleavage of the template prepared by boiling DNA for PCR amplification of gel electrophor-esis and direct results.PCR results also showed that, CTAB method, Lysozyme and proteinase K enzyme mixture cleavage boiling method to get the products segment, but the template CTAB extraction method is relatively longer, Lysozyme and proteinase K enzyme mixture extraction boiling lysis method with the same CTAB, and the extraction time is shorter, so the optional enzyme Lysozyme and proteinase K enzyme mixture lysis boiling extraction template.4,Improved 25μlPCR optimization system:Taq enzyme dosage 2.0U/ml, Mg2 concentration of 2.0mM, the annealing temperature was 50℃. Amplification program:94℃4min into the cycle,94℃30s,50℃30s,72℃30s,35 cycles, then extension under 72℃5min.5,PCR optimized system on the five standard strains (L.brevis,L.Plantarum,L.buchneri, L. cheese, L.lactis) and 3 strains isolated from the spoilage bacteria (L.brevis,L.Plantarum,L.buchneri) compared with PCR results. The results showed that three spoilage (L.brevis,L.Plantarum,L.buchneri) and the three standard strains (L.brevis,L.Plantarum,L.buchneri) can produce amplified product. The other two standard strains (L. cheese, L.lactis) without amplification results.