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Primer and detection method as well as kit for rapidly detecting bacillus subtilis

A technology for Bacillus subtilis and a detection method is applied in the field of a primer for rapid detection of Bacillus subtilis, a detection method and a kit, which can solve the problems of complicated operation, long time consumption and the like, and achieve the effects of simple operation, high sensitivity and low cost.

Active Publication Date: 2014-01-08
BRIGHT DAIRY & FOOD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The technical problem to be solved by the present invention is to provide a method for detecting Bacillus subtilis with fast detection speed, high sensitivity, strong specificity and low cost in view of the current situation that the current detection of Bacillus subtilis generally adopts a time-consuming and complicated biochemical identification method. method

Method used

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  • Primer and detection method as well as kit for rapidly detecting bacillus subtilis

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Experimental program
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Effect test

Embodiment 1

[0024] Detection of Bacillus subtilis in embodiment 1 fresh milk

[0025] Take 10ml of fresh milk, centrifuge at 6000g for 10min, discard the supernatant, and take the precipitate. The precipitate was washed twice with deionized water, and the bacterial genomic DNA was extracted with a lysis reagent (Lysis Buffer for Microorganism to Direct PCR, purchased from TaKaRa Company). Then PCR reaction was carried out using the extracted DNA as a template.

[0026] The PCR reaction system was: 3 μL of 5 μmol / L upstream primer, 3 μL of 5 μmol / L downstream primer, 2.4 μL of 2.5 mmol / L dNTP (TaKaRa company), 10×PCR Buffer (Taq TM , TaKaRa Company) 3 μL, template DNA 1 μL, Taq DNA polymerase (Taq TM , TaKaRa Company) 1U, add deionized water to 30 μL.

[0027] The PCR reaction program was: preheating at 95°C for 5 minutes, 35 cycles (95°C, 30S; 55°C, 30S; 72°C, 30S), extension at 72°C for 5min, and storage at 4°C.

[0028] Finally, electrophoresis detection was carried out, and 5 μL of...

Embodiment 2

[0030] The bacillus subtilis inspection of colony on the embodiment 2 culture plate

[0031] The sample to be tested is soybean milk purchased from a supermarket. After gradient dilution of the purchased soybean milk, select 2 to 3 appropriate dilutions, spread 0.1 mL each on an LB agar plate, and incubate aerobically at 37°C for 48 hours. Pick 3 or more suspicious colonies, use lysis reagent (Lysis Buffer for Microorganism to Direct PCR, TaKaRa Company) to extract bacterial genomic DNA and dilute it for future use. Then the PCR reaction was carried out using the extracted bacterial genome DNA as a template.

[0032] The PCR reaction system was: 3 μL of 5 μmol / L upstream primer, 3 μL of 5 μmol / L downstream primer, 2.4 μL of 2.5 mmol / L dNTP (TaKaRa company), 10×PCR Buffer (Taq TM , TaKaRa Company) 3 μL, template DNA 1 μL, Taq DNA polymerase (Taq TM , TaKaRa Company) 1U, add deionized water to 30 μL.

[0033] The PCR reaction program was: preheating at 95°C for 5 min, 35 cyc...

Embodiment 3

[0037] Embodiment 3 detection method sensitivity test

[0038] The pure cultured Bacillus subtilis was diluted and counted under a microscope with a hemocytometer. Dilute the bacterial solution to a copy number of 3×10 5 / mL, 3×10 4 / mL, 3×10 3 / mL, 3×10 2 / mL, 3×10 1 / mL before the experiment. Genomic DNA of each gradient cell was extracted with a lysis reagent (Lysis Buffer for Microorganism to Direct PCR, TaKaRa Company) and diluted for later use. Then the PCR reaction was carried out using the extracted bacterial genome DNA as a template.

[0039] The PCR reaction system was: 3 μL of 5 μmol / L upstream primer, 3 μL of 5 μmol / L downstream primer, 2.4 μL of 2.5 mmol / L dNTP (TaKaRa company), 10×PCR Buffer (Taq TM , TaKaRa Company) 3 μL, template DNA 1 μL, Taq DNA polymerase (Taq TM , TaKaRa Company) 1U, add deionized water to 30 μL.

[0040] The PCR reaction program was: preheating at 95°C for 5 min, 35 cycles (95°C, 30s; 55°C, 30s; 72°C, 30s), extension at 72°C for 5...

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Abstract

The invention discloses a primer pair and detection method and as well as kit for specifically detecting bacillus subtilis. The primer pair provided by the invention can specifically amplify a gene segment of the bacillus subtilis. The detection method comprises the following steps of: carrying out PCR (Polymerase Chain Reaction) amplification on a genome DNA of a sample to be tested, wherein the sample is obtained through seperation and extraction, carrying out agrose gel electrophoresis detection on a product, and judging whether the bacillus subtilis exists in the sample according to the existence or inexistence and the size of an electrophoretic band. The detection method serving as a molecule detection method of the bacillus subtilis has the advantages of high sensitivity, strong specificity, low detection cost and the like and has very strong practical application value.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a primer for rapid detection of bacillus subtilis, a detection method and a kit. Background technique [0002] Bacillus subtilis is a Gram-positive bacterium with typical characteristics of Bacillus. It can liquefy gelatin, peptonize milk, reduce nitrate, hydrolyze starch, and is a typical aerobic bacteria. The distribution of Bacillus subtilis is very wide, and it mainly exists in soil or rotten straw. Because of its ability to form spores, Bacillus subtilis can resist high temperature, low temperature, dryness, radiation and other adverse environments. It is one of the main polluting bacteria in the food industry. It usually causes food corruption and is extremely harmful. The current commonly used detection method for Bacillus subtilis is the aerobic plate culture method. After the bacteria grow out, the biochemical identification is carried out. In general, the isolation...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 张红发任婧刘景游春苹顾瑾麟
Owner BRIGHT DAIRY & FOOD CO LTD
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