Burkholderia gladioli molecular standard sample and preparation method thereof
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A Burkholderia Pope, Burkholderia technology, applied in biochemical equipment and methods, DNA preparation, DNA/RNA fragments, etc., to ensure quality control, good uniformity, and stability high effect
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Problems solved by technology
At present, there is no research on the standard sample of this pathogen at home and abroad.
Method used
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Embodiment 1
[0033] Burkholderia gladiolus standard strain selection Burkholderia gladioli CGMCC 1.3824;
[0034] (1) Extraction of genomic nucleic acid
[0035] ①The Burkholderia gladiolus was activated and enriched;
[0036] ②Centrifuge the enrichment culture solution obtained in step ① at 4000g, 4°C for 15min;
[0037] ③ Discard the supernatant, take 1-3 mL of the precipitate, add 10 mL of TE solution with pH 8.0 to suspend, add 0.5 mL of SDS with a concentration of 100 g / L and 50 μL of proteinase K with a concentration of 20 mg / mL, mix well, and incubate at 37 °C for 1 h;
[0038] ④Add 2mL NaCl with a concentration of 5mol / L, mix well, add 1.5mL CTAB-NaCl mixed solution, mix well, and incubate at 65°C for 20min; among them, CTAB-NaCl mixed solution is 100g / L CTAB and 0.7mol / L NaCl;
[0039] ⑤Take the supernatant, add the same volume of phenol-chloroform-isoamyl alcohol mixture as the supernatant, mix well, and centrifuge at 6000g for 10min; wherein, the volume of phenol:chloroform:is...
Embodiment 2
[0054] Carry out DNA integrity, uniformity, stability test to the standard sample that embodiment 1 makes:
[0055] 1. DNA quality and integrity testing
[0056] The prepared DNA was taken for gel electrophoresis, purity and concentration detection.
[0057] (1) DNA Integrity Detection: Direct method checks by direct electrophoresis of extracted DNA, 120V, 1.5% agarose gel electrophoresis, observes the integrity of the bands, see the test results figure 1 , the band is complete without smearing, indicating that the integrity of the DNA band is good.
[0058] (2) DNA concentration and purity detection: UV spectrophotometer method - absorb 1 μl of DNA and measure the absorbance at 260nm and 280nm with a spectrophotometer, then judge the DNA purity according to the OD260 / OD280 value, and calculate its concentration according to the OD260. Calculate according to the following formula: DNA concentration (ng / μl) = OD260 × 50 When OD260 / OD280 2.0, it means that the RNA content is h...
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Abstract
The invention discloses a preparation method of a burkholderia gladioli molecular standard sample. The preparation method comprises the step of carrying out freeze drying on extracted genome nucleic acid of burkholderia gladioli, wherein the freeze drying conditions are as follows: a freeze dryer is started, and a cooling trap is started when temperature of a drying chamber is lowered to -35 DEG C; when the temperature of the cooling trap is lowered to -40 DEG C, a nucleic acid sample prefrozen for 2 hours at the temperature of -80 DEG C is put into the drying chamber and then vacuumized; when vacuum degree is reduced to 0.5Torr, freezing is finished; and the sample is dried at the temperature of 15 DEG C, the sample is taken out when the vacuum degree is reduced to 0.1Torr, a penicillin bottle is tightened to obtain a burkholderia gladioli genome nucleic acid standard sample, and the sample is kept in a dark place and stored at the temperature of 20 DEG C. The molecular standard sample disclosed by the invention has good physical and chemical properties and can be detected by adopting a specific primer through PCR process, and sensitivity reaches 10<-4>. Detection test shows that the standard sample has good DNA integrity, uniformity and stability, can be applied to import and export detection as a positive reference material, and accuracy of a detection result is improved.
Description
technical field [0001] The invention belongs to the technical field of a standard sample of pathogenic bacteria for detection and a preparation method thereof, and in particular relates to a standard sample of Burkholderia gladiolus and a preparation method thereof. Background technique [0002] Burkholderia gladioli is a plant pathogenic bacterium, an opportunistic pathogenic bacterium in humans (which can cause pneumonia symptoms in humans), and is also a key poisoning and pathogenic bacterium in food. Food poisoning caused by contamination of coconut poisonous fermented food in Indonesia, fermented rice noodles in Northeast China and fresh white fungus has attracted widespread attention. The fatality rate of food poisoning caused by it is extremely high. In the food poisoning incidents of fermented rice noodles in northeast and southwest my country, the mortality rate reached more than 40%. Due to the existence of such bacteria in nature, long-term storage of jelly and r...
Claims
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