Botox testing in food

Botox is a pathogenic bacteria that grows in hypoxia, known as Botox, and has a strong survivability in canned and sealed pickled foods. The toxins it secretes during reproduction are extremely toxic, 10,000 times more toxic than KCN, and one of the most toxic toxins. Purified crystalline botulinum toxin 1 mg kills 200 million mice and is 40 IU/kg of semi-lethal to human beings.
Botulinum Toxin pregenuity is a non-toxic compound produced in Botox cells, which are released after Botox death and become toxic after activation by trypsin or proteases produced by bacteria in the intestines.
Botox is to block nerve ending secretion of acetylcholine, which allows muscles to contract, thereby paralysing the muscles. When people eat and absorb the toxin, the nervous system is damaged and there are symptoms such as dizziness, difficulty breathing and muscle weakness.
New Zealand dairy giant Fonterra announced on August 3rd that some of its products, such as baby milk powder and sports drinks, could be "contaminated" and contain Botox. A number of food manufacturers' products were affected. RephiLe technicians based on GB/T4789.12-2003 food hygiene
microbiology
test Botox and endotoxin testing" and "SN/T 2525-2010 food Botox
PCR
testing" summed up the food Botox test method.I. Experimental principle
the sample after the sterilization of the flat plate to isolate the single bacterios, pick suspected bacteria fell to trypsin glucose yeast-soaked broth (TPGY)
culture
-base culture, dna extraction for the culture
reagement
box to extract DNA, PCR amplification,
agar
sugar gel electrophoresis to test whether the PCR products contain Botox characteristic strips, so as to initially determine whether food is contaminated with Botox.II, Instruments and Reagents
Ultraviolet Detector NanoDrop1000 (Eppendorf), Desktop Microfuge 22R (Beckman Counter), Gel DocTM XR (Bio-RAD), PCR My Cycler (Bio-RAD), Direct-Pure UP Ultra-Pure Water System (RephiLe), Nuclear Acid Electrophoresis 100 Bacterial genome DNA extraction kit (Axygen), dNTP (Takara)
, test method
1.Sample preparation and sterilization culture
Before inoculation, bring the sterilization
filler
to a boil of 10 to 15 min to eliminate the oxygen dissolved in the media and cool quickly, never shake. Inoculate 1 to 2 g solid foods or 1 to 2 ml liquid foods for every 15 ml of sterile broth, and slowly connect the inoculations below the broth surface. Each sample is inoculated with two tubes of meat culture, at 35 degrees C±1 degrees C, and two tubes of TPYG culture, with 28 degrees C±1 degrees C and anaerobic culture of 5 d. Check the turbidity of the culture, gas production, digestion of meat particles and the odors produced. If there is growth, separate the purified culture in step 2. If no growth is seen, continue to culture 10 d.
2.Separation of pure culture
1 to 2 ml culture liquid placed in a sterile test tube, add the same amount of sterile sterile waterless ethanol, mix well, place room temperature 1 h. Inoculation with a ring of 1 to 2 treated cultures was lined on anaerobic yolk agar, and anaerobic culture at 35 ± 1 degree C was 48 h. Inoculation of suspected bacteria falls on the TPGY culture base, 35 ± 1 degree C anaerobic culture 24 h.
3.DNA template preparation and concentration determination
1.4 ml TPGY culture transferred to a 1.5 ml centrifuge tube, 14000 r/min centrifugation 2 min, discarded. Precipitation uses commercialized bacterial genome DNA extraction kits to extract DNA and follow the kit instructions. The extracted DNA is measured for purity and concentration and stored at -20 degrees C.
4.PCR Amplification
multiple PCR amplifications using type-specific prototyping designed for Thyrobacter Botulinum Toxin genes A, B, E, and F respectively (see Schedule 1), each PCR reaction tube detects one type of Botox. The PCR reaction system and parameters are set out in Schedules 2 and 3. The amount of each reagent in the reaction system can be adjusted according to the specific situation or the total volume of the reaction. The reaction system should set positive control, negative control and blank control when PCR is tested. The genome DNA of Botox type A, B, E and F was used for positive control, non-Botox genome DNA was used for negative control, and sterile ultra-pure water was used for blank control.
5.Gel electrophoresis detection PCR product
Prepare 1.2% to 1.5% agarose gel with 0.5 X TBE Buffer (gel heating melts and cools to about 60 degrees C) From EB to 0.5 μg/ml), mix the PCR product of 10 μL with the 2.0 μL 6 X sample buffer, above, with DNA Marker on the far left hole. 0.5 X TBE Buffer, 10 V/cm constant-pressure electrophoresis, electrophoresis time determined according to the movement position of bromophenol blue, electrophoresis test results are read by gel imaging system.
6.PCR results determined that
-negative control and blank control did not appear stripe, positive control appeared the expected size of the amplification strip, the sample to be tested appeared the expected size of the amplification strip, determined to be positive PCR results, according to GB/T4789.12-2003 for the corrogical test.
the expanded strip of the expected size did not appear in the sample to be tested, the PCR result was determined to be negative and the result was reported directly.
negative control, blank control and positive control PCR test results set in the test should conform to the above conditions, otherwise, either control should be redoded if the above-mentioned
normal results occur.Table 1 Lead Sequence for Botox Toxin Gene PCR DetectionTable 2 Response System for Botox PCR DetectionNote: The amount of each reagent in the reaction system can be adjusted according to the overall product of the reaction system.Table 3 Reaction Parameters for Botox Botulinum Toxin Gene PCR DetectionNote: PCR Reaction Parameters can be adjusted appropriately according to the type of gene amplifyer Introduction to other Botox detection methods:
1.PCR-RFLP (Restrictive Fragment Length Polymorphic Polymerase Chain Reaction)
The basic principle of PCR-RFLP is to amplification the purpose of DNA with PCR, and then digest and cut into different size fragments with specific endoenzymes to distinguish directly on the gel electrophoresis. The restrictive enzyme cut-in points of different alleases are distributed differently, resulting in different lengths of DNA fragment strips.
2.16S rRNA analysis
16S rRNA gene between different species of real bacteria and paleobacterium is highly conservative. It is often used to study various biological species.in nucleic acid detection, various pollutants in the water, especially nucleases, have a very large impact on the test results. DNase and RNase degrade nucleic acids; enzyme activity requires a buffer with the right concentration of ions, especially Mg2 plus, which plays a decisive role in the activity of Taq enzymes; organic matter acts as non-competitive inhibitors; and bacteria continuously release ions and small molecules of organic matter, whose gene amplification can also affect the results.RephiLe is a special recommendation for users of Direct-Pure UP ultra-pure water all-in-one machine, which feeds tap water while preparing reverse osmosis RO water and high-quality ultra-pure water, one machine and two water for customers with different needs. The terminal can be equipped with ultrafiltration
filtering
, effectively controlling bacteria < 0.1 cfu/ml, thermogen < 0.001 Eu/ml, to meet the special requirements of life science experiments.about RephiLe:
RephiLe is a professional manufacturer and supplier of water purification and laboratory separation purification products with a deep technical background in the field of pure water and filtration in the laboratory. RephiLe is available in three main categories: 1.Laboratory pure water system 2.Millybo pure water meter compatible with
supplies3. Needle filters, disc membranes, stainless steel filters and other sample preparation products RephiLe products have been exported to more than 60 countries around the world. more RephiLe product information, please visit:
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