Introduction

1. Why do we detect Aflatoxin?

   Gutter oil contains aflatoxin which is very toxic and will accumulate in people’s liver and kidney.

   In Taiwan, there are many people who are the asymptomatic carriers of hepatitis B, therefore if they be poisoned oil again, it will be easier for them to get liver disease or resulting in kidney dialysis.

   In our experiment, we try to detect Afb1 because it is the most toxic of aflatoxin. Our experiment was to find out more convenient detection method, so that everyone can easily detect their own oil if it is safe to eat.

2. Taiwanese regulations
   
3. National regulations
   1. groundnuts, nuts, dried fruit and processed products of such products or food additives: B1 aflatoxin not exceed 2ug / kg (ppb), and B1, B2, G1, G2 sum not exceeding 4ug / kg.
   2. for the storage or not for direct human consumption's peanuts: B1 aflatoxin 8ug / kg, and B1, B2, G1, G2 sum shall not exceed 15ug / kg.
   3. for the storage or not for direct human consumption's nuts and dried fruits: B1 aflatoxin 5ug / kg, and B1, B2, G1, G2 sum shall not exceed 10ug / kg.
   4. Grains class for the direct human consumption: B1 aflatoxin 2ug / kg, or B1, B2, G1, G2 sum not exceeding 4ug / kg.
   5. Dairy: M1 aflatoxin not exceed 0.05 ug / kg.[4]

Circuit Design

When aflatoxin goes into the cell, CYP1A2 will oxidize the Aflatoxin B1, and then it will turn into Aflatoxin oxidation AFBO[5].

Single-strand DNA(ssDNA) can activate protein RecA. Ecoli has a sos response system, and there is a repressor LexA which inhibits the next reaction, so the fluorescent gene won’t express.But when the activated protein RecA comes and hydrolysis the represser LexA, the next reaction can work.[7]

The Relationship between Cytochrome CYP3A4 in Vivo and the Toxicity of Aflatoxin B1 in Feedstuffs
ZHENG Lili1, ZHU Yujing1, SHAO Caimei2, ZHANG Yong1

Result

1. Whether Aflatoxin can enter e.coli or not
   1. Method

      Detection of the amount of toxins in the e.coli.

      1. Add 100μl of DH5α and 900μl of LB broth into the tube and incubate for 1hr.
      2. Centrifuge at 4000rpm for 3min and clicard 800μl of the supernatant

      3. Plate each 100μl of the bacteria onto the dishes and spread.

         Incubate the plates at 37℃ overnight

      4. Prepare each concentration of the toxin.

         Statutory standards *100 / *10 / *1 / *0.1 / *0.01

      Next day

      1. Prepare 16 microcentrifuge tubes.(5 kinds of concentration *3 timings+control)

         Add 500μl of DH5α to each tube.

         Centrifuge all tubes at 4000rpm for 3min.

         Remove the supernatent.

      2. Add 1000μl of the toxic solution each time.

         Follow the concentration and 3 timings(0.5hr / 1hr / 1.5hr).

      3. 1. Add 0.5cc of ddH₂O and mix with the bacterias
         2. Centrifuge at 13000rpm for 30 sec
         3. Remove the water
         4. Repeat step1~step3 for three times

      4. Add 1cc of ddH₂O and mix with the bacterias

         Centrifuge at 13000rpm for 30sec.

         Remove 700μl of the supernatant

      5. Kill the bacteria:

         1. Put all the tubes in the Liquid nitrogen
         2. When they freeze,heat them at 100℃
         3. Repeat step1~step2 for 3 times
2. Whether e.coli is alive in the poisons, condition or not
   1. Method

      DH5α-Pretest

      Procedure

      Because we must test E.coli’s Survival in the environment there is Aflatoxin by counting the colonies,First we test how much concentration is the best.

      1. culture

         STEP1:take 1μL DH5α to spread the plate(no Antibiotic)

         STEP2:put in 37 degree Celsius 12~16hr

      2. liquid culture

      3. STEP1:put 80μL into 2ml LB broth

         STEP2:recovering

         STEP3: After 2hr,dilute it to 10^-4,10^-5,10^-6,10^-7,and then go to spread the plate (no Antibiotic)

         STEP4: After 4hr dilute it to 10^-4, 10^-5 ,10^-6 ,10^-7 ,and then go to spread the plate (no Antibiotic), 6hr and 8hr Similarly

         STEP5:Take 200μL out from the tube and spread the plate(AMP+)

         STEP6: put in 37 degree Celsius 12~16hr

      Survival

      Procedure

      First we culture DH5α with LB only plate for 15hr. Then,pick one colony in the LB broth,and liquid culture for 15hr.

      We divided two categories A and B.

      A:

      Take 80μL into 2ml LB broth × 6 tubes and then culture 1 hr.

      After 1hr,add 20μL Aflatoxin into three tubes(conc. Is 2000ppb(A thousand times the standard value))

      And add 20μL DMSO into the other tubes.Then,culture for 3hr.

      After 3hr,dilute the broth to 10^-6

      And take 200μL to spread the plate.

      B:

      Take 80μL into 2ml LB broth in a tube And then culture 1 hr.

      After 1hr, put them into 6 tubes equally.

      Dilute the broth to 5×10^-4

      Add 0.4μL Aflatoxin(2×10^-4) in three tubes.

      Add 0.4μL DMSO in the other three tubes.

      Go to 37 degree Celsius shaking for 10min.

      Take 200μL to spread the plate.

      

Reference

• [1]Veronica S. Mary a, Ana Valdehita b, Jose M. Navas b, Hector R. Rubinstein a, Maria L. Fernandez-Cruz. Effects of aflatoxin B1, fumonisin B1 and their mixture on the aryl hydrocarbon receptor and cytochrome P450 1A induction
• [2]Yan-Ping Du Hui-Lian Zhu. The possible mechanism of Aflatoxin B1 Hepatocarcinogenesis and preventive effects of phytochemicals
• [3]Food hygiene standards - Food in aflatoxin limits (human) 82 1.4.4 Health Department Announcement fresh word No. 8189322
• [4]EU animal and plant inspection and quarantine of food hygiene regulations cum Profile
• [5]Veronica S. Mary a, Ana Valdehita b, Jose M. Navas b, Hector R. Rubinstein a,*Maria L. Fernandez-Cruz b. Effects of aflatoxin B1, fumonisin B1 and their mixture on the aryl hydrocarbon receptor and cytochrome P450 1A induction
• [6]ZHENG Lili, ZHU Yujing, SHAO Caimei, ZHANG Yong The Relationship between Cytochrome CYP3A4 in Vivo and the Toxicity of Aflatoxin B1 in Feedstuffs
• [7]Nejc Paulič2,†,Adrijana Leonardi1, Vesna Hodnik2,Gregor Anderluh2,3, Zdravko Podlesek2, Darja Žgur-Bertok2,Igor Križaj1,4,5 Matej Butala2. Structural insight into LexA–RecA* interaction Lidija Kovačič1