Series: Ernestly measurement
Measurement of EHEC O157:H7 and EHEC O26
Carried out in cooperation with Zensho Holdings Co., Ltd. and Nippon Genetics Co., Ltd.
Enterohemorrhagic E. coli (EHEC) represented by EHEC O157:H7 is E. coil that produces verotoxin (VT).
This type of bacteria is resistant to dryness, low temperatures, and freezing environment and EHEC O157:H7 is also resistant to acids. It is highly infectious and even a small amount can develop symptoms, which makes it threatening.
We tested if the gene that produces verotoxin can be measured using a mobile PCR device.
Sample A: EHEC O157 obtained from the surface of beef was placed in mEC culture medium to give a concentration of approx. 10 cfu/L.
Sample B: EHEC O26 obtained by the same procedure was placed in EC culture medium to give a concentration of approx. 10cfu/L, and contaminating bacteria was added (approx. 3000 cfu/L).
The samples were cultured at 42 degrees Celsius for 4 hours (1), 8 hours (2), and 11 hours (4). Furthermore, the solution cultured for 8 hours (3) was diluted tenfold.
*(3) is equivalent to a 7-hour culture.
Heat treatment at 98 degrees Celsius for 8 minutes after culture
Enzyme: KAPA 3G Plant (Kapa Biosystems)
Primer and probe: Equivalent to specifications in Notification No. 1120-3 issued by the Food Inspection and Safety Division
Other compositions were determined by Nippon Genetics Co., Ltd.
Detection time was approx. 25 min. (by Nippon Genetics Co., Ltd.)
After 8-hour pre-culture, 10 cfu/L of enterohemorrhagic EHEC was measured with pretreatment using only heat treatment. It is assumed that measurement is possible with a 7-hour pre-culture which indicates that determination can be provided within 8 hours including the time required for measurement (within working hours).
Measurement was carried out by a multi-mix of VT1 and VT2.
|VT1-Primer F 5’-GGA TAA TTT GTT TGC AGT TGA TGTC-3’ (25mer)|
|VT1-Primer R 5’-CAA ATC CTG TCA CAT ATA AAT TAT TTC GT-3’ (29mer)|
|VT1-probe 5’FAM-CCG TAG ATT ATT AAA CCG CCC TTC CTC TGG A-3’BHQ1 (31mer)|
|VT2-Primer F 5’- GGGCAGTTATTTTGCTGTGGA -3’ (21mer)|
|VT2-Primer R 5’-GAAAGTATTTGTTGCCGTATTAACGA-3’ (26mer)|
|VT2-probe 5’FAM-ATGTCTATCAGGCGCGTTTTGACCATCTT-BHQ2’ (29mer)|