2. Detection of Viable micro-organism
• By heat-treating foods, the foods can be sterilized. Retort foods are those treated as above.
In such retort foods, the microorganisms are killed, but the DNA of the micro-oganisms still
exists in the food. Therefore, when PCR is performed, PCR products from the DNA would be
detected. This leads an ambiguous conclusion: the micro-organisms are in the foods, though
it is not known whether they are viable or not.
• Based on the fact that RNA is quickly degraded in cocking conditions, it is considered that
RNA in the micro-organisms, when they are killed by heat-treatment, would be degraded.
The degradation gives no PCR product. Taking this logics into consideration, RT-PCR for RNA
of micro-organisms is considered to be the best choice for judgment whether viable micro-
organisms or not in the foods.
• Here, we have designed Reverse Transferase(RT)/PCR system for ribosomal RNAs of
pathogenic micro-organisms classified in 13 genera. Then, as a trial, we examined whether
or not the system worked well for ribosomal RNAs of the pathogenic micro-organisms
classified in 4 genera, i.e., Escherichia coli/Shigella, Salmonella, Clostridium, and
Campylobacter.
5. The Condition of Reverse
Transcriptase PCR of Salmonella
and E.coli
11.1.15
6. Detection Strategy
Salmonella RNA
RT primer + tag primer
Salmonella RNA/DNA
cDNA synthesis
Salmonella specific Primer
PCR
Salmonella RNA specific PCR product
tag primer
7. SRT ERT SD ED C P N NC M
RT Temp – 55℃
PCR Temp – 65℃
9.1.15,Salmonella and E coli
RNA
8.1.15 cDNA
SRT: Salmonella RNA with Salmonella specific primer
ERT: E.coli RNA with Salmonella specific primer
SD: Salmonella genomic DNA
ED: E.coli genomic DNA
C: cattle genomic DNA, P: pork genomic DNA, N: chicken genomic DNA
NC : non template control
M: 100bp ladder marker
8. Srt : Salmonella RNA with Salmonella specific primer
SCNrt: Salmonella, Cattle and Poultry RNA with Salmonella specific primer
C: cattle genomic DNA,
P: pork genomic DNA,
N: chicken genomic DNA
Sr : Salmonella RNA with Random6er primer
NC : non template control
M: 100bp ladder marker
RT Temp – 65℃
PCR Temp – 65℃
Primer – Forward & Tag
Srt SCNrt C P N Sr M
Salmonella, Cattle,
poultry
16th, Jan, 2015
10. Cl1 Cl2 Cl1r Cl2r W M Cl1 Cl2 Cl1r Cl2r W
F18 T F20T
RT Temp – 65 ℃
PCR Temp – 65 ℃
Cl1 : Clostridium RNA
Cl2 : Clostridium RNA
Cl1r : Clostridium RNA with
Random 6er
W : non template control
M: 100bp ladder marker
9.2.15 Clostridium detection using F18
and Tag pair and F20 and Tag pair
ポジティブの下のバンドは消え
た。
ランダム6merのfalseバンドは消
えなかった。
11. Cl1 Cl2 Cl1r Cl2r W M Cl1 Cl2 Cl1r Cl2r W
F18 T F20T
RT Temp – 65 ℃
PCR Temp – 68 ℃
Cl1 : Clostridium RNA
Cl2 : Clostridium RNA
Cl1r : Clostridium RNA with Random 6er
W : non template control
M: 100bp ladder marker
10.2.15 Clostridium detection using F18
and Tag pair and F20 and Tag pair
Annealing temperatureを上
げたら、
ランダム6merのfalseバンドも
消えた。
13. 15.2.15,FallT,(RT 100000)
RT Temp – 65 ℃
PCR Temp – 68 ℃
Cp21 : Campylobacter RNA
Cp23 : Campylobacter RNA
E : E coli RNA, S : Salmonella RNA ,C: cattle RNA
N : chicken RNA, Cl2 : Clostridium RNA
Cp21r : Campylobacter RNA with Random 6er
Cp23r : Campylobacter RNA with Random 6er
Cp21d : Campylobacter genomic DNA
W : non template control, M : 100bp ladder marker
cDNA
Cp21 Cp23 E S C N Cl2 Cp21r Cp23r Cp21d W M