We describe here the detection system for viable pathogenic microorganisms in foods.

1. 病原微生物の検出
Detection of Pathogenic microorganism
http://www.tsukuba-genetech.com/

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.

3. 生菌の検出
• 食品等において、加熱処理をすることにより。殺菌することが出来る。それを
利用した食品としては、レトルト食品
• これらの食品中には、死菌が存在し、菌のDNAを鋳型とした場合は、PCRで
検出されてしまう
生菌だけを検出する方法が必要
• RNAは、加熱と2価イオン(マグネシウム)存在化で、短時間に、分解される。
従って、対象検体から、菌RNAが検出されれば、生菌が存在している可能性
は高い。そこで、菌のRNAを検出する方法を開発。この方法は、～３時間で結
果が得られる。
• 現在、同時に１３属の病原菌の検出に必要なシステムを「ドライ」で構築した。
これが、機能するかどうかを確かめるために、手始めとして、４属の菌種で検
証。 その結果、問題なく検出できることが判った。
従来の方法
菌の培養をして、DNA抽出、PCRを行うため、３日を要する。

4. 菌 strain 範囲 range
Arcobacter butzuli Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter skirrowii
Bacillus cereus Bacillus cereus
Staphylococcus
aureus
Staphylococcus aureus
Mycobacterium
Mycobacterium bovis、Mycobacterium avium、Mycobacterium intracellulare,Mycobacterium
tuberculosis, Mycobacterium ulcerans, Mycobacterium avium ssp. paratuberculosis
Listeria
monocytogenes
Listeria monocytogenes
Yersinia
pseudotuberculois
Yersinia pestis, Yersinia enterocolitica, Yersinia pseudotuberculosis,
Legionella
pneumophila
Legionella pneumophila
Cronobacter spp
Cronobacter sakazakii, Cronobacter muytjensii, Cronobacter dublinensis, Cronobacter turicensis,
Cronobacter malonaticus,
Escherichia
coli/Shigella
Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Shigella boydii, Shigella sp, Escherichia coli
Salmonella
Salmonella enterica subsp. Null, Salmonella enterica subsp. enterica serovar Choleraesuis
Salmonella enterica subsp. enterica serovar Typhi, Salmonella enterica subsp. enterica serovar Infantis
Salmonella enterica subsp. enterica serovar Bovis-morbificans, Salmonella enterica subsp. enterica
serovar Enteritidis, Salmonella enterica subsp. enterica serovar Paratyphi A, Salmonella enterica subsp.
enterica serovar Paratyphi B, Salmonella enterica subsp. enterica serovar Paratyphi C, Salmonella
enterica subsp. Salamae, Salmonella enterica subsp. Diarizonae. Salmonella enterica subsp. arizonae
Salmonella enterica subsp. Houtenae, Salmonella bongori, Salmonella subterranea, Salmonella bongori
Salmonella subterranea
Campylobacter Campylobacter jejuni、Campylobacter coli、Campylobacter fetus
Vibrio cholerae、
Vibrio
parahaemolyticus
Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio fluvialis, Vibrio furnissii, Vibrio mimicus,
Vibrio anguillarum
Clostridium
perfringens,
Clostridium
botulinum
Clostridium botulinum, Clostridium perfringens,
病原微生物 (pathogenic micro-organisms)

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

9. The condition of Clostridium
DNA,RNA and Primers
5.2.14

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。

12. The Condition of Campylobacter
with RT-PCR
11-2-15 to 15-2-15

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