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Detection of pathogenic microorganisms

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

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病原微生物の検出
Detection of Pathogenic microorganism
http://www.tsukuba-genetech.com/
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.
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。
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)
The Condition of Reverse
Transcriptase PCR of Salmonella
and E.coli
11.1.15
Detection Strategy
Salmonella RNA
RT primer + tag primer
Salmonella RNA/DNA
cDNA synthesis
Salmonella specific Primer
PCR
Salmonella RNA specific PCR product
tag primer
Ad

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Detection of pathogenic microorganisms

  • 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