Limits...
Development of a novel detection system for microbes from bovine diarrhea by real-time PCR.

Tsuchiaka S, Masuda T, Sugimura S, Kobayashi S, Komatsu N, Nagai M, Omatsu T, Furuya T, Oba M, Katayama Y, Kanda S, Yokoyama T, Mizutani T - J. Vet. Med. Sci. (2015)

Bottom Line: The resulting limits of detection (LOD) for virus-spiked samples, bacteria and DNA fragments were 0.16-1.6 TCID50 (PFU/reaction), 1.3-13 CFU/reaction and 10-100 copies/reaction, respectively.All reactions showed high sensitivity in pathogen detection.The results revealed that bovine coronavirus had infected all diarrheic adult cattle and that bovine torovirus had infected the diarrheic calf.

View Article: PubMed Central - PubMed

Affiliation: The United Graduate School of Veterinary Sciences, Gifu University, Yanagito, Gifu 501-1193, Japan.

ABSTRACT
Diarrhea in cattle is one of the most economically costly disorders, decreasing milk production and weight gain. In the present study, we established a novel simultaneous detection system using TaqMan real-time PCR designed as a system for detection of microbes from bovine diarrhea using real-time PCR (referred to as Dembo-PCR). Dembo-PCR simultaneously detects a total of 19 diarrhea-causing pathogens, including viruses, bacteria and protozoa. Specific primer-probe sets were newly designed for 7 pathogens and were synthesized on the basis of previous reports for 12 pathogens. Assays were optimized to react under the same reaction conditions. The PCR efficiency and correlation coefficient (R(2)) of standard curves for each assay were more than 80% and 0.9766, respectively. Furthermore, the sensitivity of Dembo-PCR in fecal sample analysis was measured with feces spiked with target pathogens or synthesized DNA that included specific nucleotide target regions. The resulting limits of detection (LOD) for virus-spiked samples, bacteria and DNA fragments were 0.16-1.6 TCID50 (PFU/reaction), 1.3-13 CFU/reaction and 10-100 copies/reaction, respectively. All reactions showed high sensitivity in pathogen detection. A total of 8 fecal samples, collected from 6 diarrheic cattle, 1 diarrheic calf and 1 healthy cow, were tested using Dembo-PCR to validate the assay's clinical performance. The results revealed that bovine coronavirus had infected all diarrheic adult cattle and that bovine torovirus had infected the diarrheic calf. These results suggest that Dembo-PCR may be a powerful tool for diagnosing infectious agents in cattle diarrhea.

No MeSH data available.


Related in: MedlinePlus

Dembo-PCR workflow. To prepare each sample for assay, 10% fecal suspensions were madein PBS (−). The suspensions were then used directly for the extraction of bacteria andprotozoa nucleic acids with a QIAamp Fast DNA Stool Mini Kit. For virus detection, thesuspensions were centrifuged for 15 min at 10,000 rpm, and viral DNA and RNA wereextracted from the supernatants with a High Pure Viral Nucleic Acid Kit. Afterpathogen RNA and DNA were extracted, samples, reagents and each primer and probe weremixed in individual reaction tubes. Samples were applied at 2 µl pertube. A LightCycler Nano was used for all qPCR reactions performed in this study. Aone step PrimeScript RT-PCR Kit (Perfect Real time) was used for amplification ofextracts from RNA viruses, and Premix Ex Taq (Perfect Real time) was used foramplification of extracts from DNA viruses, bacteria and protozoa.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4829504&req=5

fig_001: Dembo-PCR workflow. To prepare each sample for assay, 10% fecal suspensions were madein PBS (−). The suspensions were then used directly for the extraction of bacteria andprotozoa nucleic acids with a QIAamp Fast DNA Stool Mini Kit. For virus detection, thesuspensions were centrifuged for 15 min at 10,000 rpm, and viral DNA and RNA wereextracted from the supernatants with a High Pure Viral Nucleic Acid Kit. Afterpathogen RNA and DNA were extracted, samples, reagents and each primer and probe weremixed in individual reaction tubes. Samples were applied at 2 µl pertube. A LightCycler Nano was used for all qPCR reactions performed in this study. Aone step PrimeScript RT-PCR Kit (Perfect Real time) was used for amplification ofextracts from RNA viruses, and Premix Ex Taq (Perfect Real time) was used foramplification of extracts from DNA viruses, bacteria and protozoa.

Mentions: Dembo-PCR workflow: Figure 1Fig. 1.


Development of a novel detection system for microbes from bovine diarrhea by real-time PCR.

Tsuchiaka S, Masuda T, Sugimura S, Kobayashi S, Komatsu N, Nagai M, Omatsu T, Furuya T, Oba M, Katayama Y, Kanda S, Yokoyama T, Mizutani T - J. Vet. Med. Sci. (2015)

Dembo-PCR workflow. To prepare each sample for assay, 10% fecal suspensions were madein PBS (−). The suspensions were then used directly for the extraction of bacteria andprotozoa nucleic acids with a QIAamp Fast DNA Stool Mini Kit. For virus detection, thesuspensions were centrifuged for 15 min at 10,000 rpm, and viral DNA and RNA wereextracted from the supernatants with a High Pure Viral Nucleic Acid Kit. Afterpathogen RNA and DNA were extracted, samples, reagents and each primer and probe weremixed in individual reaction tubes. Samples were applied at 2 µl pertube. A LightCycler Nano was used for all qPCR reactions performed in this study. Aone step PrimeScript RT-PCR Kit (Perfect Real time) was used for amplification ofextracts from RNA viruses, and Premix Ex Taq (Perfect Real time) was used foramplification of extracts from DNA viruses, bacteria and protozoa.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4829504&req=5

fig_001: Dembo-PCR workflow. To prepare each sample for assay, 10% fecal suspensions were madein PBS (−). The suspensions were then used directly for the extraction of bacteria andprotozoa nucleic acids with a QIAamp Fast DNA Stool Mini Kit. For virus detection, thesuspensions were centrifuged for 15 min at 10,000 rpm, and viral DNA and RNA wereextracted from the supernatants with a High Pure Viral Nucleic Acid Kit. Afterpathogen RNA and DNA were extracted, samples, reagents and each primer and probe weremixed in individual reaction tubes. Samples were applied at 2 µl pertube. A LightCycler Nano was used for all qPCR reactions performed in this study. Aone step PrimeScript RT-PCR Kit (Perfect Real time) was used for amplification ofextracts from RNA viruses, and Premix Ex Taq (Perfect Real time) was used foramplification of extracts from DNA viruses, bacteria and protozoa.
Mentions: Dembo-PCR workflow: Figure 1Fig. 1.

Bottom Line: The resulting limits of detection (LOD) for virus-spiked samples, bacteria and DNA fragments were 0.16-1.6 TCID50 (PFU/reaction), 1.3-13 CFU/reaction and 10-100 copies/reaction, respectively.All reactions showed high sensitivity in pathogen detection.The results revealed that bovine coronavirus had infected all diarrheic adult cattle and that bovine torovirus had infected the diarrheic calf.

View Article: PubMed Central - PubMed

Affiliation: The United Graduate School of Veterinary Sciences, Gifu University, Yanagito, Gifu 501-1193, Japan.

ABSTRACT
Diarrhea in cattle is one of the most economically costly disorders, decreasing milk production and weight gain. In the present study, we established a novel simultaneous detection system using TaqMan real-time PCR designed as a system for detection of microbes from bovine diarrhea using real-time PCR (referred to as Dembo-PCR). Dembo-PCR simultaneously detects a total of 19 diarrhea-causing pathogens, including viruses, bacteria and protozoa. Specific primer-probe sets were newly designed for 7 pathogens and were synthesized on the basis of previous reports for 12 pathogens. Assays were optimized to react under the same reaction conditions. The PCR efficiency and correlation coefficient (R(2)) of standard curves for each assay were more than 80% and 0.9766, respectively. Furthermore, the sensitivity of Dembo-PCR in fecal sample analysis was measured with feces spiked with target pathogens or synthesized DNA that included specific nucleotide target regions. The resulting limits of detection (LOD) for virus-spiked samples, bacteria and DNA fragments were 0.16-1.6 TCID50 (PFU/reaction), 1.3-13 CFU/reaction and 10-100 copies/reaction, respectively. All reactions showed high sensitivity in pathogen detection. A total of 8 fecal samples, collected from 6 diarrheic cattle, 1 diarrheic calf and 1 healthy cow, were tested using Dembo-PCR to validate the assay's clinical performance. The results revealed that bovine coronavirus had infected all diarrheic adult cattle and that bovine torovirus had infected the diarrheic calf. These results suggest that Dembo-PCR may be a powerful tool for diagnosing infectious agents in cattle diarrhea.

No MeSH data available.


Related in: MedlinePlus