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Development of TaqMan-based quantitative PCR for sensitive and selective detection of toxigenic Clostridium difficile in human stools.

Kubota H, Sakai T, Gawad A, Makino H, Akiyama T, Ishikawa E, Oishi K - PLoS ONE (2014)

Bottom Line: CDSC detected C. difficile in 9 of the 12 specimens, and toxigenic types of the isolates from the 9 specimens were consistent with those identified by qPCR, supporting the validity of our qPCR method.Moreover, the qPCR examination revealed that the carriage rate of whole C. difficile and that of toxigenic strains in the 82 subjects over a 6-month period ranged from 2.4 to 6.8% and 1.2 to 3.8%, respectively.Our qPCR method should be an effective tool for both clinical diagnosis and epidemiological investigation of C. difficile.

View Article: PubMed Central - PubMed

Affiliation: Yakult Honsha European Research Center for Microbiology ESV, Gent-Zwijnaarde, Belgium; Yakult Central Institute, Tokyo, Japan.

ABSTRACT

Background: Clostridium difficile is the main cause of nosocomial diarrhea, but is also found in asymptomatic subjects that are potentially involved in transmission of C. difficile infection. A sensitive and accurate detection method of C. difficile, especially toxigenic strains is indispensable for the epidemiological investigation.

Methods: TaqMan-based quantitative-PCR (qPCR) method for targeting 16S rRNA, tcdB, and tcdA genes of C. difficile was developed. The detection limit and accuracy of qPCR were evaluated by analyzing stool samples spiked with known amounts of C. difficile. A total of 235 stool specimens collected from 82 elderly nursing home residents were examined by qPCR, and the validity was evaluated by comparing the detection result with that by C. difficile selective culture (CDSC).

Results: The analysis of C. difficile-spiked stools confirmed that qPCR quantified whole C. difficile (TcdA+TcdB+, TcdA-TcdB+, and TcdA-TcdB- types), TcdB-producing strains (TcdA+TcdB+ and TcdA-TcdB+ types), and TcdA-producing strains (TcdA+TcdB+ type), respectively, with a lower detection limit of 103 cells/g of stool. Of the 235 specimens examined, 12 specimens (5.1%) were C. difficile-positive by qPCR: TcdA+TcdB+ strain in six specimens and TcdA-TcdB- strain in the other six. CDSC detected C. difficile in 9 of the 12 specimens, and toxigenic types of the isolates from the 9 specimens were consistent with those identified by qPCR, supporting the validity of our qPCR method. Moreover, the qPCR examination revealed that the carriage rate of whole C. difficile and that of toxigenic strains in the 82 subjects over a 6-month period ranged from 2.4 to 6.8% and 1.2 to 3.8%, respectively. An average qPCR count of C. difficile detected was 104.5 cells/g of stool, suggesting that C. difficile constituted a very small fraction of intestinal microbiota.

Conclusion: Our qPCR method should be an effective tool for both clinical diagnosis and epidemiological investigation of C. difficile.

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Related in: MedlinePlus

qPCR quantification of C. difficile DSM 1296T (A+B+ strain) spiked into a human stool.Stool samples taken from a healthy adult and supplemented with serial dilutions of C. difficile DSM 1296T (A+B+ strain) at final concentrations ranging from 103 to 108 cells/g of stool were examined by qPCR using CD16SrRNA-F/R/P (A), tcdA-F/R/P (B), or tcdB-F/R/P (C). Cell counts of the spiked C. difficile were determined by DAPI staining. The obtained analytical curve of the C. difficile-spiked stool (□) was compared with the standard analytical curve of the C. difficile pure culture (○).
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pone-0111684-g001: qPCR quantification of C. difficile DSM 1296T (A+B+ strain) spiked into a human stool.Stool samples taken from a healthy adult and supplemented with serial dilutions of C. difficile DSM 1296T (A+B+ strain) at final concentrations ranging from 103 to 108 cells/g of stool were examined by qPCR using CD16SrRNA-F/R/P (A), tcdA-F/R/P (B), or tcdB-F/R/P (C). Cell counts of the spiked C. difficile were determined by DAPI staining. The obtained analytical curve of the C. difficile-spiked stool (□) was compared with the standard analytical curve of the C. difficile pure culture (○).

Mentions: The lower detection limit and detection accuracy of qPCR for C. difficile in stools were evaluated by analyzing stool samples spiked with C. difficile vegetative cells at a final concentration of 103 to 108 cells/g of stool. The analytical curve of the C. difficile-spiked stool was compared with the standard analytical curve of the C. difficile pure culture (Figure 1A–C). In the case of all the three primers-probe sets, the obtained analytical curves were nearly equivalent over the range of 103 to 108 cells, confirming that qPCR with the standard analytical curve of the pure culture allowed accurate detection of C. difficile in stools. These results also indicated that our qPCR method enabled quantitative detection of C. difficile in stools with a lower detection limit of 103 cells/g of stool.


Development of TaqMan-based quantitative PCR for sensitive and selective detection of toxigenic Clostridium difficile in human stools.

Kubota H, Sakai T, Gawad A, Makino H, Akiyama T, Ishikawa E, Oishi K - PLoS ONE (2014)

qPCR quantification of C. difficile DSM 1296T (A+B+ strain) spiked into a human stool.Stool samples taken from a healthy adult and supplemented with serial dilutions of C. difficile DSM 1296T (A+B+ strain) at final concentrations ranging from 103 to 108 cells/g of stool were examined by qPCR using CD16SrRNA-F/R/P (A), tcdA-F/R/P (B), or tcdB-F/R/P (C). Cell counts of the spiked C. difficile were determined by DAPI staining. The obtained analytical curve of the C. difficile-spiked stool (□) was compared with the standard analytical curve of the C. difficile pure culture (○).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4216139&req=5

pone-0111684-g001: qPCR quantification of C. difficile DSM 1296T (A+B+ strain) spiked into a human stool.Stool samples taken from a healthy adult and supplemented with serial dilutions of C. difficile DSM 1296T (A+B+ strain) at final concentrations ranging from 103 to 108 cells/g of stool were examined by qPCR using CD16SrRNA-F/R/P (A), tcdA-F/R/P (B), or tcdB-F/R/P (C). Cell counts of the spiked C. difficile were determined by DAPI staining. The obtained analytical curve of the C. difficile-spiked stool (□) was compared with the standard analytical curve of the C. difficile pure culture (○).
Mentions: The lower detection limit and detection accuracy of qPCR for C. difficile in stools were evaluated by analyzing stool samples spiked with C. difficile vegetative cells at a final concentration of 103 to 108 cells/g of stool. The analytical curve of the C. difficile-spiked stool was compared with the standard analytical curve of the C. difficile pure culture (Figure 1A–C). In the case of all the three primers-probe sets, the obtained analytical curves were nearly equivalent over the range of 103 to 108 cells, confirming that qPCR with the standard analytical curve of the pure culture allowed accurate detection of C. difficile in stools. These results also indicated that our qPCR method enabled quantitative detection of C. difficile in stools with a lower detection limit of 103 cells/g of stool.

Bottom Line: CDSC detected C. difficile in 9 of the 12 specimens, and toxigenic types of the isolates from the 9 specimens were consistent with those identified by qPCR, supporting the validity of our qPCR method.Moreover, the qPCR examination revealed that the carriage rate of whole C. difficile and that of toxigenic strains in the 82 subjects over a 6-month period ranged from 2.4 to 6.8% and 1.2 to 3.8%, respectively.Our qPCR method should be an effective tool for both clinical diagnosis and epidemiological investigation of C. difficile.

View Article: PubMed Central - PubMed

Affiliation: Yakult Honsha European Research Center for Microbiology ESV, Gent-Zwijnaarde, Belgium; Yakult Central Institute, Tokyo, Japan.

ABSTRACT

Background: Clostridium difficile is the main cause of nosocomial diarrhea, but is also found in asymptomatic subjects that are potentially involved in transmission of C. difficile infection. A sensitive and accurate detection method of C. difficile, especially toxigenic strains is indispensable for the epidemiological investigation.

Methods: TaqMan-based quantitative-PCR (qPCR) method for targeting 16S rRNA, tcdB, and tcdA genes of C. difficile was developed. The detection limit and accuracy of qPCR were evaluated by analyzing stool samples spiked with known amounts of C. difficile. A total of 235 stool specimens collected from 82 elderly nursing home residents were examined by qPCR, and the validity was evaluated by comparing the detection result with that by C. difficile selective culture (CDSC).

Results: The analysis of C. difficile-spiked stools confirmed that qPCR quantified whole C. difficile (TcdA+TcdB+, TcdA-TcdB+, and TcdA-TcdB- types), TcdB-producing strains (TcdA+TcdB+ and TcdA-TcdB+ types), and TcdA-producing strains (TcdA+TcdB+ type), respectively, with a lower detection limit of 103 cells/g of stool. Of the 235 specimens examined, 12 specimens (5.1%) were C. difficile-positive by qPCR: TcdA+TcdB+ strain in six specimens and TcdA-TcdB- strain in the other six. CDSC detected C. difficile in 9 of the 12 specimens, and toxigenic types of the isolates from the 9 specimens were consistent with those identified by qPCR, supporting the validity of our qPCR method. Moreover, the qPCR examination revealed that the carriage rate of whole C. difficile and that of toxigenic strains in the 82 subjects over a 6-month period ranged from 2.4 to 6.8% and 1.2 to 3.8%, respectively. An average qPCR count of C. difficile detected was 104.5 cells/g of stool, suggesting that C. difficile constituted a very small fraction of intestinal microbiota.

Conclusion: Our qPCR method should be an effective tool for both clinical diagnosis and epidemiological investigation of C. difficile.

Show MeSH
Related in: MedlinePlus