Development of TaqMan-Based Quantitative PCR for Sensitive and Selective Detection of Toxigenic <i>Clostridium difficile</i> in Human Stools

<div><p>Background</p><p><i>Clostridium difficile</i> is the main cause of nosocomial diarrhea, but is also found in asymptomatic subjects that are potentially involved in transmission of <i>C. difficile</i> infection. A sensitive and accurate detection method of <i>C. difficile</i>, especially toxigenic strains is indispensable for the epidemiological investigation.</p><p>Methods</p><p>TaqMan-based quantitative-PCR (qPCR) method for targeting 16S rRNA, <i>tcdB</i>, and <i>tcdA</i> genes of <i>C. difficile</i> was developed. The detection limit and accuracy of qPCR were evaluated by analyzing stool samples spiked with known amounts of <i>C. difficile</i>. 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 <i>C. difficile</i> selective culture (CDSC).</p><p>Results</p><p>The analysis of <i>C. difficile</i>-spiked stools confirmed that qPCR quantified whole <i>C. difficile</i> (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 <i>C. difficile</i>-positive by qPCR: TcdA⁺TcdB⁺ strain in six specimens and TcdA⁻TcdB⁻ strain in the other six. CDSC detected <i>C. difficile</i> 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 <i>C. difficile</i> 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 <i>C. difficile</i> detected was 10^4.5 cells/g of stool, suggesting that <i>C. difficile</i> constituted a very small fraction of intestinal microbiota.</p><p>Conclusion</p><p>Our qPCR method should be an effective tool for both clinical diagnosis and epidemiological investigation of <i>C. difficile</i>.</p></div

Comparison of qPCR analytical curves among <i>C. difficile</i> strains.

a<p>Each analytical curve of different <i>C. difficile</i> strains was generated with serial dilutions ranging from 10 to 10⁵ cells per reaction. X-axis is bacterial cells applied to the reaction (log₁₀ cells/reaction) and Y-axis is the <i>C_T</i> values obtained.</p>b<p>Differences in <i>C_T</i> values compared with that of the type strain (DSM 1296^T) are indicated.</p><p>Comparison of qPCR analytical curves among <i>C. difficile</i> strains.</p

Comparison of detection results of toxigenic <i>C. difficile</i> or toxins by qPCR, <i>C. difficile</i> selective culture (CDSC), and enzyme immunoassay (EIA).

a<p>Toxigenic types were identified on the basis of qPCR counts for the three genes, according to the criteria described in Materials and Methods.</p>b<p>The toxigenic type of isolates was determined on the basis of PCR amplification of <i>tcdA</i> and <i>tcdB</i> by using the method of Kato <i>et al</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111684#pone.0111684-Kato1" target="_blank">[15]</a>.</p>c<p>na, not applicable.</p><p>Comparison of detection results of toxigenic <i>C. difficile</i> or toxins by qPCR, <i>C. difficile</i> selective culture (CDSC), and enzyme immunoassay (EIA).</p

Comparison of detection results of <i>C. difficile</i> between qPCR and <i>C. difficile</i> selective culture (CDSC).

a<p>“<i>C. difficile</i> positive/negative” was defined by presence/absence of qPCR amplification with the 16S rRNA primers-probe set.</p>b<p>“<i>C. difficile</i> positive/negative” was defined by presence/absence of <i>C. difficile</i> isolation by means of stool culture.</p><p>Comparison of detection results of <i>C. difficile</i> between qPCR and <i>C. difficile</i> selective culture (CDSC).</p

<i>C. difficile</i> carriage rates in four nursing home populations.

<p>On the basis of the qPCR counts for three genes (16S rRNA, <i>tcdA</i>, and <i>tcdB</i>), the toxigenic types (A⁺B⁺, A⁻B⁺, or A⁻B⁻) of <i>C. difficile</i> predominating in individual specimens were identified. The rates of carriage of each toxigenic type of <i>C. difficile</i> at three stool samplings (S1, S2, and S3) were calculated with respect to each nursing home (n = 11, 14, 24, and 33, respectively) and the total population (n = 82).</p

Specific detection of target <i>C. difficile</i> strains by qPCR with newly developed oligonucleotide sets.

a<p>na, not applicable.</p>b<p>The reactivity of qPCR for the target bacteria with each primers-probe set was investigated by using DNA extracts corresponding to 10⁵ cells per reaction from each pure culture of the listed strains. Reactivity was judged by using the criteria described in the Materials and Methods. In addition, negative PCR results were obtained for the following bacterial strains, representing the major intestinal bacteria: <i>Blautia productus</i> JCM 1471^T, <i>Faecalibacterium prausnitzii</i> ATCC 27768^T, <i>Bacteroides vulgatus</i> ATCC 8482^T, <i>Bacteroides ovatus</i> JCM 5824^T, <i>Fusobacterium varium</i> ATCC 8501^T, <i>Collinsella aerofaciens</i> ATCC 25986^T, <i>Prevotella melaninogenica</i> ATCC 25845^T, <i>Veillonella parvula</i> GIFU 7884^T, <i>Bifidobacterium longum</i> ATCC 15707^T, <i>Bifidobacterium adolescentis</i> ATCC 15703^T, <i>Bifidobacterium catenulatum</i> ATCC 27539^T, <i>Lactobacillus gasseri</i> DSM 20243^T.</p><p>Specific detection of target <i>C. difficile</i> strains by qPCR with newly developed oligonucleotide sets.</p

Oligonucleotide sequences used in this study.

a<p>GenBank accession number NR074454.</p>b<p>GenBank accession number M30307.</p>c<p>GenBank accession number X53138.</p><p>Oligonucleotide sequences used in this study.</p

Primer sets for MLST analysis.

<p>Primers targeting species belonging to <i>B. adolescentis</i>, <i>B. bifidum</i>, <i>B. catenulatum</i>, <i>B. pseudocatenulatum</i> were designed by Delétoile <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078331#pone.0078331-Deltoile1" target="_blank">[30]</a>, and <i>B. longum</i> subsp. <i>longum</i> primers were designed by Makino <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078331#pone.0078331-Makino1" target="_blank">[21]</a>.</p

Detection of mother-infant monophyletic <i>Bifidobacterium</i> strains among 16 families.

<p>“•” represents that at least one mother-infant monophyletic <i>Bifidobacterium</i> strain was isolated from the indicated family. Mother no. 5 gave birth to twins (A, B).</p

The numbers of bifidobacteria in infants from 0 day to 7 days after birth.

<p>Infant no.^a The <i>B. adolescentis</i> group consists of <i>B. adolescentis</i> genotypes A and B. ^b The <i>B. catenulatum</i> group consists of <i>B. catenulatum</i> and <i>B. pseudocatenulatum</i>.</p