Rapid Detection of Enterotoxigenic Clostridium perfringens by Real-Time Fluorescence Resonance Energy Transfer PCR

Cruz, Wilfred P. dela; Gozum, Mary M. A.; Lineberry, Sarah F.; Stassen, Sarah D.; Daughtry, Marianne; Stassen, Nicholas A.; Jones, Mary Smith McCrory; Johnson, Oswald L.

doi:10.4315/0362-028x-69.6.1347

Cited by 11 publications

(3 citation statements)

References 29 publications

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“…The assay developed by Kaneko et al [ 13 ] could detect >10 3 cfu/g of cpe -positive C. perfringens in meat samples, but it required enrichment culture specimens of spiked samples. dela Cruz et al [ 29 ] developed a rapid real-time fluorescence resonance energy transfer PCR targeting plc and cpe genes of C. perfringens . However, the assay could detect about 20 copies of target sequence per PCR, whereas our assay could detect even a single copy per PCR using pure culture.…”

Section: Discussionmentioning

confidence: 99%

Sensitive quantification of Clostridium perfringens in human feces by quantitative real-time PCR targeting alpha-toxin and enterotoxin genes

et al. 2015

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BackgroundClostridium perfringens is a widespread pathogen, but the precise quantification of this subdominant gut microbe remains difficult due to its low fecal count (particularly in asymptomatic subjects) and also due to the presence of abundant polymerase-inhibitory substances in human feces. Also, information on the intestinal carriage of toxigenic C. perfringens strains in healthy subjects is sparse. Therefore, we developed a sensitive quantitative real-time PCR assays for quantification of C. perfringens in human feces by targeting its α-toxin and enterotoxin genes. To validate the assays, we finally observed the occurrence of α-toxigenic and enterotoxigenic C. perfringens in the fecal microbiota of healthy Japanese infants and young adults.MethodsThe plc-specific qPCR assay was newly validated, while primers for 16S rRNA and cpe genes were retrieved from literature. The assays were validated for specificity and sensitivity in pre-inoculated fecal samples, and were finally applied to quantify C. perfringens in stool samples from apparently healthy infants (n 124) and young adults (n 221).ResultsThe qPCR assays were highly specific and sensitive, with a minimum detection limit of 103 bacterial cells/g feces. Alpha-toxigenic C. perfringens was detected in 36 % infants and 33 % adults, with counts ranging widely (103-107 bacterial cells/g). Intriguingly, the mean count of α-toxigenic C. perfringens was significantly higher in infants (6.0 ± 1.5 log10 bacterial cells/g), as compared to that in adults (4.8 ± 1.2). Moreover, the prevalence of enterotoxigenic C. perfringens was also found to be significantly higher in infants, as compared to that in adults. The mean enterotoxigenic C. perfringens count was 5.9 ± 1.9 and 4.8 ± 0.8 log10 bacterial cells/g in infants and adults, respectively.ConclusionsThese data indicate that some healthy infants and young adults carry α-toxigenic and enterotoxigenic C. perfringens at significant levels, and may be predisposed to related diseases. Thus, high fecal carriage of toxigenic C. perfringens in healthy children warrants further investigation on its potential sources and clinical significance in these subjects. In summary, we present a novel qPCR assay for sensitive and accurate quantification of α-toxigenic and enterotoxigenic C. perfringens in human feces, which should facilitate prospective studies of the gut microbiota.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0561-y) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Sensitive quantification of Clostridium perfringens in human feces by quantitative real-time PCR targeting alpha-toxin and enterotoxin genes

et al. 2015

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“…The critical point in enterotoxin (CPE) determination is enterotoxin expression. As is known, this toxin is produced only upon sporulation and, if the environment is not good enough for this, a possible false negative can take place (15,16). However, in addition to the positive and negative controls from the RPLA kit, we use two enterotoxigenic strains as positive controls for the culture medium.…”

Section: Azoreductase Activity Determinationmentioning

confidence: 99%

Characterization of the Plasmidic or Chromosomal cpe Gene and Metabolic Activities in Clostridium perfringens Isolates from Food in San Luis - Argentina

Corigliano¹,

Guzmán²,

Stagnitta³

2011

Cent Eur J Public Health

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Food poisoning and non-food poisoning illnesses due to C. perfringens (by enterotoxin production) have been associated to chromosomal or plasmidic location of the cpe gene, respectively. Clostridial pathogenicity has been correlated to protease and azoreductase production. The aim of this work was: i) to assess the sanitary-hygienic quality of dehydrated soups (100 samples) consumed in San Luis-Argentina; ii) to verify the presence of C. perfringens in these food products using the "Most Probable Number" method (MPN) and plate-counting methods; iii) to characterise enterotoxigenicity in strain isolates by RPLA; iv) to determine the chromosomal or plasmidic location of the cpe gene in enterotoxigenic strains previously isolated from food in our lab, using PCR; v) to correlate chromosomal cpe and spore heat-resistance; vi) to compare protease activity in cpe+ and cpe-strains; and vii) to compare azoreductase activity in cpe+ and cpe-strains. Twenty-six isolates had a count a 3-43 bacteria g-1 count using MPN; 7.7% exceeded the Argentine Food Code (CAA) limit. All isolates showed protease activity: enterotoxigenic isolates had higher protease activity than non-enterotoxigenic isolates. All isolates showed azoreductase activity: enterotoxigenic isolates had higher activity and shorter reducing times. Enterotoxigenic isolates showed chromosomal location for the gene responsible for the enterotoxin.

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“…The R.A.P.I.D. has been used to detect Salmonella (Van Kessel,, Clostridium perfringens(dela Cruz et al, 2006), and Bacillus anthracis…”

mentioning

confidence: 99%

Impact of bacterial genomics on determining quality and safety in the dairy production chain

Marco

Wells-Bennik

2008

International Dairy Journal

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Rapid Detection of Enterotoxigenic Clostridium perfringens by Real-Time Fluorescence Resonance Energy Transfer PCR

Cited by 11 publications

References 29 publications

Sensitive quantification of Clostridium perfringens in human feces by quantitative real-time PCR targeting alpha-toxin and enterotoxin genes

Sensitive quantification of Clostridium perfringens in human feces by quantitative real-time PCR targeting alpha-toxin and enterotoxin genes

Characterization of the Plasmidic or Chromosomal cpe Gene and Metabolic Activities in Clostridium perfringens Isolates from Food in San Luis - Argentina

Impact of bacterial genomics on determining quality and safety in the dairy production chain

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