Development of a real-time fluorescence resonance energy transfer PCR to detect arcobacter species

Abstract

A real-time PCR targeting the gyrase A subunit gene outside the quinolone resistance-determining region has been developed to detect Arcobacter species. The species identification was done by probe hybridization and melting curve analysis, using fluorescence resonance energy transfer technology. Discrimination between Arcobacter species was straightforward, as the corresponding melting points showed significant differences with the characteristic melting temperatures of 63.5 degrees C, 58.4 degrees C, 60.6 degrees C, and 51.8 degrees C for the Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter cibarius, and Arcobacter nitrofigilis type strains, respectively. The specificity of this assay was confirmed with pure cultures of 106 Arcobacter isolates from human clinical and veterinary specimens identified by phenotypic methods and 16S rRNA gene sequencing. The assay was then used to screen 345 clinical stool samples obtained from patients with diarrhea. The assay detected A. butzleri in four of these clinical samples (1.2%). These results were confirmed by a conventional PCR method targeting the 16S rRNA gene with subsequent sequencing of the PCR product. In conclusion, this real-time assay detects and differentiates Arcobacter species in pure culture as well as in the competing microbiota of the stool matrix. The assay is economical since only one biprobe is used and multiple Arcobacter species are identified in a single test.

FIG. 1.

Representation of the internal nucleotide sequences from the gyrA genes of five Arcobacter species and locations of the FRET probes used for real-time PCR amplification. The sequences from nucleotide 1984 to nucleotide 2083 are shown and compared to the numbering for the Arcobacter butzleri D2682 type strain (GenBank accession number DQ464331). The sensor probe (boxed) was labeled at the 5′ end with LC-Red 640-N-hydroxysuccinimide ester, and a 3′ terminal phosphate block was added. The anchor probe (underscored) was labeled at the 3′ end with fluorescein. Asterisks indicate type strains. M indicates nucleotide mismatches with the sensor probe inducing different melting points (with FRET). X indicates silent anchor probe nucleotide mismatches. •, spontaneous point mutation outside the interest region. Nucleotides that have replaced the expected nucleotides are indicated in bold. Ab, Arcobacter butzleri; Ac, Arcobacter cryaerophilus; Aci, Arcobacter cibarius; As, Arcobacter skirrowii; and An, Arcobacter nitrofigilis. The GenBank accession numbers corresponding to the gyrA sequences of A. butzleri strains D2686, Cipolla 4, 1340-2003, 1137-2003, 520H-2004, 235-2004, 1285-2003, 1426-2003, 1172-2003, 1188-2003, and 1477-2003 are DQ464331, DQ464335, DQ464334, DQ464333, DQ464332, EF176585, EF176586, EF176587, EF176588, EF176589, and EF176590, respectively. The GenBank accession numbers corresponding to the gyrA sequences of A. cryaerophilus strains A169/B, PC249, PC367, 322H-2004, 492-2004, and 622H-2004 are DQ464336, EF176591, EF176592, EF176593, EF176594, and EF176595, respectively, and those for A. cibarius strain CCUG 48482, Arcobacter skirrowii strain 449/80, and Arcobacter nitrofigilis strain C1 are DQ464337, DQ464338, and DQ464339, respectively.

FIG. 2.

MCA of the 352-bp amplicon of the gyrA genes of Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter cibarius, and Arcobacter nitrofigilis obtained with the real-time PCR assay. The melting peaks generated from the dissociation of the fluoroprobes from the Arcobacter species with four different T_ms can be observed. Values on the y axis represent the ratio of the first negative derivative of the change in fluorescence [d(F2/F1)] to the variation in temperature (dT).

FIG. 3.

PCR analysis of DNA extracted from stool samples. (A) MCA of the 352-bp amplicon of the gyrA gene. (B) Agarose gel electrophoresis of PCR products from Arcobacter butzleri strain D2686 and human stool samples obtained with primers ARCO and BUTZ for amplification of 401 bp from the 16S rRNA gene. M, 1-kb ladder; lane 1, A. butzleri strain D2686 as a reference; lanes 2 to 5, human stool samples 51669, 52654, 54941, and 59372, respectively; lane 6, negative control; d(F2/F1)/dT, ratio of the first negative derivative of the change in fluorescence to the variation in temperature.