Multiplex PCR method for detection of three Aeromonas enterotoxin genes.

A novel multiplex PCR method using three sets of specific primers was developed for the detection of the cytotoxic (act), heat-labile (alt), and heat-stable (ast) enterotoxin genes in Aeromonas spp. This assay was used to characterize 35 reference strains as well as 537 food-borne isolates. A total of seven gene pattern combinations were encountered, including act, alt, act/alt, act/alt/ast, act/alt/148-bp amplicon, alt/ast, and alt/148-bp amplicon. The alt gene was detected with 34 reference strains (97%) and occurred singly in 14% of these strains. The frequency of occurrence of the act/alt, act/alt/ast, and alt/ast gene patterns in reference strains was 14 (40%), 2 (6%), and 2 (6%), respectively. An unpredicted amplicon was detected in 11 reference strains (31%). Characterization of this amplicon showed that its size was 148 bp, as generated by the AHLF and AHLR primers, and that it uniquely aligned with the Aeromonas salmonicida A449 genome sequence (GenBank accession number CP000644). This amplicon was named Aeromonas salmonicida subsp. salmonicida hypothetical protein amplicon (AssHPA). In the 537 food-borne isolates, the act and alt genes were most dominant and were detected in 349 (65%) and 452 (84%) isolates, respectively, either alone or in combinations. The act and alt genes occurred singly in 30 (6%) and 128 (24%) of these strains, respectively. The act/alt gene pattern occurred in 315 isolates (59%), whereas the ast gene was always linked to strains exhibiting the act/alt/ast and alt/ast gene combinations in 4 (0.7%) and 5 (0.9%) isolates, respectively. The uniplex amplification of three enterotoxin genes separately confirms the specificity of the unique selected primers. This multiplex PCR is rapid and simple and can detect the presence of three Aeromonas enterotoxin genes in a single assay.

Molecular strategies for the detection, identification, and differentiation between enteroinvasive Escherichia coli and Shigella spp.

A strategy for the detection, identification, and differentiation of enteroinvasive Escherichia coli (EIEC) and Shigella spp. has been developed. The strategy includes (i) a multiplex PCR for the amplification of two virulence genes, i.e., iuc (222 bp) and ipaH (629 bp); (ii) amplification of the ial gene (a 1,038-bp amplicon) located within a large plasmid; and (iii) restriction fragment length polymorphism (RFLP) of the ial gene amplicon. The multiplex PCR provided three patterns. Pattern 1 (iuc-/ ipaH+) was found in 10 (67%) of 15 EIEC strains tested, pattern 2 (iuc+/ipaH-) in only 2 (4.4%) of 46 non-EIEC isolates, whereas pattern 3 (iuc+/ipaH+) was observed in all Shigella spp. and also in 5 (33%) of 15 EIEC strains tested. The pattern 3 EIEC strains were all positive for the ial gene. The PCR-RFLP of the ial gene amplicon using the endonuclease AclI was used to differentiate Shigella spp. from the EIEC strains that belonged to pattern 3. The ial gene was present in 21 (38%) of 56 and 6 (40%) of 15 Shigella spp. and EIEC strains tested, respectively. The PCR-RFLP of the ial gene amplicon divided the strains in two types. Type 1 did not contain the restriction enzyme site and was found in 6 (100%) of 6 EIEC strains, 4 (80%) of 5 Shigella boydii, and 4 (100%) of 4 Shigella dysenteriae strains tested. Type 2, which gave two fragments of 286 and 752 bp, was observed in 5 (83%) of 6 Shigella fiexneri strains and 6 (100%) of 6 Shigella sonnei strains. Detection, identification, and differentiation of Shigella spp. and EIEC were achieved by analyses of the PCR patterns and RFLP types. To our knowledge, this is the first study to demonstrate a simple and rapid method for detecting, identifying, and differentiating, at the molecular level, Shigella spp. and EIEC strains. This method will have tremendous utility as an epidemiological tool and in helping to develop policies, risk assessments, and national and international methods for Shigella spp.