The 65 serotypes of human enteroviruses are classified into four species, Human enterovirus (HEV) A to D, based largely on phylogenetic relationships in multiple genome regions. The 39-non-translated region of enteroviruses is highly conserved within a species but highly divergent between species. From this information, species-specific RT-PCR primers were developed that can be used to rapidly screen collections of enterovirus isolates to identify species of interest. The four primer pairs were 100 % specific when tested against enterovirus prototype strains and panels of isolates of known serotype (a total of 193 isolates). For evaluation in a typical application, the species-specific primers were used to screen 186 previously uncharacterized non-polio enterovirus isolates. The HEV-B primers amplified 68?3 % of isolates, while the HEV-A and HEV-C primers accounted for 9?7 and 11?3 % of isolates, respectively; no isolates were amplified with the HEV-D primers. Twelve isolates (6?5 %) were amplified by more than one primer set and eight isolates (4?3 %) were not amplified by any of the four primer pairs. Serotypes were identified by partial sequencing of the VP1 capsid gene, and in every case sequencing confirmed that the species-specific PCR result was correct; the isolates that were amplified by more than one species-specific primer pair were mixtures of two (11 isolates) or three (one isolate) species of viruses. The eight isolates that were not amplified by the species-specific primers comprised four new serotypes (EV76, EV89, EV90 and EV91) that appear to be unique members of HEV-A based on VP1, 3D and 39-non-translated region sequences. INTRODUCTIONThe 65 human enterovirus serotypes (family Picornaviridae) are classified into four species, Human enterovirus (HEV)-A, HEV-B, HEV-C and HEV-D, largely on the basis of phylogenetic analysis of multiple genome regions (King et al., 2000). Analysis of the complete genome sequences of all the prototype strains has shown that the species remain phylogenetically coherent in all genome regions except in the 59-non-translated region (NTR) (Brown et al., 2003; Oberste et al., 2004a, d). Seven of the 13 simian enterovirus prototype strains are provisional members of two HEV species, with six strains in HEV-A and one in HEV-B (Oberste et al., 2002). The remaining six strains are proposed to define three new species in the genus Enterovirus (Oberste et al., 2002), while the bovine and porcine enteroviruses comprise two additional species (King et al., 2000). Recombination, a major mechanism of enterovirus evolution, has been observed to occur in nature only among members of the same species, except in the 59-NTR (Brown et al., 2003; Lindberg et al., 2003;Lukashev et al., 2003 Lukashev et al., , 2004 Oberste et al., 2004a, d, e;Oprisan et al., 2002). Species identity therefore appears to have some biological significance integral to virus survival.Genus-specific primers are widely used for the amplification of enteroviruses from clinical specimens by RT-PCR (Rotbart & Ro...

Ureaplasma, spp. Mycoplasma genitalium, and Mycoplasma hominis are associated with infection of the genitourinary tract, reproductive failure, and neonatal morbidity and mortality. We have developed a multiplex PCR for the detection of these Mycoplasmatales in a single amplification reaction. The analytical sensitivities of this assay were 10.8, 10.8, and 8.8 CFU for each organism, respectively. This multiplex PCR was compared to culture on 26 cervical swabs, 2 vaginal swabs, 4 female urine specimens, 49 semen samples, 2 male urine specimens, and 1 nonspecified sample. A total of 21 specimens were culture positive (25%); 17 of these were PCR positive. An additional 11 specimens were PCR positive but culture negative. Of the 21 culture-positive specimens, 17 (81%) grew Ureaplasma spp. and 4 (19%) grew Mycoplasma spp. Of the 28 PCR-positive specimens, Ureaplasma spp. was detected in 23 (82%), M. hominis was detected in 3 (11%), and both were detected in 2 (7%). In a confirmatory analysis, all samples were tested by amplification of a second target of the ureaplasma genome. True-positive cases were defined as a positive result by culture or by both amplification assays. The multiplex PCR detected organisms in 26 of the 30 true-positive specimens, as well as in 2 other specimens. Based on a 36% prevalence of infection, the sensitivity, specificity, and positive and negative predictive values of multiplex PCR analyses were 87, 96, 94, and 93%, respectively. Multiplex PCR offers a rapid, sensitive, and easy method to detect genital mycoplasmas.

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The recent discovery of recombinant circulating vaccine-derived poliovirus (recombinant cVDPV) has highlighted the need for enhanced global poliovirus surveillance to assure timely detection of any future cVDPV outbreaks. Six pairs of Sabin strain-specific recombinant primers were designed to permit rapid screening for VDPV recombinants by PCR.