BACKGROUND: Enteroviruses are a leading cause of aseptic meningitis in adult and pediatric populations. We describe the development of a real-time RT-PCR assay that amplifies a small target in the 5' nontranslated region upstream of the classical Rotbart enterovirus amplicon. The assay includes an RNA internal control and incorporates modified nucleotide chemistry. METHODS: We evaluated the performance characteristics of this design and performed blinded parallel testing on clinical samples, comparing the results with a commercially available RT-PCR assay (Pan-Enterovirus OligoDetect kit) that uses an enzyme immunoassay-like plate end detection. RESULTS: We tested 778 samples and found 14 discrepant samples between the 2 assays. Of these, the real-time assay detected 6 samples that were negative by the OligoDetect kit, 5 of which were confirmed as positive by sequence analysis using an alternative primer set. Eight discrepant samples were positive by the OligoDetect kit and real-time negative, with 6 confirmed by sequencing. Overall, detection rates of 97% and 96% were obtained for the OligoDetect kit and real-time assays, respectively. Sequence analysis revealed the presence of a number of single nucleotide polymorphisms in the targeted region. The comparative sensitivities of the 2 assays were equivalent, with the limit of detection for the real-time assay determined to be approximately 430 copies per milliliter in cerebrospinal fluid. CONCLUSIONS: This novel real-time enterovirus assay is a sensitive and suitable assay for routine clinical testing. The presence of single nucleotide polymorphisms can affect real-time PCR assays.
BACKGROUND: Enteroviruses are a leading cause of aseptic meningitis in adult and pediatric populations. We describe the development of a real-time RT-PCR assay that amplifies a small target in the 5' nontranslated region upstream of the classical Rotbart enterovirus amplicon. The assay includes an RNA internal control and incorporates modified nucleotide chemistry. METHODS: We evaluated the performance characteristics of this design and performed blinded parallel testing on clinical samples, comparing the results with a commercially available RT-PCR assay (Pan-Enterovirus OligoDetect kit) that uses an enzyme immunoassay-like plate end detection. RESULTS: We tested 778 samples and found 14 discrepant samples between the 2 assays. Of these, the real-time assay detected 6 samples that were negative by the OligoDetect kit, 5 of which were confirmed as positive by sequence analysis using an alternative primer set. Eight discrepant samples were positive by the OligoDetect kit and real-time negative, with 6 confirmed by sequencing. Overall, detection rates of 97% and 96% were obtained for the OligoDetect kit and real-time assays, respectively. Sequence analysis revealed the presence of a number of single nucleotide polymorphisms in the targeted region. The comparative sensitivities of the 2 assays were equivalent, with the limit of detection for the real-time assay determined to be approximately 430 copies per milliliter in cerebrospinal fluid. CONCLUSIONS: This novel real-time enterovirus assay is a sensitive and suitable assay for routine clinical testing. The presence of single nucleotide polymorphisms can affect real-time PCR assays.
Authors: K E Hanson; E S Slechta; J A Killpack; C Heyrend; T Lunt; J A Daly; A C Hemmert; A J Blaschke Journal: J Clin Microbiol Date: 2015-12-30 Impact factor: 5.948
Authors: Erin H Graf; Keith E Simmon; Keith D Tardif; Weston Hymas; Steven Flygare; Karen Eilbeck; Mark Yandell; Robert Schlaberg Journal: J Clin Microbiol Date: 2016-01-27 Impact factor: 5.948
Authors: Jeffrey J Germer; Irina Ankoudinova; Yevgeniy S Belousov; Walt Mahoney; Chen Dong; Jihong Meng; Jayawant N Mandrekar; Joseph D Yao Journal: J Clin Microbiol Date: 2017-02-22 Impact factor: 5.948