Real-time reverse transcription-PCR assay for comprehensive detection of human rhinoviruses

Abstract

Human rhinoviruses (HRVs) are important contributors to respiratory disease, but their healthcare burden remains unclear, primarily because of the lack of sensitive, accurate, and convenient means of determining their causal role. To address this, we developed and clinically validated the sensitivity and specificity of a real-time reverse transcription-PCR (RT-PCR) assay targeting the viral 5' noncoding region defined by sequences obtained from all 100 currently recognized HRV prototype strains and 85 recently circulating field isolates. The assay successfully amplified all HRVs tested and could reproducibly detect 50 HRV RNA transcript copies, with a dynamic range of over 7 logs. In contrast, a quantified RNA transcript of human enterovirus 68 (HEV68) that showed the greatest sequence homology to the HRV primers and probe set was not detected below a concentration of 5 x 10(5) copies per reaction. Nucleic acid extracts of 111 coded respiratory specimens that were culture positive for HRV or HEV were tested with the HRV real-time RT-PCR assay and by two independent laboratories that used different in-house HRV/HEV RT-PCR assays. Eighty-seven HRV-culture-positive specimens were correctly identified by the real-time RT-PCR assay, and 4 of the 24 HEV-positive samples were positive for HRV. HRV-specific sequences subsequently were identified in these four specimens, suggesting HRV/HEV coinfection in these patients. The assay was successfully applied in an investigation of a coincidental outbreak of HRV respiratory illness among laboratory staff.

FIG. 1.

Alignment of partial 5′NCR sequences of 100 HRV and 52 HEV serotypes in regions corresponding to primers and probes used for the HRV real-time RT-PCR assay. Consensus sequences and nucleotide variations from the consensus are shown for each alignment. A blank square indicates that that base is identical to the consensus sequence base. The dash indicates the presence of an indel at position 367. ∼, Not determined. The nucleotide position numbering is based on the sequence of HRV1B (accession no. D00239). Letters in the Sequences column indicate the following HRV strains: A, 3, 4, 6, 13, 14, 17 to 19, 27, 37, 41, 48, 49, 53, 61, 73, 79, 82 to 84, 90, 92, 93, 96, and 97; B, 2, 8 to 11, 15, 16, 20, 21, 23 to 25, 29, 30, 32, 34, 38, 40, 44, 46, 50, 54 to 57, 60, 62, 66 to 68, 74, 76, 80, 81, 85, 95, 98, and 100; C, 1A, 1B, 22, 43, 51, 64, 71, 75, 86, and 94; D, 7, 12, 31, 36, 39, 45, 47, 58, and 89; E, 5, 35, 42, 52, 65, 69, and 91; F, 26 and 99; G, 59 and 63; H, 28; I, 33; J, 70; K, 72; L, 77; M, 78; and N, 88. Letters in the Sequences column indicate the following HEV types: O, coxsackievirus types A1, A2, A11, A13, A15, A17 to A22, and A24, HEV68 and HEV70, and poliovirus types 1 and 3; P, coxsackievirus types A3 to A7, A10, A12, and A14, coxsackievirus types B1, B3, and B5, echoviruses 1, 6 to 8, 13, 16 to 21, 27, 29, 31, and 32, and enterovirus types 69 and 71; Q, echovirus types 4, 14, and 15; R, poliovirus type 2; S, coxsackievirus type A8; T, echovirus type 11; U, echovirus type 26; and V, echovirus type 33. Ref. Acc. No., reference accession number.

FIG. 2.

Representative real-time RT-PCR amplification plot obtained with serial 10-fold dilutions (5 × 10¹ to 5 × 10⁷ copies per reaction) of HRV14 RNA transcript. The top panel shows a baseline subtractive curve fit (CF) view of the data, with relative fluorescence units (RFU) plotted against cycle numbers. The default setting of 10 times the standard deviation of the RFU measured in all wells over the baseline cycles was used to calculate the C_T for a positive reaction (horizontal line). The bottom panel shows a standard curve analysis of the DNA amplification plots with C_T values plotted proportionately against the logarithm of the input copy number. The dynamic range of assay spans 7 logs, with an R² value of 0.999.

FIG. 3.

HRV detected by real-time RT-PCR in serial nasal and throat swab specimens from four laboratory staff members (A, B, C, and D) with acute respiratory illness. C_T values are plotted against the number of days after the onset of illness. Neg, HRV not detected; black inverted triangle, the end of reported respiratory symptoms.