A One-Step, Real-Time PCR Assay for Rapid Detection of Rhinovirus

Abstract

One-step , real-time PCR assays for rhinovirus have been developed for a limited number of PCR amplification platforms and chemistries , and some exhibit cross-reactivity with genetically similar enteroviruses. We developed a one-step , real-time PCR assay for rhinovirus by using a sequence detection system (Applied Biosystems; Foster City , CA). The primers were designed to amplify a 120-base target in the noncoding region of picornavirus RNA , and a TaqMan (Applied Biosystems) degenerate probe was designed for the specific detection of rhinovirus amplicons. The PCR assay had no cross-reactivity with a panel of 76 nontarget nucleic acids , which included RNAs from 43 enterovirus strains. Excellent lower limits of detection relative to viral culture were observed for the PCR assay by using 38 of 40 rhinovirus reference strains representing different serotypes , which could reproducibly detect rhinovirus serotype 2 in viral transport medium containing 10 to 10,000 TCID 50 (50% tissue culture infectious dose endpoint) units/ml of the virus. However, for rhinovirus serotypes 59 and 69, the PCR assay was less sensitive than culture. Testing of 48 clinical specimens from children with cold-like illnesses for rhinovirus by the PCR and culture assays yielded detection rates of 16.7% and 6.3%, respectively. For a batch of 10 specimens, the entire assay was completed in 4.5 hours. This real-time PCR assay enables detection of many rhinovirus serotypes with the Applied Biosystems reagent-instrument platform. Rhinoviruses are the most common cause of viral upper respiratory tract infections and have been associated with more severe lower tract infections in compromised patients.1-3 Several real-time, RT-PCR assays have been developed; these have improved the diagnosis of rhinovirus infection over traditional culture methods, which are slow and insensitive. 4 -7 However, only a few published PCR assays have combined reverse transcription and PCR in the same real-time reaction (ie, one-step assay). 8,9 The advantages of the one-step assay over the two-step assay include improved workflow, reduction in assay preparation time, and elimination of cross contamination from the transfer of cDNA from the reverse transcription reaction into the PCR reaction. One-step assays have been developed for a limited number of PCR amplification platforms and chemistries and may not necessarily perform optimally with other platforms.8 In addition, cross-reactivity with genetically similar enteroviruses has been reported with some assays. 8,9A one-step, real-time PCR assay has not been described for the ABI Prism Sequence Detection System (Applied Biosystems; Foster City, CA) though this platform is widely used in clinical and research laboratories. High PCR efficiency with this platform generally requires the use of primers and a TaqMan probe (Applied Biosystems) with melting temperatures of 58°C to 60°C and 68°C to 70°C, respectively, and an amplicon size of 50 to 150 bp. The 5Ј noncoding region of the rhinovirus genome is most commonly targe...

Recent advances in the detection of respiratory virus infection in humans

“…In contrast, the one‐step, real‐time PCR assay detects RVs by using a sequence detection system. This assay improves workflow, reduces preparation time, and eliminates cross‐contamination that might be introduced during cDNA transfer from the reverse transcription reaction into the PCR reaction step 40 . Semi‐nested RT‐PCR assay is based on an extremely sensitive PCR to detect airborne RVs, which has a limit of detection (LOD) of approximately 0.8 median tissue culture infectious dose (TCID 50 ) 41 .…”

Critical Analysis of Rhinovirus RNA Load Quantification by Real-Time Reverse Transcription-PCR

“…However, there are inconsistent data about whether viral load is correlated with disease severity, and the threshold cycle (C T ) values from real-time RT-PCR are converted too frequently into viral copies per milliliter without any validation. This is particularly important because many published assays include primers with degenerate nucleotides or probe sequences that are biased toward a given genotype or species (4,14,22,24).…”

“…However, there are inconsistent data about whether viral load is correlated with disease severity, and the threshold cycle (C T ) values from real-time RT-PCR are converted too frequently into viral copies per milliliter without any validation. This is particularly important because many published assays include primers with degenerate nucleotides or probe sequences that are biased toward a given genotype or species (4,14,22,24).We validated previously a two-step real-time RT-PCR assay, named Panenterhino/Ge/08, which could detect all known HRV genotypes and, to a lesser extent, human respiratory enteroviruses (HEVs) (22). In this study, we first validated the Panenterhino/ Ge/08 assay in a one-step quantitative format using an internal extraction control and serial dilutions of an in vitro-transcribed rhinovirus RNA reference standard.…”

A Rapid Microscopic Method for the Confirmation of Rhinoviruses in Cell Culture

“…Although PCR is considered more sensitive than culture, one study demonstrated the lack of PCR sensitivity for serotypes 59 and 69 when compared with the latter [7] . Also, heightened PCR sensitivity may hinder the proper association of PCR-identified rhinovirus RNA with infectious virus production and clinical symptoms of respiratory illness.…”

“…Sensitivity/ specificity issues, along with high reagent costs and the need for specialized equipment and training limit the widespread use of rhinovirus molecular techniques [4,6,7] . Despite these obstacles, the potential benefits of viral detection in lower respiratory tract disease will necessitate the continued development and improvement of these molecular assays.…”