Development of a RT Real-Time PCR for the Detection and Quantification of Human Rhinoviruses

Gambarino, Stefano; Costa, Cristina; Elia, Mariateresa; Sidoti, Francesca; Mantovani, Samantha; Gruosso, V.; Bergallo, Massimiliano; Cavallo, Rossana

doi:10.1007/s12033-009-9164-x

Cited by 22 publications

(19 citation statements)

References 21 publications

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“…DNA plasmid standards were constructed as previously described (7). As regards cRNA standards, a large amount of HRV plasmid were obtained using PureLink™ HiPure Plasmid Midiprep Kit (Invitrogen, Carlsbad, USA) following manufactures' instructions.…”

Section: Methodsmentioning

confidence: 99%

“…Two microliters of ccDNA were added to 18μl of the reaction mix, giving a final reaction volume of 20μl using the thermal profile previously described (7). Different features were analyzed including the quantification capability, efficiency, sensitivity, linearity, detection limit, repeatability and reproducibility as described elsewhere (7). A total of 67 BAL specimens were extracted using an automated extraction of combined DNA/RNA using NucliSens easyMAG platform (bioMerièux).…”

Section: Methodsmentioning

confidence: 99%

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Improvement of HRV Quantification Using cRNA-Based Standards for Real Time RT-PCR

et al. 2010

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Real Time RT-PCR developed in recent years represents an useful tool in the diagnosis of RNA viruses. In order to accurately quantify and normalize a RNA target, efficiency of reverse-transcription must be considered. In this study a cRNA-standard based quantitative Real Time RT-PCR have been developed for HRV quantification on bronchoalveolar lavage (BAL) specimens. Results has been compared to a quantitative plasmid standard-based Real Time RT-PCR previously developed by us.Large amount of pHRV was linearized and purified. Blunt ends were generated and cRNA production was carried out. Dilutions of cRNA were generated and dynamic range, intraand inter-test variability, sensitivity and limit of detection were evaluated. Sixty-seven BAL, previously resulted positive to our plasmid standard-based method, were evaluated using cRNA-standard quantification. cRNA curve showed a broad dynamic range with a good intra-and inter-test variability, with an average of 3.23 threshold cycles more in comparison to plasmid standard-based curve. In terms of specimen quantification, a difference of 1.07log was found, showing a significant underrate using plasmid standard-based quantification.The method for cRNA-standard construction seems more suitable for quantification of RNA viruses, in order to normalize the quantification in reverse-transcription.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Improvement of HRV Quantification Using cRNA-Based Standards for Real Time RT-PCR

et al. 2010

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“…cDNA templates were evaluated by using a quantitative PCR (qPCR) assay with primers amplifying the 59 untranslated region of rhinovirus. 16 A separate qPCR for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to evaluate RNA quality. 17 Sequence analysis of the 59 nontranslated region of rhinovirus was performed by pyrosequencing with the use of a seminested assay that used our previously reported rhinovirus real-time qRT-PCR amplimer.…”

Section: Molecular Virological Studiesmentioning

confidence: 99%

Duration of Rhinovirus Shedding in the Upper Respiratory Tract in the First Year of Life

Loeffelholz¹,

Trujillo

Pyles

et al. 2014

Pediatrics

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WHAT'S KNOWN ON THIS SUBJECT: Rhinoviruses are commonly detected in both acutely ill and asymptomatic infants and children. The finding may represent new infection or prolonged presence of rhinovirus RNA in the respiratory tract. WHAT THIS STUDY ADDS:In young, otherwise healthy infants, shedding of RNA from the same rhinovirus strain rarely persisted longer than 30 days. abstract BACKGROUND: Current molecular diagnostic methods have detected rhinovirus RNA in a high proportion of asymptomatic infants and children, raising the question of the clinical significance of these findings. This study investigates the prevalence of prolonged rhinovirus RNA presence in the upper respiratory tract of infants during the first year of life. METHODS:In a longitudinal study, infants were followed from birth up to 12 months. Nasopharyngeal specimens were collected monthly (months 1-6 and month 9) and during an upper respiratory infection. Rhinoviruses were detected by quantitative reverse-transcription polymerase chain reaction. Presence of repeated rhinovirus RNA was evaluated by nucleotide sequence analysis. RESULTS:A total of 2153 specimens from 362 infants were studied; 341 distinct rhinovirus infections in 216 infants were identified. Follow-up specimens were available within 30 days for 179 infections, creating the sample set to assess prolonged rhinovirus presence. Of the 179 infections, 46 involved the detection of the same rhinovirus strain in repeated specimens, including 8 events of prolonged presence of the same strain (detected in specimens collected .30 days apart), representing 4.5% of the evaluable rhinovirus infections. There were 26 events in which a rhinovirus strain was replaced by a different strain within a 30-day interval, representing 14.5% of the 179 infections. CONCLUSIONS:Although rhinovirus infections are common in healthy infants, prolonged presence of rhinovirus RNA in the respiratory tract after an upper respiratory infection was uncommon (,5%). Detection of rhinovirus RNA in an infant most likely represents an infection within a 30-day period. Dr Loeffelholz co-conceptualized and co-designed the study and wrote the initial manuscript; Dr Trujillo coordinated data collection; Dr Pyles conceptualized and supervised molecular virology studies and reviewed and revised the manuscript; Mr Miller performed molecular virology studies and provided interpretation of the data for inclusion in the manuscript; Dr Alvarez-Fernandez supervised data collection; Dr Pong performed molecular virology studies and performed data collection and analysis; Dr Chonmaitree conceptualized and designed the study, provided overall study supervision, and revised the manuscript; and all authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work. 4,5 A study in adult volunteers showed that cultivatable rhinoviruses were routinely recovered for ∼2 weeks after infection. 6 Recurrent rhinovirus infection has been described in patients with asthma 7 and in those at risk of developing...

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“…Real-time RT-PCR assays have been developed for HRVs and/or HEVs (20,22,38,39). Tapparel et al (18) also sequenced recent clinical strains to design two double-dye DNA probes that could specifically detect HRVs.…”

Section: Discussionmentioning

confidence: 99%

Simultaneous Detection and Differentiation of Human Rhino- and Enteroviruses in Clinical Specimens by Real-Time PCR with Locked Nucleic Acid Probes

et al. 2013

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Development of a RT Real-Time PCR for the Detection and Quantification of Human Rhinoviruses

Cited by 22 publications

References 21 publications

Improvement of HRV Quantification Using cRNA-Based Standards for Real Time RT-PCR

Improvement of HRV Quantification Using cRNA-Based Standards for Real Time RT-PCR

Duration of Rhinovirus Shedding in the Upper Respiratory Tract in the First Year of Life

Simultaneous Detection and Differentiation of Human Rhino- and Enteroviruses in Clinical Specimens by Real-Time PCR with Locked Nucleic Acid Probes

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