Real-time PCR for rapid diagnosis of entero- and rhinovirus infections using LightCycler

Kares, Saara

doi:10.1016/s1386-6532(03)00093-3

Cited by 67 publications

(54 citation statements)

References 20 publications

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“…Whereas the locations of these conserved sequences offer considerable flexibility for designing targeted primers/probes for HEV real-time RT-PCR assays (25,38,47), the development of comparable assays for HRVs has been hampered by their greater genetic variability and the paucity of published HRV sequence data from the 5ЈNCR. The few realtime assays that have been described for HRVs were not evaluated against or failed to detect all known HRV serotypes (8, 9, 44, 51), or they used Sybr green instead of probe-based fluorescence detection (8); the results of Sybr green testing can be difficult to interpret (49).…”

Section: Discussionmentioning

confidence: 99%

“…However, most of these assays require postamplification processing of the amplicon by gel electrophoresis, probe hybridization, sequencing, or restriction analysis to confirm and differentiate HRVs from HEVs (1,3,4,5,14,24,31,34,41). More recently, real-time RT-PCR assays have been described for HRVs/HEVs (8,9,25,38,44) that offer potentially rapid, sensitive, and quantitative results and that are less prone to amplicon contamination. However, because of the more extensive genetic variability of the HRVs and the lack of available sequence data in the public domain, few real-time RT-PCR assays have been described specifically for the HRVs (8,9,44,51), and none to our knowledge has been shown to successfully detect all recognized HRV prototype strains.…”

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confidence: 99%

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Real-Time Reverse Transcription-PCR Assay for Comprehensive Detection of Human Rhinoviruses

Lu¹,

Holloway

Dare³

et al. 2008

J Clin Microbiol

265

229

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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 ؋ 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 HRVculture-positive specimens were correctly identified by the real-time RT-PCR assay, and 4 of the 24 HEVpositive 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.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Real-Time Reverse Transcription-PCR Assay for Comprehensive Detection of Human Rhinoviruses

Lu¹,

Holloway

Dare³

et al. 2008

J Clin Microbiol

265

229

View full text Add to dashboard Cite

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“…GXEA was evaluated in parallel with an in-house real-time PCR assay, as described previously (8). Briefly, after RNA isolation from 140 l of CSF by use of the QIAamp viral RNA mini kit (Qiagen, Hilden, Germany), 5 l of RNA was amplified with previously described primers targeting 120 bp of the 5Ј noncoding region and with a LightCycler RNA master hybridization probes kit (Roche Applied Science, Mannheim, Germany) on a LightCycler (version 2.0) instrument (Roche Applied Science).…”

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confidence: 99%

GeneXpert Enterovirus Assay: One-Year Experience in a Routine Laboratory Setting and Evaluation on Three Proficiency Panels

et al. 2008

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A prospective unblinded comparative evaluation of three assays for the detection of enteroviral RNA performed on 83 positive and 79 negative cerebrospinal fluid samples showed initial and resolved sensitivities of 90.4% and 98.8%, respectively, for the Cepheid GeneXpert enterovirus assay; 94.0% and 97.6%, respectively, for the Argene enterovirus consensus kit; and 100% and 100%, respectively, for an in-house real-time PCR. The initial and resolved specificities were 100% for all three assays.Enteroviruses comprise a large group of immunologically distinct serotypes of viruses belonging to the family Picornaviridae (18). Infection with enteroviruses is associated with protean clinical manifestations, ranging from asymptomatic or mild febrile illness to severe and potentially fatal syndromes, including paralysis, aseptic meningitis, encephalitis, myocarditis, and neonatal systemic infection (1). Enteroviruses are the most common cause of aseptic meningitis in both children and adults and may cause up to 90% of cases of aseptic meningitis for which an etiology is identified (1,14,18,19). The rapid detection and the rapid characterization of enteroviral meningitis are essential for making decisions for patient management and treatment (5,15,17,20). The provision of enterovirus test results on a daily basis can have a substantial impact on health care and has been shown to be highly cost-effective (9,12,17). Since the conventional means of diagnosis of enteroviral meningitis by cell culture from cerebrospinal fluid (CSF) is timeconsuming and expensive and lacks sensitivity, some commercial and several in-house PCR protocols as well as nucleic acid sequence-based amplification assays have been developed during the last 15 years in order to improve the ability to detect enterovirus from CSF (2-4, 6, 7, 11, 13, 16, 20-22, 24-26). The most widely used PCR-based assays for the routine diagnosis of enteroviral meningitis are those that detect PCR products in microtiter wells (6,21,22) and those that use the real-time PCR technology (2,3,13,(24)(25)(26). However, all of these approaches require specially trained laboratory staff, thus limiting the ability of the "real-time" technology to deliver patient results that would be most useful for making patient management decisions STAT.The latest development in the field of the molecular diagnosis of enteroviral meningitis is a fully automated real-time multiplex, reverse transcription-PCR assay, the GeneXpert enterovirus assay (GXEA; Cepheid, Sunnyvale, CA) (10). GXEA is, at present, the only FDA-approved assay for the qualitative detection of enterovirus RNA in CSF. It combines automated nucleic acid sample preparation, amplification, and real-time detection of enteroviral RNA in a disposable, macro/microfluidic cartridge using the GeneXpert Dx system instrument. To date, only one evaluation of GXEA has been published in peer-reviewed journals. A multicenter beta trial with 102 CSF samples obtained from patients with suspected meningitis (34 of whom were enterovirus positive) ...

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“…Further, the rRT-PCR assay increased the detection sensitivity to 10-fold that of the conventional RT-PCR assays [27] . Simultaneous detection and quantification of both the enteroviruses and rhinoviruses was carried out efficiently by rRT-PCR assay [28] . Diagnosis of Hepatitis A and C viruses (HAV, HCV) employs rRT-PCR assays for rapidity and accuracy.…”

Section: Applications Of Rrt-pcr As Diagnosticmentioning

confidence: 99%

Applications of Real-time Reverse Transcription Polymerase Chain Reaction in Clinical Virology Laboratories for the Diagnosis of Human Diseases

Manojkumar¹,

Mrudula²

2006

American J. of Infectious Diseases

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Early diagnosis that prevents further expansion of the causative agent and thereby aids control and eradication of infectious agents is critical for countries trying to obtain a particular disease free status. In these diagnostics speed is paramount, including the assay's applicability, sensitivity, specificity, cost effectiveness and patentability. Real-time Reverse Transcription PCR (rRT-PCR) has revolutionized the field of molecular biology and is being utilized increasingly in novel clinical diagnostic assays. This technique has been applied for rapid detection of point mutation, drug resistant variant and genotyping in several viruses. The combination of excellent sensitivity, specificity, low contamination risk, and speed has made this technique an appealing alternative to cell culture or immunoassay-based testing methods for diagnosing infectious diseases. rRT-PCR assays are most established for the detection of viral load and therapy monitoring. In this review, the usefulness or applications of rRT-PCR assays in the diagnosis of some of the important human viral infections have been summarized.

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Real-time PCR for rapid diagnosis of entero- and rhinovirus infections using LightCycler

Cited by 67 publications

References 20 publications

Real-Time Reverse Transcription-PCR Assay for Comprehensive Detection of Human Rhinoviruses

Real-Time Reverse Transcription-PCR Assay for Comprehensive Detection of Human Rhinoviruses

GeneXpert Enterovirus Assay: One-Year Experience in a Routine Laboratory Setting and Evaluation on Three Proficiency Panels

Applications of Real-time Reverse Transcription Polymerase Chain Reaction in Clinical Virology Laboratories for the Diagnosis of Human Diseases

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