Direct and uninterrupted RNA amplification of enteroviruses with colorimetric microwell detection

Kao, Shaw Yi; Niemiec, Tina M.; Loeffelholz, Mike J.; Dale, Beverly A.; Rotbart, Harley A.

doi:10.1016/s0928-0197(94)00041-7

Cited by 8 publications

(6 citation statements)

References 25 publications

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“…EV serotypes other than those isolated in our study are reported to be less common causes of CNS disease. All 15 EV serotypes which were found in our study, including the EV serotypes isolated from the PCR-negative CSF specimens, have been reported to be detectable by the Amplicor EV PCR test (14). With our own collection of EV serotypes isolated from fecal specimens, we could demonstrate that a positive signal in the Amplicor EV PCR test was obtained with serotypes EC6 and CB4, which initially could not be detected in the CSF specimens.…”

Section: Discussionsupporting

confidence: 47%

“…Detection is performed colorimetrically on a microwell plate with an immobilized oligonucleotide probe specific for enteroviruses (22). According to the manufacturer the Amplicor EV PCR test detects most of the 66 different serotypes of EV at a sensitivity of Յ1 50% tissue culture infective dose (TCID 50 ) (14). Three serotypes, coxsackievirus A2 (CA2), CA4, and CA8, have not been tested.…”

Section: Methodsmentioning

confidence: 99%

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Multicenter Evaluation of the Amplicor Enterovirus PCR Test with Cerebrospinal Fluid from Patients with Aseptic Meningitis

Vliet¹,

Glimåker

Lebon

et al. 1998

J Clin Microbiol

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The Amplicor Enterovirus PCR test was compared with viral culture for the detection of enteroviruses in cerebrospinal fluid (CSF) specimens. In a multicenter study in which nine laboratories participated, a total of 476 CSF specimens were collected from patients with suspected aseptic meningitis. Sixty-eight samples were positive by PCR (14.4%), whereas 49 samples were positive by culture (10.4%), demonstrating that the Amplicor Enterovirus PCR test was significantly more sensitive than culture (P < 0.001). After discrepancy analysis the sensitivity and specificity of the Amplicor Enterovirus PCR test obtained by using viral culture as the “gold standard” were 85.7 and 93.9%, respectively. Our results with the CSF specimens collected in different countries demonstrate that the Amplicor test is capable of detecting a large variety of enterovirus serotypes and epidemiologically unrelated isolates in CSF specimens from patients with aseptic meningitis. The Amplicor Enterovirus PCR test is a rapid assay which can be routinely performed with CSF samples and is an important improvement for the rapid diagnosis of enteroviral meningitis.

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Section: Discussionsupporting

confidence: 47%

Section: Methodsmentioning

confidence: 99%

Multicenter Evaluation of the Amplicor Enterovirus PCR Test with Cerebrospinal Fluid from Patients with Aseptic Meningitis

Vliet¹,

Glimåker

Lebon

et al. 1998

J Clin Microbiol

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“…In this way, the instruments and reagents involved in the PCR procedure, as well as the technicians performing the assay, were validated before processing of clinical specimens. The AMPLICOR EV test procedure has been described elsewhere (9). In short, viral RNA from specimens was extracted by mixing 100 l of CSF with 400 l of lysis solution, with incubation at room temperature for 10 min.…”

Section: Methodsmentioning

confidence: 99%

“…In addition, both RT-PCR methods failed to detect three specimens which had been positive by culture, six specimens were AMPLICOR negative and culture positive, and three specimens were in-house negative and culture positive, for a total of 12 false-negative specimens. After typing, none of these 12 enteroviruses were found to be Echo-1, Echo-5, Echo-22, or Echo-23, four enteroviral types often missed by the AMPLICOR test (9). Moreover, the in-house PCR assay has previously been shown to be highly sensitive in detecting the enterovirus types involved in these false-negative results (5).…”

Section: Among Our 29 Patients With Sporadic Am the Percentage Of Pomentioning

confidence: 99%

Evaluation of a Commercially Available Reverse Transcription-PCR Assay for Diagnosis of Enteroviral Infection in Archival and Prospectively Collected Cerebrospinal Fluid Specimens

Pozo¹,

Casas²,

Tenorio³

et al. 1998

J Clin Microbiol

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A commercially available reverse transcription (RT)-PCR method (AMPLICOR EV; Roche Diagnostic Systems, Inc., Branchburg, N.J.) was evaluated for detection of enteroviruses in cerebrospinal fluid from patients with neurological disease. This assay was compared with virus isolation in cell culture and an in-house RT-PCR method designed with a nonoverlapping region of the enteroviral genome. A panel of 200 cerebrospinal fluid specimens prospectively collected from patients with a wide variety of neurological symptoms, including 50 patients involved in three different outbreaks of acute aseptic meningitis, was assayed. A second panel of 97 archived cerebrospinal fluid specimens, stored for 2 to 5 years, from patients with aseptic meningitis associated with several enterovirus outbreaks was also studied. From the first panel, enteroviruses were detected in 13 of 50 specimens by cell culture (26%), in 43 of 50 specimens by AMPLICOR EV (86%), and in 46 of 50 specimens by the in-house assay (92%) from patients with aseptic meningitis associated with outbreak and 1 of 29, 3 of 29, and 4 of 29 specimens, respectively, from sporadic cases of aseptic meningitis. The remaining 121 cerebrospinal fluid specimens from patients with other neurological syndromes were negative by all tests. From the second panel, enteroviral RNA was detected by the AMPLICOR test (31 of 97 specimens, 32%) and the in-house assay (39 of 97 specimens, 40%). According to our results, patients with aseptic meningitis should be analyzed for enteroviral infection in cerebrospinal fluid by RT-PCR methods, and the AMPLICOR EV test is a suitable tool for performing such studies. Archival cerebrospinal fluid specimens are less suitable for evaluation of the performance of RT-PCR methods designed for enterovirus detection.

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“…RT-PCR will therefore assume a major role in the laboratory diagnosis of enterovirus infections. An enterovirus PCR test kit for the diagnosis of enteroviral meningitis, now commercially available from Roche Molecular Systems, will assist in the transition to PCR-based diagnosis (199,382,478). This test is based on single-step reverse transcription and amplification of enterovirus RNA, and it incorporates enzymatic elimination of amplicon contamination, thus harmonizing and simplifying laboratory procedures and reducing the risk of false positivity due to carryover of previously amplified nucleic acid.…”

Section: Enterovirus Detection By Pcrmentioning

confidence: 99%

Molecular Typing of Enteroviruses: Current Status and Future Requirements

et al. 1998

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Human enteroviruses have traditionally been typed according to neutralization serotype. This procedure is limited by the difficulty in culturing some enteroviruses, the availability of antisera for serotyping, and the cost and technical complexity of serotyping procedures. Furthermore, the impact of information derived from enterovirus serotyping is generally perceived to be low. Enteroviruses are now increasingly being detected by PCR rather than by culture. Classical typing methods will therefore no longer be possible in most instances. An alternative means of enterovirus typing, employing PCR in conjunction with molecular genetic techniques such as nucleotide sequencing or nucleic acid hybridization, would complement molecular diagnosis, may overcome some of the problems associated with serotyping, and would provide additional information regarding the epidemiology and biological properties of enteroviruses. We argue the case for developing a molecular typing system, discuss the genetic basis of such a system, review the literature describing attempts to identify or classify enteroviruses by molecular methods, and suggest ways in which the goal of molecular typing may be realized.

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Direct and uninterrupted RNA amplification of enteroviruses with colorimetric microwell detection

Cited by 8 publications

References 25 publications

Multicenter Evaluation of the Amplicor Enterovirus PCR Test with Cerebrospinal Fluid from Patients with Aseptic Meningitis

Multicenter Evaluation of the Amplicor Enterovirus PCR Test with Cerebrospinal Fluid from Patients with Aseptic Meningitis

Evaluation of a Commercially Available Reverse Transcription-PCR Assay for Diagnosis of Enteroviral Infection in Archival and Prospectively Collected Cerebrospinal Fluid Specimens

Molecular Typing of Enteroviruses: Current Status and Future Requirements

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