Evaluation of a one-tube RT-PCR system for detection of enteroviruses

Manayani, Darly J.; Cherian, Thomas; Murali, N.; Finny, GJ; Green, Jonathan; Brown, David W.; Ravi, Vasanthapuram; Abraham, Mutluri; Sridharan, Gopalan

doi:10.1016/s1386-6532(01)00234-7

Cited by 6 publications

(5 citation statements)

References 15 publications

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“…[1] If virus-induced CPE was observed, the infected cells were frozen and thawed, and the cell culture supernatant was used for RNA extraction. [13] Negative cultures were incubated for 10 days.…”

Section: Methodsmentioning

confidence: 99%

“…The cDNA obtained was then subjected to a nested polymerase chain reaction (PCR). [13] The outer primers were 5’-CGGTACCTT TGTACGCCTGTT- 3’ and 5’-CCGCATTCAGGGGCCGGAGGACT-3’, while the inner primers were 5’-GCACTTCTGTTTCCCC-3’ and 5’-CATTCAGGGGCCGGAGGA-3’.…”

Section: Methodsmentioning

confidence: 99%

“…Furthermore, EV isolates (one per patient) were subjected to typing with consensus EV sequencing primers, which spanned the VP1-2A region of the EV genome. [13] The typing primers used included 5’-ATGTAYGTXCCXCCXGGXGG-3’, 5’-ATGTAYRTXCCXMCXGGXGC-3’, and 5’-GCXCCXGAYTGXTGXCCRAA-3’. All the primers used in our study were from published sources.…”

Section: Methodsmentioning

confidence: 99%

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Virological investigation of hand, foot, and mouth disease in a tertiary care center in South India

Vijayaraghavan

Chandy

Selvaraj

et al. 2012

J Global Infect Dis

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Context:Hand, foot, and mouth disease (HFMD) remains a common problem in India, yet its etiology is largely unknown as diagnosis is based on clinical characteristics. There are very few laboratory-based molecular studies on HFMD outbreaks.Aim:The aim of this study was to characterize HFMD-related isolates by molecular techniques.Settings and Design:Between 2005 and 2008, during two documented HFMD outbreaks, 30 suspected HFMD cases presented at the Outpatient Unit of the Department of Dermatology, Christian Medical College (CMC), Vellore. Seventy-eight clinical specimens (swabs from throat, mouth, rectum, anus, buttocks, tongue, forearm, sole, and foot) were received from these patients at the Department of Clinical Virology, CMC, for routine diagnosis of hand, foot, and mouth disease.Materials and Methods:Samples from these patients were cultured in Vero and rhabdomyosarcoma (RD) cell lines. Isolates producing enterovirus-like cytopathogenic effect (CPE) in cell culture were identified by a nested reverse transcription–based polymerase chain reaction (RT-PCR) and sequenced. The nucleotide sequences were analyzed using the BioEdit sequence program. Homology searches were performed using the Basic Local Alignment Search Tool (BLAST) algorithm.Statistical Analysis used:The statistical analysis was performed using Epi Info version 6.04b and Microsoft Excel 2002 (Microsoft Office XP).Results:Of the 30 suspected HFMD cases, only 17 (57%) were laboratory confirmed and Coxsackievirus A16 (CVA16) was identified as the etiological agent in all these cases.Conclusions:Coxsackievirus A16 (CVA16) was identified as the virus that caused the HFMD outbreaks in Vellore between 2005 and 2008. Early confirmation of HFMD helps to initiate control measures to interrupt virus transmission. In the laboratory, classical diagnostic methods, culture and serological tests are being replaced by molecular techniques. Routine surveillance systems will help understand the epidemiology of HFMD in India.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Virological investigation of hand, foot, and mouth disease in a tertiary care center in South India

Vijayaraghavan

Chandy

Selvaraj

et al. 2012

J Global Infect Dis

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“…Rapid diagnostic tests have been developed in recent years and are usually based on nucleic acid amplification technology, such as reverse transcription-PCR and nucleic acid sequencebased amplification (3,7,9,12,20,24,31). These assays have proven to be sensitive and specific but, unfortunately, are often in a (semi-)nested format or require specific detection methods, which poses serious hazards for amplification product carryover.…”

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confidence: 99%

Rapid and Sensitive Routine Detection of All Members of the Genus Enterovirus in Different Clinical Specimens by Real-Time PCR

et al. 2002

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We developed a rapid and sensitive method for the routine detection of all members of the enterovirus genus in different clinical specimens by using real-time TaqMan quantitative PCR. Multiple primer and probe sets were selected in the highly conserved 5-untranslated region of the enterovirus genome. Our assay detected all 60 different enterovirus species tested, whereas no reactivity was observed with the viruses from the other genera of the picornaviridae family, e.g., hepatovirus and parechovirus. Weak cross-reactivity was observed with 7 of the 90 different high-titer rhinovirus stocks but not with rhinovirus-positive clinical isolates. Analysis of a well-characterized reference panel containing different enteroviruses at various concentrations demonstrated that the enterovirus real-time TaqMan PCR is as sensitive as most of the currently used molecular detection assays. Evaluation of clinical isolates demonstrated that the assay is more sensitive than the "gold standard" method, i.e., viral culture. Moreover, the PCR assay can be used on different clinical specimens, such as plasma, serum, nose and throat swabs, cerebrospinal fluid, and bronchoalveolar lavage, without apparent inhibition. Our data demonstrate that the real-time TaqMan PCR is a rapid and sensitive assay for the detection of enterovirus infection. The assay has a robust character and is easily standardized, which makes it an excellent alternative for the conventional time-consuming viral culture.

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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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Evaluation of a one-tube RT-PCR system for detection of enteroviruses

Cited by 6 publications

References 15 publications

Virological investigation of hand, foot, and mouth disease in a tertiary care center in South India

Virological investigation of hand, foot, and mouth disease in a tertiary care center in South India

Rapid and Sensitive Routine Detection of All Members of the Genus Enterovirus in Different Clinical Specimens by Real-Time PCR

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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