Evaluation Of Reverse Transcription Research Articles

Evaluation of reverse transcription – polymerase chain reaction assays for detecting Apple chlorotic leaf spot virus in certification and quarantine programs

Apple chlorotic leaf spot virus (ACLSV), the type member of the genus Trichovirus, family Flexiviridae, occurs worldwide in a wide range of rosaceous hosts and is an important pathogen targeted in certification and quarantine programs. Detection of ACLSV in infected hosts has been based on inoculation by grafting (indexing), enzyme-linked immunosorbent assays (ELISA), and more recently, on reverse transcription – polymerase chain reaction (RT–PCR). The latter has the potential to become a reliable molecular indexing tool. However, its suitability for routine diagnosis needs to be verified for a broad spectrum of virus isolates in a wide range of hosts. In this study, oligonucleotide primers targeting the coat-protein (CP) region and the replicase region of ACLSV were compared in RT–PCR assays and applied to detection of virus isolates from natural hosts originating from domestic and imported sources of plant germplasm. Primers CLS6860 and CLS7536 amplifying a 676-bp (base pair) fragment in the CP-coding region of ACLSV were the most reliable, detecting all isolates, in all tested hosts. The forward/reverse primer pair 4F/4R, which amplifies a 390-bp fragment in the replicase-coding region, detected most of the tested ACLSV isolates, but generated some nonspecific amplifications with several other fruit-tree viruses in the family Flexiviridae. Alignment of the associated nucleotide sequences of the respective viruses indicated high levels of identity with the targeted replicase region, supporting the use of this region for broad-spectrum detection of distantly related viruses. However, this region is less useful for accurate virus identification. Comparison of the RT–PCR data with the ELISA and indexing results highlighted the advantages of a reliable molecular assay for problematic hosts.

Evaluation of reverse transcription and polymerase chain reaction (RT/PCR) for the detection of rota viruses: applications of the assay

SummaryOur aim was to evaluate the reverse transcription and polymerase chain reaction (RT/PCR) technique for the detection of rotavirus shedding by infected children as a routine diagnostic procedure, in comparison to the enzyme-linked immunosorbent assay (ELISA), electron microscopy (EM) and polyacrylamide gel etectrophoresis (PAGE) of rotavirus double stranded RNA.Two-hundred and twenty stool specimens were collected from infants and young children with diarrhoea, and 10–20% faecal suspensions were made. Several methods of rotavirus dsRNA extraction were assayed. Electrophoretic analysis of viral RNA was carried out on 10% polyacrylamide gols followed by silver staining. RT/PCR was performed using oligonucleotide primers specific for both 3′ and 5′ ends of the rotavirus gene encoding VP7 which ere highly conserved among group A rotaviruses.Following RNA extraction with phenol-chloroform and ethanol precipitation, RT/PCR could detect rotaviral RNA in only 11 of 25 samples known to contain rotaviruses by conventional methods. The purification of RNA extracts by CF11 cellulose and the application of the RNAID method were equally effective in extracting RNA and/or removing inhibitory substances from the faecal samples. RT/PCR led to the detection of 66 positive samples from 220 specimens tested (30%). whilst 64 specimens were positive by EUSA (29%), 59 (26.3%) by PAGE and 56 (25.4%) by EM. In our study, RT/PCR was 100 times more sensitive than the ELISA test in detecting rotaviruses serially diluted in a faecal suspension. Although RT/PCR is theoretically much more sensitive than ELISA, PAGE and EM for detection of rotaviruses, great care must be taken to remove inhibitory substances from the enzymatic reactions. We do not consider that RT/PCR should replace immunoassays with high sensitivity and specificity for rotavirus testing in faecal samples, although this technique has other applications, like the search for rotavirus in different clinical specimens (sera, cerebrospinal fluid, respiratory secretions, etc.) and in environmental samples, as well as the typing of viral strains using serotype-specific primers.