Removal of inhibitory substances from human fecal specimens for detection of group A rotaviruses by reverse transcriptase and polymerase chain reactions

Abstract

A method was developed for the purification of rotavirus RNA from fecal extracts in order to permit the sensitive identification of group A rotavirus in fecal specimens by the polymerase chain reaction. Sequential reactions with reverse transcriptase and Taq polymerase with directed primers from rotavirus gene 6 yielded characteristic 259-base-pair fragments that were then visualized by silver stain on a polyacrylamide gel. As few as 500 genomic copies of purified rotavirus RNA could be detected in this manner. However, when the method was applied to fecal samples with added rotavirus virions, inhibition was noted in many of the fecal extracts which were tested. The inhibition could be reversed by dilution of the fecal extract, but sensitivity was also reduced by a corresponding dilutional factor. The inhibition was quantitatively removed by an added step in the extraction process that utilized chromatographic cellulose fiber powder (CF11 powder) to purify the rotavirus RNA during a series of rapid washing and elution steps. After CF11 purification, rotavirus RNA could be detected in experimental fecal samples at dilutions 1,000to 10,000-fold beyond the detection limits of standard techniques such as enzyme immunoassay and the direct visualization of RNA following polyacrylamide gel electrophoresis. Furthermore, following purification by CF11, rotavirus RNA could be detected in all of seven enzyme-linked immunosorbent assay-positive fecal samples obtained from a child with rotavirus gastroenteritis; when CF11 purification was not performed, rotavirus RNA could be detected in only four of these samples, even after the removal of inhibitors by dilution of the extracts. Large-scale identification of rotavirus in fecal specimens may be possible by use of CF11 purification of viral RNA prior to sequential reactions with reverse transcriptase and Taq polymerase in a modified polymerase chain reaction.

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

“…In a preliminary set of assays, 25 faecal samples previously found to be rotavirus-positive by ELISA, PAGE and EM were tested using differ-ent methods of RNA extraction in order to determine the most reliable and efficient procedure. The results of these assays are shown in table I. Phenol-chloroform extraction followed by binding of RNA to CFl 1 cellulose in ST"&ethanol and elution with STE, as described by Wilde et al (1990), was an efficient method to recover rotaviral RNA from stool samples free of inhibitory activity. All 25 samples analysed by RT/FCR were positive when phenol-extracted and purified by CFl 1 cellulose, whereas only 11 (44%) were positive when purification with CFll was omitted.…”

“…RNAs extracted from faecal samples with phenol-chloroform were purified using CFll cellulose fibre powder as described by Wilde et al (1990). After phenoI&loroform/isoamyl alcohol extraction (25:24: l), the aqueous phase was mixed with 95 % ethanol to bring the final solution to 15% ethanol (vol/vol).…”

Microbiological Quality Control for Laboratory Rodents and Lagomorphs

“…Dramatic advances in molecular biology during recent years have coincided with a shift from antigen immunoassays to molecular assays for microbial genomic sequences. This shift has been most prohounced for tests performed directly on clinical specimens, because molecular methods, particularly the PCR, have proven to be substantially more sensitive than their immunoassay counterparts (Wilde et al, 1990). Molecular techniques are better able to differentiate among strains or isolates of microbial species than the traditional strain typing procedures, such as serotyping Tenover, 1998;Tenover et al, 1994;Ushijima et al, 1992).…”

“…Various measures are taken to prevent cross-contamination, including physical separation of preand postamplification procedures, decontamination of work surfaces with chemicals or UV irradiation, and enzymatic digestion or chemical inactivation of amplified template. Conversely, PCR sensitivity can be diminished by specimens such as feces or whole blood that alter the reaction environment or otherwise inhibit target amplification by the Taq polymerase (Wilde et al, 1990). This inhibition can be detected by including an internal assay control or by spiking a duplicate reaction with control template; inhibition is prevented by purifying DNA or RNA from the specimen.…”

Viral Infections of Laboratory Mice

“…Electron microscopy of faeces of diarrhoeic pups should reveal typical wheel-shaped rotavirus particles, 60-80 nm in diameter. RT-PCR also can be used to detect rotavirus RNA in faecal samples [404]. Good timing is critical for establishing the diagnosis from faeces because virus is shed for only a few days in immunocompetent mice.…”