RNA extraction for molecular detection of Newcastle disease virus - comparative study of three methods

RNA extraction is one of the essential factors influencing the sensitivity of real-time reverse transcription polymerase chain reaction (rRT-PCR) as a diagnostic test for Newcastle disease virus (NDV). The separation of RNA from the other biological substances present in the clinical sample and subsequent removal of reagent residues are particularly important because of the possible inhibitory effect on the PCR. The aim of the present study was to compare the sensitivity of rRT-PCR following Newcastle disease virus RNA extraction with Qiagen (R) RNeasy Mini Kit, Mag MAX (TM)-96 AI/ND Viral RNA Isolation Kit and TRIZOL (R) LS. The length of time required to carry out the extraction of a certain number of samples as well as the presence of any cross contamination was also evaluated in this study. A set of ten avian paramyxovirus 1 (APMV 1) samples provided by the National Veterinary Services Laboratories - United States Department of Agriculture, Ames, Iowa was used for this comparison. For each sample, RNA was extracted in triplicate using each of the three methods. The isolated RNAs were tested by Matrix-gene rRT-PCR using the AgPath-ID (TM) One-Step RT-PCR and the mean Ct values were analyzed statistically. The lowest Ct values in the rRT-PCR were observed using the Qiagen (R) RNeasy Mini Kit. Mag MAX (TM)-96 AI/ND Viral RNA Isolation Kit in combination with an automated system allowed processing of a greater number of samples in a shorter time period, which would be especially beneficial during a Newcastle disease outbreak. Cross contamination was not observed for any of the utilized methods.