Detection of human rhinovirus RNA in nasal washings by PCR

Abstract

A PCR assay was developed to detect human rhinovirus (HRV) RNA in nasal washings from individuals experimentally infected with HRV-39 or HRV strain Hanks. Total RNA was purified from samples stored in the presence of vanadyl ribonucleoside complex (VRC) by one of two methods: proteinase K digestion followed by multiple extractions with phenol/chloroform (PK-PC); or denaturation with guanidinium thiocyanate followed by one phenol/chloroform extraction (GTC). The limit of detection of HRV in nasal washings spiked with HRV-39 was lower with the GTC method (1 TCID50) than with the PK-PC method. In a study of 31 nasal washings extracted by the PK-PC method, the sensitivity (93%) and negative predictive value (94%) were sub-optimal in comparison to cell culture. In a study of 60 nasal washings extracted by the GTC method, the number of samples positive by PCR (25) exceeded by two the number positive by isolation in cell culture. A GTC-based method for HRV RNA extraction in nasal washings was superior to a proteinase K-phenol/chloroform-based method in regard to sensitivity, consumption of reagents, material and time.