Multicenter Comparison of Nucleic Acid Extraction Methods for Detection of Severe Acute Respiratory Syndrome Coronavirus RNA in Stool Specimens

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Abstract
The emergence of a novel coronavirus (CoV) as the cause of severe acute respiratory syndrome (SARS) catalyzed the development of rapid diagnostic tests. Stool samples have been shown to be appropriate for diagnostic testing for SARS CoV, although it has been recognized to be a heterogeneous and difficult sample that contains amplification inhibitors. Limited information on the efficiency of extraction methods for the purification and concentration of SARS CoV RNA from stool samples is available. Our study objectives were to determine the optimal extraction method for SARS CoV RNA detection and to examine the effect of increased specimen volume for the detection of SARS CoV RNA in stool specimens. We conducted a multicenter evaluation of four automated and four manual extraction methods using dilutions of viral lysate in replicate mock stool samples, followed by quantitation of SARS CoV RNA using real-time reverse transcriptase PCR. The sensitivities of the manual methods ranged from 50% to 100%, with the Cortex Biochem Magazorb method, a magnetic bead isolation method, allowing detection of all 12 positive samples. The sensitivities of the automated methods ranged from 75% to 100%. The bioMérieux NucliSens automated extractor and miniMag extraction methods each had a sensitivity of 100%. Examination of the copy numbers detected and the generation of 10-fold dilutions of the extracted material indicated that a number of extraction methods retained inhibitory substances that prevented optimal amplification. Increasing the volume of sample input did improve detection. This information could be useful for the extraction of other RNA viruses from stool samples and demonstrates the need to evaluate extraction methods for different specimen types.

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