[Significance of nucleic acid extraction for the performance of COBAS AMPLICOR Mycobacterium-PCR assay]

On an AMPLICOR-PCR assay for the diagnosis of mycobacterial infections, it has been found that approximately 10% of the routinely performed assays appear as indefinable cases with internal control (IC)-negative, which is caused by contaminant PCR-inhibitory substances in the extracted nucleic sample. To decrease the number of indefinable cases, we studied the IC performance of four nucleic acid extraction methods in one manual method(A) and three commercial kits(B, C and D). Thirty samples from 10 kinds of specimens without mycobacterial infections were subjected to the four nucleic extraction methods and the nucleic extracts were compared on PCR-inhibitory. The frequency of IC-negative samples was different among the four methods and the rates were 33.3%(n = 10) in A, 30.0%(n = 9) in B, 16.7%(n = 5) in C and 6.7%(n = 2) in D. In the PCR-inhibitory experiments using serially diluted IC-negative samples, the recovery of IC positivity was also different according to the method used. Although the IC-negative nucleic samples by D method easily became IC-positive at the 1/5 range of dilution, some of the IC-negative nucleic samples by A, B or C method were still IC-negative in the 1/10 range. These results indicate that the occurrence rate of indefinable cases with IC-negative is markedly different in the nucleic extraction method, suggesting that utilizing a suitable nucleic acid extraction method is important when using the diagnostic AMPLICOR-PCR assay.