Comparison of two DNA extraction methods on inhibitory sputum samples

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The polymerase chain reaction (PCR) is a promising method in the rapid diagnosis of Mycobacterium tuberculosis infections. However, ongoing problems such as contamination and inhibition have prevented its widespread use in routine practice. Contamination occurs primarily from two sources: total genomic DNA from other samples or from cultures in the same laboratory, and the small amplified products carried over from previous PCR assays. Inhibition is mainly due to some, as yet unknown, substances still present with DNA after extraction. Inhibition, which has been reported as 10–20%, must be monitored by internal or external controls [1,2]. These extra tests or repetitions of the assay increase the costs. Contamination and inhibition are the problems of the DNA extraction phase. Complex extraction procedures are more prone to contamination; on the other hand, simple methods fail to eliminate the inhibitors. Therefore, the ideal extraction method should be simple enough to avoid contamination, yet effective in eliminating the inhibitory substances. Several DNA extraction methods have been proposed for routine practice, but none of these has yet been tested for effectiveness in eliminating the inhibitors.

We compared two DNA extraction methods applied to samples which had shown inhibition: a simple chloroform extraction [3] and a guanidium thiocyanate-silica (GuSCN-silica) method [4]. Briefly, 50 μL of chloroform was added to 200 μL aliquots, followed by heat inactivation at 80°C for 20 min in 1.5-mL Eppendorf tubes. The heat-inactivated samples were centrifuged at 12 000 g for 2 min. Ten-microliter amounts of the aqueous supernatants were subjected to PCR. The second method is incorporated in a new kit (QIAamp; DiagenGmbH Gilden/Germany), designed for nucleic acid extraction from clinical samples. The method depends mainly on the lytic effect of the chaothropic agent GuSCN and proteinase K and the capture of nucleic acids (NAs) under high-salt conditions by silica. Briefly, 200 μL of the sample is mixed with 200 μL of lysis solution and incubated at 80°C for 20 min. After a wash step, NA is eluted by distilled water. The procedure can be completed within 30 min. Genomic DNA binds to silica in the presence of GuSCN and so it has been suggested that inhibitors would be removed during the washing steps. To determine the samples with inhibitory effects, we seeded 50 sputa with 107M. tuberculosis and prepared DNA simply by boiling half the sediment in distilled water. The remainder was stored at –20°C. Nine of these 50 samples inhibited the PCR reaction, as judged by negative amplification results. To compare the effectiveness of the two extraction methods (chloroform and GuSCN-silica methods), the stored sediments from inhibitory sputum samples were thawed and divided into equal aliquots and were processed by the two extraction methods separately. We amplified a 123-bp fragment of IS6110 repetitive insertion element. The conditions for the PCR have been described in detail elsewhere [5]. The extracts that still inhibited the reaction after the chloroform method were subjected to a second application, this time by the silica method.

The chloroform method eliminated the inhibitors from five, and the silica method from four, of nine sputa. The silica method was only beneficial in two samples that were still inhibitory after chloroform. However, these two samples had already been successfully cleared of inhibitors by the silica method in the first round. Finally, inhibitors from three sputa could be cleared only by the chloroform and two only by the silica method. Both methods were effective on two samples and neither on the remaining two. We concluded that the silica method, a more complex and multistep procedure, is not superior to the simple one-step chloroform method. Both methods are capable of eliminating inhibitors from different samples and so can be used one after the other for problem samples.

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