Comparison of Six Methods of Extracting Mycobacterium tuberculosis DNA from Processed Sputum for Testing by Quantitative Real-Time PCR
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微生物临床杂志 2005年第5期
ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, Utah 84108
ABSTRACT
Six methods of extracting Mycobacterium tuberculosis DNA from sputum for testing by quantitative PCR were compared: Tris-EDTA (TE) buffer, PrepMan Ultra, 2% sodium dodecyl sulfate (SDS)-10% Triton X with and without sonication, Infectio Diagnostics, Inc. (IDI) lysing tubes, and QIAGEN QIAamp DNA mini kit; all included a 15-min boiling step. Pooled digested and decontaminated sputum was spiked with M. tuberculosis ATCC 27294. Each extraction method was repeated eight times. Quantitative PCR was performed on the Smart Cycler and Rotor-Gene 3000 using primers targeting an 83-bp fragment of IS6110. An minor grove binding Eclipse probe with a fluorescent label was used for detection. An internal control was included to detect amplification inhibition. The limit of detection of M. tuberculosis DNA was 0.5 fg with both instruments. Calculated DNA concentrations (picograms) extracted using IDI, PrepMan, QIAGEN, and TE were 42.8, 30.4, 28.2, and 7.4, respectively, when run on the Smart Cycler, and 51.7, 20.1, 14.9, and 8.6, respectively, when run on Rotor-Gene. All extractions using SDS/Triton X with or without sonication were inhibited. Of the extraction methods evaluated, IDI lysis tubes provided the greatest yield of mycobacterial DNA, and the procedure can be completed in less than 1 h versus 2.5–3 h for the QIAGEN extraction.
TEXT
Tuberculosis is a public health problem worldwide, and for optimal control, early diagnosis is necessary (4, 6, 7). Several researchers have developed real-time PCR assays that provide rapid detection of various target sequences of Mycobacterium tuberculosis complex (MTBC) and drug resistance genes in patient specimens (1, 3, 5, 16-19). The ability of these assays to detect MTBC in clinical samples is dependent on both the target sequence selected and the efficiency of the DNA extraction procedure. Several methods of mycobacterial cell wall lysis and DNA extraction have been evaluated, including detergents, proteolytic enzymes, mechanical disruption, and temperature changes alone and in various combinations (1, 2, 5, 8-15, 17, 20). The objective of this study was to compare six methods of extracting M. tuberculosis DNA from respiratory specimens: Tris-EDTA (TE) boil extraction (10), PrepMan ultra extraction (Applied Biosystems, Inc., Foster City, CA), Infectio Diagnostics, Inc. (IDI) lysis extraction (Infectio Diagnostics, Inc. Quebec, Canada), QIAGEN QIAmp DNA mini kit (QIAGEN, Inc., Valencia, CA), sodium dodecyl sulfate (SDS)-Triton X extraction (9), and SDS-Triton X plus sonication. The effectiveness of each extraction method was assessed using two quantitative real-time PCR assays.
Sample preparation. Digested and decontaminated (N-acetyl-cysteine-2% NaOH) sputum specimens that were culture negative for mycobacteria were pooled for use as the standard respiratory specimen. A suspension of M. tuberculosis ATCC 27294 was prepared in sterile saline and adjusted to the density of a 1.0 McFarland standard. The suspension was diluted 1:10 in saline and used to spike the pooled respiratory specimen. Spiked specimens were stored in 200-μl aliquots at –70°C until extracted.
DNA extraction. For all six extraction procedures, each of which was repeated eight times, the spiked respiratory specimen was first thawed and centrifuged at 6,000 x g for 1 min. The supernatant was discarded, and the pellet was processed for each procedure as follows. (i) TE boil extraction. A 200-μl aliquot of TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA) was added, and the mixture was briefly mixed on a vortex mixer. The suspension was placed in a boiling water bath for 15 min to destroy any viable mycobacteria and then centrifuged at 16,000 x g for 5 min. A 100-μl aliquot of the supernatant was transferred to a sterile tube and stored at –20°C until PCR testing. (ii) PrepMan extraction. A 200-μl aliquot of PrepMan Ultra sample preparation reagent were added to the pellet, and the suspension was treated as described for TE buffer. (iii) IDI extraction. Pellets were treated as described for TE buffer with the following additional steps. The TE suspension was placed into an IDI lysis tube (Infectio Diagnostic, Inc., Quebec, Canada), which contains a glass bead matrix. Tubes were vigorously mixed for 5 min on the highest setting of a Vortex Genie 2 (Scientific Industries, Inc., Bohemia, NY) in a microtube foam insert and then placed into a boiling water bath. (iv) QIAGEN extraction. Pellets were processed using the QIAGEN QIAamp DNA mini kit (QIAGEN, Valencia, CA) tissue protocol, with the following modifications: an enzymatic digestion step with 30 mg/ml lysozyme added to the tissue lysis buffer was followed by boiling for 15 min, and the proteinase K step was incubated at 56°C for 1 h. DNA was purified per manufacturer's recommendations through a spin column. (v) SDS-Triton X extraction. Pellets were treated as described for TE buffer, except 200 μl of the nonionic detergent mix (2% SDS-10% Triton X-100) was substituted for the TE buffer (9). (vi) SDS-Triton X plus sonication extraction. A 200-μl aliquot of 2% SDS-10% Triton X-100 was added to the pellet, and the mixture was briefly agitated on a vortex mixer. The suspension was sonicated for 15 min and then treated as described for TE buffer.
Primers and probes. Primers and probes were designed using minor grove binding (MGB) Eclipse Design Software (Epoch Biosciences, Bothell, WA). Primers were purchased from IT BioChem (Idaho Technologies, Salt Lake City, UT), and the minor grove binding (MGB) Eclipse probes were from Epoch Biosciences. MTBC-specific primers were used to amplify an 83-bp fragment of the IS6110 target. The forward primer corresponds to the region from base 801 to base 818 (sequence, 5'-CTAACCGGCTGTGGGTA-3'; base numbering referenced with GenBank accession no. X52471). The reverse primer corresponds to the region of IS6110 from base 866 to base 884 (sequence, 5'-CGTAGGCGTCGGTGACAAA-3'). The MTBC-specific Eclipse probe consists of an oligonucleotide (sequence, 5'-MGB-NFQ-AGACCTCACCTATGT-FAM-3') labeled with a nonfluorescent quencher molecule and the MGB moiety at the 5' end and a FAM fluorescent dye label at the 3' end. Bases with asterisks () are proprietary "superbases," manufactured by Epoch Biosciences for increased hybridization stability.
An internal control (IC) plasmid containing genomic DNA from nonhuman sources was used to detect PCR inhibition in the extracts. The IC primers and Eclipse probe have been described previously (J. B. Stevenson, K. C. Carroll, and D. R. Hillyard, Abstr. 18th Ann. Clin. Virol. Symp., abstr. M35, 2002).
Real-time PCR conditions. Each extract was tested on two real-time PCR instruments: the Smart Cycler II System (Cepheid, Sunnyvale, CA) and the Rotor-Gene 3000 real-time DNA analysis system (Corbett Research, Sydney, Australia). All reactions on each instrument were optimized to obtain the best amplification kinetics. For the Smart Cycler and the Rotor-Gene, the PCR mixture (25 μl) contained 12.5 μl of reconstituted OmniMix HS (3 U TaKaRa hot start Taq polymerase, 200 μM deoxynucleoside triphosphates, 4 mM MgCl2, and 25 mM HEPES buffer [pH 8.0 ± 0.1]), 0.5 μM of each MTBC primer, 200 nM MTBC Eclipse probe, 0.25 μM of each IC primer, 200 nM IC Eclipse probe, 20 pg IC DNA, and 2 μl extracted DNA template with the following exception. The Rotor-Gene PCR mixture contained 300 nM MTBC Eclipse probe. Water was used as a no-template control. Cycling conditions were identical for both instruments: 1 cycle at 95°C for 2 min, 50 cycles at 95°C for 5 s, 58°C for 20 s, and 76°C for 20 s. The crossing threshold cycle (Ct) is the cycle at which there is a significant increase in fluorescence above the background or a specified threshold. The Ct was determined from a curve generated from a plot of cycle number versus fluorescence with a manual threshold set above the background fluorescence of the no template control. The Ct is inversely proportional to the logarithm of the initial number of template molecules.
DNA standards. M. tuberculosis ATCC 27294 DNA was extracted using the IDI procedure. The concentrations of M. tuberculosis and IC DNA were determined by using the PicoGreen dsDNAQuantitation kit (Molecular Probes, Inc., Eugene, OR). Serial 10-fold dilutions of the M. tuberculosis DNA, ranging from 25 pg/μl to 2.5 fg/μl, were used as external standards. These standards were run in triplicate on both instruments. A standard curve was included or imported for each Rotor-Gene run. To generate the standard curve, the Ct value in the logarithmic linear phase was plotted against the logarithm of the known concentrations of M. tuberculosis DNA. The concentrations of mycobacterial DNA in the spiked respiratory samples were calculated by comparing the average Ct value from the logarithmic linear phase of the fluorescence curve with the line generated from external standards on that instrument.
Statistical analysis. The efficiency of each DNA extraction method (n = 8) was compared using the data analysis package included within Microsoft Excel 2000 software (Microsoft Corporation, Redmond, WA). All methods on both instruments were evaluated by a two-tailed Student's t test paired two-sample means.
The limit of detection of both instruments, using known concentrations of M. tuberculosis DNA to create a standard curve, was 0.5 fg of DNA in the presence of an IC. The PCR assay was inhibited on both instruments when specimens were extracted using 2% SDS-10% Triton X with and without sonication, as indicated by the lack of amplification of the IC target in the extracted samples and amplification of IC in the no-template control samples. These methods were included in our evaluation based on data from a report by Khan and Yadav (9), which showed that the SDS and Triton X concentrations we used were optimal for extracting DNA from a mycobacterial colony. In retrospect, our results are not totally unexpected, since an excess of SDS above 0.01% has been shown to inhibit PCR, primarily due to denaturation of the Taq polymerase (Critical Factors for Successful PCR manual, QIAGEN, Inc.). Although there were methodological differences between our study and that of Khan and Yadav (e.g., standard versus real-time PCR, different polymerases, different ratios of template to reaction volume), they do not adequately explain the complete inhibition observed in our evaluation and the amplification in theirs. Reasons for the striking difference between the results of the two studies are unclear.
Mean (± standard deviation [SD]) Ct values for the other four extraction methods evaluated (illustrated in Fig. 1) were 25.3 ± 0.64 (range, 24.2 to 26.6) for IDI, 25.9 ± 0.62 (range, 24.8 to 26.7) for QIAGEN, 25.8 ± 0.56 (range, 24.7 to 26.6) for PrepMan, and 27.9 ± 0.79 (range, 26.7 to 29.6) for TE when testing was performed on the Smart Cycler. When testing was done on the Rotor-Gene, mean (±SD) Ct values were 25.1 ± 1.82 (range, 21.7 to 27.5) for IDI, 26.9 ± 1.61 (range, 23.9 to 29.4) for QIAGEN, 26.5 ± 1.52 (range, 26.3 to 29.7) for PrepMan, and 27.7 ± 1.10 (range, 23.7 to 29.8) for TE. The differences in Ct values were significant (P = 0.05) for IDI and TE, QIAGEN and TE, and PrepMan and TE when testing was performed on the Smart Cycler, but there were no significant differences in Ct values between any of the methods when testing on the Rotor-Gene.
The amount of DNA extracted by each method, which is inversely proportional to Ct, was calculated from the mean Ct. When testing on the Smart Cycler, the DNA yield was 42.8 pg for IDI, 28.2 pg for QIAGEN, 30.4 pg for PrepMan, and 7.4 pg for TE. DNA yields for these same methods when testing on the Rotor-Gene were 51.7 pg, 14.9 pg, 20.1 pg, and 8.6 pg, respectively.
Column-purified DNA theoretically should be the cleanest, containing the fewest PCR-inhibitory substances. The goal of our study was to find a DNA extraction method that was quick but did not compromise the PCR or assay sensitivity, using the QIAGEN extraction method as the gold standard for PCR purification. Our results showed that column purification is not necessary for the extraction of DNA from sputum samples to be tested for M. tuberculosis by PCR. The time required to complete each extraction was less than 1 h for TE buffer, PrepMan, and IDI and involved two or three transfers of material to new tubes, compared to 2.5 to 3 h for QIAGEN with extensive hands-on processing.
Although not the primary aim of our study, we evaluated two different real-time PCR instruments for the detection of M. tuberculosis from respiratory samples in a clinical laboratory setting. PCR on the Smart Cycler was completed in 55 min, and the Rotor-Gene PCR runs were 95 min in duration. The Smart Cycler can run 16 reactions wells per block with up to six processing blocks connected to one computer for a 96-tube capacity. The Rotor-Gene has a 72 tube capacity for a 25-μl PCR volume. The Smart Cycler is a totally closed system with a lower chance of amplicon tubes opening for possible amplicon contamination.
A final point to be considered when selecting an extraction method for use with the M. tuberculosis PCR assay is cost. For the kits or reagents evaluated in this study, the cost per extraction is $3.00 for IDI, $2.30 for QIAGEN, $1.05 for PrepMan, and approximately $0.03 for TE buffer.
In summary, of the six extraction methods evaluated, IDI lysis tubes provided the greatest yield of DNA on both the Smart Cycler and Rotor-Gene instruments. Additionally, the IDI procedure was technically simple and was completed in less than 1 h, with approximately 20 min of hands-on time. Extraction with TE buffer, although very easy to do and inexpensive, provided the poorest DNA yield. SDS-Triton X consistently inhibited the PCR on both real-time instruments and, therefore, cannot be recommended for extraction of mycobacterial DNA.
ACKNOWLEDGMENTS
We thank Andrea Stetler for secretarial assistance, Mark Kushnir for the statistical advice, and Jeffery Stevenson for providing the internal control plasmid. Yevgeniy S. Belousov at Epoch Bioscience designed the MGB primers and probe used in this study.
This study was funded by the Associated Regional and University Pathologists Institute for Clinical and Experimental Pathology.
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ABSTRACT
Six methods of extracting Mycobacterium tuberculosis DNA from sputum for testing by quantitative PCR were compared: Tris-EDTA (TE) buffer, PrepMan Ultra, 2% sodium dodecyl sulfate (SDS)-10% Triton X with and without sonication, Infectio Diagnostics, Inc. (IDI) lysing tubes, and QIAGEN QIAamp DNA mini kit; all included a 15-min boiling step. Pooled digested and decontaminated sputum was spiked with M. tuberculosis ATCC 27294. Each extraction method was repeated eight times. Quantitative PCR was performed on the Smart Cycler and Rotor-Gene 3000 using primers targeting an 83-bp fragment of IS6110. An minor grove binding Eclipse probe with a fluorescent label was used for detection. An internal control was included to detect amplification inhibition. The limit of detection of M. tuberculosis DNA was 0.5 fg with both instruments. Calculated DNA concentrations (picograms) extracted using IDI, PrepMan, QIAGEN, and TE were 42.8, 30.4, 28.2, and 7.4, respectively, when run on the Smart Cycler, and 51.7, 20.1, 14.9, and 8.6, respectively, when run on Rotor-Gene. All extractions using SDS/Triton X with or without sonication were inhibited. Of the extraction methods evaluated, IDI lysis tubes provided the greatest yield of mycobacterial DNA, and the procedure can be completed in less than 1 h versus 2.5–3 h for the QIAGEN extraction.
TEXT
Tuberculosis is a public health problem worldwide, and for optimal control, early diagnosis is necessary (4, 6, 7). Several researchers have developed real-time PCR assays that provide rapid detection of various target sequences of Mycobacterium tuberculosis complex (MTBC) and drug resistance genes in patient specimens (1, 3, 5, 16-19). The ability of these assays to detect MTBC in clinical samples is dependent on both the target sequence selected and the efficiency of the DNA extraction procedure. Several methods of mycobacterial cell wall lysis and DNA extraction have been evaluated, including detergents, proteolytic enzymes, mechanical disruption, and temperature changes alone and in various combinations (1, 2, 5, 8-15, 17, 20). The objective of this study was to compare six methods of extracting M. tuberculosis DNA from respiratory specimens: Tris-EDTA (TE) boil extraction (10), PrepMan ultra extraction (Applied Biosystems, Inc., Foster City, CA), Infectio Diagnostics, Inc. (IDI) lysis extraction (Infectio Diagnostics, Inc. Quebec, Canada), QIAGEN QIAmp DNA mini kit (QIAGEN, Inc., Valencia, CA), sodium dodecyl sulfate (SDS)-Triton X extraction (9), and SDS-Triton X plus sonication. The effectiveness of each extraction method was assessed using two quantitative real-time PCR assays.
Sample preparation. Digested and decontaminated (N-acetyl-cysteine-2% NaOH) sputum specimens that were culture negative for mycobacteria were pooled for use as the standard respiratory specimen. A suspension of M. tuberculosis ATCC 27294 was prepared in sterile saline and adjusted to the density of a 1.0 McFarland standard. The suspension was diluted 1:10 in saline and used to spike the pooled respiratory specimen. Spiked specimens were stored in 200-μl aliquots at –70°C until extracted.
DNA extraction. For all six extraction procedures, each of which was repeated eight times, the spiked respiratory specimen was first thawed and centrifuged at 6,000 x g for 1 min. The supernatant was discarded, and the pellet was processed for each procedure as follows. (i) TE boil extraction. A 200-μl aliquot of TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA) was added, and the mixture was briefly mixed on a vortex mixer. The suspension was placed in a boiling water bath for 15 min to destroy any viable mycobacteria and then centrifuged at 16,000 x g for 5 min. A 100-μl aliquot of the supernatant was transferred to a sterile tube and stored at –20°C until PCR testing. (ii) PrepMan extraction. A 200-μl aliquot of PrepMan Ultra sample preparation reagent were added to the pellet, and the suspension was treated as described for TE buffer. (iii) IDI extraction. Pellets were treated as described for TE buffer with the following additional steps. The TE suspension was placed into an IDI lysis tube (Infectio Diagnostic, Inc., Quebec, Canada), which contains a glass bead matrix. Tubes were vigorously mixed for 5 min on the highest setting of a Vortex Genie 2 (Scientific Industries, Inc., Bohemia, NY) in a microtube foam insert and then placed into a boiling water bath. (iv) QIAGEN extraction. Pellets were processed using the QIAGEN QIAamp DNA mini kit (QIAGEN, Valencia, CA) tissue protocol, with the following modifications: an enzymatic digestion step with 30 mg/ml lysozyme added to the tissue lysis buffer was followed by boiling for 15 min, and the proteinase K step was incubated at 56°C for 1 h. DNA was purified per manufacturer's recommendations through a spin column. (v) SDS-Triton X extraction. Pellets were treated as described for TE buffer, except 200 μl of the nonionic detergent mix (2% SDS-10% Triton X-100) was substituted for the TE buffer (9). (vi) SDS-Triton X plus sonication extraction. A 200-μl aliquot of 2% SDS-10% Triton X-100 was added to the pellet, and the mixture was briefly agitated on a vortex mixer. The suspension was sonicated for 15 min and then treated as described for TE buffer.
Primers and probes. Primers and probes were designed using minor grove binding (MGB) Eclipse Design Software (Epoch Biosciences, Bothell, WA). Primers were purchased from IT BioChem (Idaho Technologies, Salt Lake City, UT), and the minor grove binding (MGB) Eclipse probes were from Epoch Biosciences. MTBC-specific primers were used to amplify an 83-bp fragment of the IS6110 target. The forward primer corresponds to the region from base 801 to base 818 (sequence, 5'-CTAACCGGCTGTGGGTA-3'; base numbering referenced with GenBank accession no. X52471). The reverse primer corresponds to the region of IS6110 from base 866 to base 884 (sequence, 5'-CGTAGGCGTCGGTGACAAA-3'). The MTBC-specific Eclipse probe consists of an oligonucleotide (sequence, 5'-MGB-NFQ-AGACCTCACCTATGT-FAM-3') labeled with a nonfluorescent quencher molecule and the MGB moiety at the 5' end and a FAM fluorescent dye label at the 3' end. Bases with asterisks () are proprietary "superbases," manufactured by Epoch Biosciences for increased hybridization stability.
An internal control (IC) plasmid containing genomic DNA from nonhuman sources was used to detect PCR inhibition in the extracts. The IC primers and Eclipse probe have been described previously (J. B. Stevenson, K. C. Carroll, and D. R. Hillyard, Abstr. 18th Ann. Clin. Virol. Symp., abstr. M35, 2002).
Real-time PCR conditions. Each extract was tested on two real-time PCR instruments: the Smart Cycler II System (Cepheid, Sunnyvale, CA) and the Rotor-Gene 3000 real-time DNA analysis system (Corbett Research, Sydney, Australia). All reactions on each instrument were optimized to obtain the best amplification kinetics. For the Smart Cycler and the Rotor-Gene, the PCR mixture (25 μl) contained 12.5 μl of reconstituted OmniMix HS (3 U TaKaRa hot start Taq polymerase, 200 μM deoxynucleoside triphosphates, 4 mM MgCl2, and 25 mM HEPES buffer [pH 8.0 ± 0.1]), 0.5 μM of each MTBC primer, 200 nM MTBC Eclipse probe, 0.25 μM of each IC primer, 200 nM IC Eclipse probe, 20 pg IC DNA, and 2 μl extracted DNA template with the following exception. The Rotor-Gene PCR mixture contained 300 nM MTBC Eclipse probe. Water was used as a no-template control. Cycling conditions were identical for both instruments: 1 cycle at 95°C for 2 min, 50 cycles at 95°C for 5 s, 58°C for 20 s, and 76°C for 20 s. The crossing threshold cycle (Ct) is the cycle at which there is a significant increase in fluorescence above the background or a specified threshold. The Ct was determined from a curve generated from a plot of cycle number versus fluorescence with a manual threshold set above the background fluorescence of the no template control. The Ct is inversely proportional to the logarithm of the initial number of template molecules.
DNA standards. M. tuberculosis ATCC 27294 DNA was extracted using the IDI procedure. The concentrations of M. tuberculosis and IC DNA were determined by using the PicoGreen dsDNAQuantitation kit (Molecular Probes, Inc., Eugene, OR). Serial 10-fold dilutions of the M. tuberculosis DNA, ranging from 25 pg/μl to 2.5 fg/μl, were used as external standards. These standards were run in triplicate on both instruments. A standard curve was included or imported for each Rotor-Gene run. To generate the standard curve, the Ct value in the logarithmic linear phase was plotted against the logarithm of the known concentrations of M. tuberculosis DNA. The concentrations of mycobacterial DNA in the spiked respiratory samples were calculated by comparing the average Ct value from the logarithmic linear phase of the fluorescence curve with the line generated from external standards on that instrument.
Statistical analysis. The efficiency of each DNA extraction method (n = 8) was compared using the data analysis package included within Microsoft Excel 2000 software (Microsoft Corporation, Redmond, WA). All methods on both instruments were evaluated by a two-tailed Student's t test paired two-sample means.
The limit of detection of both instruments, using known concentrations of M. tuberculosis DNA to create a standard curve, was 0.5 fg of DNA in the presence of an IC. The PCR assay was inhibited on both instruments when specimens were extracted using 2% SDS-10% Triton X with and without sonication, as indicated by the lack of amplification of the IC target in the extracted samples and amplification of IC in the no-template control samples. These methods were included in our evaluation based on data from a report by Khan and Yadav (9), which showed that the SDS and Triton X concentrations we used were optimal for extracting DNA from a mycobacterial colony. In retrospect, our results are not totally unexpected, since an excess of SDS above 0.01% has been shown to inhibit PCR, primarily due to denaturation of the Taq polymerase (Critical Factors for Successful PCR manual, QIAGEN, Inc.). Although there were methodological differences between our study and that of Khan and Yadav (e.g., standard versus real-time PCR, different polymerases, different ratios of template to reaction volume), they do not adequately explain the complete inhibition observed in our evaluation and the amplification in theirs. Reasons for the striking difference between the results of the two studies are unclear.
Mean (± standard deviation [SD]) Ct values for the other four extraction methods evaluated (illustrated in Fig. 1) were 25.3 ± 0.64 (range, 24.2 to 26.6) for IDI, 25.9 ± 0.62 (range, 24.8 to 26.7) for QIAGEN, 25.8 ± 0.56 (range, 24.7 to 26.6) for PrepMan, and 27.9 ± 0.79 (range, 26.7 to 29.6) for TE when testing was performed on the Smart Cycler. When testing was done on the Rotor-Gene, mean (±SD) Ct values were 25.1 ± 1.82 (range, 21.7 to 27.5) for IDI, 26.9 ± 1.61 (range, 23.9 to 29.4) for QIAGEN, 26.5 ± 1.52 (range, 26.3 to 29.7) for PrepMan, and 27.7 ± 1.10 (range, 23.7 to 29.8) for TE. The differences in Ct values were significant (P = 0.05) for IDI and TE, QIAGEN and TE, and PrepMan and TE when testing was performed on the Smart Cycler, but there were no significant differences in Ct values between any of the methods when testing on the Rotor-Gene.
The amount of DNA extracted by each method, which is inversely proportional to Ct, was calculated from the mean Ct. When testing on the Smart Cycler, the DNA yield was 42.8 pg for IDI, 28.2 pg for QIAGEN, 30.4 pg for PrepMan, and 7.4 pg for TE. DNA yields for these same methods when testing on the Rotor-Gene were 51.7 pg, 14.9 pg, 20.1 pg, and 8.6 pg, respectively.
Column-purified DNA theoretically should be the cleanest, containing the fewest PCR-inhibitory substances. The goal of our study was to find a DNA extraction method that was quick but did not compromise the PCR or assay sensitivity, using the QIAGEN extraction method as the gold standard for PCR purification. Our results showed that column purification is not necessary for the extraction of DNA from sputum samples to be tested for M. tuberculosis by PCR. The time required to complete each extraction was less than 1 h for TE buffer, PrepMan, and IDI and involved two or three transfers of material to new tubes, compared to 2.5 to 3 h for QIAGEN with extensive hands-on processing.
Although not the primary aim of our study, we evaluated two different real-time PCR instruments for the detection of M. tuberculosis from respiratory samples in a clinical laboratory setting. PCR on the Smart Cycler was completed in 55 min, and the Rotor-Gene PCR runs were 95 min in duration. The Smart Cycler can run 16 reactions wells per block with up to six processing blocks connected to one computer for a 96-tube capacity. The Rotor-Gene has a 72 tube capacity for a 25-μl PCR volume. The Smart Cycler is a totally closed system with a lower chance of amplicon tubes opening for possible amplicon contamination.
A final point to be considered when selecting an extraction method for use with the M. tuberculosis PCR assay is cost. For the kits or reagents evaluated in this study, the cost per extraction is $3.00 for IDI, $2.30 for QIAGEN, $1.05 for PrepMan, and approximately $0.03 for TE buffer.
In summary, of the six extraction methods evaluated, IDI lysis tubes provided the greatest yield of DNA on both the Smart Cycler and Rotor-Gene instruments. Additionally, the IDI procedure was technically simple and was completed in less than 1 h, with approximately 20 min of hands-on time. Extraction with TE buffer, although very easy to do and inexpensive, provided the poorest DNA yield. SDS-Triton X consistently inhibited the PCR on both real-time instruments and, therefore, cannot be recommended for extraction of mycobacterial DNA.
ACKNOWLEDGMENTS
We thank Andrea Stetler for secretarial assistance, Mark Kushnir for the statistical advice, and Jeffery Stevenson for providing the internal control plasmid. Yevgeniy S. Belousov at Epoch Bioscience designed the MGB primers and probe used in this study.
This study was funded by the Associated Regional and University Pathologists Institute for Clinical and Experimental Pathology.
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