Culture and Cell Suspension Preparation
Colonies, from five sputum samples cultured on (Lowenstein Jensen) medium slants for seven weeks, were harvested to prepare a two loopfuls of colony per mL suspension in BBL™ Middlebrook ADC Enrichment broth.
For each DNA extraction protocol, an aliquot (200 μL) of each cell suspension was transferred into a sterile 1.5 mL screw-capped tube and centrifuged for 3 min at 10,000 × g. The supernatant was carefully removed and the pellet washed twice with sterile distilled water, centrifuging each time as above. The five resultant pellets were then subjected to total nucleic acid extraction.
Nucleic Acid Extraction
The resultant pellets were carefully re-suspended in 200 μL of sterile distilled water, mixed by vortex, and placed on a heating block at 95°C for 30 min. After cooling at room temperature and centrifuging at 5000 × g for 10 min; the supernatants were transferred to a new sterile 1.5 mL tube and stored at 4°C.
A suspension of each pellet in 200 μL of sterile distilled water was mixed by vortex. To this, 22.2 μL of 10% SDS was added and mixed gently by inversion, and thereafter incubated on a heating block at 65°C for 30 min. The resultant lysate was allowed to cool to room temperature. Subsequent to lysate incubation on ice for 10 min and centrifugation at 5000 × g for 10 min, the supernatant was recovered to a new 1.5 mL tube. The supernatant was incubated with 2 volumes of absolute ethanol on ice for at least 1 hour, and centrifuged at 12,000 × g for 10 min; the resultant supernatant was decanted and pellets air dried. The pellets were mixed gently in 50 μL of warm sterile distilled water and stored at 4°C.
Cell pellets suspended in 200 μL of sterile distilled water, were frozen at -20°C. Upon thawing on ice, the suspensions were incubated at 100°C for 30 min. Lysates were mixed and centrifuged at 6000 × g for 10 min. The supernatants were recovered to a new 1.5 mL tube and stored at 4°C.
Cell deposits were incubated at 95°C for 30 min as a suspension in 200 μL of lysis buffer (10 mM Tris-HCl and 1 mM EDTA) and the resulting lysates were gently mixed and centrifuged at 5,000 × g for 10 min. The supernatants were recovered and stored at 4°C.
Cell pellets in 200 μL of lysis buffer (10 mM Tris-HCL and 1 mM EDTA), were mixed gently with 22.2 μL of 10% SDS by inversion, and incubated at 65°C for 30 min. The lysates were allowed to cool to room temperature before incubating on ice for 10 min. Following centrifugation at 5,000 × g for 10 min, recovered supernatants were then incubated with 2 volumes of absolute ethanol on ice for at least 1 hour. DNA was recovered by centrifugation at 12,000 × g for 10 min, air-drying pellet, and re-suspending pellet in 50 μL of warm 1× TE buffer. These were stored at 4°C.
Aliquots (150 μL) of lysis buffer (10 mM TRIS-HCl pH 8/1 mM EDTA/1% Triton-X 100) were added to cell pellets and incubated 95°C for 30 min. Resultant lysates were then mixed by vortex and centrifuged for 8 min at 7,000 × g. Subsequently, supernatants were carefully recovered and stored at 4°C.
This was a repeat of protocol 6 above with 1% Tween-20 replacing TritonX-100.
PCR amplification of Mycobacterium tuberculosis DNA
Five microlitres (5.0 μL) of each extract was used with Qiagen PCR kit for the gene amplification. Commercially purified DNA of Mycobacterium tuberculosis H37RV standard strain and PCR water were used as positive and negative controls respectively. The following primers were used;
(rpoB For; TGGTCC GCTTGC ACGAGG GTCAGA and rpoB Rev; CTCAGG GGTTTC GATCGG GCACAT).
(RTB39; TGGCCG CGGCGG TCGACA TT and RTB38; GGTCAG TGGCCA GCATCG TC).
(STR53; TCACCA TCGACG AAGCTC CG and STR31; CTAGAC GCGTCC TGTGCA TG).
(STR52; GTGAAG ACCGCG GCTCTG AA and STR34; TTCTTG ACACCC TGCGTA TC).
The cycling programme was: initial denaturation at 95°C for 15 min; followed by 45 cycles of denaturation at 94°C for 1 min, annealing 60 - 64°C for 1 min and an extension at 72°C for 1 min; a final extension step at 72°C for 10 min was performed.
Amplicons were resolved on an ethidium bromide stained 2% agarose gel.
In avoiding contaminations, culture processing was performed in a separate biosafety cabinet located in separate room; nucleic acids extracts and PCR mixes were prepared in PCR work stations in separate rooms; and disposable plugged micropipette tips were used.
Relative Quantification of Nucleic Acids
Extracts obtained by each protocol was incubated with 2 volumes of absolute ethanol on ice for at least 1 hour and centrifuged at 12,000 × g for 10 min. Supernatant were decanted and pellets washed with 70% ethanol. The centrifugation was repeated; the pellets were air-dried and resuspended in 50 μL of warm 1× TE buffer.
The yield of total nucleic acid for each protocol was determined by measuring and calculating the average absorbance at 260 nm of the five extracts. Also, absorbance at 280 nm was measured and the ratio of UV absorbance 260/280 nm calculated.