Identification of mycobacterial isolates have been traditionally based on biochemical tests and phenotypic results. Because these methods are slow and time consuming, laboratories are increasingly using molecular methods for identification of species.
Several methods of DNA extraction have been developed including enzymatic lysis using detergents, mechanical disruption, and heat lysis–based methods.1–3 Previous studies have reported that standard DNA extraction protocols do not completely inactivate Mycobacterium tuberculosis4,5 and that laboratory-acquired tuberculosis infections are evident.6,7
The technical complexities and the expense of equipment and consumables make the use of enzymatic lysis, detergents, and other commercial extraction kits inappropriate for use in developing countries. In an attempt to reduce the steps, the cost, and the risk of acquiring a laboratory infection, we evaluate the use of inactivated whole mycobacterial cells for direct detection of mycobacterial DNA using polymerase chain reaction (PCR).
A collection of 44 M. tuberculosis complexes were isolated from samples randomly obtained from patients attending Abuo Anga Hospital in Khartoum State. They were identified and confirmed by standard conventional methods.8 For inactivation, old cultures that were > 3 weeks were inoculated onto Lowenstein-Jensen slants for 3 weeks, and one colony from each isolate was suspended in 500 μL of 70% ethanol for 2 hours. Mycobacterial cells were centrifuged at 13,000 rpm for 10 minutes, the supernatant was discarded, and the pellet was washed twice with sterile distilled water. After washes, the pellet was resuspended in 500 μL of sterile distilled water in a 1.5-mL Eppendorf tube containing one glass bead ~3–4 mm in diameter. The tube was vortexed to disperse clumps of organisms. Viability of mycobacteria was determined by culturing 0.2 mL of the previous suspension in Lowenstein-Jensen slants and incubating it for 8 weeks at 37°C. No growth was observed after an 8-week incubation at 37°C. Growth in the negative control was observed, which were cultured in parallel under the same conditions. Our results are in agreement with previous studies that reported M. tuberculosis seeded into sputum sediments was efficiently killed when treated for 1 hour with 50%, 70%, or 95% ethanol.9
The PCR was based on the amplification of the insertion sequence IS6110.10 Three microliters of the inactivated suspension and 1.0 μL of each primer (10 pmol) were added to the PCR mixture, which contained 1 unit of Taq DNA polymerase (Fermentas, St. Leon-Rot, Germany), 0.6 μL DNTPs mixture (10 mmol/L), and 2.5 μL PCR buffer (Qiagen, Hilden, Germany). The volume was adjusted to 25 μL with distilled water. Thermal cycling was performed on a Biometra DNA thermal cycler with an initial cycle of 5 minutes at 95°C followed by 35 cycles each consisting of 1.0 minute at 94°C, 2.0 minutes at 65°C, and 1.0 minute at 72°C, followed by a 10-minute final extension step at 72°C. Water was used as a negative control to rule out carry over of the amplicon throughout the steps of the protocol. A positive amplicon was obtained with all 44 isolates tested as shown in Figure 1. Our result are in agreement with Sritharan and Barker11 regarding the exclusion of organic extraction and with Kocagoz et al12 regarding the exclusion of enzymatic lysis before amplification. However, in these studies, heat was used as the only agent to break down the bacteria to release the DNA before PCR amplification. In our study, we excluded the organic, enzymatic lysis and heat before DNA amplification by PCR, and we used whole inactivated Mycobacterium cells.
Previous studies have raised concerns that lysis of M. tuberculosis before PCR may not be necessary for amplification to occur.13 The issue of sample safety and risk of infection to the staff workers has not been addressed in most previous studies. The application of molecular technique necessitates the removal of material derived from this organism out of a containment level 3 laboratory to perform the work. Therefore, protecting the laboratory workers conducting these procedures is necessary.
To determine sensitivity, we estimated the concentration of M. tuberculosis in the suspension by adjusting the turbidity to McFarland No. 1 as described by McFarland.14 In a test tube, 0.1 mL of a 1% solution of anhydrous barium chloride was mixed with 9.9 mL of a cold solution of 1% sulfuric acid. The tube was kept in the refrigerator until a fine white precipitate of barium sulfate became visible after vigorous shaking. At that time, the tube had a density corresponding to ~3 × 108 mycobacteria/mL of suspension. We prepared a solution of M. tuberculosis corresponding to McFarland No. 1 standard, which is equivalent to 3 × 108 mycobacteria/mL. Ten-fold serial dilution of this suspension was performed until we ended up with concentrations of M. tuberculosis equivalent to 10−1 bacilli/mL. Five microliters of each dilution was used for PCR reaction. The endpoint of the protocol was equivalent to 300 bacilli. The sensitivity of the protocol may be higher or lower than the result obtained. The number of organisms was adjusted depending on the turbidity of the suspension, which may contain clumps of cells. For this, the accurate estimation of sensitivity is difficult. To our knowledge this is the first method that uses ethanol-inactivated colonies directly without lysis to the cells before PCR reaction. This protocol reduces the risk of infection to workers and is free of any inhibitory substance for the PCR reaction. The routine method used in our laboratory for DNA extraction is based on chemical and enzymatic lysis of mycobacterial cells. Inactivation of colonies before PCR described in this method can be performed in < 3 hours. Considering its safety, low cost, and simplicity, we recommend it to be used as a replacement for the expensive conventional extraction protocols currently used.
Address correspondence to Haitham Elbir, 1304 Khartoum 11111, Khartoum, Sudan. E-mail:
Acknowledgments: The authors thank Salah Eldine Gumaa at the Department of Epidemiology, Tropical Medicine Research Institute, National Center for Research, Sudan, for service and cooperation that have made this work possible.
Financial support: This work was funded by the Tropical Medicine Research Institute, National Center for Research, Sudan.
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