Optimized Method for Preparation of DNA from Pathogenic and Environmental Mycobacteria

Abstract

Genomic studies on pathogenic and environmental mycobacteria are of growing interest for understanding of their evolution, distribution, adaptation, and host-pathogen interaction. Since most mycobacteria are slow growers, material from in vitro cultures is usually scarce. The robust mycobacterial cell wall hinders both experimental cell lysis and efficient DNA extraction. Here, we compare elements of several DNA preparation protocols and describe a method that is economical and practical and reliably yields large amounts-usually 10-fold increased compared to earlier protocols-of highly pure genomic DNA for sophisticated downstream applications. This method was optimized for cultures of a variety of pathogenic and environmental mycobacterial species and proven to be suitable for direct mycobacterial DNA extraction from infected insect specimens.Mycobacterial diseases are a major health concern for humans (i.e., Mycobacterium tuberculosis, M. leprae, M. ulcerans, M. avium, and M. paratuberculosis) (4, 13, 18, 29, 30) (13,20). Efficient methods for DNA preparation are required both for the identification and genotyping of such pathogens and for population genomics, which is developing into an important tool to study bacterial evolution, virulence, and epidemiology.Extraction of mycobacterial genomic DNA is especially demanding since (i) many mycobacterial species are among the most extreme slow growers, accounting for small amounts of starting material, and (ii) a robust and waxy cell wall renders mycobacteria difficult to lyse. Published protocols for mycobacterial DNA preparations and commercially available extraction kits are mainly designed for the isolation of small amounts of genomic material suitable for conventional PCR application (2,7,9,11,14,15,23,24,27,28,33), such as for testing of potentially contaminated milk (6,8,17). However, such DNA quantities and qualities are usually not sufficient for more sophisticated molecular analyses.M. ulcerans, the causative agent of the devastating human skin disease Buruli ulcer, is one of the slowest growers among mycobacterial species, and the development of molecular tools is crucial for studying its transmission and microepidemiology. The objective of this study was to develop an optimized extraction protocol for DNA of both high quantity and quality from scarce material of in vitro-cultivated M. ulcerans disease isolates. We compared elements of several protocols and developed a DNA preparation method that is optimized in each individual step and thus ready to use for virtually all mycobacterial species to yield a maximum of pure genetic material. In addition, we applied the established method to cultures of a variety of pathogenic and environmental mycobacterial species and tested it by isolating DNA from insects experimentally infected with M. ulcerans. MATERIALS AND METHODSMycobacterial strains and preparation of mycobacterial cell suspensions. The strains used for this investigation and their origins are as follows: M. ulcerans Agy99, Malaysia 1615, and Japa...

Standardization of a TaqMan-Based Real-Time PCR for the Detection of Mycobacterium tuberculosis-Complex in Human Sputum

“…A pure culture of Mycobacterium tuberculosis reference strain Mt14323 10 was used to determine the detection limit (analytical sensitivity). Ten-fold serial dilutions from 10 7 to 1 fg (equivalent to 10 6 -10 genome copies) were prepared in triplicate from the genomic DNA, 11 and each of them was mixed with 5.4 ng of human DNA derived from 1.5 To assess the specificity of the qPCR all three runs included duplicate negative samples (no template control) to discriminate possible background. The external quality assessment panel also contained two negative control pooled specimens negative for MTBc.…”

Lack of Insertional-Deletional Polymorphism in a Collection of Mycobacterium ulcerans Isolates from Ghanaian Buruli Ulcer Patients

“…Bacterial pellets of about 20 mg (wet weight) were heat inactivated for 1 h at 95°C, followed by cell wall disruption and digestion. DNA was extracted from the supernatants by phenol-chloroform (Fluka, Buchs, Switzerland) extraction and subjected to ethanol precipitation as described previously (30). DNA was measured by the optical density at 260 nm using a NanoDrop 1000 spectrophotometer (Thermo Fisher, Waltham, MA).…”

“…Genomic DNA was extracted from bacterial pellets using an optimized method for mycobacterial DNA preparation (30). Bacterial pellets of about 20 mg (wet weight) were heat inactivated for 1 h at 95°C, followed by cell wall disruption and digestion.…”

Mechanical Disruption of Lysis-Resistant Bacterial Cells by Use of a Miniature, Low-Power, Disposable Device

“…Many common pathogens can be lysed through chemical agents, such as detergents and chaotropic salts, or by enzymatic treatment (8,31). However, lysis is a significant challenge for thick-walled microorganisms such as Bacillus anthracis spores and Mycobacterium tuberculosis cells (13,18,22). The multilayer structure of Bacillus spores includes an outer cortex and coat that is resistant to chemical and physical treatments (5,23).…”