Molecular and Other Rapid Methods
Our laboratory is equipped to perform a number of rapid molecular tests for identification of Mycobacteria and other aerobic Actinomycetes. Testing is performed by highly trained technologists to provide extremely accurate and reliable results in the shortest amount of time possible.
We offer the following rapid methods:
16S rDNA Sequencing
The objective of this test is to identify Mycobacteria and other aerobic Actinomycetes to the species level by sequencing the first 500 base pairs of the 16S ribosomal gene. The genomic DNA is extracted from bacterial cells grown in liquid or solid media. The 16S rDNA fragment of interest is amplified by PCR and purified. Cycle sequencing is performed on the purified PCR product, labeling the appropriate nucleotide with one of four fluorescent tags (dye terminators).
After the extension product is purified using a gel filtration cartridge, electrophoresis is performed on a genetic analyzer. The resulting forward and reverse sequences are aligned and edited to resolve base pair ambiguities in the consensus sequence using the Microseq software from Applied Biosystems. The edited consensus sequence is compared to a sequence database library revealing a list of close matches. The final species identification is determined based on the percent divergence from the compared sequences in the library, the mycolic acid profile (HPLC), growth characteristics of the organism (including colony morphology, pigmentation and temperature sensitivity), and if required, biochemical tests.
GenProbe
The GenProbe test is a commercially produced kit that can identify several different species of Mycobacteria. Currently, GenProbe produces kits for M. tuberculosis complex, M. avium intracellulare complex, M. kansasii, M. gordonae, M. avium, and M. intracellulare. The method, unlike the MTD kit (also made by GenProbe) does not use an amplification step. The test, like HPLC, requires a fully-grown culture to work. Ribosomal RNA is first extracted from the culture. A chemiluminescent labeled, DNA probe, specific to the target organism, is added and a positive result in a luminometer indicates a match.
Mycobacterium Direct Test (MTD)
The MTD is used to detect M. tuberculosis complex directly from clinical specimens. The test is a target-amplified nucleic acid probe test for the in vitro diagnostic detection of rRNA, utilizing Transcription Mediated Amplification and the Hybridization Protection Assay. Nucleic acids are released from the mycobacterial cells by sonication. Heat denatures the nucleic acids and disrupts the secondary rRNA structure. At a constant 42°C, the rRNA target is reproduced in multiple copies by transcription of DNA intermediates. The specific sequences (amplicons) are then detected by the same hybridization principle used in the AccuProbe test manufactured by GenProbe.
The MTD is not performed on blood, feces, urine, or specimens grossly contaminated with blood. The assay can be performed on formalized tissue samples embedded in paraffin. Minimum volume of specimen required is 1.0 ml.
There can be limitations in the MTD assay such as false negatives due to specimen inhibitors in the sample. The inhibitors inactivate the amplification enzymes. Our laboratory tests each sample for specimen inhibitors. The laboratory report will indicate detection of inhibitors if present in the sample. In addition, the laboratory performs smear and culture isolation as an internal quality assurance control for the MTD test. Any acid-fast positive cultures will be reported to the submitting facility.
Results from the MTD are available for cerebral spinal fluid 24 hours from receipt by the laboratory. Results for all other specimens are available 72 hours from receipt by the laboratory.
High Performance Liquid Chromatography (HPLC)
The objective of this test is to identify Mycobacteria and other aerobic Actinomycetes. The method used is a modified version of the CDC protocol developed in the early 90s. Cells from culture are first saponified in a strong base. The mycolic acids (from which all Mycobacteria species derived their namesake) are acidified, then dissolved and extracted in an organic solvent. Extraction effectively separates the cellular mycolic and other fatty acids from all other cellular material present in the original sample. These fatty acids are derivitized for detection by phospholuminescence. Each sample is run through a C-18 column on a HPLC instrument and the resulting chromatogram is examined.
HPLC chromatograms for M. tuberculosis complex are distinct and conserved, making it an invaluable tool in the clinical identification of TB in culture. Likewise, various other Mycobacteria are readily identified by HPLC, as described in the section Identification of NTM.
DNA Fingerprinting by Diversilab System
DNA fingerprinting is performed using the Diversilab system from Biomerieux. All bacterial genomes studied to date contain a unique pattern of repetitive sequences that, when amplified, can be used to differentiate between species and strains. Our fingerprinting system amplifies these repetitive elements from a pure culture using a repetitive PCR method and then separates the amplicons by size. This creates a distinct DNA fingerprint that can be used for identification and strain typing.
We are currently validating this method and incorporating it in to our workflow. At this time we are not accepting clinical samples.
