Nontuberculous Mycobacteria DNA Detection

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General Information

Lab Name
Nontuberculous Mycobacteria DNA Detection
Lab Code
Epic Ordering
Nontuberculous Mycobacteria DNA Detection

Detection and identification of Nontuberculous Mycobacteria DNA.

While Mycobacterium tuberculosis complex members are the most commonly known human pathogens, Non-tubercolosis Mycobacteria (also known as environmental mycobacteria, atypical mycobacteria and mycobacteria other than tuberculosis (MOTT)) are also implicated in tuberculosis-like disease, localized lymphadenitis, gastrointestinal disease, and disseminated infections. Acid fast bacilli can be very difficult to grow due to their nutritional requirements. Some specimens may never reveal the presence of a pathogen because of low abundance and/or lack of viability. The use of PCR to detect Mycobacterial DNA extracted directly from clinical specimens facilitates the identification of these pathogens.

For an accurate detection of the presence of Mycobacteria in clinical specimens, the UWMC Molecular Diagnosis Section utilizes a combination of several techniques. The detection of Mycobacterium tuberculosis and Mycobacterium avium complexes, for example, relies on fluorescence resonance energy transfer (FRET) probes in a nested PCR protocol that targets the heat shock protein 65 gene (hsp65). Utilization of MTB and MAI complex specific fluorescent probes on a real-time PCR platform facilitates rapid and highly sensitive detection of MTB and MAI complex DNA in clinical specimens.

For other Non-tuberculous Mycobacteria (including rapidly growing mycobacteria (RGM)) and Mycobacterium leprae, detection and speciation involves a multi-locus PCR strategy. Identifications are performed by DNA sequencing, which provides direct, unambiguous data and can distinguish medically relevant subspecific phylogenetic lineages.

In many specimens acid fast bacilli can be seen microscopy of tissue sections but are very difficult to grow due to their fastidious nature, or are not viable as a result of antimicrobial therapy. Some specimens may never reveal the presence of a pathogen because of low abundance and/or lack of viability. The use of PCR to detect this DNA extracted directly from clinical specimens facilitates the identification of these pathogens.

For more information on this test, to download a requisition form, and for a list of acceptable and unacceptable specimens, please refer to: Molecular Diagnosis Microbiology Section Website

AFB PCR, Atypical Mycobacteria PCR, broad range AFB PCR, broad range Mycobacteria PCR, hsp65, M. avium complex, MAC, MAI PCR, Molecular AFB, Molecular Mycobacteria, Molecular Mycobacterium, MOTT, Mycobacterial identification, Mycobacterial sequencing, Mycobacterium identification, Mycobacterium leprae, Mycobacterium sequencing, Nontuberculous Mycobacterium PCR, rpoB, universal AFB PCR, universal Mycobacterium PCR



DNA extraction, nucleic acid purification, polymerase chain reaction (PCR), sequencing

Reference Range
See individual components
Interferences and Limitations

Indeterminate Results Interpretation

This Nontuberculous Mycobacteria DNA Detection test can detect and identify Nontuberculous Mycobacteria with 3 gene targets: 16S, hsp65, and rpoB.

The presence of non-Mycobacterial DNA in these targets can interfere with our ability to rule out Nontuberculous Mycobacteria.

In this scenario, the corresponding target will be reported as "Indeterminate. Unable to rule out due to interfering templates."

  • 16S: indeterminate results may be resolved by additional testing if it is noted in the results.
    Resolution may require Bacterial DNA Detection by PCR (BCTDNA) and may also require reflexive 16S rRNA Next Generation Sequencing (NGS16S) if there is evidence of multiple bacterial templates.
  • rpoB: is a more specific target for Nontuberculous Mycobacteria. There currently is not any additional testing available to resolve and indeterminate result for rpoB.
  • hsp65: is the most specific target for Nontuberculous Mycobacteria. There currently is not any additional testing available to resolve and indeterminate result for hsp65.

If there is not any interference seen in a target it will be reported as "Not Detected. Not applicable."


Ordering & Collection

Specimen Type
Tissue (Fresh frozen or paraffin-embedded), Fluid (see Acceptable Specimens for details)

Acceptable specimens are listed below. Please see this page for complete specimen collection and handling instructions.

Specimens should be collected into a DNA free container labeled with at least two identifiers.

Acceptable Specimens

  • Fresh frozen tissue
  • Fresh frozen fluid: any body fluid is acceptable if it is not listed under Unacceptable Specimens. See common examples below.
  • Formalin Fixed Paraffin-embedded tissues (FFPE, PET): blocks, scrolls, and unstained slides are acceptable
  • eSwabs and UTM (universal transport media)
  • Sodium polyanethol sulfonate (SPS, Wampole Isolator Tubes) acceptable with disclaimer

Common acceptable body fluid examples: cerebrospinal fluid, pleural fluid, pericardial fluid, urine, bronchial lavage, joint fluid, bone marrow, vitreous fluid, etc.

Unacceptable Specimens

  • Sputum*, blood, serum, plasma, stool
  • No citrated or heparinized solutions
  • Tissues floating in formalin
  • Swab/fluid collected in tube containing agar

*Mycobacterium avium complex DNA Detection (MAVDNA) can be ordered on sputum.

Specimen Processing Technicians: see 'Processing' tab fo what to do if NTMDNA is ordered on a sputum.

Handling Instructions

Fresh frozen tissue/fluid specimens should be submitted and maintained on dry ice.

Formalin Fixed Paraffin-embedded tissues (FFPE, PET) can be sent ambient or with ice packs during warmer summer months to prevent melting.

Optimal Quantity:

  • Fresh Tissue: 0.3-1.0 cm^3
  • Fluid: 0.2-1 mL
  • Formalin Fixed Paraffin-embedded Tissue (FFPE/PET): blocks are preferred and will be sent back to client upon completion of testing
  • Scrolls/unstained slides: cross-sectional area >1cm^2 send 10 sections of 10µm thickness, if <1cm^2 send 20 sections if available

Please note: We do not need a separate specimen aliquot for each test ordered. Only a single specimen aliquot or block of optimal quantity is necessary for performing multiple tests. If multiple aliquots or blocks of optimal quantity are sent, up to 2 will be pooled.

requested: See Optimal Quantity above
minimum: Specimens below optimal quantity are acceptable for testing, however, diagnostic yield is generally proportional to specimen size.



Specimen Processing Technicians: If NTMDNA is ordered on sputum please cancel/credit as YSTYP. Then order MAVDNA if the following verbiage is noted on the requisition (see below). If the following verbiage is not noted on requisition/order form, client needs to be contacted to add on MAVDNA.

Requisition Verbiage If one of the following boxes is checked off, then cancel/credit as YSTYP and order MAVDNA:

[ ] AFB (if sputum sample, only TBCDNA and MAVDNA will be performed)
[ ] Nontuberculous Mycobacteria PCR NTMDNA, Not Acceptable NTMDNA: Sputum (see MAVDNA)


UWMC/HMC – store and send specimen refrigerated and freeze specimens at -20°C upon arrival in UW-ML Microbiology. If specimen storage and transport will exceed 8 hours, freeze at -20°C.

Freeze specimens at -20°C upon arrival

Outside Laboratories: Freeze specimens in sterile containers at -20°C. Transport all specimens on dry ice.


LIS Dept Code
Micro Molecular Diag (MMD)
Performing Location(s)
UW-MT Microbiology, Molecular Diagnostics

Clinical Lab, Room # NW177,
University of Washington Medical Center,
1959 NE Pacific street, Seattle, WA 98195

Fresh frozen tissues/fluids result in 2-3 business days after receipt of specimen. Formalin Fixed Paraffin-embedded tissues result in 3-4 business days after receipt of specimen.
Available STAT?

Billing & Coding

CPT codes
Interfaced Order Code