Bacterial PCR reflex NGS

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General Information

Lab Name
Bacterial PCR reflex NGS
Lab Code
BCTDNA
Epic Ordering
Bacterial DNA detection by PCR
Description

Detection and identification of Bacterial DNA (16S rRNA target).

Accurate identification of bacterial isolates is an essential task of the clinical microbiology laboratory that enables initiation of proper antimicrobial therapy. For many organisms, traditional phenotypic identification may be difficult, laborious and time-consuming. This issue is further confounded by phenotypic variation within species, many newly described pathogenic species and the limited battery of phenotypic tests available to distinguish among established and potential bacterial pathogens. It is sometimes essential to identify bacterial isolates to species level in order to rule out species that are drug resistant or more pathogenic, to detect unsuspected pathogens, ascribe pathogenicity to species so far considered to be nonpathogenic and to identify new bacterial species.

With more than 20,000 sequences available in public databases (e.g. NCBI), 16S ribosomal RNA gene (rRNA) sequencing is considered by current taxonomists to be the gold standard in bacterial identification and classification. 16S rRNA gene contains conserved regions useful for the design of broad-range PCR primers that can amplify various fragments of the 16S rRNA gene from pathogenic and nonpathogenic bacteria. These fragments include hypervariable regions containing species-specific signature sequences useful for bacterial identification to species level.

In many specimens bacteria can be seen by 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 bacterial 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.

Reflexive testing

When suspected pathogenic microorganisms are detected, identification procedures are performed, as appropriate for the organism and specimen, including evaluation of polymicrobial specimens using Next Generation Sequencing.

Forms & Requisitions

Molecular Microbiology Order Form

Synonyms
16S PCR, 16S sequencing, bacteria 16S, bacteria identification, bacteria PCR, bacteria sequencing, bacterial 16S, bacterial identification, bacterial PCR, bacterial sequencing, broad range bacteria PCR, broad range bacterial PCR, molecular bacteria, molecular bacterial
Components

Interpretation

Method

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

Reference Range
See individual components
Guidelines

Ordering & Collection

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

Acceptable specimens are listed below. Please see Molecular Microbiology Specimen Submission for complete specimen collection and handling instructions.

Shipping/Handling

  • Fresh frozen tissue/fluid specimens should be collected into a DNA free container labeled with at least two identifiers and 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.

Acceptable Specimens

*Mycobacterium avium complex DNA Detection [MAVDNA] can be ordered on sputum

**Fungal PCR reflex NGS [FUNDNA] and Fungal DNA Detection by PCR (without reflex to NGS) [NRFDNA] may have interference due to some lots of eSwabs which have been found to contain Saccharomyces cerevisiae DNA, resulting in false positive detection. Clinical correlation and/or retesting with a different collection method is advised. The detection of S. cerevisiae from eSwab specimens can interfere with our ability to rule out other fungal DNA.

    Unacceptable Specimens

    • Blood, serum, plasma, stool/rectal swabs
    • No citrated or heparinized solutions
    • Tissues floating in formalin
    • Swab/fluid collected in tube containing agar

    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.

    Forms & Requisitions

    Molecular Microbiology Order Form

    Handling Instructions

    Please see Molecular Microbiology Specimen Submission for complete specimen collection and handling instructions.

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

    Processing

    Processing

    UWMC/HMC: Store and send fresh tissue/fluid specimens refrigerated, if specimen storage and transport will exceed 8 hours, freeze at -20°C. Freeze all fresh tissue/fluid specimens at -20°C upon arrival in UW Molecular Microbiology.

    Performance

    LIS Dept Code
    Micro Molecular Diag (MMD)
    Performing Location(s)
    UW-MT Microbiology, Molecular Diagnostics
    206-520-4600

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    Shipping Address

    Attn: Molecular Microbiology
    UW CLSPS
    1601 Lind Ave SW Room 117
    Renton, WA 98057
    Phone: 206-520-4600
    Alternate phone: 206-598-6147

    Performing Lab Address

    Clinical Microbiology Lab, NW177
    University of Washington Medical Center
    1959 NE Pacific Street
    Seattle, WA 98195
    Phone: 206-598-5735
    Alternate phone: 206-598-6147

    Contact Information

    Please e-mail us with any questions or comments you may have. Your inquiry will be answered as soon as possible.

    email: molmicdx@uw.edu

    The Molecular Microbiology lab is open from Monday-Friday, 7am-4pm PDT.

    Billing inquiries and requests for faxed reports can be made to our Client Services Department at (206) 520-4600 or (800) 713-5198.

    For results or other inquiries, we can be reached by phone at the following numbers:

    • Phone: (206) 598-5735
    • Alternate phone: (206) 598-6147
    • FAX: (206) 520-4903

    For assistance during weekends, holidays and after hours, please contact Lab Medicine Resident at (206) 598-6190
    Frequency
    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?
    No

    Billing & Coding

    CPT codes
    87801
    LOINC
    76575-0
    Interfaced Order Code
    UOW4294