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Test ID: 16S RT-PCR
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16S Real Time Polymerase Chain Reaction
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Useful For
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16S rRNA sequencing is a broad-range PCR used in identifying bacterial pathogens to the species level by sequencing the highly conserved bacterial 16S rRNA region.
This technology is especially beneficial, but not limited, to more invasive, difficult-to-diagnose and complicated culture-negative infections where pathogen identification is of high importance.
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Method name and description
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Taqman Real Time Polymerase Chain Reaction (PCR) targeting 16S Ribosomal RNA Gene using an in-house protocol, followed by 16S Ribosomal RNA Gene Sequencing with next-generation sequencing (NGS) using a commercially available kit based on nanopore technology.
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Clinical information
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16S rRNA gene amplicon sequencing has become a pivotal tool for the characterization of bacterial diversity and community structure of almost any environment. Targeted amplification of specific regions of the 16S rRNA gene followed by NGS is a powerful strategy for identifying bacteria in a given sample.
The 16S real time PCR test is a semi-quantitative method using a Taqman probe which is used to initially estimate if sufficient 16S target is present in the specimen to proceed with the subsequent sequencing step. For specimens where the cycle threshold (Ct) value is lower than the cut-off, the sample has enough 16S target to be sequenced. If the Ct is higher, sequencing is expected to give negative results.
https://www.mdpi.com/2079-6382/11/2/159
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Aliases
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16S rRNA gene sequencing
Bacterial sequencing
16S
Ribosomal RNA
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Specimen type / Specimen volume / Specimen container
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Specimen type:
- Pus from abscesses
- CSF and other sterile body fluids such as joint fluid
- Tissues
- Other specimen types where infection is suspected and cultures are negative
Specimen volume: Preferably more than 200 uL.
Specimen container: Sterile container
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Collection instructions / Special Precautions / Timing of collection
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Collection as per organization guidelines
No special precautions / Timing of collection
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Relevant clinical information to be provided
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Clinical presentation and history
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Storage and transport instructions
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Transport immediately to Central Specimen Receiving and Processing area (CSRP) in the Qatar Rehabilitation Institute (QRI), 3rd floor, Hamad bin Khlaifa Medical City (HBKMC), in a cool box with ice pack.
If specimen(s) cannot be delivered immediately to the CSRP, store in a refrigerator (2- 8◦C) for no more than 6 hours.
Note: For specimens collected in non-HBKMC facilities, please ensure the specimen is sent within the above timeframes to the nearest laboratory specimen receiving area in order to prevent deterioration in specimen quality.
For any likely delays beyond 6 hours and/or enquiries about the patient specimen or testing, please contact the virology secretary on extension 44395125 or 44395124 during normal working hours (Sunday to Thursday 7:00 to 15:00) or the on-call virologist out of hours.
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Specimen Rejection Criteria
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- Quantity of specimen not sufficient (QNS).
- The specimen has leaked
- The specimen is in the wrong container
- Improper storage of specimen before submission to the laboratory.
- Clots detected in plasma visually or by testing instruments (applicable for blood/plasma specimens only)
- Patient identifiers on request do not match those on the container.
- Gross hemolysis of the specimen (applicable for blood/plasma specimens only)
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Biological reference intervals and clinical decision values
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- 16S RT-PCR with Ct value > cutoff, no sequencing is performed, and the test is reported as negative.
- 16S RT-PCR with Ct value < cutoff, sequencing is performed. The result is reported as positive, with the identification of the organism.
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Factors affecting test performance and result interpretation
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Quality of sample received in the laboratory
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Turnaround time / Days and times test performed / Specimen retention time
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Turnaround time: within 14 calendar days
Days and times test performed: once a week in the Virology laboratory
Specimen retention time: 1 month
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