Bacteria – rRNA gene PCR Test

Consistent with O. Reg. 671/92 of the French Language Services Act, laboratory testing information on this page is only available in English because it is scientific or technical in nature and is for use only by qualified health care providers and not by members of the public.

Background
The 16S rRNA gene PCR assay is a non-specific molecular assay that can help identify bacteria that are unable to be cultured through traditional means. The 16S rRNA gene consists of highly conserved nucleotide sequences that are shared by all bacteria. By using PCR primers that are targeted to conserved regions of the 16S rRNA gene, broad-range PCRs can be used to detect DNA from almost any bacterial species in culture or in certain specimen types. The identity of the bacteria is determined by nucleotide sequencing of the PCR product followed by comparison of the sequence with known sequences in the GenBank or other databases.

This assay is typically performed when a bacterial infection is suspected but all cultures from a specimen have been negative. It is performed on specimens that are normally considered sterile sites.

For CSF, PHO Laboratory also offers specific real-time PCR assays for Streptococcus pneumoniae and Neisseria meningitidis. These tests must be ordered separately or in addition to test 16S rRNA gene PCR. If either of these organism-specific tests is positive, the 16S rRNA gene PCR test (if requested) will be cancelled.
For information regarding other testing options, refer to:

Updates
Effective September 02, 2025, submission of the 16S rRNA gene PCR Testing Intake Form is mandatory along with General Test Requisition and specimen.

*Uncontrolled print copy. Valid only on day of print: 03 Sep 2025.

Testing Indications

This test is intended to be used on clinical specimens from normally sterile sites in which bacterial infection is suspected and only after all other routine methods (culture and/or specific molecular panels) have been negative. The assay not suitable for identifying multiple species in a single specimen.

16S rRNA gene PCR testing on clinical samples requires submission of the intake form which provides information that determines whether testing will proceed.

This test should only be requested after all other diagnostic tests have been exhausted

This is NOT a STAT test.

Acceptance/Rejection Criteria

Specimens received without the appropriate forms (See: Submission and Collection Notes) are subject to cancellation.

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
16S rRNA gene PCR	General Test Requisition 16S rRNA Gene PCR Testing Intake Form	Tissue	0.5 grams	Sterile container Virus-Enteric kit Order # 390087
16S rRNA gene PCR	General Test Requisition 16S rRNA Gene PCR Testing Intake Form	CSF	250 µl	Sterile container Virus-Enteric kit Order # 390087
16S rRNA gene PCR	General Test Requisition 16S rRNA Gene PCR Testing Intake Form	Sterile body fluids	500 µl	Sterile container Virus-Enteric kit Order # 390087
16S rRNA gene PCR	General Test Requisition 16S rRNA Gene PCR Testing Intake Form	Swabs from sterile sites	500 µl	Nasopharyngeal Collection Kit for Viral PCR order#: 390082

Submission and Collection Notes

Complete all fields of the requisition form, including:

Test(s) requests and indications for testing
Patient setting/population/source

For clinical specimens, label the specimen container(s) with the patient’s first and last name, date of collection, and one other unique identifier such as the patient’s date of birth or Health Card Number. For additional information see: Criteria for Acceptance of Patient Specimens. Failure to provide this information may result in rejection or testing delay.

Compete all fields on the mandatory 16S rRNA gene PCR Testing Intake Form and submit with the General Test Requisition and sample. Provide information regarding sample type, clinical history, any other testing performed.

Formalin-Fixed Paraffin-Embedded (FFPE) and blood specimens are not acceptable for testing.

Sample types not listed above (e.g. fixed tissue, blood samples) must be discussed with the Microbiologist before submitting samples for testing.

This is NOT a STAT test

Timing of Specimen Collection

For best results, it is important that specimens submitted for this test are those that are collected:

before antibiotic treatment has started.
as soon as possible after symptom onset and ideally within 3 days.

Other specimen types (e.g. CSF, tissues) may be considered acceptable beyond 3 days if they are kept frozen

Storage and Transport

All clinical specimens must be shipped in accordance with the Transportation of Dangerous Goods Act/Regulations.

Place each specimen type in an individual biohazard bag and seal. Insert the corresponding requisition in the pocket on the outside of each sealed biohazard bag. Specimens can be shipped as Category B (UN3373).
Clotted blood/serum specimens should be stored at 2-8°C following collection and shipped to PHO on ice packs.

All specimens submitted for molecular testing should be stored at 2-8°C following collection and shipped to PHO on ice packs. If a delay in transport to PHO is anticipated (more than 72 hours), specimens should be frozen (at -80°C if possible) and shipped on dry ice.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

16S rRNA gene PCR testing is performed twice a week.

TAT is up to 5 business days from the time of receipt at PHO Laboratory.

Test Methods

16S rRNA gene PCR testing is a laboratory-developed assay that has not been Health Canada cleared or approved.

It is an endpoint PCR test followed by Sanger sequencing if an amplicon of the correct size is observed after the PCR step.

Failure to detect bacterial 16S rRNA gene PCR does not rule-out the possibility of bacterial infection.

Samples with multiple species of bacteria present may be uninterpretable.

Identification of bacterial DNA from one species does not eliminate the possibility of DNA from other species being present in the sample (typically in lower amounts that may go undetected).

Test results must be correlated with patient’s clinical presentation, treatment history and other findings.

Algorithm

Note: For CSFs, PHO Laboratory also offers specific real-time PCR assays for Streptococcus pneumoniae and Neisseria meningitidis.

These tests must be requested separately or in addition to test 16S rRNA gene PCR.

If either of these organisms are suspected, these tests will be performed first and if positive, the 16S rRNA gene PCR test (if requested) will be cancelled

Interpretation

All results should be interpreted in the context of the specific clinical scenario. Given the overlap in the distribution of disease, testing for other potential co-pathogens should be considered.

Result	Interpretation	Comment
Not Detected	16S rRNA gene NOT DETECTED by PCR	This test cannot be used to rule-out the presence of bacteria or bacterial infection. It is possible that bacterial DNA may be present below the limit of detection of this assay. Test results should be correlated with patient symptoms/clinical presentation and other findings. This is an end-point PCR and Sanger sequencing-based assay that has been verified at the Public Health Ontario Laboratory for clinical use. It has not been cleared or approved by Health Canada.
Detected	16S rRNA gene detected by PCR. Sequence homologous to: Genus species	Test results should be correlated with patient symptoms/clinical presentation and other findings. This is an end-point PCR and Sanger sequencing-based assay that has been verified at the Public Health Ontario Laboratory for clinical use. It has not been cleared or approved by Health Canada.
Detected – Unable to speciate	16S rRNA gene detected by PCR. Sequence homologous to: Genus only	Test results should be correlated with patient symptoms/clinical presentation and other findings. This is an end-point PCR and Sanger sequencing-based assay that has been verified at the Public Health Ontario Laboratory for clinical use. It has not been cleared or approved by Health Canada.
Unable to be interpreted	16S rRNA gene detected by PCR; sequence is unable to be interpreted	Sequence may be mixed or of low quality. Test results should be correlated with patient symptoms/clinical presentation and other findings. This is an end-point PCR and Sanger sequencing-based assay that has been verified at the Public Health Ontario Laboratory for clinical use. It has not been cleared or approved by Health Canada.

Reporting

Results are reported to the physician, authorized health care provider (General O. Reg 45/22, s.18) or submitter as indicated on the requisition.

Bacterial species that are of public health significance are reported to Medical Officer of Health as per the Ontario Health Protection and Promotion Act.

*Uncontrolled print copy. Valid only on day of print: 03 Sep 2025.

Contact Laboratory Customer Service

Microbiology and Laboratory Services

customerservicecentre@oahpp.ca

Updated 2 Sep 2025