Lyme Disease - PCR

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.

This page provides information on PCR testing for Lyme disease (or Lyme Borreliosis). Test requests are received at Public Health Ontario (PHO) and referred to the National Microbiology Laboratory (NML).

For information regarding other testing options, refer to the our Lyme Disease – Serology webpage.

Lyme disease is caused by spirochetal bacteria of the genus Borrelia. In North America, the main causative agent is Borrelia burgdorferi sensu stricto, while in Europe, B. burgdorferi, B. afzelii and B. garinii are the relevant genospecies. In Russia and Northern Asia, B. garinii is the most common species causing Lyme disease. As a vector-borne zoonotic disease, the bacterium is transmitted through the bite of a species of Ixodes ticks, thus most cases occur during the warmer months when the ticks are active.

Initial symptoms may vary from asymptomatic to a combination of fever, headache, myalgia, stiff neck and fatigue, and may be accompanied by a characteristic circular or bull’s eye rash called erythema migrans (EM). Untreated, the disease may progress to the joints, the heart, and the nervous system.

Updates

Effective October 1, 2025, the National Microbiology Laboratory (NML) discontinued routine nucleic acid testing (molecular/PCR) for Borrelia burgdorferi sensu stricto.
Molecular/PCR testing may be considered only in specialized cases, following review and prior approval by a PHO Microbiologist in consultation with the NML. For additional information, contact PHO Laboratory Customer Service at 416-235-6556 or 1-877-604-4567.

*Uncontrolled print copy. Valid only on day of print: 06 Dec 2025.

Testing Indications

PCR testing is not sensitive on specimens other than synovial fluid.
Serology is the primary test to diagnose Lyme disease and should always be the appropriate specimens submitted.
Testing of asymptomatic patients, including individuals following a tick bite, is not recommended.
Testing for individuals with erythema migrans, acute phase (seasonal occurrence and exposure in an endemic/risk area) is generally not recommended.

Acceptance/Rejection Criteria

Samples submitted without prior approval will not be accepted.

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
Borrelia spp. PCR testing^1,2,3,4 B. burgdorferi sensu stricto B. afzelii B. garinii B. mayonii B. spielmanii	General Test Requisition	CSF	0.5 ml CSF	Collect CSF into a sterile container without additives.
Borrelia spp. PCR testing^1,2,3,4 B. burgdorferi sensu stricto B. afzelii B. garinii B. mayonii B. spielmanii	General Test Requisition	Synovial fluid	0.5 ml synovial fluid	Collect synovial fluid into a sterile container without additives.
Borrelia spp. PCR testing^1,2,3,4 B. burgdorferi sensu stricto B. afzelii B. garinii B. mayonii B. spielmanii	General Test Requisition	Tissue	1 to 5 mg fresh tissue	Fresh or paraffin-embedded tissue: see NML website.

Submission and Collection Notes

Lyme PCR testing must be pre-approved by a PHO Microbiologist, with the clinical context and rationale for the request clearly indicated on the requisition. Contact PHO’s Laboratory Customer Service Centre at 416-235-6556/1-877-604-4567 for pre-approval before submission.

Note: This test is performed for investigational or research purposes only.

Complete all fields of PHO’s General Test Requisition Form. Include the patient’s full name, date of birth, and Health Card Number (must match the specimen label), physician name and address, and mandatory clinical information:

Enter Borrelia spp PCR or Lyme disease PCR under ‘Test description’.
Date of symptom onset
Date of collection
Clinical signs and symptoms
Recent Travel history (within 12 months)
Exposure history (e.g. tick bite)

Note: Specimens may be delayed or rejected if they are not the appropriate specimen type, have insufficient volume, or are not accompanied by the mandatory patient information.

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. Failure to provide this information may result in rejection or delay in testing.

Specimens are sent to NML for PCR testing. Refer to the NML website for details: Molecular Detection by PCR and Sequencing - Guide to Services - CNPHI (canada.ca)

Limitations

Initiation of antibiotic treatment prior to testing may result in decreased bacterial genome which will affect the outcome of PCR testing.

PCR testing assay is generally not sensitive for many specimen types; Serum is a low-yield specimen type and will be rejected.

Storage and Transport

Store specimens refrigerated, up to 5 days, and ship them with freezer packs. If greater than 5 days, store at – 20 °C and ship on dry ice.

All clinical specimens must be shipped in accordance to the Transportation of Dangerous Good Act.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

In special cases, PCR testing can be performed at the NML, with s a TAT of up to 21 days from receipt at NML.

Test Methods

With prior approval, Borrelia species PCR can be referred out to NML for testing.

Extracted DNA is screened by a real-time PCR assay specific for Borrelia species. If positive by real-time PCR, specimens are tested by melt-curve analysis, conventional PCR and sequencing for confirmation and species determination.

Reporting

NML results are received back at PHO’s laboratory, and reports are forwarded to the ordering physician or health care provider indicated on the requisition.

Specimens that are reactive for Borrelia species are reported to the Medical Officer of Health as per Ontario Health Protection and Promotion Act.

Data and Analysis