Polymerase Chain Reaction (PCR)

DETECTION OF BORRELIA BURGDORFERI BY PCR—OVERVIEW

The polymerase chain reaction (PCR) is a sensitive method in which minute amounts of DNA in clinical specimens are amplified.24 In this method, extracted DNA (target DNA from B. burgdorferi) is "denatured" by being heated at high temperatures. This separates the DNA and makes the individual strands accessible for hybridization to specific oligonucleotide primers.

The mixture is cooled and the primers bind or hybridize to their complementary single-stranded DNA sequences in a process called "annealing." In the final step of the cycle, the primers are "extended" on the DNA template by DNA polymerase.

[Figure 8]

Figure 8. First Step in PCR

For analogy, the primers are specific antibodies and the target DNA is B. burgdorferi antigen. The advantage of the PCR is that B. burgdorferi-specific DNA sequences can be exponentially amplified by performing multiple heating and cooling cycles in the presence of nucleotides, DNA polymerase, and primers. In a system with 100% efficiency, 31 cycles would produce a billion copies of the DNA from a single copy of the gene.

[Figure 9]

Figure 9. Subsequent cycles create multiple copies of target DNA

The positive control utilized in this system is that of multiple dilutions (103, 102, 101, 100) of DNA from a boiled B31 (ATCC 35210) bacterial culture. The assay is repeated if the 100 control B. burgdorferi is negative. A negative control of distilled water is used in all assays.

A negative result with the PCR assay implies only that B.burgdorferi DNA was not detected in the patient sample. While the PCRs potential sensitivity is tremendous, the sample tested must contain at least one recoverable organism for the genomic assay and pieces or blebs of antigen for the plasmid assay.

Precaution: In order to minimize DNA contamination, separate workstations are used for the set-up, reagent preparation, cycling, and gel electrophoresis. In addition, hot start procedures are employed.

Sample Type: Theoretically, the PCR can be performed on any type of sample: serum, whole blood, urine, CSF, tissue biopsy, joint fluid, and ticks. To date, the highest recovery has been from EDTA whole blood, CSF, and joint fluid.25 Samples have excellent stability if mixed with equal parts of 95% ethanol, special IGeneX PCR buffer, or are kept at -70°C. However, most samples are acceptable even at room temperature. (ee the section on Specimen Preparation and Transport for more details.)

MULTIPLEX PCR ASSAY FOR B. burgdorferi

The standard PCR test is not always sensitive enough because of the very low numbers of organisms present. In addition, it is well known that the PCR sensitivity is reduced in the presence of inhibitors. Therefore, IGeneX has developed a nucleic acid-based Multiplex PCR diagnostic assay that simultaneously detects genomic and plasmid B.burgdorferi DNA in clinical samples. The multiplex PCR offers enhanced performance, when compared to currently available microbiological, immunological, and amplified tests for the detection of microorganisms in test samples. Some of the advantages are:

  • Increased sensitivity: The multiplex PCR can detect a bacterium or part of one in a given sample more frequently.

    While the genomic PCR gives excellent sensitivity and specificity if one recoverable bacterium or the DNA from one is obtained, the plasmid PCR does not require a whole organism but recognizes pieces or blebs of B. burgdorferi antigen. Dorward et al.23 reported the presence of pieces or blebs of B. burgdorferi antigen in urine and other tissues. In addition, Nocton et al.27 detected (by PCR) B. burgdorferi plasmid DNA in 96% of the patients with persistent Lyme arthritis. Based on this information, the plasmid PCR may have greater potential for sensitivity than the genomic PCR.

    Therefore, a multiplex PCR test that simultaneously detects genomic and plasmid DNA provides much higher sensitivity than either one performed by itself.

  • Increased specificity: Accurate identification of biochemically unusual strains of B. burgdorferi, and those with dramatically different outer membrane proteins, is possible.
  • The multiplex PCR is a direct assay for the presence of the microorganism, and has the consequent potential to identify the etiological agent.
  • The assay is independent of the host's immune response schedule. Therefore, much earlier detection of the microorganism is possible.
  • Direct testing allows the monitoring of the efficacy of an antibiotic regime.
  • The assay has the potential to detect the etiological agent in samples of tissue normally low in antibody titers (such as skin).
  • The assay can be performed on all types of samples: EDTA whole blood, serum, cerebral spinal fluid, synovial fluid, urine, breast milk, tissue biopsy, and ticks.

The multiplex PCR-based diagnostic test for B. burgdorferi, performed directly on the clinical specimen, is a three-step assay (Figure 1). The steps are:

  • Hybridization/Selection
  • Amplification of the specific DNA (genomic and plasmid)
  • Detection of B. burgdorferi-specific amplified DNA fragments

Hybridization/Selection

The hybridization/selection step specifically removes the "common PCR inhibitors" from the clinical sample and at the same time selects, purifies, and concentrates the DNA fragment of interest, thereby improving sensitivity. The DNA probes used for the selection of B. burgdorferi genomic and plasmid DNA fragments are designed from published sequences.25

PCR Amplification

During the second step, the purified B. burgdorferi fragment is PCR amplified with B.burgdorferi-specific primers - a genomic set (derived from Rosa et al.25) and an Osp A specific set (derived from Bayer et al.45) A B. burgdorferi-specific primer, as used herein, refers to a synthetically produced nucleic acid sequence that, by design and selection, contains specific nucleotide sequences. Under predetermined PCR conditions, this sequence "hybridizes" or binds specifically to B. burgdorferi DNA but not to DNA from other Borrelia species or to human DNA. Therefore, only B. burgdorferi-specific DNA is amplified.

Detection of Amplified Products

In the third step, the PCR amplified B. burgdorferi DNA fragments are detected by agarose gel electrophoresis. Any of the "right size" amplified product detected by agarose gel electrophoresis is B. burgdorferi specific.

The combination of these three steps imparts very high specificity and sensitivity to the test. It has been suggested that very small pieces (rather than whole organisms) of B. burgdorferi are present in some clinical samples. Therefore, the multiplex PCR has potentially greater sensitivity than the standard PCR.

IGeneX, Inc.
795 San Antonio Rd., Palo Alto, CA 94303 USA
Tel. 650.424.1191 / 800.832.3200 Fax. 650.424.1196