DNA Sequence Testing

DNA sequencing, namely the Sanger sequencing technology, is the commonly used “gold standard” for validation of the results of all other nucleic acid-based clinical tests. Ötzi the Iceman who died 5,300 years ago had Lyme disease which was diagnosed in 2012 by DNA sequencing of the genomic DNA remnants of Borrelia burgdorferi preserved in the cadaver. We are using the same approach to diagnose Lyme disease and Lyme disease-like borrelioses when the pathogens are still in the blood, in the joint fluid or in the cerebrospinal fluid of a patient. This technology is especially useful in testing for Borrelia miyamotoi infections because its “diagnosis currently relies on the use of tests to detect DNA of the organism”, as stated in a CDC guideline [3]. 

The principle of DNA sequencing-based tests for Lyme Borreliosis

We perform nested PCR amplification of a highly conserved segment of the 16S ribosomal RNA gene (16S rDNA) with hypervariable regions of the pathogenic borreliae isolated from the patient’s samples. All bacteria have 16S rDNA which is needed to initiate protein synthesis. Each bacterial species has its own unique 16S rDNA “signature sequence”. If the sample is positive for this unique segment of DNA, a Sanger sequencing of the PCR amplicon is performed for validation of the PCR products. The sequencing data are submitted to the GenBank for sequence alignment analysis. The GenBank report confirms the molecular diagnosis.

However, the bacteria causing Lyme borrelioses usually contain only one copy of this DNA segment per spirochete, and there are very few Lyme bacteria in the circulating blood even at the spirochetemic stage of the infection. For this reason, we have adopted a nested PCR technology to increase the PCR sensitivity, using a high-fidelity low temperature PCR system for DNA target amplification [4].  

The results of DNA sequencing versus the two-tier serology test results in diagnosing Borrelia burgdorferi and Borrelia miyamotoi infections during spirochetemia, are shown in the following two actual cases based on testing the patient’s split serum sample. The two-tier serology results were ambiguous. The DNA sequencing results were not. 

*Special Note: Serum is a very poor sample for routine detection of infectious agents. Most spirochetes are trapped in the blood clot.

Case 1:  Lyme disease with Borrelia burgdorferi

The patient presented with clinical symptoms of possible Lyme disease and a two-tier serology test reporting an ELISA value of 0.72, an IgM Western Blot positive for the 41 and 23 bands, and an IgG Western Blot positive for the 66 and 41 bands. Residual bacteria of Borrelia burgdorferi were found in the serum sample, and confirmed by PCR and DNA sequencing as follows.

Case 2:  Atypical “Lyme disease” with Borrelia miyamotoi 

The patient presented with clinical symptoms of possible Lyme disease and a two-tier serology test reporting an ELISA value of 0.65, a negative IgM Western Blot, and an IgG Western Blot positive for a 41 band. Residual bacteria of Borrelia miyamotoi were found in the serum sample, and confirmed by PCR and DNA sequencing as follows.

References