The pathogens, which causes Lyme disease are bacteria, so called borrelia (spiral-shaped bacteria). The genetic material of borrelia is DNA.
The cause of TBE is a virus. The genetic material of the TBE-virus is RNA.
It is now possible to detect both TBE and Lyme disease pathogens with a DNA test done directly on the tick.
In preparation for work done in the school laboratory, the captured ticks are stored for several weeks in 70% alcohol at a temperature of -70°C. After this period of time we can be sure that the alcohol has killed all disease-causing microorganisms, so that there is no danger of infection. Storing the ticks at such an extremely low temperatur assures that the genetic material of the pathogens won’t be broken down by enzymes. A professional laboratory with a staff trained to handle pathogens in a safe manner would begin right away to work with the ticks.
We start by detecting borrelia and TBE viruses in the ticks with the help of the DNA\RNA analytics. This means that a test is done to see if the genetic material of the given pathogen can be detected in the sample, (in this case being the tick).
The first step in extracting the genetic material of the pathogens is to mechanically destroy the chitin shell of the ticks either by pulverizing it in liquid nitrogen or with the use of an Ultraturrax, a special kind of mini turbo-powered hand-held blender with over 20,000 rotations per minute. The cells are then lysed with special enzymes. Fats, proteins, cell-organelle and salts that would otherwise interfere are removed in several consecutive purification processes. After the isolation of the genetic material is complete, potential borrelia-DNA and TBE-virus-RNA is identified with the help of the so called PCR method.
Borrelia can be relatively easily detected using the PCR method (ist relativ einfach mit einem PCR –Schritt möglich), because the genetic material in this case consists of DNA. A suitable sequence for the amplification of a borrelia-gene was chosen from various sources of scientific writings.
In order to detect TBE, the virus-RNA must first be transcribed into DNA (reverse transcription), so that they can subsequently be amplified in a “nested PCR-process”. In the „nested-PCR” two PCR-rounds are carried out, one after the other, so that even the least possible amounts of the desired information can be traced. A somewhat smaller fragment of the first batch is duplicated in the second PCR-round. The „nested PCR” inhances the sensitivity und specificity of the detection process.
This process is termed RT-PCR (Reverse Transcriptase - Polymerase-Chain Reaction): First the Reverse Transkriptase (RT) is employed to transcribe the RNA in cDNA (c stands for complementary). This cDNA can subsequently be used in a PCR to amplify the sequences in question.
The products of the PCR are then analysed on an agarose gel, through which the DNA-fragments can be sorted and evaluated according to size.
The subsequent interpretation of the results is relatively easy. A negative PCR-result indicates that genetic material from the pathogens cannot be found in the tick. Risk of infection can thereby be almost completely ruled out. A positive result on the other hand proves the presence of borrelia bacteria or TBE-viruses resp. and the risk of a consequent transmission of the disease-causing microorganism in the case of a tick bite.
A positive control was carried out in order to assure that no mistakes were made in the process of the diagnostic analysis. RNA from the tick’s mitochondria from each sample was also transcribed into cDNA (reverse transcription) and was then amplified with a PCR. In order to assure reliable results all control reactions had to provide a visible PCR-product, whereas in the case of the borrelia and TBE-PCR only infected ticks would have to provide a signal when analysed on the agarose gel. In the case of a mistake in the laboratory procedure no positive control would be detectable. This would indicate that the procedure would have to be repeated.
The entire process of detection was carefully verified using original borrelia-DNA, TBE-RNA and TBE c-DNA. Dr. Stephan Aberle, from the Clinical Institute for Virology at the Medical University of Vienna provided us with reference samples of TBE-virus genetic material. Dr. Arnold Bito, from the Cell Biology department at the University of Salzburg provided us with the reference samples of genetic material from borrelia.
1. A Summary of the Test Procedure:
· Isolation of the ticks’ DNA and RNA
2. The Experimental Procedure – The Laboratory Protocol
2.1 DNA and RNA Isolation
· Disrupt the chitin shell of the ticks using either liquid nitrogen or the Ultraturrax for mechanical disruption
4.2.2 Amplification of the Borrelia DNA using PCR
Sample (DNA) 1 µl MgCl2
2.3 Amplification of TBE-RNA and positive control through RT-PCR
(RevertAid First Strand cDNA Synthesis Kit, Fa. Fermentas)
The first step was to transcribe the RNA through reverse transcription into cDNA. An extremely high amount of randomly generated primers garauntees that all of the available RNA (TBE-virus specific as well as tick-inherent) is transcribed for the positive control.
5 µl sample (RNA)
· Vortex briefly
4 µl reaction buffer
· Vortex briefly
In the next step an ordinary PCR follows, in which the newly created cDNA is used as a template. In this part the desired cDNA sequence is selectively amplified using the respective primers for the first time. Erst bei diesem Schritt wird selektiv mit passenden Primern die gesuchte cDNA Sequenz amplifiziert
The following takes place:
a) Amplification of TBE-cDNA
1. PCR TBE:
b) The Amplification of mitochondrial ticks’ cDNA as Positive Control.
To assure that the isolation of the RNA as well as the reverse transcription were succesfully carried out, a certain segment of the mitochondrial tick-RNA and the respective cDNA is duplicated.:
2.4 Visualization of all PCR-Products by Agarose Gel Electrophoresis
In order firstly to make the PCR-products visible and secondly to determine their size, an Agarose-gel-elektrophoresis is carried out.
1 % Agarose gel in 0,5x Tris-Borat-buffer pH 8,6 (TBE)
DNA-dye (Gelstar): stained reagent that forms adducts on the DNA is induced to florescence.
Preparation of the sample for the Elektrophoresis:
Sample buffer: contains a dye (= bromphenol blue), which makes it possible to follow the migration of the DNA in the gel and glycerin to “weigh down” the DNA, which subsequently sinks into the gel-pockets.
The DNA migrates from the negative to the positive pole; The migration of the DNA in the gel can be traced based on observations of the dye front. After approximately 45 minutes the gel is removed from the dye gel chamber and placed on the illuminator. The gel is observed and photographed on the blue-light illuminator. The stained DNA-bands show a greenish-yellow glow.