PCR has become a common application in medical diagnostics, in areas ranging from the detection of microbes to genetic testing. The demand for new tests is huge, given that there is a seemingly endless list of applications for which PCR can be used. dsl-Labor für Molekulargenetische Diagnostik specialises in the development of PCR tests and also gives advice and training to medical laboratory personnel. Veterinarians are among the company’s major clients since the rapid and reliable diagnosis of disease plays a particularly important role in animal husbandry.
Polymerase chain reaction (PCR) is playing an increasing role in medical diagnostics, for example for the detection of viral and bacterial pathogens. PCR is used to amplify and detect tiny amounts of pathogen DNA or RNA in an infected blood or urine sample. The technique is highly sensitive and thus permits the reliable detection of relatively small amounts of pathogen. In addition to its high level of sensitivity and its ability to measure target sequence quantity, real-time PCR provides a result within just a few hours.
Since separate tests have to be developed for each individual pathogen and given that there is a seemingly endless list of disease-causing microbes, it is evident that suitable PCR tests are not available for the majority of human and animal diseases. “There is therefore huge demand for individual PCR tests, especially for the detection of microorganisms,” says Dr. Sabine Lautenschläger, managing director of dsl-Labor, which provides assistance for setting up PCR laboratories, accreditation procedures and the development of PCR tests. dsl-Labor also offers training and support for all aspects of molecular diagnostics. The small company located in the city of Eriskirch on Lake Constance has already developed more than one hundred PCR tests for clients from Germany and abroad, including tests that enable the detection of common pathogens such as salmonella or avian flu viruses as well as more exotic ones such as the egg drop syndrome virus.
Test development starts with a comprehensive literature review, which in fact is the largest part of Sabine Lautenschläger’s work. She first looks for scientific publications with information on tests for a particular pathogen in which she is interested. “This gives us an idea of which microbial gene segment is likely to be suitable for PCR detection,” she says. Once she has identified a suitable DNA sequence, she then goes on to determine whether it is possible to optimise the primers used for replication and the labelling probes to render them suitable for the reliable detection of even the smallest amounts of pathogen. This review of scientific literature serves an additional purpose. “We also come across research groups that are working on a pathogen we are interested in, and we can ask them for samples which we then use as a positive control in whichever test we are developing,” says Lautenschläger.
Actual laboratory work is only a small part of Lautenschläger’s work. Once a suitable gene segment has been identified and suitable primers and probes have been selected, Lautenschläger evaluates the performance of the test in the laboratory by varying the temperature or the concentration of the substrates used in order to establish optimal reaction conditions. “This is particularly important for quantitative real-time PCR analyses, but finding optimal reaction conditions can be a rather time-consuming process,” says Lautenschläger. Once all this is done, the clients will receive information relating to the test’s technical validation, work instructions and a short protocol that enables them to carry out the test. “I often visit my clients in their laboratories in order to show them personally how the test works. And I also teach their staff how to perform the test efficiently and reliably,” says Lautenschläger.
Lautenschläger knows from experience that new PCR tests are not only required in human medicine, but that they also increasingly applied in veterinary medicine. “The tests can also quite easily be performed by small veterinary laboratories that are equipped with PCR devices; in-depth knowledge of molecular biology is not required,” says Lautenschläger highlighting the advantages of her PCR tests. Moreover, animal breeding farms need to carry out regular controls to detect potential pathogens and limit the extent of possible infection. PCR is the cheapest and quickest technique available for this purpose, as it identifies the presence of a suspected pathogen within around an hour.
Standard antibody tests are often unsuitable for satisfactorily dealing with veterinary issues. Such assays are unable to differentiate potential vaccination strains from their pathogenic field strain counterparts. “The animals are usually vaccinated against relevant diseases. They therefore possess antibodies against such diseases; the presence of antibodies can therefore not be used as evidence of infection,” says Lautenschläger.
Likewise, animals might reveal disease symptoms shortly after they have been vaccinated, a reaction that is due to the antigens contained in the vaccine. “A specific PCR test is able to discriminate the vaccine pathogen from the disease-causing wild-type pathogen,” says Lautenschläger.
In addition to being used for the detection of pathogens, PCR tests also have a broad range of other applications in diagnosis and research. Classical genetic testing is used for the identification of hereditary diseases and individuals by their respective DNA profiles. DNA profiling or DNA fingerprinting, as such techniques are called, can thus be used to solve paternity issues as well as identify features such as an animal’s coat colour or coat length in order to select particular animals for breeding. “At present, there are around 50 such tests on the market. Progress in research such as the deciphering of the dog genome opens up further possibilities for new tests,” says Lautenschläger.
dsl-Labor does not offer “off-the-shelf” tests. “All the tests that we develop are tailored to the individual requirements of clients and the microbes and applications that they are interested in,” says Lautenschläger. Whereas tests that enable the detection of intracellular Leishmania parasites that also infect humans play a huge role in the south of Germany, the demand for such tests is relatively limited in the north. As a general rule, many variables need to be taken into account; each test needs to be tailored to pathogen, pathogen strain, host animal species, PCR equipment used and assay conditions. “Our tests are therefore developed and optimised in each case for a specific application,” concludes Sabine Lautenschläger.
Further information:dsl-LaborMozartstraße 3688097 EriskirchTel.: +49 (0)7541/950527-2Fax: +49 (0)7541/950527-3E-mail: lautenschlaeger(at)dsl-labor.com