Until now, however, LAMP PCR was clearly inferior to the classical variant. It was not as robust and not as sensitive. The method has since been improved by researchers worldwide. "LAMP PCR gives a reliable yes or no answer, indicating whether the sample contains the suspected pathogen or not," Kabisch says. In future, the technique could become a good alternative to the classical PCR method. However, it does not provide exact information about the amount of viruses or bacteria contained.
Bioproduced enzymes are key
The enzymes needed for LAMP PCR are produced by bacteria of the Bacillus subtilis variety. The genetic material of these bacteria is genetically modified to produce the enzymes "reverse transcriptase" or a "polymerase".
do all the work
The first enzyme is needed for the detection of RNA viruses such as Sars-CoV-2. It translates RNA into DNA, which can then be amplified with the second enzyme, a kind of molecular copier, and thus made visible. Bacteria also produce these enzymes in a conventional production process. However, they are then purified from the disrupted bacterial cells. "In the production method we have chosen, the bacteria do all the work," says Johannes Kabisch. They make the enzymes and are then induced to enter a steady state, the bacterial spore, by altering culture conditions. The key feature of this technique is that "the spore carries the desired enzyme on its surface, it even kills its mother cell itself, and a simple centrifugation is all that is needed to purify the enzymes attached to the spores," says the researcher from Darmstadt.
The enzymes are not only easier to purify and cheaper to produce, they are also more stable because they are attached to the spores and last for about three months even at room temperature. However, before enzymes derived from such bioproduction arrive in diagnostics, all sorts of fine-tuning work must be done, test protocols must be checked and found to be reliable. Kabisch is confident: "By carefully adjusting the production process and testing the obtained enzymes, we will be able to develop reliable test systems". Following a successful transfer of knowledge from Darmstadt and Cambridge, he hopes to be able to produce enough enzymes in Addis Ababa for 10,000 prototypes of a Sars-CoV-2 test kit in the not-too-distant future.