Development of a LAMP technique for rapid detection of Listeria ivanovii in the field

L. ivanovii is one of the significant bacterial pathogens that causes abortion, stillbirth and neonatal septicemia in ruminants. L. ivanovii can spread rapidly within a herd. It is important to quickly diagnose the pathogen prevent the infection of more susceptible animals in the herd. The aim of thesis is to develop a LAMP technique, which enables rapid and sensitive diagnosis of L. ivanovii in the field. In the thesis study, firstly, 6 LAMP primers specific for the L. ivanovii hemD gene were designed. Studies were carried out to evaluate the specificity and sensitivity of the developed LAMP technique, and the sensitivity of the technique was determined by after comparison of the results with real-time PCR carried out with the same samples and targeting the same gene. As a result of specificity studies, it was determined that the LAMP technique gave positive results only with DNAs belonging to L. ivanovii strains. In sensitivity studies, the first application of the LAMP technique was performed as specified in the user manual of the LAMP kit, and the LAMP technique showed lower sensitivity than the real-time PCR technique. Therefore, an optimization study was carried out and optimal reaction conditions for the LAMP technique were determined. The detection limits of the optimal LAMP technique in terms of the number of target bacteria extracted in 10-fold bacterial dilutions and artificially contaminated fetal liver, cotyledon and fetal stomach content samples were 25 x 102 cfu/25 μl, 25 x 103 cfu/25 mg, 25 x 102 cfu/25 mg and 25 x 102 cfu/25 mg, respectively and the detection limits of the technique in terms of the number of target bacteria in reaction were 37,5 cfu/3 μL, 375 cfu/3 μL, 37,5 cfu/3 μL and 37,5 cfu/3 μL, respectively. These results revealed that the sensitivity of the technique is similar to the real-time PCR technique in terms of the number of target bacteria extracted, while the LAMP technique is more sensitive in terms of the number of target bacteria in the reaction. After optimization, the technique was also validated using a colorimetric LAMP kit, and amplification product were observed with the naked eye. Thus, the applicability of the developed LAMP technique in the field has been demonstrated. In addition, a validation study of the technique was carried out with 50 field samples sent to the diagnostic laboratory of the Microbiology Department, and L. ivanovii was detected in 2 of these samples. As a result, in the thesis study, a specific and sensitive LAMP technique was developed for the field diagnosis of L. ivanovii. In the literature review, it was seen that there was a LAMP technique developed for the detection of L. ivanovii for the first time in the field of Veterinary Microbiology and for the second time in the general literature. For this reason, it is thought that the presented thesis study will contribute to the literature.