Colorimetric LAMP PCR kit design for rapid diagnosis of Bovine coronavirus, Bovine rotavirus group A, Escherichia coli K99+ and Cryptosporidium parvum in Neonatal calf diarrhea

Reducing calf deaths and diseases are important for the future and profitability of cattle herds. Early diagnosis and treatment of calf diarrhea can reduce mortality rates and eliminate the need for cattle imports. It is important to produce local molecular kits based on the colorimetric Loop-mediated isothermal amplification (LAMP) method and to use them in the field, instead of using immunocrotographic tests with low sensitivity. With this study, four important enteric pathogens responsible for calf diarrhea (Bovine coronavirus [BCoV; formerly Betacoronavirus 1], group A Bovine rotavirus [BRV], Escherichia coli K99+ and Cryptosporidium parvum) were isolated in farm and field conditions without requiring qualified personnel and complicated devices. It is aimed to design a routine molecular kit based on the colorimetric LAMP method that can be detected in tubes in the range of 15-30 minutes. In addition, it is aimed to reveal the test kit performances by comparing the results of these LAMP PCR kits, which we will design, with in-house PCR and immunochromatographic test kits. A total of 100 calves stool samples were included (these samples were confirmed as positive for BcoV (n:25), BRV group A (n:25), Escherichia coli K99+ (n:25) and Cryptosporidium parvum (n:25) by in-house PCR). Nucleic acid (DNA/RNA) isolations from Neonatal Calf stool samples were performed using the commercial isolation kit. Results from LAMP kit and rapid antigen kits were compared according to in-house PCR results. Results of this study, especially E. coli K99 diagnostic performance were found to be closer to each other, while performance differences in viral parameters such as BRV and BcoV were found to be significantly different. It was determined that the LAMP PCR method performed better than rapid antigen test. As a conclusion, It is very important for early diagnosis to use the colorimetric LAMP method, which can be detected in separate tubes in 15-30 minutes in farm and field conditions without requiring qualified personnel and complicated devices. We believe that, thanks to early and reliable diagnosis, the calf mortality rates will be greatly reduced, as the infected animals will be treated early.