Development of burkholderia pseudomallei detection based on lamptts1 gene in crude soil sample

Abstract

Melioidosis is the third most common cause of death from infectious diseases, after HIV/AIDS and tuberculosis in Northeastern Thailand. It caused by gram negative bacterium Burkholderia pseudomallei which their natural habitats are soil and surface water. Detection of B. pseudomallei in soil is important for defining geographical areas and indicating a risk of melioidosis in human and animals. Bacterial culture is conventional method for B. pseudomallei detection from soil sample. Although, it is simple but laborious and time consuming for large number of soil sample such as soil surveys or screening. Loopmediated isothermal amplification (LAMP) targeting the TSS1 gene was developed to detect B. pseudomallei in soil sample collected from Northeastern Thailand. 10 g. of soil was suspended in 10 ml TBSS-C50 broth at 40 °C for 48 hr. 250 μl of soil suspension was boiled at 100 °C for 10 min and then used for direct detection of B. pseudomallei without prior extraction. In addition, we performed methods to eliminate inhibitors in soil by adding 10 mg/ml bovine serum albumin (BSA) into the reaction. The results demonstrated that the detection of LAMP product was clearly seen when adding BSA with boiling methods.