Abstract
Mycoplasma synoviae (MS) remains a serious concern in production of poultry and affects world production of chickens and turkeys. Loop-mediated isothermal amplification (LAMP) of DNA has been recently used for the identification of different economically important avian pathogens. The aim of this study was to develop LAMP for simple and inexpensive detection of MS strains in poultry using specifically designed primers targeting hemagglutin A (vlh) gene. The assay was conducted in a water bath for 1 h at 63 °C. The results were visualized after addition of SYBR Green® fluorescent dye. LAMP was specific exclusively for MS without cross-reactivity with other Mycoplasma species. The sensitivity of LAMP was determined as 10−1 CFU/ml and was 1,000 times higher than MS-specific polymerase chain reaction. LAMP assay was conducted on 18 MS field strains to ensure its reliability and usefulness. This is the first report on LAMP development and application for the rapid detection of MS isolated from chickens. This simple method may be applied by diagnostic laboratories without access to expensive equipment.
Highlights
In order to determine the optimal conditions of Loop-mediated isothermal amplification (LAMP), Mycoplasma synoviae (MS) reference strain (ATCC 25204) was used as the target template
LAMP assays were incubated under isothermal condition between 56 and 70 °C
The specific colour change, greenish fluorescence and amplicons were observed at 66.3 °C, but in case of field MS strains, the optimal temperature was 63.0 °C
Summary
The aim of this study was to develop LAMP for simple and inexpensive detection of MS strains in poultry using designed primers targeting hemagglutin A gene. The objective of this study was to develop LAMP for the detection of MS without the need of thermal cycler application using water bath or heating block
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