Construction And Application Of Loop-mediated Isothermal Amplification And PCR For Rapid Detection Of Lawsonia Intracellularis

Porcine proliferative enteropathy (PPE) is an acute contagious intestinal haemorrhage disease in naive adult pigs and a wasting disease in growing pigs, caused by Lawsonia intracellularis (LI). It was first reported by Bieste and Sohwarce in 1931, and confirmed by Rowland et al. in 1974. However, it has been one of the most popular swine diseases around the world. Although mortality rate caused by PPE is not high, because the main performance of PPE is chronic, this disease leads to sick pigs grow slow, the average daily gain and feed utilization rate significantly decrease, and the elimination rate rise, which can cause serious economic loss to pig farms. PPE is a common disease in the whole world in recent years. In the developed pig industry countries and regions, due to disable or limit in feed with antibodies, PPE incidence increased year by year. In China, clinical PPE suspected cases has a gradually increase tendency, but research about this disease is still in its infancy, and the overall grasp for this disease is absence, therefore, it is necessary to conduct in-depth study of PPE as soon as possible to provide a scientific reference for prevention and cure.This study, according to the design principles of primer, designed a specific primer for the AM18025.2.1 gene sequences of Lawsonia intracellularis. After the optimization of the reactive conditions, a PCR method and a LAMP method for Lawsonia intracellularis have been established successfully. Sensitivity tests showed that the polymerase chain reaction established could be detected 103 copies, and LAMP method could be detected 101 copies. Besides, specific tests showed using these two methods to detect E.coli、Salmonella、PEDV、PRV, the results were negative, and they all had good stability and repeatability. LAMP products can be analyzed by a visual inspection. Positive reaction tube became turbid under natural light, and it appeared the green fluorescence after adding SYBR Green I, under the UV light shining.Fecal specimens from different pig farms in Sichuan Province (n=114) suspected of having LI infection were tested by LAMP and PCR method.60 (50%) were positive by both PCR and LAMP,41 (34.2%) were negative by both tests. However,13 (10.8%) were positive by LAMP and negative by PCR. The coincidence rate of PCR reached 76.0% and LAMP reached 92.4% when using FIRST kit to review at the same time.Although, Porcine proliferative enteropathy caused by Lawsonia intracellularis does not have a high disease mortality, the economic losses is huge, and the infection rate of this disease is high in China at present. In this experiment, the PCR method and LAMP method have the characteristics of quick reaction, easy to handle. Additionally, the LAMP method does not need complicated equipment, is cheap, and reaction conditions are simple, which is suitable for rapid detection in laboratory and field. The methods established in this experiment can provide a certain reference to detecte Lawsonia intracellularis.