Rapid visual detection of Spiroplasma eriocheiris by loop-mediated isothermal amplification with hydroxynaphthol blue dye

Abstract

Abstract In recent years, a new type of Spiroplasma has been found, which can cause “tremor disease” of Chinese mitten crab Eriocheir sinensis. The outbreak of epidemic “tremor disease” has caused a serious setback in the Chinese mitten crab farming industry, with an incidence rate of more than 30% and a mortality rate of 80% − 100%. Therefore, finding a sensitive method to detect tremor disease of E. sinensis has become the current research focus. In this research, a loop-mediated isothermal amplification detection method coupled with hydroxynaphthol blue dye (LAMP-HNB) was developed and used to rapidly detect Spiroplasma eriocheiris. Firstly, we designed and synthesized the specific outer primers, inner primers and loop primers based on the 16S ribosomal RNA gene of S. eriocheiris. Secondly, LAMP-HNB detection method of S. eriocheiris was successfully established by screening primers, adjusting the temperature and time of the reaction, and optimizing the concentration of Mg2+ and dNTP. In the specific tests, only samples infected with S. eriocheiris showed a positive result, and other infections caused by bacteria and parasites were tested negative, thus proving that the test has high specificity. The detection limit is 2.5 × 10− 6 ng/µL, indicating high sensitivity. The method for detecting S. eriocheiris is a high-speed visual inspection method based on LAMP-HNB detection. It is a simple, fast, sensitive, and inexpensive method that can be applied to a wide range of field investigations.