Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Mycoplasma hyorhinis

Abstract

To establish a method for sensitive and rapid diagnosis of Mycoplasma hyorhinis in clinical specimens, a simple, sensitive loop-mediated isothermal amplification (LAMP) assay was designed and evaluated. Three sets of four special primers, recognizing distinct sequences of the target, were designed for sensitive, specific amplification of nucleic acid under isothermal conditions. The LAMP assay was carried out using 35 clinical specimens of bronchoalveolar lavage fluid (BALF) from pigs. For comparison, these specimens were also tested using conventional PCR, real-time PCR, and nested PCR assays. After optimization of the reaction condition and reaction system, the LAMP reaction successfully detected Mycoplasma hyorhinis within 40 minutes at 61 degrees C. The LAMP assay achieved a sensitivity of 10(1) copies per microL at 61 degrees C in 40 minutes, compared to real-time PCR and nested PCR, and was over 10(3) times more sensitive than conventional PCR. In the test for the specificity of the LAMP assay, only Mycoplasma hyorhinis genomic DNA was positive and no other microorganisms were positive with the primers, indicating that the LAMP assay is specific to Mycoplasma hyorhinis. Mycoplasma hyorhinis was detected in 32 samples using the LAMP and real-time PCR assays and in 27 and 11 samples using the nested PCR assay and conventional PCR assay, respectively. All the positive samples detected by real-time PCR, nested PCR and conventional PCR assays were positive in the LAMP assay. The LAMP assay is inexpensive, easy to perform, shows a rapid reaction and does not require complex instruments like PCR. Therefore, LAMP is a simple, accurate, fast, and economical assay suitable as an alternative in veterinary practices.