Abstract
In order to establish a rapid detection method for Mycoplasma ovipneumoniae, this study used the loop-mediated isothermal amplification (LAMP) technique to carry out nucleic acid amplification and chromatographic visualization via a lateral flow dipstick (LFD) assay. The M. ovipneumoniae elongation factor TU gene (EF-TU) was detected using a set of specific primers designed for the EF-TU gene, and the EF-TU FIP was detected by biotin labeling, which was used in the LAMP amplification reaction. The digoxin-labeled probe specifically hybridized with LAMP products, which were visually detected by LFD. Here, we established the M. ovipneumoniae LAMP-LFD rapid detection method and tested the specificity, sensitivity, and clinical application of this method. Results showed that the optimized LAMP performed at 60 °C for 60 min, and LFD can specifically and visually detect M. ovipneumoniae with a minimum detectable concentration at 1.0 × 102 CFU/mL. The sensitivity of LAMP-LFD was 1000 times that of the conventional PCR detection methods, and the clinical lung tissue detection rate was 86% of 50 suspected sheep infected with M. ovipneumoniae. In conclusion, LAMP-LFD was established in this study to detect M. ovipneumoniae, a method that was highly specific, sensitive, and easy to operate, and provides a new method for the prevention and diagnosis of M. ovipneumoniae infection.
Highlights
Mycoplasma ovipneumoniae (M. ovipneumoniae) is an important pathogen that causes atypical pneumonia in goats and sheep (Nicholas et al 2002; Parham et al 2006; Xin et al 2012; Besser et al 2013)
The loop-mediated isothermal amplification (LAMP)-lateral flow dipstick (LFD) method uses a biotin LAMP product hybridized with a digoxin-labeled DNA probe that is complexed with a gold-labeled anti-digoxin antibody
Sensitivity of LAMP-LFD was investigated with different concentrations of M. ovipneumoniae genomic DNA as template and sterile water as negative control, LAMP amplification was carried out using the reaction system and reaction conditions of the above optimization
Summary
Mycoplasma ovipneumoniae (M. ovipneumoniae) is an important pathogen that causes atypical pneumonia in goats and sheep (Nicholas et al 2002; Parham et al 2006; Xin et al 2012; Besser et al 2013). M. ovipneumoniae show respiratory disorders, runny noses, weight loss, growth retardation, and primary infection within 1–3 months, depending on the age of the sheep (Besser et al 2017). There is an urgent need to develop a rapid and accurate method to detect M. ovipneumoniae. Such a method provides a reference for early prevention, diagnosis, and epidemiological investigation and has a certain core value for farmers. The main methods for detecting M. ovipneumoniae are pathogen diagnosis, enzyme linked immunosorbent assay (ELISA), and PCR (Jiang et al 2016) (Jiang et al 2016; Ziegler et al 2014; Li et al 2016; Yang et al 2014; Kılıc et al 2013).
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