Loop-mediated isothermal amplification-lateral-flow dipstick (LAMP-LFD) to detect Mycoplasma ovipneumoniae

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 × 10 2 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. Electronic supplementary material The online version of this article (10.1007/s11274-019-2601-5) contains supplementary material, which is available to authorized users.

Development and validation of the isothermal recombinase polymerase amplification assays for rapid detection of Mycoplasma ovipneumoniae

“…Previous studies had demonstrated the efficacy of the PCR and LAMP to detect 16S rRNA and other conserved regions of genomic DNA of M. ovipneumoniae in different clinical specimens, including the nasal swabs and lung samples [4,9,14,15]. In this study, the RPA primers and probes were also designed based on the 16S rRNA gene.…”

“…Since first confirmed in Australia in 1972, infections by M. ovipneumoniae have been an endemic problem worldwide and have caused severe economic losses to the sheep and goat industry [10][11][12]. Bacteriological culture of M. ovipneumoniae is currently the gold standard for diagnosis, however, the culture is cumbersome and time-consuming due to the fastidious nature of the bacterium as well as that the follows required species identification by biochemical or serological tests, which make the assay burdensome for the routine applications [13][14][15]. In addition, the bacterial isolation may be hampered by sample contamination and prior antibiotic treatments received by the diseased animals.…”

“…Different nucleic acid amplification-based methods have been described to be sensitive and specific for M. ovipneumoniae, i.e. PCR, real-time PCR, and loopmediated isothermal amplification (LAMP) [9,14,15]. PCR assays require a well-equipped laboratory, expensive equipment and trained personnel, which limits their application in the under-equipped laboratories and the point-of-need (PON) diagnosis [9,15].…”

Development and validation of the isothermal recombinase polymerase amplification assays for rapid detection of Mycoplasma ovipneumoniae

“…Bacteriological culture of M. ovipneumoniae is currently the gold standard for diagnosis, however, the culture is cumbersome and time-consuming due to the fastidious nature of the bacterium as well as that the follows required species identification by biochemical, serological or molecular tests, which make the assay burdensome for the routine applications [13][14][15]. In addition, the bacterial isolation may be hampered by sample contamination and prior antibiotic treatments received by the diseased animals.…”

“…However, seroconversion to M. ovipneumoniae is often delayed after natural infection, which makes the serology less effective in detecting early-stages of infection in herds, and unsuitable for detecting acute mycoplasmal pneumonia in the field [9,14]. Different nucleic acid amplification-based methods have been described to be sensitive and specific for M. ovipneumoniae, i.e.…”