Recombinase polymerase amplification assay combined with a dipstick-readout for rapid detection of Mycoplasma ovipneumoniae infections

Abstract

Mycoplasma ovipneumoniae infects both sheep and goats causing pneumonia resulting in considerable economic losses worldwide. Current diagnosis methods such as bacteriological culture, serology, and PCR are time consuming and require sophisticated laboratory setups. Here we report the development of two rapid, specific and sensitive assays; an isothermal DNA amplification using recombinase polymerase amplification (RPA) and a real-time PCR for the detection of M. ovipneumoniae. The target for both assays is a specific region of gene WP_069098309.1, which encodes a hypothetical protein and is conserved in the genome sequences of ten publicly available M. ovipneumoniae strains. The RPA assay performed well at 39°C for 20 min and was combined with a lateral flow dipstick (RPA-LFD) for easy visualization of the amplicons. The detection limit of the RPA-LFD assay was nine genome copies of M. ovipneumoniae per reaction and was comparable to sensitivity of the real-time PCR assay. Both assays showed no cross-reaction with 38 other ovine and caprine pathogenic microorganisms and two parasites of ruminants, demonstrating a high degree of specificity. The assays were validated using bronchoalveolar lavage fluid and nasal swab samples collected from sheep. The positive rate of RPA-LFD (97.4%) was higher than the real-time PCR (95.8%) with DNA as a template purified from the clinical samples. The RPA assay was significantly better at detecting M. ovipneumoniae in clinical samples compared to the real-time PCR when DNA extraction was omitted (50% and 34.4% positive rate for RPA-LFD and real-time PCR respectively). The RPA-LFD developed here allows easy and rapid detection of M. ovipneumoniae infection without DNA extraction, suggesting its potential as a point-of-care test for field settings.

Development of a Rapid Sex Identification Method for Newborn Pigeons Using Recombinase Polymerase Amplification and a Lateral-Flow Dipstick on Farm

“…In female samples, the test band appeared after a reaction time of approximately 15 min and became darkest after 25 min ( Figure 2 A). In a study examining the detection of Mycoplasma ovipneumoniae infections using RPA-LFD, the darkest band was obtained at 39 °C using 0.4 mM of both probes and 0.12 mM probe after a 10 min reaction, which was earlier than the present test (for which a 15 min reaction was necessary [ 30 ]). For the detection of Salmonella, the test band appeared after 5 min and became the darkest after 20 min at 39 °C, with 0.4 µM of both primers and 0.1 μM probe [ 27 ].…”

“…In the male samples, no signal was detected for any quantity, even on the 100 ng strip ( Figure 3 B). The sensitivity of the RPA-LFD for different DNA resources has varied, reported at values of 0.2 ng, 80 pg, 10 fg and 50 pg [ 28 , 30 , 31 , 32 ]. The RPA-LFD detector used in this study, displayed a higher sensitivity than many other similar techniques.…”

A Novel Sample-to-Answer Visual Nucleic Acid Detection System for Adenovirus Detection

“…The ALP FINA module not only reduces cost and simplifies the steps but also has an approximate extraction efficiency that is comparable to that of a traditional centrifugal column, endowing it with great advantages in field applications. The RPA-LFD technology can be operated at room temperature with impressive advantages, such as high sensitivity, good specificity, rapid reaction, and electricity-independence, making it ideal for field detection ( 26 , 27 ). However, the main drawback of its traditional open-mode operation was the production of nucleic acid contamination.…”

Microfluidic chip and isothermal amplification technologies for the detection of pathogenic nucleic acid

“…Strikingly, this assay displays a detection limit of 20 copies/μL as well as a sensitivity four times as high as that of real-time quantitative PCR (qPCR). Sandeep et al [ 57 ] established an RPA-LFD assay for detecting Mycoplasma ovipneumoniae , which helped obtain visual test results within 20 min at 39°C. In particular, this assay showed a detection limit of 9 copies/reaction, comparable to the sensitivity of real-time fluorescence quantitative PCR, while it had a higher specificity.…”