关键词: 重组聚合酶扩增检测, 犬耳痒螨, 灵敏度, 特异性

Abstract:

To establish a recombinant polymerase amplification (RPA) method for detecting Otodectes cynotis, this study used the conserved 18S-ITS1 gene sequence of O. cynotis as the target sequence, designed and synthesized RPA amplification primers and probes, established an RPA reaction system, and evaluated the sensitivity and specificity of the method. After identifying 150 clinical samples of suspected O. cynotis infection in canine earwax using fluorescence quantitative PCR, the RPA detection method and microscopic examination method were used to detect the difference in positive detection rate between the two methods. The results showed that the RPA reaction system constructed consisted of 5 mg recombinant polymerase, 10 μl reaction buffer, 2 μl OC18-F primers, 2 μl OC18-R primers, 1 μl labelled fluorescent probe, 1 μl DNA template, 0.5 μl 500 mmol/L magnesium acetate, and sterilized ultrapure water added to make up to 50 μl. The amplification conditions were 39 ℃ for 30 minutes, and the sensitivity of detecting O. cynotis was 1 copy/μl, with high specificity. There was no cross-reactivity with other mite species such as O. cati, Psoroptes cuniculi, Sarcoptes scabiei, Demodex brevis, D. folliculorum and P. natalensis. The positive detection rate of RPA on clinical samples of canine earwax suspected of infection with O. cynotis was 71.33% (107/150), which was higher than the detected by microscopy with 42.67% (64/150) (χ² = 10.813, P < 0.05). The positive consistency rate between RPA detection and fluorescence quantitative PCR detection is 100%. The RPA method established in this study for detecting O. cynotis has the characteristics of fast detection speed, high sensitivity, strong specificity and high accuracy, which provid reference for the clinical diagnosis of O. cynotis disease.

Key words: Recombinant polymerase amplification detection, Otodectes cynotis, Sensitivity, Specificity