一种重组聚合酶扩增检测犬耳痒螨的方法建立及应用  

Establishment and application of a recombinant polymerase amplification method for detecting Otodectes cynotis

作　　者：宋绍征 孟雅琴 武颖超 于康英 周哲 SONG Shaozheng;MENG Yaqin;WU Yingchao;YU Kangying;ZHOU Zhe(Department of Basic Medicine,School of Health and Nursing,Wuxi Taihu University,Wuxi 214064,Jiangsu,China;Jiangyin Lingfeng Pet Hospital,Wuxi 214400,Jiangsu,China)

机构地区：[1]无锡太湖学院健康与护理学院基础医学系,江苏无锡214064 [2]江阴市领峰宠物医院,江苏无锡214400

出　　处：《中国寄生虫学与寄生虫病杂志》2024年第6期802-805,共4页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：江苏省高校“青蓝工程”优秀青年骨干教师项目(苏教师函[2021] 11号);江苏省高校自然科学基金(19KJB180030,23KJD180008);企业委托横向课题(23WURD088)。

摘　　要：为建立一种利用重组聚合酶扩增(recombinant polymerase amplification, RPA)检测犬耳痒螨(Otodectes cynotis)的方法,本研究以犬耳痒螨18S核糖体DNA第一内转录间隔区(18S-ITS1)保守基因序列作为靶标序列,设计合成RPA扩增引物和探针,建立RPA反应体系,并评价该方法的灵敏度和特异性。将150例疑似犬耳痒螨感染的犬耳垢临床样品经荧光定量PCR鉴定后,采用RPA检测法和镜检法分别检测,并比较2种方法的阳性检出率差异。结果显示,构建的RPA反应体系为:重组聚合酶5 mg,反应缓冲液10μl,OC18-F引物2μl,OC18-R引物2μl,标记荧光探针1μl,DNA模板1μl,500 mmol/L醋酸镁0.5μl,灭菌超纯水补齐至50μl。扩增条件为39℃30 min。该方法检测犬耳痒螨的灵敏度为1拷贝/μl,且具有较高的特异性,与猫耳痒螨、兔耳痒螨、人疥螨、皮脂蠕形螨、毛囊蠕形螨、水牛痒螨等其他螨种均无交叉反应。RPA检测法对疑似感染犬耳痒螨的犬耳垢临床样品的阳性检出率为71.33%(107/150),高于镜检法的42.67%(64/150)(χ^(2)=10.813,P <0.05),与荧光定量PCR检测结果的阳性一致率为100%。本研究建立的RPA检测犬耳痒螨的方法具有检测速度快、灵敏度高、特异性强、精准度高等特点,为犬耳痒螨病的临床诊断提供参考。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,10μl reaction buffer,2μl OC18-F primers,2μl OC18-R primers,1μl labelled fluorescent probe,1μl DNA template,0.5μl 500 mmol/L magnesium acetate,and sterilized ultrapure water added to make up to 50μl.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)(χ^(2)=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.

关 键 词：重组聚合酶扩增检测 犬耳痒螨 灵敏度 特异性 

分 类 号：R384.429[医药卫生—医学寄生虫学]