摘要
目的建立适合快速检测利什曼原虫无症状感染者的分子生物学方法。方法选择利什曼原虫k DNA小环保守区的2对快速诊断特异性引物RV1-RV2、K13A-K13B,以杜氏利什曼原虫山东分离株前鞭毛体抽提的k DNA为模板进行PCR扩增,并通过对扩增条带测序比对来鉴定方法的可靠性。运用该法对四川省黑水县105例无症状家犬和新疆喀什地区部分乡镇75例无症状易感人群的静脉血样进行检测,并同时对上述地区确诊的部分病犬及病人(均为7例)进行检测,以验证该方法的可行性及准确性。结果 RV1-RV2、K13A-K13B两对引物扩增出与预期片段大小一致的条带,序列比对结果显示扩增产物在利什曼原虫种内保守性高;该方法对105例无症状家犬及75例无症状居民静脉血样的阳性检出率分别为37.14%(39/105)和82.67%(62/75),且对同地区确诊病犬及病人血样本检测的阳性率均为100%(7/7)。结论该方法适于目前我国黑热病流行区利什曼原虫无症状感染者的检测,且灵敏快速准确,具有较好的推广应用价值。
Objective To develop a rapid molecular biological method for detection of the asymptomatic infection of Leish- mania. Methods Two pairs of primers named RV1-RV2 and K13A-K13B were selected to be the fast diagnosis primers since they were designed according to the conserved region of Leishmania kinetoplast DNA （kDNA） minicircles. The PCR amplifica- tion products of Leishmania donovani promastigote from Shandong Province were sequenced to compare their conservatism. The method was applied to detect 105 venous blood samples from healthy home canine and 7 venous blood samples from home canine suffered from Kala-azar in Heishui County of Sichuan Province, and 75 venous blood samples from susceptible population （no leishmaniasis symptoms） and 7 venous blood samples from patients in Xinjiang Kashi area in order to verify the feasibility and accuracy of the method. Results The size of PCR products was consistent with the expected fragments with high conservative among Leishmania species. The positive rates of 105 home canine samples and 75 susceptible population samples were 37.14% （39/105） and 82.67% （62/75） rspectively, and the positive rates of home canine suffered from Kala-azar and patients were all 100% （7/7）. Conclusion Thisrapiddiagn^sismeth^dissuitab^ef^rdetecti^n~fasympt^maticinfecti^n~fLeishmaniainKa^a- azar endemic areas of China with high sensitive and specific, thus it has bright perspective to be used.
出处
《中国血吸虫病防治杂志》
CAS
CSCD
北大核心
2015年第1期45-48,52,共5页
Chinese Journal of Schistosomiasis Control
基金
科技部重大科技专项基金(2008IPB202)
山东省医学科学院青年基金项目
关键词
利什曼原虫
无症状感染
kDNA
PCR
检测
Leishmania
Asymptomatic infection
Kinetoplast DNA （kDNA）
PCR
Detection
