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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 establish a molecular biological method suitable for rapid detection of Leishmania asymptomatic infection. Methods Two pairs of rapid diagnostic primers RV1-RV2 and K13A-K13B of Leishmania kringle conserved region were selected and PCR amplified with k DNA extracted from Leishmania donovani isolates Increase, and by comparing the amplified bands sequencing to identify the reliability of the method. The method was used to detect the venous blood samples from asymptomatic 105 dogs in Heishui county of Sichuan province and 75 asymptomatic susceptible populations in some villages in Kashgar region of Xinjiang province. At the same time, ) To test the feasibility and accuracy of the method. Results Two pairs of primers RV1-RV2 and K13A-K13B amplified the same size bands as the expected fragments. Sequence alignment showed that the amplified products were highly conserved among Leishmania species. The positive rates of venous blood samples from dogs and 75 asymptomatic residents were 37.14% (39/105) and 82.67% (62/75), respectively. The positive rates of blood samples from dogs and patients were all 100% (7/7). Conclusion This method is suitable for the detection of Leishmania asymptomatic infection in the endemic areas of kala-azarin in China at present, and is sensitive, rapid and accurate, and has good popularization and application value.