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目的建立一种能替代传统免疫学方法用于按蚊人血指数测定的分子生物学检测技术。方法根据人核糖体DNA序列设计1对特异性引物,建立聚合酶链反应(PCR)鉴定按蚊胃内人血的方法。同时,对猪血、牛血、羊血、小鼠血以及未吸血按蚊中提取的DNA进行检测,验证该检测方法的特异性,并对吸饲人血后不同时间(1、6、12、18、24、27、30、33、36、40、44、48h)的按蚊进行检测,测试该方法的检测敏感性。结果该方法可从人血提取的DNA中扩增得到519bp大小的特异性条带,对其他动物血样及未吸血按蚊中所提取的DNA均未能扩增出特异性条带;所有吸人血24h内的中华按蚊均能扩增出特异性条带,在吸人血后27、30、33、36h的各5只中华按蚊中,分别有4、4、2、1只能扩出特异性条带,吸血40h后的中华按蚊均不能扩增出特异性条带。Logistic回归分析表明,吸血后24～40h,PCR检测阳性按蚊数与吸血后的时间呈负相关关系(P<0.01)。结论本研究所建立的PCR方法可准确鉴定吸血24h内的中华按蚊胃内的人血液来源,可替代传统免疫学方法用于按蚊人血指数的测定。
Objective To establish a molecular biological detection technique that can replace the traditional immunological methods for the determination of blood index of anopheles. Methods According to human ribosomal DNA sequence, a pair of specific primers was designed to establish a method for the identification of human blood in Anopheles stephensi by polymerase chain reaction (PCR). At the same time, the DNA extracted from pig blood, bovine blood, sheep blood, mouse blood and non-blood-sucking Anopheles sinensis was tested to verify the specificity of the test method, , 18,24,27,30,33,36,40,44,48h) Anopheles mosquitoes were tested to test the detection sensitivity of the method. Results The method could amplify the 519bp specific band from the DNA extracted from human blood and did not amplify specific bands for the DNA extracted from other blood samples and Anaesthesiomyoideae Anopheles sinensis within 24 hours of blood could amplify the specific bands. Among the 5 Chinese anopheles mosquitoes at 27, 30, 33 and 36 hours after inhaling blood, only 4, 4, 2 and 1 could expand A specific band, after 40h blood-sucking Anopheles sinensis can not be amplified specific bands. Logistic regression analysis showed that there was a negative correlation between the number of positive Anopheles mosquitoes detected by PCR and the time after blood-sucking (P <0.01) 24 to 40 hours after blood-sucking. Conclusion The PCR method established in this study can accurately identify the source of human blood in the stomach of Anopheles sinensis within 24h of blood sampling, which can replace the traditional immunological method for the determination of blood index of Anopheles.