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目的 寻找一种高度特异、敏感的马来丝虫的检测方法 ,并将其用于现场检测。 方法 根据马来丝虫 Hha 重复序列 ,合成马来丝虫特异性引物 1对和寡核苷酸探针 1条 ,采用地高辛加尾标记试剂盒对人工合成寡核苷酸探针进行非放射性标记 ,并对标记后的探针进行标记效率检测 ;将血液标本和蚊虫标本分别用蛋白酶 K等处理液和 Chelex- 10 0处理后 ,结合 PCR技术 ,对该探针的特异性和敏感性进行检测 ;并将此探针用于平远县马来丝虫病的监测。 结果 经测定 ,标记后探针的有效浓度为 1.5～ 2 fmol/μl;结合 PCR技术 ,该探针可检出 10 0 μl阳性血样中 1条微丝蚴 ;5 0只蚊中含有 1只只感染 1条幼丝虫阳性蚊亦能准确检出。班氏丝虫等其它寄生虫标本的检测结果均为阴性 ;采自平远县的 2 4 0份血液标本和 10 0 8只中华按蚊标本均为阴性。 结论 该探针杂交检测方法特异性强 ,敏感性高 ,可作为一种新的马来丝虫病监测方法在现场中使用。
Objective To find out a highly specific and sensitive detection method of Malayan filariasis and to use it for on-site testing. Methods Based on the H chain of Malayan silkworm, 1 pair of specific primers and 1 oligonucleotide probe were synthesized, and the synthetic oligonucleotide probes were digested with digoxigenin labeling kit Radiolabeling and labeling the labeled probe to detect the efficiency of the labeling; the blood samples and the mosquito samples were respectively treated with proteinase K and Chelex-10 0, combined with PCR, the specificity and sensitivity of the probe The probe was used to monitor Malayan filariasis in Pingyuan County. The results showed that the effective concentration of the labeled probe was 1.5 ~ 2 fmol / μl. In combination with PCR, the probe could detect 1 microfilariae in 100 μl of positive blood samples and 1 in 50 mosquitoes Infection with a small silkworm positive mosquito can be accurately detected. Bancroftian filaria and other parasite specimens were negative; 240 blood samples collected from Pingyuan County and 108 specimens of Anopheles sinensis were negative. Conclusion The hybridization detection method of this probe is highly specific and sensitive and can be used in the field as a new monitoring method of malayian filariasis.