马来丝虫非放射性寡核苷酸探针的制备及现场应用研究

PREPARATION OF A DIG-OLIGONUCLEOTIDE PROBE OF BRUGIA MALAYI AND APPLICATION IN THE FIELD

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 search a specific and sensitive method for detection of Brugia malayi infection and applicate in the field research. Methods An oligonucleotide probe was synthesized and labeled with digoxin. A pair of primers based on the HhaⅠ repeated sequence were also synthesized. The infection of B. malayi was detected by combining with hybridization using this nonradiative probe and polymerase chain reaction. For estimating the yield and the detection of speciality and sensitivity of the probe, double distilled water and other five negative control parasite DNA samples, two positive parasite DNA samples were detected. The blood samples and mosquito samples collected from Pingyuan were also detected with this system. Results The efficacious probe concentration was 1.5 -2 fmol/μl. Only DNA of B. malayi microfilariae and DNA of B. malayi larvae were positive, the other samples include DNA of W. bancrofti microfilariae, DNA of T. gondii , DNA of S. japonicum , DNA of P. vivax , DNA of C. sinensis and double distilled water were negative. Using this method, even 1 microfilariae per 100 μl blood can be detected, a mosquito infected by a filarial single larvae can also be detected from a group of 50 mosquitoes. On the other hand, 240 blood samples and 1 008 An. sinensis collected from the surveillant point in Guangdong Province were examined with this methods, all samples were negative. Conclusion The probe is very specific and sensitive, and is valuable in the field investigation of B. malayi infection.