RESUMO
Objective To explore the application of PCR in detection and diagnosis of Echinococcus granulosus and develop a diagnostic technique by DNA hybridization labeled with DIG.Methods A pair of primers were designed according to Echinococcus granulosus gene fragment sequence, and the special primers were as: P1 5′-GGAATGGAGAGAAGTTAC-3′ P2 5′-GCAACCTCCGGAACTTGC-3′. The hydatid fluid, secondary hydatid and protoscolex of Echinococcus granulosus were used as template, 417 bp special band was got after being amplified by PCR. The PCR product was purified and labeled with DIG. The special DNA probe was successfully got and was used to detect the Echinococcus granulosus. Results The DNA from Escherichia coli, Shigella, Tubercle, Cysticercus granulosus and healthy human leukocyte were extended by PCR and hybrided by DNA probe labeled with DIG, and only Cysticercus granulosus expressed 471 bp special band. The sensitivity of PCR was that one Cysticercus granulosus can be detected or 100-10 fg DNA, while the dot hybridization was 2500 fg. Heterologous DNA did not be positive reaction, even increasing the spotting membrane dosages. Conclusion The technique of PCR labeled with DIG showed good specificity, high sensitivity, more accuracy and quickness and so it can provide scientific basis for early diagnosis and epidemiological investigation of Echinococcus granulosusEXPERIMENTAL RESEARCH WITH PCR AND DNA HYBRIDIZATION TECHNIQUE IN DETECTION AND DIAGNOSIS OF ECHINOCOCCUS GRANULOSUS$$$$ Feng Xiaomei, Ji Hugang, Cao Yinfang, Wang Wenhao (Department of Clinical Laboratory, Inner Mongolia Autonomous Region Hospital, Huhhot 010017, China) Abstract Objective To explore the application of PCR in detection and diagnosis of Echinococcus granulosus and develop a diagnostic technique by DNA hybridization labeled with DIG.Methods A pair of primers were designed according to Echinococcus granulosus gene fragment sequence, and the special primers were as: P1 5′-GGAATGGAGAGAAGTTAC-3′ P2 5′-GCAACCTCCGGAACTTGC-3′. The hydatid fluid, secondary hydatid and protoscolex of Echinococcus granulosus were used as template, 417 bp special band was got after being amplified by PCR. The PCR product was purified and labeled with DIG. The special DNA probe was successfully got and was used to detect the Echinococcus granulosus. Results The DNA from Escherichia coli, Shigella, Tubercle, Cysticercus granulosus and healthy human leukocyte were extended by PCR and hybrided by DNA probe labeled with DIG, and only Cysticercus granulosus expressed 471 bp special band. The sensitivity of PCR was that one Cysticercus granulosus can be detected or 100-10 fg DNA, while the dot hybridization was 2500 fg. Heterologous DNA did not be positive reaction, even increasing the spotting membrane dosages. Conclusion The technique of PCR labeled with DIG showed good specificity, high sensitivity, more accuracy and quickness and so it can provide scientific basis for early diagnosis and epidemiological investigation of Echinococcus granulosus[infections.