[Detection of Schistosomia japonicum 5D gene by polymerase chain reaction and genechip technique].
OBJECTIVE
In order to develop the diagnostic genechip for specific detection of Schistosoma japonicum (Chinese mainland strain).


METHODS
Probe and primers were designed based on the Schistosoma japonicum 5D gene encoding an immunogenic miracidial antigen. The probe for the conservative and specific gene sequence was spotted onto the specially treated glass slides by pin-based spotting robot Pixsys 5500 and was employed to make genechips. A polymerase chain reaction (PCR) protocol was designed to effectively amplify the 5D gene fragment containing the probe sequence from cercaria, egg, adult worm and infected Oncomelania DNA as well as other flukes DNA, respectively. After 35 cycles by PCR, the products were then labeled with fluorescent Cy3-labeled primer, using dissymmetrical PCR. The labeled PCR products of the target genes were hybridized to the diagnostic genechips for detection of Schistosoma japonicum and a fluorescent scanner (ScanArray 3000) was used to observe and record the hybridization signals.


RESULTS
The result obtained from the study showed that a 262 bp DNA fragment was amplified from cercaria, egg and adult worm with the designed primers and enable the genechip be applied to detect a single cercaria, egg and adult worm. When the genechip was used to detect Clonorchis sinensis, Fasciolopsis busk, and Paragonimus westermani DNA, the results showed negative, indicating that the genechip had good specificity.


CONCLUSIONS
The genchip technique for detection of Schistosoma japonicum was established successfully and having the characteristics of high sensitivity and specificity.
Zhou
Jun
J
Department of Pathogenic Biology, Nanjing Medical University, Nanjing 210002, China.
Tao
Kai-hua
KH
Li
Yue-xi
YX
Qian
Wan-hong
WH
Zhang
Jin-hai
JH
Wang
Yong
Y
Zhang
Zhao-song
ZS
D000818:Animals; D002681:China; D018509:DNA, Helminth / Q000235:genetics; D017238:Genes, Helminth / Q000235:genetics*; D005821:Genetic Techniques; D016133:Polymerase Chain Reaction / Q000379:methods; D012549:Schistosoma japonicum / Q000235:genetics*; D012680:Sensitivity and Specificity
D018509:DNA, Helminth