title: An effective sequence characterized amplified region-PCR method derived from restriction site-amplified polymorphism for the identification of female Schistosoma japonicum of zoonotic significance.
abstract: In the present study, restriction site-amplified polymorphism (RSAP) markers were used to examine the genetic variability of Schistosoma japonicum isolates from different endemic provinces in mainland China. Of the 45 pairs of primers screened, 10 RSAP markers showed a clear banding pattern with good resolution; however, only six exhibited a polymorphism among different isolates. Among six RSAP markers, one pair of primers (R8+R10) was able to differentiate male and female parasites, and amplified one constant specific band for female S. japonicum isolates. The specific band was recovered, re-amplified and sequenced, and a sequence of 162 bp was obtained. Based on this sequence, a pair of specific primers was designed and used to develop sequence characterized amplified region (SCAR)-PCR assay for identification and differentiation of female S. japonicum isolates. The SCAR-PCR assay allowed the specific identification of female S. japonicum, with no amplicons being amplified from male S. japonicum, Fasciola hepatica, Clonorchis sinensis, S. mansoni (male and female parasite). DNA sequencing confirmed the identity of the amplified products. The minimum amount of DNA detectable using SCAR-PCR assay was 0.3 ng for female S. japonicum. The SCAR-PCR was able to differentiate effectively the male and female S. japonicum worms collected from 12 geographical origins in eight endemic provinces, the gender of which was known based on the morphological and biological features. These results showed that SCAR-PCR provides an effective tool for the sex differentiation studies of S. japonicum, identification of female S. japonicum, diagnosis and epidemiological survey of S. japonicum infections in animals and human.
authors.affiliation: Laboratory of Parasitology, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, Guangdong Province, P R China.
authors.forename: Guang-Hui
authors.initials: GH
authors.lastname: Zhao
authors.forename: Juan
authors.initials: J
authors.lastname: Li
authors.forename: Rui-Qing
authors.initials: RQ
authors.lastname: Lin
authors.forename: Feng-Cai
authors.initials: FC
authors.lastname: Zou
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.forename: Zi-Guo
authors.initials: ZG
authors.lastname: Yuan
authors.forename: Xi-Hao
authors.initials: XH
authors.lastname: Mo
authors.forename: Hui-Qun
authors.initials: HQ
authors.lastname: Song
authors.forename: Ya-Biao
authors.initials: YB
authors.lastname: Weng
authors.forename: Xing-Quan
authors.initials: XQ
authors.lastname: Zhu
mesh_terms: D000818:Animals; D002681:China; D004587:Electrophoresis, Agar Gel; D005260:Female; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012549:Schistosoma japonicum / Q000235:genetics*; D012554:Schistosomiasis japonica / Q000469:parasitology*; D012680:Sensitivity and Specificity; D012732:Sex Determination Analysis / Q000379:methods*; D015047:Zoonoses / Q000469:parasitology*