title: [Detection of Plasmodium sporozoites in mosquitoes by using fluorescent quantitative PCR].
abstract: OBJECTIVE
  To establish a fluorescent quantitative PCR (FQ-PCR) method for quantitative detection and species identification of Plasmodium sporozoites in Anopheles mosquitoes.
  
  
  METHODS
  One pair of human Plasmodium genus-specific primers based on 18S rRNA genes were used and the reaction system and reaction condition of FQ-PCR were optimized by using the mixture of Plasmodium 18S rRNA gene recombinant plasmids and Anopheles DNA as a template. The specificity was verified by using four Plasmodium spp. 18S rRNA gene plasmid DNA as well as mosquito DNA and the Plasmodium species was identified according to the value of melting temperature (Tm). The standard curve was made by using P. vivax 18S rRNA gene recombinant plasmids which were serially diluted by negative Anopheles DNA as a template. The sensitivity was analysed by using plasmid DNA and laboratory infected sporozoite positive mosquito DNA, respectively. The different parts and different amounts of Anopheles DNA were added into the reaction system to investigate the influence of Anopheles DNA on the assessment.
  
  
  RESULTS
  There was no specific amplification for mosquito DNA and human blood DNA. There was specific amplification for Plasmodium 18S RNA gene recombinant plasmids and the Tm(s) of P. malariae, P. falciparum, P. ovale and P. vivax were 71.0, 72.7, 73.9 degrees C and 75.9 degrees C, respectively, which were easy to be identified. The standard curve indicated a good linear relationship between the cycle threshold (Ct) and template concentration (r = -0.99). The sensitivity was 50 copies of plasmid DNA or one sporozoite positive mosquito DNA diluted by 32 times of mosquito DNA. Anopheles DNA could inhibite the FQ-PCR reaction. The Ct value of amplification showed a good reproducibility both within the same experiment and among different experiments.
  
  
  CONCLUSIONS
  The novel SYBR Green I based FQ-PCR method developed in this study shows a high sensitivity and specificity and it can be used for quantitative detection and species identification of sporozoites in mosquitoes.
authors.affiliation: Jiangsu Institute of Parasitic Diseases, Key Laboratory on Technology for Parasitic Disease Prevention and Control, Ministry of Health, Wuxi 214064, China.
authors.forename: Yao-Bao
authors.initials: YB
authors.lastname: Liu
authors.forename: Hua-Yun
authors.initials: HY
authors.lastname: Zhou
authors.forename: Sheng-Qiang
authors.initials: SQ
authors.lastname: Wang
authors.forename: Ju-Lin
authors.initials: JL
authors.lastname: Li
authors.forename: Han-Wu
authors.initials: HW
authors.lastname: Zhu
authors.forename: Guo-Ding
authors.initials: GD
authors.lastname: Zhu
authors.forename: Ya-Ping
authors.initials: YP
authors.lastname: Gu
authors.forename: Wei-Ming
authors.initials: WM
authors.lastname: Wang
authors.forename: Jun
authors.initials: J
authors.lastname: Cao
authors.forename: Qi
authors.initials: Q
authors.lastname: Gao
mesh_terms: D000818:Animals; D000852:Anopheles / Q000235:genetics / Q000469:parasitology*; D004247:DNA / Q000032:analysis; D005453:Fluorescence; D010961:Plasmodium / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012680:Sensitivity and Specificity; D034101:Sporozoites / Q000166:cytology*
chemical_list: D012337:RNA, Ribosomal, 18S; D004247:DNA