title: Capture and Ligation Probe-PCR (CLIP-PCR) for Molecular Screening, with Application to Active Malaria Surveillance for Elimination.
abstract: BACKGROUND
  Malaria control programs have achieved remarkable success during the past decade. Nonetheless, sensitive and affordable methods for active screening of malaria parasites in low-transmission settings remain urgently needed.
  
  
  METHODS
  We developed a molecular screening method, capture and ligation probe-PCR (CLIP-PCR), which achieved the sensitivity of reverse-transcription PCR but eliminated the reliance on RNA purification and reverse transcription. In this method, 18S rRNA of genus Plasmodium is released from blood, captured onto 96-well plates, and quantified by the amount of ligated probes that bind continuously to it. We first used laboratory-prepared samples to test the method across a range of parasite densities and pool sizes, then applied the method to an active screening of 3358 dried blood spot samples collected from 3 low-endemic areas in China.
  
  
  RESULTS
  Plasmodium falciparum diluted in whole blood lysate could be detected at a concentration as low as 0.01 parasites/μL, and a pool size of ≤36 did not significantly affect assay performance. When coupled with a matrix pooling strategy, the assay drastically increased throughput to thousands of samples per run while reducing the assay cost to cents per sample. In the active screening, CLIP-PCR identified 14 infections, including 4 asymptomatic ones, with <500 tests, costing <US$0.60 for each sample. All positive results were confirmed by standard quantitative PCR.
  
  
  CONCLUSIONS
  CLIP-PCR, by use of dried blood spots with a pooling strategy, efficiently offers a highly sensitive and high-throughput approach to detect asymptomatic submicroscopic infections with reduced cost and labor, making it an ideal tool for large-scale malaria surveillance in elimination settings.
authors.affiliation: Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing, China.
authors.forename: Zhibin
authors.initials: Z
authors.lastname: Cheng
authors.affiliation: National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, WHO Collaborating Center for Malaria, Schistosomiasis, and Filariasis, Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai, China;
authors.forename: Duoquan
authors.initials: D
authors.lastname: Wang
authors.affiliation: Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing, China.
authors.forename: Xiaoyi
authors.initials: X
authors.lastname: Tian
authors.affiliation: Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Yunnan Institute of Parasitic Diseases, Puer, Yunnan, China. zhizheng10@yahoo.com sxdsxyab@163.com ningxiao116@126.com.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Sun
authors.affiliation: National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, WHO Collaborating Center for Malaria, Schistosomiasis, and Filariasis, Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai, China; zhizheng10@yahoo.com sxdsxyab@163.com ningxiao116@126.com.
authors.forename: Ning
authors.initials: N
authors.lastname: Xiao
authors.affiliation: Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing, China. zhizheng10@yahoo.com sxdsxyab@163.com ningxiao116@126.com.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Zheng
mesh_terms: D002681:China; D059788:Dried Blood Spot Testing / Q000379:methods; D006801:Humans; D057230:Limit of Detection; D008288:Malaria / Q000175:diagnosis*; D008403:Mass Screening / Q000379:methods*; D025202:Molecular Diagnostic Techniques / Q000379:methods; D010961:Plasmodium / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012337:RNA, Ribosomal, 18S
chemical_list: D012337:RNA, Ribosomal, 18S