›› 2013, Vol. 31 ›› Issue (3): 4-180-184.

Previous Articles     Next Articles

Development of an IMS-qPCR Method for Detection of Cryptosporidium parvum in Water

GAO Shan-shan1,2, WU Shao-qiang3, LUO Jing1, WANG Cheng-min1, ZHANG Min1, ZHAO Bao-hua2, HE Hong-xuan1 *   

  1. 1 National Research Center for Wildlife Diseases, Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101，China；2 College of Life Science, Hebei Normal University, Shijiazhuang 050016，China；3 Institute of Animal Quarantine, Chinese Academy of Inspection and Quarantine, Beijing 100123, China
  • Online:2013-06-30 Published:2013-07-17

PDF (PC)

Abstract: Objective  To develop a detection method for Cryptosporidium parvum oocysts from water samples, which combined immunomagnetic separation （IMS） with Taqman real-time PCR （qPCR）.  Methods  Conditions of sepa-ration and enrichment of IMS method by using specific streptavidin magnetic beads coated with monoclonal antibodies Cp23 directed against C. parvum oocysts were optimized. Special primers of PCR and Taqman probes were designed referring to the 18S rDNA gene of C. parvum（GenBank Accession No. AB513881.1）. The conserved genes were amplified from genomic DNA of C. parvum, and then cloned into Peasy-T1 vector. Tenfold dilutions of positive plasmids（104-108 copy/μl） were used to construct a standard curves by Taqman qPCR. The specificity of the assay was determined using genomic DNA of C. baileyi, Toxoplasma gondii, C. canis and Escherichia coli. The sensitivity of this assay was evaluated by analyzing 10-fold serially dilutions of plasmids ranging from 100 to 108 copy/μl. Both IMS-qPCR assay and indirect immunofluorescent-antibody assay（IFA） were applied to detect 50 water samples collected from the dairy cattle farms in Hebei.  Results  The optimal incubation concentration and time of antibody Cp23 were 20 ng/ml and 30 min, respectively, and the catching time was 30 min, the recovery rate was more than 95%. PCR product was 272 bp, and identified by restriction enzyme digestion and nucleotide sequencing. There was a good linear relationship between the standard plasmids and Ct value（correlation r2=0.996 1） of the Taqman qPCR. No cross-reactivity was observed with C. baileyi, T. gondii, C. canis and E. coli. The sensitivity of C. parvum-specific assay was 10 copy/μl. Compared with IFA as golden standard method, the specificity and sensitivity of IMS-qPCR for 50 water samples was 100%（18/18） and 93.8%（30/32）, respectively.  Conclution  The IMS-qPCR assay can be used to specifically detect C. parvum oocysts in water samples.

Key words: Cryptosporidium parvum, Immunomagnetic-bead, Taqman real-time PCR, 18S rDNA gene