title: Species-specific identification of ruminant components contaminating industrial crude porcine heparin using real-time fluorescent qualitative and quantitative PCR.
abstract: Ever since the emergence of bovine spongiform encephalopathy, the source of pharmaceutical heparin has been restricted to porcine intestinal mucosa. In this project, two real-time fluorescent PCR methods were developed to assist with quality control analysis. The first is a qualitative method which relies on SYBR Green I chemistry to confirm the porcine origin of industrial crude porcine heparin (ICPH), identify any ruminant contaminants, and generally control purity. The second is based on TaqMan chemistry and is able to quantitatively identify porcine, bovine, caprine, and ovine components and contaminants in ICPH. By targeting mitochondrial DNA, both PCR systems showed a detection limit of 1 pg DNA and amplification efficiencies ranging between 96% and 102%. Moreover, quantitative PCR showed a detection limit of 0.02 ppm in samples comprising porcine, bovine, caprine, and ovine DNA. The results of qualitative PCR over 27 ICPH samples showed that all samples were porcine in origin and that 17 had ruminant contaminants. The results of quantitative PCR further showed that out of all 17 samples with ruminant contaminants, seven samples had bovine, ovine, and caprine contaminants, two samples had bovine and ovine contaminants, and eight samples had only ovine contaminants. In conclusion, the qualitative PCR system was found to be a relatively inexpensive, rapid, and flexible method of identifying the porcine origin of and ruminant contaminants in ICPH, while the quantitative PCR was found suitable to accurately analyze the components and contaminants in detail. Both methods are suitable for routine control assays for the evaluation of ICPH purity and origins of contaminants.
authors.affiliation: Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Huang
authors.forename: Ting
authors.initials: T
authors.lastname: Xu
authors.forename: Gui-Yu
authors.initials: GY
authors.lastname: Wang
authors.forename: Jun-Fu
authors.initials: JF
authors.lastname: Huang
authors.forename: Han
authors.initials: H
authors.lastname: Xia
authors.forename: Richard
authors.initials: R
authors.lastname: Yin
authors.forename: Angie
authors.initials: A
authors.lastname: Tang
authors.forename: Wei-Ling
authors.initials: WL
authors.lastname: Fu
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000737:chemistry*; D001483:Base Sequence; D002417:Cattle; D004272:DNA, Mitochondrial / Q000032:analysis*; D004340:Drug Contamination; D006041:Goats; D006493:Heparin / Q000737:chemistry*; D057230:Limit of Detection; D016133:Polymerase Chain Reaction / Q000379:methods*; D012418:Ruminants; D012756:Sheep; D013045:Species Specificity; D013552:Swine
chemical_list: D000925:Anticoagulants; D004272:DNA, Mitochondrial; D006493:Heparin